Description

THYMIDINE KINASE MUTANTS

Technical Field

The present invention relates generally to mutant enzymes of the HerpesΛ'iridae and, more specifically, to compositions and methods which utilize thymidine kinase mutants.

Back-Ground of the Invention Although many bacterial diseases are, in general, easily treated with antibiotics, very few effective treatments exist for many viral, parasitic, cancerous, and genetic diseases. Cancer, for example, may be treated by surgical resection of a solid tumor. Nevertheless, a majority of patients with solid tumors also possess micrometastases beyond the primary tumor site. If treated with surgery alone, approximately 70% of these patients will experience recurrence of the cancer. Thus, cancer accounts for one-fifth of the total mortality in the United States, and is the second leading cause of death.

In addition to surgery, many cancers are now also treated with a combination of therapies involving cytotoxic chemotherapeutic drugs (e.g., vincristine, vinblastine, cisplatin, methotrexate, 5-FU, etc.) and/or radiation therapy. One difficulty with this approach, however, is that radiotherapeutic and chemotherapeutic agents are toxic to normal tissues, and often create life-threatening side effects. In addition, these approaches often have extremely high failure/remission rates (up to 90% depending upon the type of cancer). Numerous other methods have been attempted in order to bolster or augment an individual's own immune system in order to eliminate cancer cells. For example, some scientists have utilized bacterial or viral components as adjuvants, in order to stimulate the immune system to destroy tumor cells. Such agents have generally been useful as adjuvants and as nonspecific stimulants in animal tumor models, but have not yet proved to be generally effective in humans.

Lymphokines have also been utilized in the treatment of cancer (as well as viral and parasitic diseases), in order to stimulate or affect specific immune cells in the

generation of an immune response One group, for example, utilized the lymphokine Interleukin-2 in order to stimulate peripheral blood cells in order to expand and produce large quantities of cells which are cytotoxic to tumor cells (Rosenberg et al , N. Engl. J. Med. 313 1485-1492, 1985) Others have suggested the use of antibody-mediated treatment using specific monoclonal antibodies or "magic bullets" in order to specifically target and kill tumor cells (Dillman, "Antibody Therapy," Principles of Cancer Biotherapy, Oldham (ed ), Raven Press, Ltd , New York, 1987) One difficulty, however, is that most monoclonal antibodies are of murine origin, and thus hypersensitivity against the murine antibody may limit its efficacy, particularly after repeated therapies Common side effects include fever, sweats and chills, skin rashes, arthritis, and nerve palsies

One approach which has recently garnered significant interest is the use of gene therapy, which has been utilized to treat not only genetic diseases, but viral and cancerous diseases as well (see PCT Publication Nos WO 91/02805, EPO 415,731, and WO 90/07936) Briefly, specifically designed vectors which have been derived from viruses are used to deliver particular genetic information into cells Such genetic information may itself be useful to block expression of damaging proteins or antigens (e.g., antisense therapy), may encode proteins which are toxic and kill selected cells, may encode therapeutic proteins which bolster a cell's immune response, or encode proteins which replace inactive or nonexistent proteins

One protein which has recently been suggested for use in such therapies is the type 1 Herpes Simplex Virus thymidine kinase (HSVTK-1) Briefly, thymidine kinase is a salvage pathway enzyme which phosphorylates natural nucleoside substrates as well as nucleoside analogues (see Balasubramaniam et al , J. of Gen. Vir. 77:2979- 2987, 1990) This protein may be utilized therapeutically by introducing a retroviral vector which expresses the protein into the cell, followed by administration of a nucleoside analogue such as acyclovir or ganciclovir HSVTK-1 then phosphorylates the nucleoside analogue, creating a toxic product capable of killing the host cell Thus, use of retroviral vectors which express HSVTK has been suggested for not only the treatment of cancers, but for other diseases as well

The present invention provides novel thymidine kinase mutants with increased biological activities which are suitable for a variety of applications, such as gene therapy, and further provides other, related advantages

Summary of the Invention

Briefly stated, the present invention provides compositions and methods which utilize Herpeswndae thymidine kinase mutants Within one aspect of the present

invention, isolated nucleic acid molecules which encode Herpesvi dae thymidine kinase enzymes comprising one or more mutations are provided, at least one of the mutations encoding an amino acid substitution upstream from a DRH nucleoside binding site which increases a biological activity of the thymidine kinase, as compared to unmutated thymidine kinase. Within another aspect, the mutation is an amino acid substitution within a DRH nucleoside binding site which increases a biological activity of said thymidine kinase, as compared to unmutated thymidine kinase. Within yet another aspect, isolated nucleic acid molecules are provided encoding a Herpesλ'iridae thymidine kinase enzyme comprising one or more mutations, at least one of the mutations being an amino acid substitution downstream from a DRH nucleoside binding site (e.g., 4, 5 or 6 nucleotides downstream) which increases a biological activity of the thymidine kinase, as compared to unmutated thymidine kinase. Representative examples of suitable Herpesviridae thymidine kinase enzymes include Herpes Simplex Virus Type 1 thymidine kinase, Herpes Simplex Virus Type 2 thymidine kinase, Varicella Zoster Virus thymidine kinase, and marmoset herpesvirus, feline herpesvirus type 1, pseudorabies virus, equine herpesvirus type 1, bovine herpesvirus type 1, turkey herpesvirus, Marek's disease virus, herpesvirus saimiri and Epstein-Barr virus thymidine kinases. Within other embodiments, the thymidine kinase may be a primate herpesvirus thymidine kinase, or a non-primate herpesvirus thymidine kinase, such as an avian herpesvirus thymidine kinase.

A wide variety of mutations are contemplated within the context of the present invention. For example, within one embodiment mutations which encode one or more amino acid substitutions from 1 to 7 amino acids upstream from the DRH nucleoside binding site are described. Within a preferred embodiment, the amino acid which is one position upstream from the DRH nucleoside binding site is substituted with an amino acid selected from the group consisting of valine, leucine, cysteine and isoleucine. Within another preferred embodiment, the amino acid alanine is substituted for the amino acid which is present seven amino acids upstream from the DRH nucleoside binding site. Within other embodiments, glutamic acid may be substituted for aspartic acid in the DRH nucleoside binding site. Within another embodiment, a histidine residue may be substituted for arginine in the DRH nucleoside binding site. Within other embodiments, the thymidine kinase enzyme is truncated, and yet retains biological activity.

Within further embodiments of the invention, isolated nucleic acid molecules are provided which encode a thymidine kinase enzyme capable of phosphorylating a nucleoside analogue at least one-fold over the phosphorylation of the nucleoside analogue by a wild-type thymidine kinase enzyme. Within other

embodiments, the thymidine kinase enzyme phosphorylates a nucleoside analogue at least x-fold over the phosphorylation of a nucleoside analogue by a wild-type thymidine kinase enzyme, wherein x is selected from the group consisting of 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5. Within yet another embodiment, the thymidine kinase enzyme is capable of phosphorylating a nucleoside analogue, wherein

(TKmNAp) /(TKmTp) z >

(TKwtNA _P ) / (TKwtT _P )

and wherein TK _nι NA _p is the rate of phosphorylation of a nucleoside analogue by a thymidine kinase mutant, TK _ιn T _p is the rate of phosphorylation of thymidine by a thymidine kinase mutant, TK _w1 NA _p is the rate of phosphorylation of a nucleoside analogue by an unmutated thymidine kinase enzyme, TK _vvt T _p is the rate of phosphorylation of a thymidine kinase enzyme by an unmutated thymidine kinase enzyme, and z is selected from the group consisting of l, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5. Representative examples of suitable nucleoside analogues include ganciclovir, acyclovir, famciclovir, buciclovir, penciclovir, valciclovir, trifluorothymidine, l-[2-deoxy, 2-fluoro, beta-D-arabino furanosyl]-5-iodouracil, ara-A, araT 1 -beta-D-arabinofuranoxyl thymine, 5-ethyl-2'-deoxyuridine, 5-iodo-5'-amino-2, 5'-dideoxyuridine, idoxuridine, AZT, AIU, dideoxycytidine and AraC. Particularly preferred mutant thymidine kinases for the increased phosphorylation of nucleoside analogues include those wherein the enzyme is a type 1 Herpes Simplex Virus thymidine kinase, and further, wherein the amino acid alanine is substituted for proline at position 155, and the amino acid valine is substituted for phenylalanine at position 161. Within other embodiments, isoleucine may be substituted for phenylalanine at position 161, and cysteine for phenylalanine at position 161.

Within other aspects of the present invention, mutant thymidine kinase enzymes which are encoded by the above-described nucleic acid molecules are provided, as well as vectors which are capable of expressing such molecules. Within one aspect, expression vectors are provided comprising a promoter operably linked to a nucleic acid molecule of the present invention. Within a preferred aspect, the vector is a viral vector capable of directing the expression of a nucleic acid molecule as described above. Representative examples of such viral vectors include herpes simplex viral vectors, adenoviral vectors, adenovirus-associated viral vectors, pox vectors, parvoviral vectors, baculovirus vectors and retroviral vectors. Within another aspect, viral vectors are provided which are capable of directing the expression of a nucleic acid molecule which encodes a thymidine kinase enzyme comprising one or more mutations, at least one of

the mutations encoding an amino acid substitution which increases a biological activity of thymidine kinase, as compared to unmutated thymidine kinase.

A wide variety of promoters may be utilized in the present invention, including, for example, promoters such as the MoMLV LTR, RSV LTR, Friend MuLv LTR, Adenoviral promoter, Neomycin phosphotransferase promoter/enhancer, late parvovirus promoter, Herpes TK promoter, SV40 promoter, Metallothionen Ila gene enhancer/promoter, Cytomegalovirus Immediate Early Promoter, Cytomegalovirus Immediate Late Promoter, as well as tissue-specific promoters such as the tyrosinase related promoters (TRP-1 and TRP-2), DF3 enhancer , SLPI promoter (secretory leucoprotease inhibitor ~ expressed in many types of carcinomas), TRS (tissue specific regulatory sequences), tyrosine hydroxylase promoter, adipocyte P2 promoter, PEPCK promoter, CEA promoter, α fetoprotein promoter, whey acidic promoter, and casein promoter. Within related aspects, the above-described vectors may be provided as pharmaceutical compositions, along with a pharmaceutically acceptable carrier or diluent.

Within other aspects of the present invention, host cells are provided which carry one of the above-described vectors. Representative examples of such cells include human cells, dog cells, monkey cells, rat cells, and mouse cells.

Within other aspects of the present invention, methods are provided for inhibiting a pathogenic agent in a warm-blooded animal, comprising the step of administering to a warm-blooded animal a vector as described above, such that the pathogenic agent is inhibited. Within various embodiments, the vector may be administered /// vivo, or to cells ex vivo, which are then transplanted (or re-transplanted) in the animal. Within other embodiments, the pathogenic agent may be viruses, bacteria, parasites, tumor cells, or autoreactive immune cells.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.

Brief Description of the Drawings

Figure 1 is a schematic outline which depicts a strategy for construction of a random nucleotide-containing library, and selection of TK mutants. Figure 2 is a photograph which shows selection of TK and AZT mutants.

Figure 3 depicts the nucleic acid and amino acid sequences of: Wild- type, TKF105, TKI208, and TKF2 TK for codons 165 to 175.

Figure 4 is a series of graphs which depict the thermostability of wild- type TK and TK mutants

Figure 5 is a graph which depicts heat-inactivation profiles for m vitro translated wild-type and TKF2 thymidine kinase Figure 6 is an autoradiograph of SDS/PAGE-fractionated in vitro translated products (wild-type and TKF2)

Figure 7 is an autoradiograph of ⁵ S-radiolabeled cell-free translation products subjected to SDS-PAGE and TCA-precipitable counts

Figures 8A and 8B are two graphs which illustrate a time course analysis of high activity(A) and low activity (B) mutants produced in a rabbit reticulocyte lysate cell-free translation system

Figures 9A and 9B are two graphs which show the thermal stability of high activity (A) and low activity (B) TK mutants

Figure 10 is a bar graph which depicts a phosphorylation of nucleosides and nucleoside analogs by mutant and wild-type thymidine kinases

Figure 1 1 is a bar graph which indicates TK activity of wild-type, TKF36, and dummy (pMDC) plasmids

Figure 12 is a graph which indicates the thymidine uptake activity of cells containing TKF36, TKF52, wild-type plasmid, TKF99, or dummy plasmids (pMDC) over time

Figure 13 is a schematic illustration of one representative example of gene therapy utilizing an HSVTK mutant

Figure 14 is an illustration which depicts the nucleotides which were randomized in the LIF-ALL library, as well as the results of selection Figure 15 is a table which shows amino acid substitutions of selected and unselected clones

Figure 16 is a table which shows the number of mutants selected from the LIF-ALL library which were sensitive to GCV or ACV

Figure 17 is a table which shows nucleotide changes in selected TK mutants

Figure 18 is a table which shows the amino acid sequence at positions 159- 161 and 168-170, and phosphorylation level of several mutant TKs

Figure 19 is a graph which shows the survival of cells grown on GCV and transfected with various TK mutants Figure 20 is a graph which shows the survival of cells grown on ACV and transfected with various TK mutants

Figure 21 illustrates a nucleotide sequence and deduced amino acid sequence representative of a human guanylate kinase.

Figure 22 illustrates a nucleotide sequence and deduced amino acid sequence of a representative murine guanylate kinase.

Detailed Description of the Invention

DEFINITIONS Prior to setting forth the invention, it may be helpful to an understanding thereof to first set forth definitions of certain terms that will be used hereinafter.

"Vector" refers to an assembly which is capable of directing the expression of the mutant Ik gene, as well as any additional sequence(s) or gene(s) of interest. The vector must include transcriptional promoter/enhancer elements, as well as another sequence which, when transcribed, is operably linked to the tk gene and/or other gene of interest. The vector may be composed of either deoxyribonucleic acids ("DNA"), ribonucleic acids ("RNA"), or a combination of the two (e.g., a DNA-RNA chimeric). Optionally, the vector may include a polyadenylation sequence, one or more restriction sites, as well as one or more selectable markers such as neomycin phosphotransferase or hygromycin phosphotransferase. Additionally, depending on the host cell chosen and the vector employed, other genetic elements such as an origin of replication, additional nucleic acid restriction sites, enhancers, sequences conferring inducibility of transcription, and selectable markers, may also be incorporated into the vectors described herein.

"Tissue-specific promoter" refers to transcriptional promoter/enhancer elements which control gene expression in a limited number of tissues, or in a single tissue. Representative examples of tissue-specific promoters include the tyrosine hydroxylase promoter, adipocyte P2 promoter, PEPCK promoter, α fetoprotein promoter, whey acidic promoter, and casein promoter.

"Biological activity" of thymidine kinase refers to the ability of the thymidine kinase enzyme to phosphorylate nucleosides (e.g., dT) and nucleoside analogues such as ganciclovir (9-{ [2-hydroxy-l-(hydroxymethyl)ethoxyl methyl} guanosine), famciclovir, buciclovir, penciclovir, valciclovir, acyclovir (9-[2-hydroxy ethoxy)methyl] guanosine), trifluorothymidine, l-[2-deoxy, 2-fluoro, beta-D-arabino furanosyl]-5-iodouracil, ara-A (adenosine arabinoside, vivarabine), 1-beta-D- arabinofuranoxyl thymine, 5-ethyl-2'-deoxyuridine, 5-iodo-5'-amino-2,5'-dideoxyuridine, idoxuridine (5-iodo-2'-deoxyuridine), AZT (3' azido-3' thymidine), ddC (dideoxycytidine), AIU (5-iodo-5' amino 2', 5'-dideoxyuridine) and AraC (cytidine

arabinoside). As utilized herein, a thymidine kinase mutant is considered to have "increased biological activity" if the level or rate of activity increases at least "y" fold over unmutated thymidine kinase, wherein y is selected from the group consisting of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5. Within preferred embodiments, thymidine kinase mutants are considered to have increased biological activity when

wherein TK _nι NA _p is the rate of phosphorylation of a nucleoside analogue by a thymidine kinase mutant, TK T _p is the rate of phosphorylation of thymidine by a thymidine kinase mutant, TK _Vt NA _p is the rate of phosphorylation of a nucleoside analogue by an unmutated thymidine kinase enzyme, TK _%vt T _p is the rate of phosphorylation of a thymidine kinase enzyme by an unmutated thymidine kinase enzyme, and z is selected from the group consisting of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5. In addition to the ability of thymidine kinase to phosphorylate nucleosides and nucleoside analogues, the phrase "biological activity" should also be understood to refer to other biological properties of thymidine kinase, such as protein stability (e.g., as measured by resistance to proteolytic enzyme degradation by enzymes such as trypsin), and thermostability (e.g., maintenance of nucleoside analogue phosphorylation upon increases in temperature).

"Pathogenic agent" refers to either a foreign organism which is responsible for a disease state, or an "altered" cell which is responsible for a disease state. Representative examples of pathogenic agents include foreign organisms such as viruses, bacteria and parasites, as well as altered cells such as tumor cells and autoreactive immune cells. As utilized herein, a pathogenic agent is considered to be "inhibited" if either the growth or spread of the pathogenic agent is slowed, or if the pathogenic agent itself is destroyed.

As noted above, the present invention provides compositions and methods which utilize Herpe.sviridae thymidine kinase mutants. Briefly, thymidine kinase mutants of the present invention may be prepared from a wide variety of Herpesviridae thymidine kinases, including for example both primate herpesviruses, and nonprimate herpesviruses such as avian herpesviruses. Representative examples of suitable herpesviruses include Herpes Simplex Virus Type 1 (McKnight et al., N c. Acids Res 5:5949-5964, 1980), Herpes Simplex Virus Type 2 (Swain and Galloway, J. Virol. 46.1045- 1050, 1983), Varicella Zoster Virus (Davison and Scott, J. Gen. Virol.

67 1759-1816, 1986), marmoset herpesvirus (Otsuka and Kit, Virology 755 316-330, 1984), feline herpesvirus type 1 (Nunberg et al , J. Virol (53 3240-3249, 1989), pseudorabies virus (Kit and Kit, U.S Patent No 4,514,497, 1985), equine herpesvirus type 1 (Robertson and Whalley, Nuc. Acids Res. 76 1 1303-1 1317, 1988), bovine herpesvirus type 1 (Mittal and Field, J. Virol 70.2901-2918, 1989), turkey herpesvirus (Martin et al., J. Virol. 63.2847-2852, 1989), Marek's disease virus (Scott et al , J. Gen. Virol. 70 3055-3065, 1989), herpesvirus saimiri (Honess et al , J. Gen. Virol. 70.3003- 3013, 1989) and Epstein-Barr virus (Baer et al , Nature (London) 310 207-311, 1984) Such herpesviruses may be readily obtained from commercial sources such as the American Type Culture Collection ("ATCC", Rockville, Maryland) Deposits of certain of the above-identified herpesviruses may be readily obtained from the ATCC, for example ATCC No VR-539 (Herpes simplex type 1), ATCC Nos VR-734 and VR-540 (Herpes Simplex type 2), ATCC No VR-586 (Varicella Zoster Virus), ATCC No VR-783 (Infectious laryngothracheitis), ATCC Nos VR-624 VR-987, VR-2103, VR-2001 , VR-2002, VR-2175, VR-585 (Marek's disease virus), ATCC Nos VR-584B and VR-584B (turkey herpesvirus), ATCC Nos VR-631 and VR-842 (bovine herpesvirus type 1), and ATCC Nos VR-2003, VR-2229 and VR-700 (equine herpesvirus type 1) Herpesviruses may also be readily isolated and identified from naturally occurring sources (e.g., from an infected animal) Any of the above-cited herpesviruses (as well as other members of the

Herpesλ'iridae) may be readily utilized in order to prepare thymidine kinase mutants of the present invention Briefly, the primary region which is believed to be responsible for nucleoside binding is found in the area surrounding Sites 3 and 4 (see Balasubramaniam et al , J. Gen. Vir. 77 2979-2987, 1990) These sites are characterized by highly conserved regions, and consist of the motif -DRH- (for Site 3), and -C(Y/F)P- (for Site 4) Although the numbering of nucleic acids may change substantially from one herpesvirus to another, as utilized herein, reference will be made to positions relative to the DRH nucleoside binding site For example, for Herpes Simplex Virus type 1 (McKnight et al , Nucl. Acids Res. 8 5949-5964, 1980), this site may be found at amino acids 162, 163 and 164 DRH nucleoside binding sites for other representative herpesviruses include 163, 164 and 165 for Herpes Simplex Virus type 2, 129, 130 and 131 for Varicella Zoster Virus, 130, 131 and 132 for Marmoset herpesvirus, and 148, 149 and 150 for Epstein-Barr virus

For herpesviruses which have not been previously sequenced, the DRH nucleoside binding site may be readily identified by sequencing the nucleic acid sequence encoding the enzyme, or by amino acid sequencing the enzyme itself, followed by alignment of the sequence to other known herpesvirus sequences (see Balasubramanian,

ibid ) To the extent that more than one -DRH- motif is identified, the proper motif may be readily identified by, for example, crystal structure analysis (Sanderson et al , J. Mol. Biol. 202.917-919, 1988, Montfort et al , Biochem 29(30) 6964-6977, 1990, Hardy et al , Science 235 448-455, 1987), or crosslinking studies (Knoll et al , Bioch. Biophys. Ada 7727.252-260, 1992)

The thymidine kinase gene from the selected herpesvirus may then be readily isolated and mutated as described below, in order to construct nucleic acid molecules encoding a thymidine kinase enzyme comprising one or more mutations which increases a biological activity of the thymidine kinase, as compared to unmutated thymidine kinase As utilized herein, it should be understood that "unmutated thymidine kinase" refers to native or wild-type thymidine kinase such as that described by McKnight et al (Nitcl. Acid Res. 8 5949-5964, 1980) The biological activity of such kinases may be readily determined utilizing any of the assays which are described herein, including for example, determination of the rate of nucleoside analogue uptake, determination of the rate of nucleoside or nucleoside analogue phosphorylation (see Examples 2-4) In addition, thymidine kinase mutants may be readily selected which are characterized by other biological properties, such as thermostability (see Examples 2-4), and protein stability

A wide variety of thymidine kinase mutations are contemplated within the scope of the present invention For example, within one embodiment of the invention, isolated nucleic acid molecules are provided which encode a Herpesvindae thymidine kinase enzyme comprising one or more mutations, at least one of the mutations encoding an amino acid substitution upstream (5') from the DRH nucleoside binding site Briefly, any amino acid position upstream (5') of the DRH nucleoside binding site may be substituted for another amino acid given the disclosure provided herein Representative amino acids which may be substituted (and their one letter symbols) include alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V)

Within one embodiment of the invention, nucleic acid molecules are provided which encode thymidine kinase mutants with one or more amino acid substitutions from 1 to 7 amino acids upstream from the -DRH- nucleoside binding site Within one embodiment, the amino acid which is one position upstream from the DRH nucleoside binding site is substituted with another amino acid, including for example, alanine (A), arginine (R), asparagine (N), aspartic acid (D), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), lysine (K), methionine (M), phenylalanine (F), proline

(P), serine (S), threonine (T), tryptophan (W), and tyrosine (Y). Particularly preferred substitutions in this regard include valine (V), leucine (L), cysteine (C) and isoleucine

(I).

Within another embodiment, amino acids which are from two to six positions upstream from the DRH nucleoside binding site are substituted with other amino acids, including for example, alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). Within other embodiments, the amino acid which is seven positions upstream from the DRH nucleoside binding site is substituted with other amino acids, including for example, arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). A particularly preferred substitution in this regard includes alanine (A).

Within other aspects of the invention, nucleic acid molecules are provided which encode thymidine kinase enzymes comprising one or more mutations which increase a biological activity of the thymidine kinase enzyme, wherein the mutation encodes an amino acid substitution w positions upstream from the DRH nucleoside binding site, wherein "w" is any integer greater than 8 (and generally less than 162). Representative amino acids which may be substituted include, for example, alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

Within another aspect of the present invention, nucleic acid molecules are provided which encode thymidine kinase mutants with one or more amino acid substitutions within the -DRH- nucleoside binding site. Within one embodiment of the invention, the asparatic acid in the -DRH-nucleoside binding site is substituted with other amino acids, including for example, alanine (A), arginine (R), asparagine (N), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). Within another embodiment of the invention, the arginine in the -DRH- nucleoside binding site is substituted with other amino acids, including for example, alanine (A), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid

(E), glycine (G), histidine (H), isoleucine (1), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

Within certain preferred aspects of the present invention, nucleic acid molecules are provided which encode thymidine kinase enzymes comprising one or more mutations which increase a biological activity of the thymidine kinase enzyme, wherein the mutation encodes one or more amino acid substitutions 1, 2 or 3 amino acids upstream (5') from the DRH nucleoside binding site, and/or one or more substitutions 4, 5 or 6 amino acids downstream from the DRH nucleoside binding site, or 1, 2 or 3 amino acids upstream from the CYP nucleoside binding site (see Figure 14). Specific illustrations of such mutants are described in more detail below in Example 8 and in Figure 18.

Within yet another embodiment of the invention, the histidine in the - DRH- nucleoside binding site is substituted with any other amino acid, including for example, alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

Within other aspects of the present invention, nucleic acid molecules are provided which encode thymidine kinase enzymes comprising one or more mutations which increase a biological activity of the thymidine kinase enzyme, wherein the mutation encodes an amino acid substitution from 1 to 11 positions downstream (3') from the DRH nucleoside binding site. These amino acids may be substituted with other amino acids, including for example, alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

Within another aspect of the present invention, nucleic acid molecules are provided which encode thymidine kinase enzymes comprising one or more mutations which increase a biological activity of the thymidine kinase enzyme, wherein the mutation encodes an amino acid substitution from 12 to "v" positions downstream (3') from the DRH nucleoside binding site, wherein "v" is any integer greater than 13 (and generally less than 202). These amino acids may be readily substituted with other amino acids, including for example, alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

Within various aspects, nucleic acid molecules of the present invention may encode several amino acid mutations. For example, within one preferred embodiment, thymidine kinase mutants are provided which encode mutations with 1, 2, 3, 4, 5 or more amino acid substitutions, as well as in-frame deletions. Particularly preferred mutants in this regard include P155A/F161V, P155A/F161C, P155A/D162E, I160L/F161L/A168V/L169M and F161L/A168V/L169Y/L170C.

Any of the above-described thymidine kinase mutants may be readily screened for increased biological activity, given the assays described herein and below in the Examples.

CONSTRUCTION OF THYMIDINE KINASE MUTANTS Thymidine kinase mutants of the present invention may be constructed using a wide variety of techniques. For example, mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes a derivative having the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Deletion or truncation derivatives of thymidine kinase mutants may also be constructed by utilizing convenient restriction endonuclease sites adjacent to the desired deletion. Subsequent to restriction, overhangs may be filled in, and the DNA religated. Exemplary methods of making the alterations set forth above are disclosed by Sambrook et al. (Molecular cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, 1989).

Thymidine kinase mutants may also be constructed utilizing techniques of PCR mutagenesis, chemical mutagenesis (Drinkwater and Klinedinst, PNAS 53:3402- 3406, 1986), by forced nucleotide misincorporation (e.g., Liao and Wise Gene 55:107- 1 1 1, 1990), or by use of randomly mutagenized oligonucleotides (Horwitz et al., Genome 3: 1 12-1 17, 1989). Particularly preferred methods for constructing thymidine kinase mutants are set forth in more detail below in Examples 1 through 4.

HSVTK VECTORS

Within the context of the present invention, the term "thymidine kinase mutant" should be understood to include not only the specific protein described herein

(as well as the nucleic acid sequences which encode these proteins), but derivatives thereof which may include various structural forms of the primary protein which retain

biological activity For example, a thymidine kinase mutant may be in the form of acidic or basic salts, or in neutral form In addition, individual amino acid residues may be modified by oxidation or reduction Furthermore, various substitutions, deletions, or additions may be made to the amino acid or nucleic acid sequences, the net effect of which is to retain or further enhance the increased biological activity of the mutant Due to code degeneracy, for example, there may be considerable variation in nucleotide sequences encoding the same amino acid sequence

Other derivatives of the thymidine kinase mutants disclosed herein include conjugates of thymidine kinase mutants along with other proteins or polypeptides This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins which may be added to facilitate purification or identification of thymidine kinase mutants (see U S Patent No 4,851,341, see also, Hopp et al., Bio/Technology 6 1204, 1988 )

Within one embodiment of the present invention, truncated derivatives of thymidine kinase mutants are provided For example, site-directed mutagenesis may be readily performed in order to delete the N-terminal 45 amino acids of a thymidine kinase mutant, thereby constructing a truncated form of the mutant which retains its biological activity

Mutations in nucleotide sequences constructed for expression of derivatives of thymidine kinase mutants should preserve the reading frame phase of the coding sequences Furthermore, the mutations will preferably not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, which would adversely affect translation of the receptor mRNA Such derivatives may be readily constructed using a wide variety of techniques, including those discussed above

As noted above, the present invention provides recombinant vectors which include either synthetic, or cDNA-derived nucleic acid molecules encoding thymidine kinase mutants or derivatives thereof, which are operably linked to suitable transcriptional or translational regulatory elements Suitable regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, insect, or plant genes Selection of appropriate regulatory elements is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art Examples of regulatory elements include a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal

Nucleic acid molecules which encode any of the thymidine kinase mutants described above may be readily expressed by a wide variety of prokaryotic and

eukaryotic host cells, including bacterial, mammalian, yeast or other fungi, viral, insect, or plant cells. Methods for transforming or transfecting such cells to express foreign DNA are well known in the art (see, e.g., Itakura et al., U.S. Patent No. 4,704,362; Hinnen et al., PNAS USA 75: 1929-1933, 1978; Murray et al., U.S. Patent No. 4,801,542; Upshall et al., U.S. Patent No. 4,935,349; Hagen et al., U.S. Patent No. 4,784,950; Axel et al., U.S. Patent No. 4,399,216; Goeddel et al., U.S. Patent No. 4,766,075; and Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989; for plant cells see Czako and Marton, Plant Physiol. 104: 1067- 1071 , 1994; and Paszkowski et al., Biotech. 24:387- 392, 1992).

Bacterial host cells suitable for carrying out the present invention include E. coli, B. sublilis, Salmonella typhimurium, and various species within the genus' Pseudomonas, Slreptomyces, and Slaphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art. Representative examples of bacterial host cells include DH5α (Stratagene, LaJolla, California).

Bacterial expression vectors preferably comprise a promoter which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication. Representative promoters include the β-lactamase (penicillinase) and lactose promoter system (see Chang et al.. Nature 275:615, 1978), the T7 RNA polymerase promoter (Studier et al., Meth. Enzymol. 755:60-89, 1990), the lambda promoter (Elvin et al., Gene 57: 123-126, 1990), the trp promoter (Nichols and Yanofsky, Meth. in Enzymology 707: 155, 1983) and the tac promoter (Russell et al., Gene 20: 231, 1982). Representative selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes. Many plasmids suitable for transforming host cells are well known in the art, including among others, pBR322 (see Bolivar et al., Gene 2:95, 1977), the pUC plasmids pUC18, pUC19, pUCl 18, pUCl 19 (see Messing, Meth. in Enzymology 707:20-77, 1983 and Vieira and Messing, Gene 79:259-268, 1982), and pNH8A, pNHlόa, pNH18a, and Bluescript M13 (Stratagene, La Jolla, Calif). Yeast and fungi host cells suitable for carrying out the present invention include, among others Saccharomyces pombe, Saccharomyces cerevisiae, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus. Suitable expression vectors for yeast and fungi include, among others, YC _p 50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3 (Turnbull, Bio/Technology 7:169, 1989). Protocols for the transformation of yeast are also well known to those of ordinary skill in the art. For example, transformation may be readily accomplished either by preparation of spheroplasts of yeast with DNA (.see Hinnen et al., PNAS USA

75 1929, 1978) or by treatment with alkaline salts such as LiCl (see Itoh et al., J. Bacteriology 153 163, 1983) Transformation of fungi may also be carried out using polyethylene glycol as described by Cullen et al. (Bio/Technology 5 369, 1987)

Mammalian cells suitable for carrying out the present invention include, among others COS (e.g., ATCC No CRL 1650 or 1651), BHK (e.g., ATCC No CRL 6281), CHO (ATCC No CCL 61), HeLa (e.g., ATCC No CCL 2), 293 (ATCC No 1573) and NS-1 cells Suitable expression vectors for directing expression in mammalian cells generally include a promoter, as well as other transcriptional and translational control sequences Common promoters include SV40, MMTV, metallothionein-1, adenovirus El a, Cytomegalovirus Immediate Early Promoter, and the Cytomegalovirus Immediate Late Promoter

Protocols for the transfection of mammalian cells are well known to those of ordinary skill in the art Representative methods include calcium phosphate mediated transfection, electroporation, lipofection, retroviral, adenoviral and protoplast fusion-mediated transfection (.see Sambrook et al , supra)

Thymidine kinase mutants may be prepared by culturing the host/vector systems described above, in order to express the recombinant thymidine kinase mutants Recombinantly produced thymidine kinase mutants may be further purified as described in more detail below As noted above, the present invention also provides a variety of both viral and non-viral vectors which are suitable for directing the expression of the nucleic acid molecules described above Within one aspect of the invention, viral vectors are provided which comprise a promoter that directs the expression of an isolated nucleic acid molecule which encodes a thymidine kinase mutant as described above A wide variety of promoters may be utilized within the context of the present invention, including for example, promoters such as MoMLV LTR, RSV LTR, Friend MuLV LTR, Adenoviral promoter (Ohno et al , Science 265 781-784, 1994), Neomycin phosphotransferase promoter/enhancer, late parvovirus promoter (Koering et al., Hum. Gene Therap. 5 457-463, 1994), Herpes TK promoter, SV40 promoter, Metallothionein Ila gene enhancer/promoter, Cytomegalovirus Immediate Early Promoter, and the Cytomegalovirus Immediate Late Promoter Within particularly preferred embodiments of the invention, the promoter is a tissue-specific promoter (see e.g., WO 91/02805; EP 0,415,731, and WO 90/07936) Representative examples of suitable tissue specific promoters include the tyrosinase related promoters (TRP-1 and TRP-2, Vile and Hart, Cane. Res. 53 962-967, 1993), DF3 enhancer (for breast cells, see Manome et al , Cane. Res. 5- 5408-5413, 1994), SLPI promoter (secretory leucoprotease inhibitor — expressed in many types of carcinomas, see Garver et al, Gene Therapy 1 46-50, 1994),

TRS (tissue specific regulatory sequences, see Dynan and Tjian, Nature 316: 114-778, 1985), albumin and α fetoprotein promoters (specific for normal hepatocytes and transformed hepatocytes, respectively), the carcino-embryonic antigen promoter (for use in transformed cells of the gastrointestinal tract, lung, breast and other tissues), the tyrosine hydroxylase promoter (for melanocytes), choline acetyl transferase or neuron specific enolase promoters for use in neuroblastomas, the regulatory sequence for glial fibroblastomas, the tyrosine hydroxylase promoter, c-erb B-2 promoter, PGK promoter, PEPCK promoter, whey acidic promoter (breast tissue), and casein promoter (breast tissue) and the adipocyte P2 promoter (Ross et al.. Genes & Dev. 1318-1324, 1993; and Lowell et al., Nature 366:140-142, 1993). In addition to the above-noted promoters, other viral-specific promoters (e.g., retroviral promoters (including those noted above, as well as others such as HIV promoters), hepatitis, herpes (e.g., EBV), and bacterial, fungal or parasitic (e.g., malarial) -specific promoters may be utilized in order to target a specific cell or tissue which is infected with a virus, bacteria, fungus or parasite. Thymidine kinase mutants of the present invention may be expressed from a variety of viral vectors, including for example, herpes viral vectors (e.g., U.S. Patent No. 5,288,641), retroviruses (e.g., EP 0,415,731 ; WO 90/07936; WO 91/0285, WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 93/1 1230; WO 93/10218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-961, 1993; Ram et al., Cancer Res. 53:83-88, 1993; Takamiya et al., J. Neurosci. Res 33:493-503, 1992; Baba et al., J. Neurosurg 79:729- 735, 1993), pseudotyped viruses, adenoviral vectors (e.g., WO 94/26914, WO 93/9191; Kolls et al., PNAS 97(1):215-219, 1994; Kass-Eisler et al., PNAS 90(24): 11498-502, 1993; Guzman et al., Circulation 55(6):2838-48, 1993; Guzman et al., Or. Res. 73(6): 1202-1207, 1993; Zabner et al., Cell 75(2):207-216, 1993; Li et al., Hum Gene Ther. -/(4):403-409, 1993; Caillaud et al., Eur. J. Neurosci. 5(10: 1287-1291, 1993; Vincent et al., Nat. Genet. 5(2): 130-134, 1993; Jaffe et al., Nat. Genet. 7(5):372-378, 1992; and Levrero et al, Gene 707(2): 195-202, 1991), adenovirus-associated viral vectors (Flotte et al., PNAS 90(22): 10613-10617, 1993), parvovirus vectors (Koering et al., Hum. Gene Therap. 5:457-463, 1994), baculovirus vectors, and pox virus vectors (Panicali and Paoletti, PNAS 79:4921 -49. \, 1982; and Ozaki et al., Biochem. Biophys. Res. Comm. 793(2):653-660, 1993). Within various embodiments, either the viral vector itself, or a viral particle which contains the viral vector may be utilized in the methods and compositions described below. Vectors of the present invention may contain or express a wide variety of additional nucleic acid molecules in addition to a thymidine kinase nucleic acid molecule as described above. For example, the viral vector may express a lymphokine, antisense

sequence, toxin or "replacement" protein (e.g., adenosine deaminase) Representative examples of ly phokines include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1, IL-12, IL-13, IL-14, 1L-15, GM-CSF, G-CSF, M-CSF, alpha-interferon, beta-interferon, gamma interferon, and tumor necrosis factors Representative examples of antisense sequences include antisense myc, antisense p53, antisense ras, as well as antisense sequences which block the expression or production of viruses such as HIV, HBV and HCV Representative examples of toxins include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A Within preferred embodiments of the invention, one or more genes which encode proteins that facilitate or increase the biological activity of thymidine kinase may be included with, and expressed by the vectors described herein For example, within one embodiment of the invention, nucleic acid molecules which encode DNA polymerase (e.g., a Herpes DNA plymerase) and/or guanylate kinase (Konrad, J. Biol. Chem 267(36) 25652-25655, 1992, Miller and Miller, J. Biol. Chem. 255(15) 7204- 7207, 1980) are expressed either from one or several separate promoters (e.g., from multiple internal ribosome binding sites) in addition to a thymidine kinase enzyme (either wild type, or thymidine kinase mutants as described above) Representative examples of such embodiments are set forth in more detail below in Examples 7 and 9 It should be understood that although certain nucleic acid molecules are disclosed which encode DNA polymerase or guanylate kinase, that the present invention is not so limited Indeed, as discussed above with respect to thymidine kinase mutants, a wide variety of nucleic acid molecules are considered to be included within the scope of the present invention which encode DNA polymerase or guanylate kinase activity (e.g., truncated nucleic acid molecules or nucleic acid molecules which are degenerate with respect to the encoded amino acid sequence)

Thymidine kinase mutants may also be expressed in non-human transgenic animals such as mice, rats, rabbits, sheep, dogs and pigs (see Hammer et al (Nature 315 680-683, 1985), Palmiter et al (Science 222 809-814, 1983), Brinster et al (Proc. Natl. Acad. Sci. USA 82 4438-4442, 1985), Palmiter and Brinster (Cell 41.343- 345, 1985) and U S Patent No 4,736,866) Briefly, an expression unit, including a nucleic acid molecule to be expressed together with appropriately positioned expression control sequences, is introduced into pronuclei of fertilized eggs, for example, by microinjection Integration of the injected DNA is detected by blot analysis of DNA from tissue samples It is preferred that the introduced DNA be incorporated into the germ line of the animal so that it is passed on to the animal's progeny Tissue-specific expression may be achieved through the use of a tissue-specific promoter, or through

the use of an inducible promoter, such as the metallothionein gene promoter (Palmiter et al., 1983, ibid), which allows regulated expression of the transgene.

HOST CELLS The above described nucleic acid molecules which encode thymidine kinase mutants of the present invention (or the vectors which contain and/or express these mutants) may readily be introduced into a wide variety of host cells. Representative examples of such host cells include plant cells, eukaryotic cells, and prokaryotic cells. Within preferred embodiments, the nucleic acid molecules are introduced into cells from a vertebrate or warm-blooded animal, such as a human, macaque, dog, cow, horse, pig, sheep, rat, hamster, mouse or fish cell, or any hybrid thereof.

The nucleic acid molecules (or vectors) may be introduced into host cells by a wide variety of mechanisms, including for example calcium phosphate-mediated transfection (Wigler et al.. Cell 14:725, 1978), lipofection; gene gun (Corsaro and Pearson, Somatic Cell Gen. 7:603, 1981 ; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 7:841-845, 1982), retroviral, adenoviral, protoplast fusion-mediated transfection or DEAE-dextran mediated transfection (Ausubel et al., (eds ), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, NY, 1987).

PREPARATION OF ANTIBODIES Antibodies to the thymidine kinase mutants or guanylate kinase protein described herein may readily be prepared given the disclosure provided herein. Within the context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab')2 as well as portions thereof that may be produced by various recombinant methods. Antibodies are understood to be reactive against a thymidine kinase mutant if it binds with a K _a of greater than or equal to 10^ M. As will be appreciated by one of ordinary skill in the art, antibodies may be developed which not only bind to a ligand such as a thymidine kinase mutant, but which also block or inhibit the biological activity of the mutant.

Briefly, polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, various fowl, rabbits, mice, or rats. Briefly, a thymidine kinase mutant (or guanylate kinase enzyme, if anti-guanylate kinase antibodies are desired) is utilized to immunize the animal through intraperitoneal, intramuscular, intraocular, or subcutaneous injections, an adjuvant such as Freund's complete or incomplete adjuvant. Following

several booster immunizations, samples of serum are collected and tested for reactivity to the thymidine kinase mutant Particularly preferred polyclonal antisera will give a signal on one of these assays that is at least three times greater than background Once the titer of the animal has reached a plateau in terms of its reactivity to the thymidine kinase mutant or guanylate kinase enzyme, larger quantities of antisera may be readily obtained either by weekly bleedings, or by exsanguinating the animal

Monoclonal antibodies may also be readily generated using conventional techniques (see U S Patent Nos RE 32,01 1, 4,902,614, 4,543,439, and 4,41 1,993 which are incorporated herein by reference, see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds ), 1 80, and Antibodies - A Lahoratoiy Manual, Harlow and Lane (eds ), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference)

Briefly, within one embodiment a subject animal such as a rat or mouse is injected with a thymidine kinase mutant or guanylate kinase enzyme as described above The thymidine kinase mutant or guanylate kinase enzyme may be admixed with an adjuvant such as Freund's complete or incomplete adjuvant in order to increase the resultant immune response Between one and three weeks after the initial immunization the animal may be reimmunized with another booster immunization, and tested for reactivity to the thymidine kinase mutant or guanylate kinase enzyme using assays described above Once the animal has plateaued in its reactivity to the mutant, it is sacrificed, and organs which contain large numbers of B cells such as the spleen and lymph nodes are harvested

Cells which are obtained from the immunized animal may be immortalized by transfection with a virus such as the Epstein-Barr virus (EBV) (see Glasky and Reading, Hybridoma 8(4) 377-389, 1989) Alternatively, within a preferred embodiment, the harvested spleen and/or lymph node cell suspensions are fused with a suitable myeloma cell in order to create a "hybridoma" which secretes monoclonal antibody Suitable myeloma lines include, for example, NS-1 (ATCC No TIB 18), and P3X63 - Ag 8 653 (ATCC No CRL 1580)

Following the fusion, the cells may be placed into culture plates containing a suitable medium, such as RPMI 1640, or DMEM (Dulbecco's Modified Eagles Medium) (JRH Biosciences, Lenexa, Kansas), as well as additional ingredients, such as Fetal Bovine Serum (FBS, i.e., from Hyclone, Logan, Utah, or JRH Biosciences) Additionally, the medium should contain a reagent which selectively allows for the growth of fused spleen and myeloma cells such as HAT (hypoxanthine, aminopterin, and thymidine) (Sigma Chemical Co , St Louis, Missouri) After about

seven days, the resulting fused cells or hybridomas may be screened in order to determine the presence of antibodies which are reactive against a thymidine kinase mutant or guanylate kinase enzyme. A wide variety of assays may be utilized to determine the presence of antibodies which are reactive against the proteins of the present invention, including for example Countercurrent Immuno-Electrophoresis, Radioimmunoassays, Radioimmunoprecipitations, Enzyme-Linked Immuno-Sorbent Assays (ELISA), Dot Blot assays, Western Blots, immunoprecipitation, Inhibition or Competition Assays, and sandwich assays (see U.S. Patent Nos. 4,376,110 and 4,486,530; see also Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Following several clonal dilutions and reassays, a hybridoma producing antibodies reactive against the thymidine kinase mutant may be isolated.

Other techniques may also be utilized to construct monoclonal antibodies (see William D. Huse et al., "Generation of a Large Combinational Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246: 1275-1281, December 1989; see also L. Sastry et al., "Cloning of the Immunological Repertoire in Escherichia coli for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region-Specific cDNA Library," Proc. Natl. Acad. Sci. USA 56:5728-5732, August 1989; see also Michelle Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas," Strategies in Molecular Biology 3:1-9, January 1990; these references describe a commercial system available from Stratacyte, La Jolla, California, which enables the production of antibodies through recombinant techniques). Briefly, mRNA is isolated from a B cell population, and utilized to create heavy and light chain immunoglobulin cDNA expression libraries in the klmmunoZap(H) and klmmunoZap(L) vectors. These vectors may be screened individually or co- expressed to form Fab fragments or antibodies (see Huse et al, supra, see also Sastry et al., supra). Positive plaques may subsequently be converted to a non-lytic plasmid which allows high level expression of monoclonal antibody fragments from E. coli.

Similarly, portions of antibodies may also be constructed utilizing recombinant DNA techniques to incorporate the variable regions of a gene which encodes a specifically binding antibody. Within one embodiment, the genes which encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using nucleotide primers for the variable region. These primers may be synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources. Stratacyte (La Jolla, Calif) sells primers for mouse and human variable regions including, among others, primers for V _Ha , V _H b, V _Hc , V _H(j , C _Hj , V _L and C _L regions. These primers may be utilized to amplify heavy or light chain

variable regions, which may then be inserted into vectors such as ImmunoZAP™ H or ImmunoZAP™ L (Stratacyte), respectively. These vectors may then be introduced into E. coli for expression. Utilizing these techniques, large amounts of a single-chain protein containing a fusion of the V _H and V _L domains may be produced (see Bird et al., Science 242:423-426, 1988). In addition, such techniques may be utilized to change a "murine" antibody to a "human" antibody, without altering the binding specificity of the antibody.

Once suitable antibodies have been obtained, they may be isolated or purified by many techniques well known to those of ordinary skill in the art (see Antibodies: A Laboratory Manual, Harlow and Lane (eds ), Cold Spring Harbor Laboratory Press, 1988). Suitable techniques include peptide or protein affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns, or any combination of these techniques.

LABELING OF ANTIBODIES

Anti-thymidine kinase or anti-guanylate kinase antibodies which are described above may be labeled with a variety of molecules, including for example, fluorescent molecules, toxins, and radionuclides. Representative examples of fluorescent molecules include fluorescein, phycoerythrin, rodamine, Texas red and luciferase. Representative examples of toxins include ricin, abrin diphtheria toxin, cholera toxin, gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A. Representative examples of radionuclides include Cu-64, Ga- 67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-I l l, 1-123, 1-125, 1-131, Re- 186, Re-188, Au-198, Au-199, Pb-203, At-21 1, Pb-212 and Bi-212. In addition, the antibodies described above may also be labeled or conjugated to one partner of a ligand binding pair. Representative examples include avidin-biotin, and riboflavin-riboflavin binding protein.

Methods for conjugating or labeling the anti-thymidine kinase or anti- guanylate kinase antibodies discussed above with the representative labels set forth above may be readily accomplished by one of ordinary skill in the art (see Trichothecene Antibody Conjugate, U.S. Patent No. 4,744,981,; Antibody Conjugate, U.S. Patent No. 5, 106,951; Fluorogenic Materials and Labeling Techniques, U.S. Patent No. 4,018,884; Metal Radionuclide Labeled Proteins for Diagnosis and Therapy, U.S. Patent No. 4,897,255; and Metal Radionuclide Chelating Compounds for Improved Chelation Kinetics, U.S. Patent No. 4,988,496; see also Inman, Methods In Enzymology, Vol. 34, Affinity Techniques, Enzyme Purification: Part B, Jakoby and Wilchek (eds.),

Academic Press, New York, p 30, 1974, see also Wilchek and Bayer, "The Avidin- Biotin Complex in Bioanalytical Applications," Anal. Biochem. 171 1-32, 1988)

PHARMACEUTICAL COMPOSITIONS As noted above, the present invention also provides a variety of pharmaceutical compositions, comprising one of the above-described thymidine kinase mutants (e.g. either the nucleic acid molecule, vector, or protein), along with a pharmaceutically or physiologically acceptable carrier, excipients or diluents Generally, such carriers should be nontoxic to recipients at the dosages and concentrations employed Ordinarily, the preparation of such compositions entails combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents

In addition, the pharmaceutical compositions of the present invention may be prepared for administration by a variety of different routes, including for example intraarticularly, intracranially, intradermally, intramuscularly, intraocularly, intraperitoneally, intrathecally, intravenously, subcutaneously or even directly into a tumor (for example, by stereotaxic injection) In addition, pharmaceutical compositions of the present invention may be placed within containers, along with packaging material which provides instructions regarding the use of such pharmaceutical compositions Generally, such instructions will include a tangible expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) which may be necessary to reconstitute the pharmaceutical composition

METHODS The present invention also provides methods for inhibiting a pathogenic agent in a warm-blooded animal, comprising administering to the warm-blood animal a vector (e.g., expression vector, viral vector, or viral particle containing a vector), as described above, such that the pathogenic agent is inhibited As will be evident to one of skill in the art, the amount and frequency of administration will depend, of course, on such factors as the nature and severity of the indication being treated, the desired response, the condition of the patient, and so forth Typically, the compositions may be administered by a variety of techniques, including for example intraarticularly, intracranially, intradermally, intramuscularly, intraocularly, intraperitoneally,

intrathecally, intravenously, subcutaneously or even directly into a tumor (for example, by stereotaxic injection)

Within other embodiments of the invention, the vectors which contain or express the nucleic acid molecules which encode the thymidine kinase mutants described above, or even the nucleic acid molecules themselves may be administered by a variety of alternative techniques, including for example administration of asialoosomucoid (ASOR) conjugated with poly (L-lysine) DNA complexes (Cristano et al , PNAS 92122-92126, 1993), DNA linked to killed adenovirus (Michael et al , J. Biol. Chem. 265(10) 6866-6869, 1993, and Curiel et al , Hum. Gene Ther. 3(2) 147-154, 1992), cytofectin-mediated introduction (DMRIE-DOPE, Vical, Calif ), direct DNA injection (Acsadi et al , Nature 352 815-818, 1991), DNA ligand (Wu et al , J. of Biol. Chem. 264 16985-16987, 1989), lipofection (Feigner et al , Proc. Nail. Acad. Sci. USA 84 7413-7417, 1989), liposomes (Pickering et al , Ore. 59(1) 13-21, 1994, and Wang et al , PNAS 84 7851-7855, 1987), microprojectile bombardment (Williams et al , PNAS 55 2726-2730, 1991), retrotransposons, transferrin-DNA complexes (Zenke), and direct delivery of nucleic acids which encode the enzyme itself either alone (Vile and Hart, Cancer Res. 53 3860-3864, 1993), or utilizing PEG-nucleic acid complexes

Within one aspect of the invention, methods are provided for inhibiting a tumor or cancer in a warm-blooded animal, comprising administering to the warm- blooded animal one of the vectors described above (or nucleic acid molecules which encode thymidine kinase mutants or guanylate kinase enzyme of the present invention), such that the tumor or cancer is inhibited Within a further embodiment, such methods further comprise the step of administering a nucleoside analogue Representative examples of such nucleoside analogues include ganciclovir, acyclovir, trifluorothymidine, l-[2-deoxy, 2-fluoro, beta-D-arabino furanosyl]-5-iodouracil, ara-A, araT 1-beta-D-arabinofuranoxyl thymine, 5-ethyl-2'-deoxyuridine, 5-iodo-5'-amino-2,5'- dideoxyuridine, idoxuridine, AZT, AIU (5-iodo-5' amino 2', 5'-dideoxyuridine), dideoxycytidine and AraC Briefly, utilizing such methods, a wide variety of tumors (both benign and malignant) may be treated Representative examples of such tumors include solid tumors such as lung carcinomas, renal cell carcinomas, breast carcinomas, colorectal carcinomas and melanomas, as well as diffuse cancers such a leukemias and lymphomas

Within other aspects of the present invention, methods are provided for treating a variety of diseases wherein a subset of cells may be characterized as "diseased" or altered utilizing the above-described nucleic acid molecules or vectors. Representative examples of such diseases include hyperkeratosis (psoriasis), prostate hypertrophy, hyperthyroidism, a wide variety of endocrinopathies, autoimmune diseases

(due to autoimmune reactive cells such as certain subsets of T cells), allergies (e.g., by modulating the activity of IgE expressing cells responsible for an allergic response), restenosis (e.g., by killing cells which are responsible for the ingrowth and/or clogging of a blood vessel), a wide array of viral diseases such as AIDS (HIV), hepatitis (HCV or HBV), and intracellular parasitic diseases. Within other embodiments of the invention, methods are provided for inhibiting the growth of or destroying cells which are not traditionally associated with a disease. For example, within certain embodiments it may be desirable to administer a vector (or nucleic acid molecule alone) which inhibits or destroys fat cells in order to initiate weight loss in an animal, or to destroy hair follicles (as a depilatory reagent).

Within other aspects of the present invention, methods are provided for utilizing the thymidine kinase mutants described above, as a negative-selection marker gene (see e.g., Czako and Marton, Plant Physiol. 70-/: 1067-1071, 1994) in prokaryotic cells, eukaryotic cells, plants (Czako and Morton, Plant Physiol. 104: 1067-1071, 1994), parasites (e.g., Trypanosomes) or viruses. Alternatively, such mutants may be utilized as a conditionally lethal marker for homologous recombination (Mansour et al., Nature 336:348-352, 1988). A representative example is set forth in more detail below as Example 6.

The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES

EXAMPLE

CONSTRUCTION OF TK MUTANTS CONTAINING MUTATIONS AT CODONS 165-175 UTILIZING A 20% RANDOM LIBRARY

Example 1 describes the construction of TK mutants containing mutations at codons 165 to 175, utilizing a 20% random library A schematic outline which depicts the strategy utilized in this example is set forth in Figure 1

A Generation of TK Mutants

1 General ion of Oligonucleotides

A 52-mer oligonucleotide with a wild-type Ik sequence (SEQUENCE ID

NO No 2) and a 56-mer that contained degenerate nucleotides spanning from codon 165 through 175 (SEQUENCE ID NO No 3) of the Ik gene (SEQUENCE ID NO 1 -

- Note that SEQUENCE ID No 1 only lists nucleotides in the open reading frame of

HSVTK-1), (where N = 80% wild-type nucleotides and a 20% mixture of the other three at each position) were synthesized by Operon Technologies (San Pablo, CA).

Both oligomers were complementary to each other along 12 bases at their 3'-ends

5'-TG GGA GCT CAC ATG CCC CGC CCC CGG CCC TCA CCC TCA TCT TCG

ATCGCCAT-3'(SEQUENCEIDNo 2)

5'-ATG AGGTAC CGN NNN NNN NNNNNNNNN NNN NNN NNNNNNNNN

NNATGGCGATCGAA-3'(SEQUENCEIDNo 3)

For the construction of pKTPD described below, two additional oligonucleotides were synthesized by Operon Technologies using phosphoramide chemistry These oligonucleotides were

5'-CCC CTC GAG CGC GGT AC-3' (SEQUENCE ID No 4) 5'-CGC GCT CGA GGG GAG CT-3' (SEQUENCE ID No 5)

2 Generation of Random Seφience-Containing Libraries a Construction of Vectors pMDC and pMCC Chimeric vectors pMDC (which produces an inactive TK gene product) and pMCC (which produces wild-type TK) were produced from plasmids pHETKl and pHETK2 essentially as described below Briefly, plasmids pHETKl and pHETK2

(Waldman et al , J. Biol. Chem. 258 1 1571-1 1575, 1983) are expression vectors that contain a HSV-1 Ik structural gene, and are derivatives of pBR322 Restriction maps of pHETKl and pHETK2 can be found in Waldman et al, J. Biol. Chem. 255 11571- 1 1575, 1983, which describes the construction of these plasmids Plasmid pHETK2 contains λPL and λPR promoters, ampR, and the cl857 temperature-sensitive repressor, whereas pHETKl contains all the above except the λPj _^ promoter Plasmids pHETKl and pHETK2 were obtained from Dr William Summers (School of Medicine, Yale University, New Haven)

In order to construct pMDC and pMCC, a dummy vector, designated pKTPD was first constructed as described by Dube et al in Biochem. 30 1 1760-11767, 1991 Briefly, oligonucleotides SEQUENCE ID Nos 4 and 5 (20 p ol of each) were first phosphorylated and then annealed to form a double-stranded oligonucleotide with Kpnl- and Sstl-compatible ends and with an internal Xho\ site In addition, pHETK2 was digested with Sst\ and Kpiή restriction endonucleases, and the large fragment isolated by agarose gel electrophoresis and subsequent electroelution Two picomoles of the large fragment was ligated with 6 pmol of the double-stranded oligonucleotide The resultant double-stranded circular DNA product (designated "pKTPD") was used to transform competent E. coli KY895 cells E. coli KY895 is a TK-deficient strain (K12 td/r, F", ilv 276) obtained from William Summers, Yale University, New Haven, CT Clones containing the recombinant plasmid pKTPD grow on LB plates containing 50 μg/mL carbenicillin The presence of recombinant plasmid DNA was verified by the cleavage at the Xhol site The inability of pKTPD to support the growth of E. coli KY895 in the thymidine kinase selection medium indicates that it does not produce a functional thymidine kinase pHETKl and pKTPD were then utilized to construct a new chimeric dummy vector, designated pMDC Briefly, upon digestion with Sphl and vwII pHETKl is cut into two fragments The larger fragment contains ampR, cI857, PR sequences, and part of the Ik gene spanning from the BamHl to the Sphl site The smaller fragment contains the remainder of the tk gene from Sphl to Pvuϊl Similarly, pKTPD upon digestion with the same two enzymes is cut into one larger and one smaller fragment The smaller SphllPvull fragment of pKTPD contains a dummy or inactive sequence within the Kpnl and Sacl sites of the tk gene Ligation of the larger fragment from pHETKl with the smaller fragment of pKTPD results in a chimeric vector, pMDC, that produces an inactive Ik gene product Another chimeric vector, pMCC, containing the wild-type tk gene was similarly constructed by ligating the larger fragment from pHETKl with the smaller fragment of pHETK2 As noted above, PMCC produces active wild-type TK

b Generation of a Library

A library containing 20% random nucleotide sequences was constructed as follows Briefly, a 52-mer oligo containing wild-type sequences (SEQUENCE ID No 2) was hybridized to a 56-mer oligo which contained degenerate sequences spanning codons 165 through 175 (Sequence ID No 3)

The hybrid was extended with the Klenow fragment of E. coli DNA polymerase I to produce a complete double-stranded DNA product This strategy was implemented in order to avoid synthesizing a long random nucleotide containing SEQUENCE ID No 3, since the locations of Kpnl and Sacl sites (insertion sites) in the vector require a long cassette The Klenow fragment generated double-stranded DNA was then subjected to polymerase chain reaction amplification by using two synthetic primers the first primer, a 5'-TGG GAG CTC ACA TGC CCC GCC-3' (SEQUENCE ID No 6) corresponds to the 21 -base sequence of 5' terminus of oligo SEQUENCE ID No 2 The second primer, b 5'- ATG AGG TAC CG-3' (SEQUENCE ID No 7) corresponds to the 1 1-base sequence of 5' terminus of oligo SEQUENCE ID No. 3. The polymerase chain reaction amplification reactions contained 20 mM Tris-HCl (pH 8 3), 25 M KC1, 1 5 mM MgCl ₂ , and 0 05% Tween 20, 0 1 mg/ml BSA, 50 μM each of the four deoxynucleoside triphosphates, 20 pmol of primer "a," 40 pmol of primer "b," approximately 1 pmol of the extended double-stranded oligonucleotide as template, and 2 units of Taq polymerase (Cetus) in 100-μl final reaction volumes Each mixture was overlaid with mineral oil and subjected to 30 rounds of temperature cycling 94°C for 1 minute, 34°C for 2 minutes, and 72°C for 7 minutes

Low molecular weight components and excess primers were removed from the polymerase chain reaction-amplified product by centrifugation with a Centricon 30 ultrafiltration unit, and the amplified DNA was digested with Kpnl and Sacl The digested double-stranded oligonucleotide containing the random sequence was again purified by a Centricon 30 unit, and ligated to the KpnllSacl digested large fragment of pMDC at 10 1 molar ratio in the presence of 1 mM ATP and 1 unit of T4 DNA ligase (BRL) in a volume of 10 μl Incubation was for 18 hours at 14°C and the reaction was terminated by phenol-CHCl3 extraction followed by ethanol precipitation

c Selection of TK Mutants

The precipitate described above was dried and dissolved in 10 μl of water, and used to transform competent E. coli KY895 by electroporation One μl of ligated product was mixed with 50 μl of competent cells and electroporated at 2 KV, 25 μF, and 400 Ohms with a Gene-pulser electroporator (Bio-Rad) After the pulse, 1 ml

of SOC medium (2% Bacto-tryptone, 0.5% Bacto yeast extract, 10 mM NaCI, 2.5 mM

KC1, 10 mM MgCl2, 10 mM MgSU4 and 20 mM glucose) was added, followed by incubation at 37°C for 1.5 hours with continuous agitation. An aliquot of each transformation solution was spread onto LB-agar medium containing 50 μg/ml of carbenicillin to determine total number of transformants. Selection for active TK clones was performed on TK selection medium that contained 50 μg/ml of carbenicillin. 10 μ g/ml of 5' fluorodeoxyuridine, 2 μg/ml of thymidine, 20 μg/ml of uridine. 2% BBL peptone, 0.5% NaCI, 0.2% glucose, and 0.8% Gel-Rite (Scott Laboratories, Inc.,

Carson, CA) (Fig. 1). Colonies on carbenicillin medium were incubated at 37°C for 14- 16 hours, whereas inoculated TK selection medium was incubated at 37°C for 24 hours.

From a total of 53,000 transformants that grew on carbenicillin medium,

190 were able to complement E. coli KY 895 for TK function.

EXAMPLE 2

CONSTRUCTION OF TK MUTANTS CONTAINING MUTATIONS AT CODONS 165-175 UTILIZING A 100% RANDOM LIBRARY

Example 2 describes the construction of TK mutants containing mutations at codons 165-175 utilizing a 100% random library. The strategy which was utilized for this example is similar to that described in Example 1 above.

A. Generation of TK Mutants

7. Generation of Oligonucleotides A 52-mer 5'-d(TG GGA GCT CAC ATG CCC CGC CCC CGG CCC

TCA CCC TCA TCT TCG ATC GCC AT)-3' (SEQUENCE ID No. 8) with a wild-type tk sequence and Kpn I site at the 5' end was synthesized by Operon Technologies (San Pablo, CA). In addition, a 56-mer containing random nucleotides corresponding to HSV-1 tk codons 165-175 and containing a Sac I site at the 3' end 5' -d(ATG AGG TAC CGN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNA TGG CGA TCG AA)-3' (SEQUENCE ID No. 3), where N = equimolar concentrations of G, A, T, or C, was also synthesized The oligonucleotides were separated by electrophoresis through a 20% denaturing polyacrylamide gel, followed by purification on a reverse-phase mini column (Glen Research, Sterling, VA) 2. Generation of a 100% Random Sequence - Containing Library

The 52-mer corresponding to the wild-type HSV-1 tk sequence was hybridized with the 56-mer containing random nucleotides. The hybrid was then

extended with the Klenow fragment of DNA polymerase I, PCR amplified, and ligated into pMDC essentially as described above in Example 1. 3. Selection of TK ⁺ Mutants

Functional TK mutants were identified by colony formation on TK- selection medium based on their ability to phosphorylate dT essentially as described below. Briefly, the ligated product was introduced into tk" E. coli strain KY895. The total number of transformants was determined by plating on LB agar containing 50 μg of carbenicillin per mL and the number of transformants that produced catalytically active thymidine kinase was determined by plating on TK-selection medium [2% BBL peptone, 0.5% NaCI, 0.2% glucose, 0.8% Gel-Rite (Scott Laboratories, Carson, CA)], 50 μg 1 mL of carbenicillin, 10 μg/mL of fluorodeoxyuridine, 2 μg/mL of dT, and 20 μ g/mL of uridine.

Two million (2 x 10 ⁶ ) transformants were screened from the 100% random library, of which 1540 formed colonies on the TK-selection medium.

B. Selection of AZT-Sensitive Mutants

A subset of 690 mutants from the 100% random library (TKI) and 190 mutants from the 20% degenerate library (TKF) (described above in Example 1) were subjected to secondary negative selection on medium containing AZT in order to identify mutants that exhibited enhanced phosphorylation of AZT. This screen is based upon the premise that mutants with increased ability to phosphorylate AZT relative to dT would be unable to form colonies on the AZT-selection medium. In particular, the product, AZT monophosphate would be further phosphorylated by the host cell's nonspecific nucleotide kinases, or possibly by the mutant TK, incorporated into bacterial DNA by host DNA polymerases, terminate DNA synthesis, and thus prevent replication of the host chromosome.

Briefly, the TK mutants were first grown as individual colonies on TK- selection medium (1.0 μg/mL of dT), and then replica plated onto AZT-selection medium (0.05 μg/mL of AZT, 1.0 μg/mL of dT). All other components in the AZT- selection medium were the same as the TK-selection medium. Those TK mutants which failed to grow on the AZT-selection medium were selected and retested for growth on both TK- and AZT-selection media separately.

Of the 880 primary selectants that were screened, only two mutants, TKF 105 (from the 20% library) and TKI 208 (from the 100% library), formed colonies on the TK-selection medium at an efficiency similar to that of E. coli harboring the wild- type plasmid but not on the AZT-selection medium (Figure 2).

The nucleotide and deduced amino acid sequences of TKF 105 and TKI 208 are presented in Figure 3. Both mutants contain a single amino acid substitution at the same position: Leu-170 was changed to He in TKF 105 and to Val in TKI 208. No other substitutions were observed in the surrounding 220 nucleotides. To ensure that the difference between TKF 105 and TKI 208 was not due to differential expression of TK in E. coli harboring mutant and wild-type plasmids, Western blots of extracts from cells containing either TKI 208 or wild-type plasmids were compared. No significant difference was observed in the amount or electrophoretic mobility of immunoreactive staining protein. Also, the rate of dT phosphorylation per mg of protein was determined, and found to be similar in extracts of E. coli harboring TKI 208, TKF 105, and wild-type plasmids.

In order to show that the lack of growth of these two mutants on AZT- selection medium was due to enhanced phosphorylation of AZT, the following experiments were conducted. 7. Rate of f ³ HJAZT Uptake

First, the rate of [ ³ H]AZT uptake relative to [ ³ H]dT into E. coli harboring wild-type and mutant plasmids was determined. These studies indicated that E. coli harboring the AZT-sensitive mutants, TKF 105 and TKI 208, exhibited a 4-fold increase in the ratio of AZT to dT uptake, as compared to E. coli with the wild-type plasmid.

2. Affinity Purification of TK

Purification of wild-type and mutant TKs was performed by affinity ehromatography on CH-Sepharose 4B (Pharmacia) coupled to -aminophenylthymidine 3 '-phosphate. Briefly, crude bacterial extract was passed three times through a 7-mL bed-volume affinity column. The column was then washed sequentially using 30 mL each of buffer A [0.1 M Tris HC1, pH 7.5/5 mM dithiothreitol (DTT)/10% glycerol], buffer B (0.1M Tris-HCl, pH 7.5/0.5 M KC1/5 mM DTT/10% glycerol), and buffer A. TK was eluted using a 60-mL linear gradient of 0-600 μM dT in buffer C (0.3 M Tris HC1, pH 7.4/50 mM KCl/10% glycerol). Active fractions were pooled and dialyzed against three changes each of 2 liters of 50 mM Tris-HCl, pH 7.4/5 mM DTT/10% glycerol. Except in the final dialysis, all the above buffers contained 50 μg/mL of aprotinin and 2 μg/mL each of pepstatin and leupeptin.

3. Kinetics of AZT Phosphorylation

Secondly, the kinetics of AZT phosphorylation by the two mutants was determined. Briefly, reactions were carried out in a final volume of 100 μl containing 50 mM Tris-HCl (pH 7.5), 5 mM ATP. 4 mM MgCl ₂ , 2.5 mM DTT, 12 mM KC1,

0.18 mg/mL of bovine serum albumin, 5% glycerol, 0.08 μCi of [ ³ H]AZT (Sigma),

.

various concentrations of unlabeled AZT (0-4 0 μM), and purified enzymes (4 and 1.2 units, respectively, for wild-type and TKI 208) (One unit of enzyme is defined as that amount that can phosphorylate 1 0 pmol of dT to TMP in 1 minute under the conditions described above ) Incubation was at 34°C ± 1°C for 10 minutes, and reactions were stopped by adding 1 0 mM unlabeled dT and cooling on ice Half of the reaction mixtures were pipetted onto a DEAE-cellulose disc (25 mm), dipped in distilled water (1 minute), followed by four washes in absolute ethanol The amount of radioactivity adsorbed to the disc was determined by scintillation spectroscopy K _m and V _max values were determined by using the Cleland SUB IN program (Cleland, Methods Enz 63' 103- 138, 1979) The values for k _ca[ were calculated using the equation V _ma = _cat [E] ₀ , where [E] ₀ = total enzyme concentration TK assays wherein phosphorylation of dT was measured were carried out in a final volume of 50 μl using 0 3 μCi ([3Η-methyl]dT 87 Ci/mmol Amersham), various concentrations of unlabeled dT (0-4 0 μM), and 1 1 and 0 5 units of TK for the wild-type and TKI 208, respectively All other components in the reaction mixtures and the incubation conditions were as described above for phosphorylation of AZT

As shown below in Table 1, the AZT-sensitive variant TKI 208 exhibits a lower K _m (4 4 μM) compared to that of the wild-type (8 5 μM) By comparing the k _cal /K _m between the two substrates (AZT vs dT), it can be seen that TKI 208 selectively phosphorylates AZT 2 3-fold more efficiently than dT Similar preliminary experiments with purified TKF 105 TK also showed lower K _m (3 7 μM) for AZT, but similar values for k _ca ,/K _m compared to the wild-type

TABLE 1

ABILITY OF WILD-TYPE AND TKI 208 TKs To PHOSPHORYLATE AZT AND DT

"cat'^m m(AZT)

Phosphorylation * _m , μM k _rαt , s s-^M ^"1 k _rαt /K _m (dT)

AZT

Wildtype 8 46 ± 1 3 3 6 \ 10' ² 4 2 x 10 ³ 1 7 x 10" ³

TKI 208 4 40 ± 0 43* 3 0 \ 10 ^"2 6 5 x 10 ³ 4 0 x 10" ³ dT

Wildtype 0 475 1 0 10 1 21 2 5 x l0 ⁶

TKI 208 0 35 1 0 008 0 56 1 57 \ 10 ⁶

>J

C. Thermostability Analysis of Mutant TKs

Mutants were analyzed for thermostability essentially as described below. Briefly, 25 μg of each extract were preincubated in 0.3 mL of 28 mM Tris-HCl, pH 7.5 containing 0.28 mg/mL of bovine serum albumin, 28 μg/mL of aprotinin, 2 μg/mL (each) of pepstatin and leupeptin, at 42°C for 0.5, 10, 20, 30, or 40 minutes. At each time point 30-μl (2.5 μg) aliquots were assayed for residual TK activity in a total reaction volume of 50 μl containing 50 mM Tris-HCl (pH 7.5), 5 mM ATP, 4 mM MgCl ₂ , 2.5 mM DTT, 12 mM KC1, 0.18 mg/mL of bovine serum albumin, 5% glycerol, and 1 μM [ ³ H-røe/by/]dT (60 x 10 ³ dpm/pmol). Incubation was at 34°C for 10 minutes. The reaction was stopped by cooling on ice, and 25 μl was pipetted onto a DEAE- cellulose disc. Wash and assay conditions for the discs were performed as described for the AZT assay above.

Assay results of unfractionated extracts of TKF 2, TKF 56, TKF 75, TKF 446 and wild-type TK are shown in Figures 4A-4D. One of the mutants, TKF 2, was more thermostable at 42°C than any of the other mutants, or than the wild-type. Except for TKF 2, all of the mutants tested, including the wild-type, had ratios of residual activity after preincubation at 42°C compared to 34°C of 0.05-0.30: TKF 2 had a ratio of 0.7. TKF 2 contains three amino acid substitutions: Pro-165 → His, Ala-167 → Ser, and Ala- 174 → Val (Figure 3). TKF 75 contained an Ala- 167 → Ser substitution, TKF 56 a Ala- 174 → Val, and TKI 440 a Pro- 165 → Ala substitution. The thermolability of mutants TKF 56 and TKF 75 with Ala- 174 → Val and Ala- 167 → Ser substitutions, respectively, was similar to that of the wild-type. Both lost >80% of their activity after incubation for 5 minutes at 42°C. TKF 440 with a Pro- 165 - Ala is more stable, but not as stable as TKF 2, the triple mutant. Two types of experiments were carried out to verify the thermostability of TKF 2. First, TK protein from TKF 2 and the wild-type plasmid harboring E. coli were purified to near homogeneity by affinity ehromatography, and assayed as described above. As before, loss of activity is less in TKF 2 than in the wild-type after preincubation at 42°C (Figure 4E). Secondly, tk genes from TKF 2 and wild-type TK were transferred into a vector with a promoter for T3 RNA polymerase. More specifically, the full-length Bgl Ϊl-Pvu I fragments of tk genes from wild-type and TKF 2 plasmids were isolated and subcloned into the pBluescript SK ⁺ (Stratagene) vector between the Spe I and EcoRI sites with the use of synthetic linkers. In vitro transcription using the T3 promoter was carried out using the Promega transcription system. In vitro translation was carried out using a reticulocyte lysate system (Promega) following the supplier's protocol. The loss of TK activity of the // vitro synthesized proteins from the wild-type and TKF 2 tk

genes as a function of preincubation at 42°C is shown in Figure 5. The protein encoded by TKF 2 lost <10% of its activity after preincubation for 45 minutes. In contrast, the protein encoded by the wild-type gene lost >80% of its initial activity. The degree of thermostability exhibited by the in vitro synthesized TKF 2 was similar to or greater than that of crude extracts harboring the original TKF 2 plasmid. For SDS/PAGE analysis, the translated products were labeled with [ ³⁵ S]methionine.

An autoradiograph of the labeled proteins after SDS/PAGE is shown in Figure 6. The arrow indicates the expected size of translated TKs as judged by molecular mass standards (Bio-Rad). From this autoradiograph it is evident that the translation products migrate as double bands, one of which corresponds to a protein of 43 kDa, which is in accord with the reported size of HSV- 1 TK expressed in E. coli. The second band could be due to the proteolytic degradation of a 32-residue fragment at the amino-terminal end, which does not detectably alter TK activity of the HSV-1 TK.

EXAMPLE 3

CONSTRUCTION AND ANALYSIS OF TK MUTANTS WITH

MUTATIONS AT CODONS 155, AND 161 TO 165 UTILIZING

A 20% RANDOM LIBRARY

This example describes the construction and analysis of TK mutants which are mutagenized at codons 155, and 161 through 165. Bacterial strains and materials which were utilized within this example are set forth below.

Bacterial Strains. E. coli strain KY895 (F", tdfc, -ilv), originally described by Igarashi et al. (Genetics 57:643-654, 1967), was used in the genetic complementation assays for thymidine kinase activity. E. coli strain NM522 (F' lacY. A (lacZ)M\ 5 proAB/supE thi A (lac proAB)Δ(b.v /MS-wcrB)5(rk-McrB-)) (NEB, Beverly, MA) was used as a recipient in all subcloning experiments. Helper phage VCM 13 (Stratagene, La Jolla, C A) was used in the production of single-stranded phage for sequencing.

Materials. L-[ ^:> S]Methionine/cysteine (specific activity, 1 140 Ci/mmol) for protein synthesis determination and [methy/-^ ] thymidine (specific activity, 87 Ci/mmol) were purchased from Amersham. Other radioisotopes [[side chain-2-^ ] acyclovir (specific activity, 28.6 Ci/mmol) and [5- ³ H]-deoxycytidine (specific activity, 29 Ci/mmol)] were purchased from Du Pont-New England Nuclear (Boston, MA), and [8- ³ H] ganciclovir (specific activity, 22 Ci/mmol) and [/?7etby/- H]-3'-azido-3' deoxythymidine (specific activity, 14 Ci/mmol) were from Moravek (Brea, CA).

Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (NEB). Promega (Madison, WI) was the source of the in vitro transcription and translation reagents except for the cap analog, ⁷ m(5')Gppp(5')G, which was purchased from NEB. Oligonucleotides used for sequencing and polymerase chain reaction amplifications were obtained from Operon (Alameda, CA). Other chemicals were purchased from Sigma (St. Louis, MO) except where designated.

A. Generation of TK Mutants

7. Generation of Oligonucleotides Two oligonucleotides were synthesized by American Synthesis, Inc.

(Pleasanton, CA): MB1 10 (70mer) 5'-TGGGAGCTCA CATGCCCCGC

CC[CCG]GCCCT CACCCTCATC [TTCGACCGCC ATCCC]ATCGC

CGCCCTCCTG-3' (SEQUENCE ID No. 9), and MB1 1 1 (38mer) 5'-ATGAGGTACC GCGCAGCTGG GTAGCACAGG AGGGCGGC-3' (SEQUENCE ID No. 10). Within these oligonucleotides, nucleotides in brackets where synthesized as 80% wild-type nucleotide, and 20% the other three nucleotides.

At the 5' end of MB 1 10 is a Sacl restriction site and, at the 5' end of MB1 1 1 , a Kpnl site. These restriction sites were utilized at a later step after second- strand synthesis occurred. Furthermore, as an internal control, a vwII site was introduced (silent change) in MB 1 1 1 in order to allow confirmation of random sequence insertion prior to sequencing. Twelve nucleotides at the 3' ends of each oligonucleotide are complementary to allow for hybridization of the two strands to each other. Each oligonucleotide was subjected to electrophoresis on a 20% acrylamide-urea gel and visualized by UV shadowing on a PEI-cellulose TLC plate (Baker, Phillipsburg, NJ), the portion of the gel containing the correct sized oligonucleotide was excised, and the oligonucleotide was eluted from the gel in 0.5M NH4Ac/10mM MgOAc2 overnight at 37°C. The eluted oligonucleotide was then ethanol-precipitated and resuspended in H2O. An OD26O measurement was taken, and the extinction coefficient for each oligo was used to determine the concentration. Equimolar amounts of MB 1 10 and MB1 1 1 (25pmol) were annealed in a small volume (20μl) in 1 x annealing buffer (10 x annealing buffer = 70mM Tris (pH 7.5)/60mM MgCl2/200mM NaCI ) for 5 minutes at 95°C, then moved to 65°C for 20 minutes, followed by slow cooling to room temperature. To the annealed oligonucleotides (20μl) were added 2μl of 10 x annealing buffer, 2.8μl of lOmM dNTPs, 0.8μl of 0.1M of dithiothreitol (DTT), 2.4μl of DNA polymerase I Klenow fragment (5 units/μL), and H2O to bring the volume to 40μL. The mixture was placed at 37°C for 30 minutes, at 65°C for 10 minutes, and finally at room temperature for 10

minutes Verification of fully extended radioactive oligonucleotides was accomplished by subjecting the samples to denaturing acrylamide gel electrophoresis and autoradiography Amplification of the extended products was performed using the polymerase chain reaction with Taq polymerase (Stratagene) The 100 uL reactions contained 20mM Tris (pH 8 3)/25mM KCl/1 5mM MgCl2/0 05% Tween 20)/0 lmg/mL BSA/50μM of each of the four deoxynucleoside triphosphates (dNTPs)/22pmol PCR primer l/20pmol PCR primer 2/2 units of Taq polymerase and 6 pmol of the extended random oligonucleotide, Primer 1 = 5' TGGGAGCTCACATGCCCCGCC-3' (SEQUENCE ID No 6) and primer 2 = 5'-ATGAGGTACCG-3" (SEQUENCE ID No 7) One drop of mineral oil was added to each tube, which was then placed in a Perkins Elmer-Cetus thermal cycler (Norwalk, CT) and programmed for 30 cycles of 95 °C for 1 minute and 34°C for 2 minutes At the end of the 30 cycles, the reactions were left at 72°C for 7 minutes, and then the cycler was maintained at 4°C After confirmation of amplification by 2% agarose gel electrophoresis, the product-containing reactions were pooled, precipitated and digested with Kpnl and Sard Doubly restricted fragments were distinguished from single cut or uncut fragments on non-denaturing acrylamide gels, and the appropriate fragment was excised and isolated as described above

2. Generation of Random Sequence - Containing Libraries Cesium chloride gradient purified pMDC ("dummy" vector) which was constructed as described above in Example 1, digested with Kpnl and Sacl restriction endonucleases, and gel-isolated from a 1% agarose/ lx TBE gel using GenClean II (Bio 101, La Jolla, CA) This vector was ligated with the gel-isolated PCR-amplified random fragment overnight at 16°C with 1 unit of T4 DNA ligase 3. Selection of TK Mutants

The ligated mixture was then used to transform KY895 by electroporation (BioRad gene pulser, 2kV, 25μF, 400 Ω) Briefly, cells were prepared for electroporation according to a protocol provided by BioRad (Richmond, CA) After each pulse, lmL of SOC (2% Bactotryptone/0 5% yeast extract/ lOmM NaCl/2 5mM KCl/lOmM MgCl2/10mM MgSO4/20mM glucose) was added to the curette and the electroporation mixture transferred to a 25 mL snap-cap Falcon tube After the tubes were shaken for 1 hour at 37°C, the cells were plated onto LB plates [per liter lOg tryptone/5g of yeast extract/1 Og NaCI (pH 7)] containing carbenicillin (50μg/mL), ("LB+ carb ⁵⁰ plates") and incubated at 37°C overnight The number of colonies was counted, picked with a toothpick, and streaked on TK selection media [2% BBL Trypticase peptone (Becton Dickenson, Cockeysville, MD)/0 5% NaCl/0 8% Gel-Rite (Scott Laboratories, Carson, CA)/0 2% glucose/50μg/mL carbenicillin/ lOμg mL 5'-

fluorodeoxyuridine/2μg/mL thymidine/ 12 5 μg/mL uridine] The basis of this selection is that 5'-fluorodeoxyuridine (FUdR) is phosphorylated by thymidine kinase to form FdUMP, an inhibitor of the de novo pathway enzyme, thymidylate synthase The requirement for dTMP can then be fulfilled only by an active thymidine kinase Uridine is supplied to inhibit thymidine phosphorylase After 16-24 hours, the TK selection plates were scored for growth, and any positives picked and restreaked on TK selection plates and LB + carb ⁵⁰ plates to confirm the phenotype

Approximately 260 random transformants were screened for their ability to complement KY895, a TK-deficient E. coli on TK selection media Of these, 82 were scored as positives and sequenced Therefore, approximately 32% of all transformants encoded functional enzymes

B Analysis of Mutants

TK mutants were isolated and sequenced as follows Briefly, mutant DNA was isolated from overnight cultures grown in 2 x YT (per liter 16g tryptone/lOg of yeast extract/5g NaCI) + carb ⁵⁰ using the Promega Magic miniprep kit according to the manufacturer's instructions, except that 3mLs of culture was used per isolation because of the low copy number of the plasmid Ten microliters of each dsDNA was alkaline-denatured, precipitated, and resuspended in Sequenase reaction buffer, H2O, and sequencing primer (5'-CATGCCTTATGCCGTGA-3') (SEQUENCE ID No 11) The primer was then annealed, and the DNA subjected to dideoxy sequencing (Sanger et al , 1977) using Sequenase according to the manufacturers instructions (USB, Cleveland, OH)

Eleven of the clones encoded wild-type amino acid sequence (13 4%), with seven of these containing the wild-type nucleotide sequence Three clones with wild-type amino acid residues contained single nucleotide changes (all different), and one contained three nucleotide changes As shown in Table IA below, a total of 49 TK positive clones containing single amino acid changes (59 8%) were identified Nineteen double amino acid mutations (23 2%), two triple (2 4%) and one clone containing four amino acid changes (1 2%) were identified Within Table IA, wild-type HSV-1 TK amino acids mutated are given in the boldface box with the residue number and the type of residue found in the majority of sequences [O = hydrophobic, I = hydrophilic, (+) = positively charged, (-) = negatively charged residues] Below the wild-type residue are the number of times a particular amino acid substitution was found In the bottom section, the percentages of each type of residue found are listed

The amino acid sequences of clones with multiple alterations are shown in Table IB The wild-type amino acids and their positions in the HSV-1 TK

polypeptide are indicated at the top of the table. Double, triple, and quadruple amino acid substitutions are shown in the respective categories. If a set of mutations was identified more than once, the number of occurrences is noted on the left in parentheses.

TABLE IA

Wild-type O O (-)i (+)I (+)I 0

Sequence P F D R H P

155 161 162 163 164 165

Substitutions 3L 41 5E 5C 3N 3L at Each 2A 4Y IG IS IT 2T

Position 2T 3C 2S iQ 2L IN

IR I S 1A

Types of 1 1%(+) 57% I 83% (-)I 100% I 100% I 10% (+)

Substitutions 33% I 43% 0 17% I 50% I 56% O 40% O

TABLE IB

C Secondary Screening and Subcloning The ability of pMCC (KY895) and 35 log-phase mutant pMDC (KY895) cultures to produce colonies on acyclovir ("ACV") or AZT plates was determined in a secondary screen as described below. Briefly, log-phase cultures of TK positive clones were serially diluted in 0.9% NaCI and spread onto acyclovir or AZT plates (TK selection plates except 1 μg/mL thymidine + 1 μg/mL acyclovir or 0.05μg/mL AZT). Mutant cultures were also spread onto duplicate TK selection and LB + carb ⁵⁰ plates. One set of TK selection plates and LB + carb ⁵⁰ plates were incubated at 42°C. All other plates were incubated at 37°C After 16-24 hours the plates were scored

Results are shown in Table II below. Briefly, only mutants that gave results which differed from those observed with the wild-type pMCC (KY895) are shown Mutants are designated with the wild-type residue and position number followed by the amino acid substitution deduced from the nucleotide sequence; e.g., F161I indicates that isoleucine replaces phenylalanine at residue 161 in this particular

40

mutant. (++) indicates that the same number of colonies were observed as compared to control plates; (+) indicates that fewer (<20% those observed with pMCC) and generally smaller (-50% smaller diameter) colonies were observed as compared to control plates; and (-) indicates that no colonies were observed.

TABLE II

As shown in Table II, all cultures formed colonies on control TK selection and LB+carb ⁵⁰ plates. In comparison to the wild-type, several mutants appeared to preferentially utilize one or both nucleoside analogues over thymidine (P155A/F161V, F161I, F161C, and R163P/H164Q). In addition, several mutants were unable to form colonies on TK selection plates at 42°C (F161L and R163P/H164Q), and one (F161I/R163H) showed a severely reduced ability to form colonies at 42°C.

D. Expression of Mutant Enzymes in a Cell-Free Translation System 7. Subcloning of Selected Mutants

In order to study the properties of the mutant TKs, the 1.07 kbp Mlul- BssHll fragment of eight mutants was subcloned into the in vitro vector pT7:HSVTKII. More specifically, DNAs of selected clones were restricted with Mlul and BssHϊl to release a 1.07kbp fragment [nucleotide numbers -335 through 1400 on the McKnight sequence (Nucl. Acids Res. 8: 5949-5964, 1980; the McKnight strain was derived from the mp strain of HSV- 1, Wagner, / V 75: 1441-1445, 1981)] . The fragments were gel-isolated from 1% agarose gels using GenCleanll, and ligated to pT7:HSVTKII vector DNA which had been restricted with Mlul and ZfosHII, treated with calf intestinal alkaline phosphatase, and gel-isolated. pT7:HSVTKII was derived from pT7:HSVTK transcription vector described by Black and Hruby in J. Biol. Chem. 267:9743-9748, 1992. Briefly, pT7:HSVTKII differs from pT7:HSVTK only by the loss of an Ncol-

Bam l fragment 3' to the end of the HSV-1 tk gene which was originally used to aid in the initial cloning of the tk gene.

2. Sequence Analysis

In the final sequence analysis of the eight mutant fragments subcloned into the pT7:HSVTKII vector, two additional amino acid differences were identified between these tk genes. The sequence of pT7:HSVTKII is exactly the same as that published by McKnight (Nuc. Acids Res 5(24):5949-5963, 1980). pMCC, the parental plasmid of pMDC and hence the vector into which the random sequences were ligated, contains two amino acid aberrations from the McKnight sequence. These are at position 434 (C— >T) and 575 (G— »A), and result in a proline-49 to leucine and an arginine-89 to glutamine change. Therefore, all mutants contain these two mutations in addition to those described. In addition, a single nucleotide difference at position 480 (C— »T) was also identified but does not result in an amino acid change.

Because all in vitro analyses were compared against pT7:HSVTKII as the wild-type, the MIul-BssY ll fragment from pMCC was subcloned into the corresponding sites of pT7:HSVTKII (now designated pT7:MCC) and the subsequent cell-free translation products compared to those derived from pT7:HSVTKII. Time course and thermal stability analyses showed no significant difference between pT7:HSVTKIl-and pT7:MCC-derived translation products. No significant difference in phosphorylation efficiency was observed between pT7:MCC and pT7:HSVTKII when thymidine (1.3-fold), deoxycytidine (1.3-fold), GCV (0.8-fold), ACV (0.95-fold), or AZT (1.1-fold) were used as substrate. Furthermore, Sanderson et al. (J. Mol. Biol. 202:917-919, 1988) reported that the K _m for thymidine and ATP and the V _m ax of TK purified from E. coli harboring pHETK2 (the parent plasmid of pMCC) and HSV-1- infected cells were indistinguishable. Therefore, the alterations observed in the properties of the mutant TKs can be attributed to the nucleotide substitutions within the target region and that any differences between the vectors (pT7:MCC and pT7:HSVTKII) exerted only minor changes in catalytic properties.

3. /// vitro Transcription and Translation The transcripts described above were then used in a rabbit reticulocyte lysate cell-free translation system to synthesize active enzymes. Cell-free translation was according to Promega using nuclease-treated rabbit reticulocyte lysates.

Expression of full-length proteins was analyzed by subjecting ³⁵ S- radiolabeled cell-free translation products to SDS-PAGE and autoradiography. Briefly, 1 μl of each radiolabeled cell-free translation in v//rø-derived mutant mRNAs was subjected to SDS-containing polyacrylamide (12%) gel electrophoresis. An autoradiograph of this gel is shown in Figure 7. The first lane contains ¹⁴ C-labeled

rainbow molecular weight markers (Amersham) with the apparent molecular weight (x 10" ³ ) given on the left The second lane corresponds to a cell-free translation performed in the absence of any added mRNA The third lane corresponds to the wild-type pT7.HSVTKII mRNA translation product All other lanes contained translation products of the mutant mRNAs produced as described above As is evident from Figure 7, the major radiolabeled translation product from each mutant transcript migrates during electrophoresis as a -43 kDa protein with the same electrophoretic mobility as that observed with translation products from wild-type pT7:HSVTKJI transcripts To quantitate the level of protein synthesis for each translation, determination of trichloroacetic acid precipitable counts from each of the same samples was performed in triplicate The amount of acid-precipitable counts roughly parallels the band intensity of each mutant in Figure 7

E Time Course Analysis of Mutant Enzymes

On the basis of TK activities, mutant TKs were classified into two subsets (1) high-activity mutants (P155A/F161V, F161I, F161 C, and D162E), (2) low- activity mutants (F161I/R163H, F161L, D162G, and R163P/H164Q) For the high- activity mutant enzymes, unlabeled translation products were diluted 1/9 and incubated for 0, 5, 10, 20, or 30 minutes at 30°C Results of this experiment are shown in Figure 8A The TK activity results (counts per minute) were adjusted to reflect equivalent protein synthesis levels using the corresponding TCA-precipitable counts ( ³⁵ S cpm). Two of the mutants (F161I and P155A/F161V) demonstrated a statistically higher affinity for thymidine than the wild-type TK Standard deviations of F161C and D162E activities (data not shown) indicate no difference in activities when compared to the wild-type TK enzyme activities

The low-activity mutants were diluted 1/5, and the rate of phosphorylation as a function of time was also determined Results of this experiment are shown in Figure 8B The time course analysis indicates that most of the mutants had less than 10% wild-type activity One, F161L, however, demonstrated a moderate ability to phosphorylate thymidine, albeit at a much reduced rate from HSVTKII

F. Thermal Stability Assays

In the assays for colony formation on TK selection plates, several mutants were unable to complement KY895 at 42°C, suggesting that these mutant TKs were temperature-sensitive To substantiate this observation, cell-free translation products were incubated at 42°C for increasing times prior to being assayed for enzyme

activity Briefly, cell free translation ("CFT") products of each high-activity mutant, -RNA, and HSVTKII samples were diluted 1/9 and incubated for 0, 5, 10, and 20 minutes at 42°C The preincubated samples were then assayed for 5 minutes (P155A/F161V and FI 611) or 20 minutes (-RNA, HSVTKII, F161C, and D162E) The percent of activity remaining was determined with the untreated samples set at 100% As shown in Figure 9A, except for F161C, all high-activity mutants displayed thermal stabilities similar to HSVTKII after 42°C preincubation periods as long as 60 minutes (data not shown) Because F161C lost greater than 90% of enzyme activity within the first 20 minutes at 42°C, shorter incubation periods at 42°C were performed (0, 5, 10, and 20 minutes) F161C was exceptionally thermolabile demonstrating a -85% activity loss after only 5 minutes at 42°C

Low-activity mutant CFT products were diluted 1/5 and incubated for 0, 20, 40, or 60 minutes at 42°C The preincubated samples were then assayed in triplicate for the thymidine phosphorylation for 60 minutes The percent of activity remaining was determined using the untreated (time 0) sample as 100% As shown in Figure 9B, for the low-activity mutant subset one translation product (F161L) was more thermolabile that HSVTKII Others in this set (R163P, F161I/R163H, H164Q, and D162G) were equivalent to HSVTKII

G Substrate Specificity Assays

Three of the mutants (P155A/F161V, F161I and F161C) were assayed in triplicate for the relative levels of phosphorylation using thymidine, deoxycytidine, ACV, GCV, or AZT as substrates Briefly, forty-eight micromoles of each tritiated substrate was used in each assay reaction Translation products were diluted for each nucleoside assay as follows (translation/H ₂ O) 1/100, thymidine, 2/3, deoxycytidine, GCV, and AZT, 4/1, ACV Each set of assays was incubated for 2 hours at 30°C and the amount of phosphorylated product determined

The counts per minute of each set of assays were adjusted, and plotted as shown in Figure 10 Briefly, both P155A/F161V and F161I displayed an elevated capacity to phosphorylate thymidine relative to HSVTKII, 2 6- and 2.2-fold, respectively Phosphorylation of deoxycytidine by the mutant enzymes ranged from 1.9- to 2 8-fold over the wild-type enzyme (F161I, 1 9-fold, F161C, 2 8-fold, P155A/F161V, 2 8-fold) Two mutants appeared to share an increased ability to phosphorylate ACV (2 4- and 2-fold over HSVTKII by F155A/F161V and F161C, respectively) All mutants demonstrated approximately wild-type levels of AZT phosphorylation All mutants assayed appeared to share a large increase in GCV phosphorylation at 3 9-5.2- fold compared to wild-type phosphorylation levels

EXAMPLE 4 ANALYSIS OF TK MUTANTS WITH ALTERED CATALYTIC EFFICIENCIES

In order to identify mutants with altered catalytic activity, 190 of the TK mutants isolated in Example 1 (TKF) were analyzed in the assays set forth below.

A. Colony Formation Ability As A Functional Thymidine Uptake

The protein content of the purified enzymes was estimated by a modification of the Bio-Rad protein assay A standard curve was established using BSA and 25 μl of Bio-Rad reagent in a final volume of 125ul The amount of protein was determined by measuring the OD at 595 nm and comparing it to that of BSA.

In order to identify mutants with altered TK activity, a secondary screening protocol was designed based on the ability of the mutants to grow on medium containing different concentrations of thymidine (Table I). Briefly, it was first established that 1.0 and 10 0 μg/mL are the minimum and maximum concentrations of thymidine in the medium that supports the growth of E. coli harboring the wild-type tk plasmid. Since E. coli harboring the wild-type plasmid are unable to form visible colonies on TK-selection medium containing low thymidine (0.05 μg/mL), it was postulated that growth at this thymidine concentration might be indicative of mutants with an increased ability to phosphorylate thymidine. Accordingly, 0.05 μg/mL thymidine was used to select for variants with high TK activity and 20 μg/ml thymidine for variants with low activity.

Table I below shows the ability of selected mutants to functionally complement lie E. coli KY 895 as a function of increasing thymidine concentration. When all the 190 TK variants and the wild-type were subjected to screening at the thymidine concentrations indicated in Table I, only one, TKF 36, formed colonies at the lowest thymidine concentration tested (0.05 μg/mL). On the other hand, only TKF 41 grew at the highest concentration of thymidine in the medium. All of the other 188 mutants and the wild-type formed visible colonies on medium containing 1 μg/mL thymidine.

TABLE I

COLONY FORMING ABILITY OF TK- E. COLI KY895 TRANSFORMED WITH WILD-TYPE

AND MUTANT PLASMIDS, AS A FUNCTION or THYMIDINE CONCENTRATION

Mutant Thymidine concentration (μg/mL) ^a

0 05 1 2 10 20

Wild-type _a +a + ± ^b -

TKF 36 + + + ± -

TKF 41 - - + + +c

TKF 52 - + + + -

TKF 99 - + + + -

TKI 208 ^d - + + + - Colony formation was determined after incubation at 7°C for 24 hours

^+ and - indicate the abil or inability of £ coli harboring different plasmids to foπn visible colonies on the indicated TK -selection media "± indicates initial cell growlh cell deatli was apparent after incubation for 20 hours and may be due to the nucleotide pool unbalance generated b\ e phosphors lation of thymidine in the mutant and wild-type clones ^c Sιnce TKF 41 seemed to be a very low activity clone. overe\pressιon of this mutant TK was necessary for the survival of E coli on TK-selection medium pMCC and pMDC expression vectors have a temperature-sensitive repressor gene cl857 which becomes inactive at 42°C and. hence, there is overcxpression of TK and subsequent cell death In order to obtain controlled expression, screening was performed at 37°C However. TKF 41 containing E coli was incubated at 42°C on 20 μg/mL thymidine-containing TK-selection medium

"TKI 208 was obtained from the library described above in Example 2

B Sequence Analysis of High and Low Activity Clone

Wild-type tk and selected mutants were sequenced as described above in Example 2 Table II shows the nucleotide and deduced amino acid sequences of the wild-type tk and selected mutants for codons 165 to 175 Briefly, TKF 36, the mutant that forms colonies on low thymidine-containing medium, contains only a single amino acid substitution (Alal68— »Ser), whereas TKF 41 contained four substitutions Pro 165 →Ser, Alal67→Gly, Leul70→Gln and Alal74→Val Interestingly, TKF 52 has a different amino acid substitution (Alal68— »Thr) at the same position as TKF 36, but is unable to form colonies on low thymidine-containing medium TKF 99 contains two amino acid substitutions (Cys 171 — Leu and Ala 174 — Thi) TKI 208 has a single nucleotide substitution which results in a Leu 170— >Val substitution

TABLE II

NUCLEOTIDE AND DEDUCED AMINO ACID SEQUENCES OF THE WILD-TYPE AND MUTANT

TK ENZYMES AT THE TARGET REGION

165 ^a 166 167 168 169 170 171 172 173 174 175 SEQ

ID

Wild-type ccc ate gec gec etc ctg tgc tac ccg gec gcg 12 pro lie Ala Ala Leu Leu Cys Tyr Pro Ala Ala 13

TKF36 ccc ate gec Tec etc ctg tgc tac ccg gec gcg 14

Pro He Ala SER Leu Leu Cys Tyr Pro Ala Ala 15

TKF41 Tec ate gGc gec ctA^ cAG tgc tac ccg gTc gcg 16

SER He GLY Ala Leu GLN Cys Tyr Pro VAL Ala 17

TKF52 ccc ate gec Ace ctg ctg tgc tac ccg gec gcg 18

Pro He Ala THR Leu Leu Cys Tyr Pro Ala Ala 19

TKF99 ccc ate gec gec TtA ctg tTA tac ccg Ace gcg 20

Pro He Ala Ala Leu Leu LEU Tyr Pro THR Ala 21

TKI208 ccc ate gec gec etc Gtg tgc tac ccg gec gcg 22 Pro He Ala Ala Leu VAL Cys Tyr Pro Ala Ala 23 ^a Shows the codon number of the target region that was degenerated The wild-type nucleotide and amino acid sequences are shown below the codon number

°The silent mutations No other nucleotide changes were observed in the region sequenced (spanning codons 140-182) Each template was sequenced twice

Substituted nucleotide and amino acid residues are shown in bold capital letters

C Thymidine Uptake in E. coli Harboring Wild-type and Mutant TK Plasmids

In order to ascertain the actual level of thymidine uptake in E. coli harboring wild-type or mutant plasmids, the following assays were performed 7. [Methyl- ³ H] thymidine uptake assay

[Λ7e/by/- ³ H]thymidine uptake in E. coli harboring wild-type or mutant plasmids was determined essentially as follows Briefly, overnight cultures of E. coli containing pMDC (inactive TK), a plasmid containing wild-type TK, or TK36 were diluted 1 100 with LB-medium containing 100 μg/mL of carbenicillin, grown to 0 1 OD at A ₅₅ , shifted to 37°C and incubated with vigorous shaking Once an OD of 1 0 was attained, the culture was brought to room temperature (~25°C) and thymidine was added to 1 0 mL aliquots at a final concentration of 0 21 μM (0 16 μCi [methyl- ³ H]thymidine) After incubation for 0, 5, 10, 20, 30 and 60 s at 22°C, 50 μl aliquots were transferred onto nitrocellulose filters (0 45 μm), washed under vacuum with 10 mL

of chilled 50 mM Tris-HCl, pH 7 4, 0 9% NaCI, dried and counted in a scintillation counter using scintiverse BD (Fisher) Results are shown in Figure 1 1 Briefly, there was essentially no thymidine uptake in E. coli harboring pMDC The amount of thymidine uptake in E. coli harboring TKF 36 was 42% greater than in E. coli harboring the wild-type plasmid (18 pmol/10 ⁸ cells compared to 12 7 pmol/10 ⁸ after incubation for 10 s)

2. Incorporation of fmethy/- ³ HJthymιdιne into acid-insoluble material

The amount of TK activity in crude E. coli extracts containing the wild¬ type and mutant plasmids was determined indirectly by measuring the incorporation of thymidine into acid-insoluble material

Briefly, cultures were grown as described above under section 1 To 0.5 mL of culture, thymidine was added to a final concentration of 1 32 μM (0 2 μCi /me/by/- ³ H]thymidine) A 30 μl aliquot was taken out after designated times of incubation and added to 2 0 mL of cold 5% perchloric acid The precipitate was washed and radioactivity incorporated into an acid-insoluble material was determined essentially as described by Dube et al., 1991

Figure 12, shows that the incorporation of /røe/by/- ³ H]thymidine into an acid-insoluble product is more rapid with TKF 36 E. coli than with E. coli harboring the wild-type plasmid or the other tk mutants tested One of the mutants, TKF 99, having two amino acid substitutions (Cysl71→Leu and Alal74— »Thr) exhibited the same rate of thymidine incorporation as did the wild-type TKF 52 contains an Alal68— >Thr substitution (compare Alal 68-»Ser in TKF 36) and is unable to form colonies in the lowest thymidine-containing TK-selection medium (Table I), yet incorporates thymidine into acid-insoluble material at a rate greater than that of wild-type but less than that of TKF 36

D Purification of Wild-type and Mutant TKS

Crude extracts of the different mutants were obtained from 11 cultures that were grown at 30°C to 0 1 OD at A ₅₅₀ , shifted to 37°C and grown to 1 0 OD The cells were harvested by centrifugation at 4°C, washed with 25 mL of a solution containing 25% (w/v) sucrose, 50 mM Tris-HCl, pH 7 5, and 5 mM ΕDTA After centrifugation the cell pellet (-5-6 g weight) was stored at -70°C The cell pellet was thawed and suspended in 20 mL of buffer I (buffer I consisted of 10 vol 50 mM Tris- HCl, pH 7.5, 10% sucrose mixed with 1 vol 0 3M spermidine-HCl, 2 0M NaCI, 10% sucrose and 0 5 mM PMSF, pH 7 5) Once resuspension was uniform, 4 0 mL of buffer I containing 6 25 mg of lysozyme was added The suspension was poured into a chilled centrifuge tube and placed on ice for 30 minutes If cells did not lyse within 30 minutes,

the tube was placed in a 37°C waterbath for 4-6 minutes to enhance lysis Once cells started to lyse as judged by increasing stringiness, 2-3 mL of chilled buffer I containing 50 μg/mL aprotinin and 2 μg/mL of each leupeptin and pepstatin, was added to a final volume of 25 mL and the mixture was centrifuged at 28,000 r p m for 1 hour at 4°C and the supernatant was stored at 70°C

The wild-type and mutant TKs were purified by affinity ehromatography on a matrix of -aminophenylthymidine 3 '-phosphate coupled to CH-Sepharose 4B (Pharmacia) as described by Kowal and Marcus (Prep. Biochem. 6 369-385, 1976) with modification by Lee and Cheng (J. Biol. Chem. 251 2600-2604, 1976) All buffers used in the purification of TK contained 5mM DTT, 50 μ/mL aprotinin, 2 μg/mL each of leupeptin and pepstatin and 1 mM PMSF unless otherwise indicated A 7 mL bed- volume column was equilibrated with buffer A (0 1 M Tris-HCl, pH 7 5, 10% glycerol) and then loaded with -25 mL of the unfractionated supernatant at a rate of 8-10 mL/h The column was recirculated with the flow-through twice and then washed sequentially with ten bed-volumes each of buffer B (0 1 M Tris-HCl, pH 7 5, 0 5 M KC1, 10% glycerol) followed by buffer A TK was eluted with a linear gradient of thymidine (0- 600 μM) using 30 mL each of buffer A and buffer C (0 3 M Tris - HCl, pH 7, 4, 50 mM KCI, 10% glycerol) TK assay was performed on all the fractions and peak TK fractions were pooled and dialyzed against three changes of 21 of dialysis buffer (50 mM Tris- HCl, pH 7 4, 5 mM DTT, 10% glycerol) In the final dialysis, protease inhibitors were omitted from the buffer and the dialyzed fractions were aliquoted and stored at -70°C. The column was washed thoroughly twice by using the same washing and elution protocols as described above prior to application of each extract preparation

The protein content of the purified enzymes was estimated by a modification of the Bio-Rad protein assay A standard curve was established using BSA and 25 μl of Bio-Rad reagent in a final volume of 125ul The amount of protein was determined by measuring the OD at 595 nm and comparing it to that of BSA [Methyl- ³ H]thymιdιne uptake

Results are shown in Figure 1 1 Briefly, there was essentially no thymidine uptake in E. coli harboring pMDC The amount of thymidine uptake in E. coli harboring TKF 36 was 42% greater than in E. coli harboring the wild-type plasmid (18 pmol/10 ⁸ cells compared to 12 7 pmol/10 ⁸ after incubation for 10 s)

The amount of TK activity in crude E. coli extracts containing the wild¬ type and mutant plasmids was determined indirectly by measuring the incorporation of thymidine into acid-insoluble material

E Kinetic Parameters Of Purified Mutant Thymidine Kinases

The three cellular parameters so far studied suggest that TKF 36 is a more active enzyme than any of the other mutant enzymes tested or the wild-type. In order to determine the kinetic parameters of catalysis, wild-type, TKF 36 and three other mutant thymidine kinases were purified to near homogeneity using affinity ehromatography as described above The purified wild-type, TKF 36 and TKI 208 were examined by electrophoresis in an SDS-PAGE system and were found to exhibit a single prominent band that migrated at 43 kDa, which was judged to be 95% homogeneous by silver staining Kinetic parameters were determined essentially as described below

Briefly, TK assay mixtures (50 μl) contained 50 mM Tris-HCl, pH 7 5, 5 mM ATP, 4 mM MgCl ₂ , 2 5 mM DTT 12 mM KC1, 0 18 mg/mL BSA, 5% glycerol, 1 μM thymidine (0 3 μCi /wetby/^Hjthymidine) and the indicated amounts of purified enzymes The kinetics of thymidine phosphorylation were determined by varying the unlabeled thymidine concentration (0-4 0 μM) and known amount of purified enzymes (the sp acts of the purified TKs were 1 1, 3 0, 0 5, 0 34 and 0 01 units for wild-type TKF 36, TKI 208, TKF99 and TKF41, respectively) One unit of enzyme is defined as the amount that phosphorylates 1 0 pmol of thymidine to thymidylic acid in 1 minute under the conditions described above Incubation was at 34 ± 1°C for 10 minutes The reaction was stopped by the addition of 1 mM cold thymidine Half of the reaction mix was pipetted onto a DEAE-cellulose disc (25 mm) and the disc was dipped in distilled water (1 minute) followed by four washes each in 10 mL of absolute ethanol The adsorbed products on the disc were counted in a scintillation counter The kinetic parameters K _m and ^ _maλ were determined by using the Cleland SUBIN program (Cleland, Methods Enzymol. 63 103-138, 1979) and the values for k _cat were calculated from the equation _max = £ _cat [E] ₀ , where [E] ₀ is the total enzyme concentration

Results of these assays are summarized in Table III Ala 168 — > Ser substitution in TKF 36 resulted in a 4 8-fold enhancement in k _cat None of the other purified mutant enzymes (TKF 41 , TKF 99 and TKI 208) that were analyzed exhibited an increase in k _ca{ compared to that of the wild-type TK A 2 2-fold decrease in k _cat results form the Leu 170 — > Val substitution in TKI 208, whereas two of the other tk mutants, TKF 99 and TKF 41 , with decreased efficiencies in the in vivo assays, exhibited a 28- and 34 700-fold decrease in r _cat Table III also presents the Michaelis constant (K _m ) for the mutants and wild-type with thymidine as a substrate The apparent K _m for the wild-type enzyme was 0 47 μM, which agrees well with previously reported values (Jamieson and Subak-Sharpe, J. Gen. Virol. 24 481-492, 1974, Elion, Am. J. Med. 73 7- 13, 1982, Waldman et al , J. Biol. Chem. 258 1 1571-1 1575, 1983) Even though TKF

36 showed a higher k _ca{ value its affinity for thymidine, as reflected in the K _m , is 6 2-fold lower than the wild-type TK TKI 208, TKF 41 and TKF 99 have a similar K _m to that of the wild-type Interestingly, the k _cal /K _m value of TKF 36 [2 0 x 10 ⁶ s^M" ¹ ] is not very different from the wild-type [2 5 x 10 ⁶ s^M" ¹ ] , while TKI 208, TKF 99 and TKF 41 exhibit lower values of 1 57 x 10 ⁶ , 0 15 x 10 ⁶ and 0 00012 x 10 ⁶ s" ¹ M- ¹ , respectively

TABLE III

COMPARISON OF KINETIC PARAMETERS OF THE THYMIDINE KINASES

^Enzyme K _m , _{v M)} A _P „ (l/»)

Wild-type 0 47 ± 0 l ^a 1.2

TKF 36 2 90 ± 0 01 5 7 ^b

TKF 41 0 28 ± 0 16 3 5 x 10- ⁵ b

TKF 99 0 29 ± 0 002 0 04 ^b

TKI 208 0 35 ± 0 008 0 5 ^{b a} Data presented as ± SE

"The P value is <0 02 compared to the wild-type

EXAMPLE 5 SELECTIVE KILLING OF CELLS TRANSFECTED WITH RETROVIRAL VECTORS CONTAINING MUTANT HSV- 1 TK

The example describes the construction of retroviral vectors which express a type 1 Herpes Simplex Virus thymidine kinase, a proline to alanine mutation at position 155, and a phenylalanine to valine mutation at position 161

Vector Construction

The thymidine kinase gene from P155A/F161V is utilized to replace the wild-type HSV tk sequences in the Moloney Murine Leukemia Virus ("MoMLV") based vector GlTkSvNa 90 from Genetic Therapy, Inc (Gaithersburg, MD, see Ram et al Cancer Research 53 83, 1993) In particular, the mutant tk gene is inserted downstream from the 5' long terminal repeat sequence, which the tk gene uses as a promoter This vector also contains an neomycin phosphotransferase gene (neo) which is expressed from an SV40 early promoter

B. Producer Cell Line

The retroviral vectors described above may then be packaged by the amphotropic retroviral packaging cell line GP+envAml2 (U.S. Patent No. 5,278,056) after calcium phosphate transfection. A vector containing the gene for β-galactosidase is used as a control vector. The cloned vector producer cells are maintained in culture containing Dulbecco's modified Eagle's medium with 10% fetal calf serum, 2mM glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin and 2.5 μg/ml Fungizone. Prior to administration, the media is removed and the cells rinsed with saline. The monolayers are trypsinized for 5-10 minutes at 37°C, collected, washed twice and resuspended at 5- 10 x 10 ⁸ cells/ml.

C. /// Vitro Sensitivity to Ganciclovir

To assess the sensitivity of cells transduced with the mutant or the wild¬ type tk gene containing vectors, rat 9L glioma cells and human U251 glioblastoma cells are transduced in vitro by exposing the cells to supernatant containing replication incompetent vector particles. The transduced cells are selected by including G148 (1 mg/ml) in the culture medium. Nontransduced, HSV tk wild-type transduced and HSV tk mutant transduced cells are then evaluated for their sensitivity to increasing levels of ganciclovir. The level of DNA synthesis is determined by tritiated thymidine incorporation after various ganciclovir exposure times and ganciclovir levels. Cell viability is determined by plating the cells in 10 cm tissue culture plates in the absence or presence of various ganciclovir concentrations, and counting the number of cells at 24 hour intervals.

D. In Vivo Transduction

The efficiency of in situ transduction of and relative level of vector gene expression in the tumor cells is determined using the β-galactosidase containing vector. Briefly, Fischer 344 rates are anesthetized and injected with 4 x 10^ syngeneic 9L gliosarcoma cells using a 10 μl Hamilton syringe connected to a stereotaxic injection apparatus. After ten days, the same stereotaxic position is used to directly inject 1.5 x 10^, 3 x 10^ or 6 x 10^ HSVtk (wild-type or mutant) β-galactosidase transduced or nontransduced producer line cells, and producer cell line supernatants into the 9L tumor. As a control, rats are injected with the same volume of sterile saline instead of cells. Ganciclovir is then administered and the rats are sacrificed to determine the anti- tumor effect. A histological examination is also performed.

E. Dose Optimization of Ganciclovir

Rats are injected intracerebrally with 4 x 10^ HSVtk (wild-type or mutant) or β-galactosidase transduced rat 9L producer cells. Seven days post inoculation, ganciclovir is administered i.p. at 5, 20 or 15 mg/kg twice daily for 7 days. Control rats receive i.p. saline injections. All rats are sacrificed after the ganciclovir treatment and the brains and tumors removed for weight determination and histological examination.

F. Tumor Regression with Wild-type and Mutant HSV tk Transduction and GCV Based on the results of the ganciclovir dose optimization, rat tumors inoculated with transduced or nontransduced producer cells or produced cell supernatant are administered ganciclovir doses for a specific time period. Antitumor effects are determined by determination of tumor weight and histological examination.

EXAMPLE 6

THE USE OF VZV TK MUTANTS AS TARGETS FOR

SELECTABLE HOMOLOGOUS RECOMBINATION

This example describes the use of a mutant Varicella Zoster Virus thymidine kinase ("VZV tk") as a target for homologous recombination in the construction of stable transfected cells lines, strains or recombinant viruses. In particular, the construction of vaccinia viruses as cloning vectors containing mutant VZV TKs for the selection of recombinant viruses in TK ⁺ cell lines is described.

A. Construction of Recombinant Vaccinia Virus Plasmids Containing VZV TK Mutants

VZV tk genes (wild-type and mutant) are cloned into a recombinant plasmid behind the vaccinia virus 7.5 K promoter for constitutive gene expression. In addition the neomycin phosphotransferase gene is cloned after the 3' end of the VZV tk gene to serve as a selectable marker. The 5' or 3' regions of the vaccinia virus encoded thymidine kinase gene flanks the 5' end of VZV tk gene and the 3' end of the neomycin phosphotransferase gene (neo). This allows for the insertion of the VZV tk gene into the viral genome and the concomitant inactivation of the vaccinia thymidine kinase gene. The remainder of the plasmid is based on pUC and contains an ampicillin resistance gene and a ColEl origin of replication for maintenance of the plasmid in E. coli.

B Construction of Recombinant Poxyiruses

The VZV tk (wild-type or mutant) + neo recombinant plasmid or recombinant plasmid containing only the neo gene is cotransfected with the wild-type vaccinia virus into BSC40 cells. Recombinant viruses are selected by resistance to G418. After several rounds of plaque purification, the recombinant viruses are subjected to plaque hybridization and DNA analysis in order to confirm the insertion and location of the foreign genes.

C. Dose Optimization of Ganciclovir Vaccinia virus infected and uninfected BSC40 cells are subjected to treatment with various doses of ganciclovir in order to determine the tolerance level. Cells infected with recombinant viruses expressing VZV TKs and neo or those expressing only neo will be grown in the presence of various levels of ganciclovir. VZV tk gene containing viruses are more sensitive to ganciclovir treatment than the cells alone or those infected with wild-type vaccinia virus. A level of ganciclovir is selected from the results of this experiment to select for the loss of sensitivity to ganciclovir for homologous recombination with other genes to be inserted into the VZV tk locus.

D. Selection of Recombinant VZV tk Poxyiruses Using Ganciclovir BSC40 is infected with the VZV tk recombinant virus in the presence of a recombinant plasmid carrying the gene to be introduced into the VV genome, abutted to the VV 7.5 K promoter cloned with VZV tk sequences flanking. Recombinant virus is selected with ganciclovir.

Any cell line stably transfected with the VZV tk gene can be the target for introduction of foreign genes by homologous recombination and for the selection of such an event by resistance to ganciclovir.

EXAMPLE 7 CONSTRUCTK )N AND ANALYSIS ( )F HSV- 1 THYMIDINE KINASE AND HSV- 1 DNA POLYMERASE VECTORS

A. Construction of Vectors

Three constructs were made containing either the HSV-1 DNA polymerase gene, HSV-1 thymidine kinase gene or both. a) pHSG576:HSVpol

The 5 5 kb H///DIII/£cr RI fragment from pGEM2-702 (David Dorsky, Univ of Conn ) was cloned into pHSG576 (Sweasy and Loeb, J. Biol. Chem 267 1407- 1410, 1992) in two steps

1 ) The 2 4 kb Pstl/EcoRl fragment was cloned into pHSG576 digested with Psl and EcoRl This clone was designated pHSG576 1/2 pol

2) The 3 lkb HtnOlll/Pstl fragment of HSV DNA polymerase was cloned into pHSG576 1/2 pol digested with H//.DIII and Pstl This clone was designated pΗSg576 HSV DNA pol b) pHSG576 HSV-l TK The XbaMBamlll fragment fro pET23d HSVTK (contains the

HSV-1 TK Ncol-Ncol fragment in pET23d, Novagen) was blunt-ended and cloned into the S al site of pHSG576 The clone was designated pHSG576 HSV-1TK c) pHSG576 HSV pol/TK

This clone contains both the HSV-1 DNA polymerase and TK genes for coexpression from the same vector It was created in a two step cloning protocol

1) The Xbal/BamHl - bluntended TK fragment was cloned into the bluntended EcoRl site of pHSG576 l/2pol (contains the 2 4kb Pstl/EcόRl fragment) 2) The 3 lkb H///DIII/ .S/I fragment (5' end of the polymerase gene) was cloned into pΗSG576 l/2pol/TK digested with H/wDIII and Pstl This clone was designated pΗSG576 HSVpol/TK

B Transformation of E. coli With A DNA Polymerase Defect E. co JS200 (polA12recA718) was transformed with pHSG576:HSV

DNA pol or pHSG576 DNA and plated on nutrient agar (NA) containing tetracycline (12 5 μg/mL) and chloramphenicol (34μg/mL) Plates were incubated at 30°C (permissive temperature) Single colonies were grown overnight in NB + tet + Cm DNA was isolated from these cultures and used to transform JS200 again From the second transformation several colonies from each were picked and used to inoculate NB + tet + Cm in the presence or absence of IPTG After overnight growth at 30°C, a single loopful of each culture was spread in a diverging spiral of increasing dilution from the center of the plate NA plates + tet + Cm +/- IPTG were incubated at 30°C (permissive) or 37°C (nonpermissive) The growth pattern of cells containing pHSG576 HSV DNA pol displayed growth of single colonies (low cell density) at 37°C, while cells containing

only the vector were unable to grow at low cell density at the nonpermissive temperature.

These results demonstrate that the Herpes DNA polymerase can complement the E. coli Poll defect in vivo.

EXAMPLE 8

CONSTRUCTION AND ANALYSIS OF TK MUTANTS WITH MUTATIONS AT CODONS 159 TO 161 AND 168 TO 170 UTILIZING A 100% RANDOM LIBRARY

This example describes the construction and analysis of TK mutants that are mutagenized at codons 159 through 161 and 168 through 170.

Bacterial Strains. SY21 1 (BL21(DE3) tdk ^" , pLysS) is cured of pLysS by repeated passages on non-selective plates (no chloramphenicol). (SY21 1 is a gift from William Summers, Yale University, New Haven, CT and is described in Summers, W. C. and Raskin, P., J. Bad. 775:6049-6051, 1993). The resulting strain BL21(DE3) tdk ^' is used in the genetic complementation assays for thymidine kinase activity. Other strains used are described in Example 3.

Cells. BHK tk ^" (tsl3) cells (ATCC No. CRL-1632) are purchased from the American Type Culture Collection and cultured in DMEM + 10% calf serum at 37°C under 6% CO ₂ .

Materials. As described in Example 3.

A. Generation of TK Mutants

7. Construction of Random Insert

Two oligonucleotides are synthesized by Operon (Alameda, CA) : MB126 (58mer) 5'-TGGGAGCTCA CATGCCCCGC CCCCGGCCCT CACCNNNNNN NNNGACCGCC ATCCCATC-3' (SEQUENCE ID No. 24) and MB 127 (51mer) 5'-ATAAGGTACC GCGCGGCCGG GTAGCANNNN NNNNNGGCGA TGGGATGGCG G-3' (SEQUENCE ID No. 25). The N designates an equimolar mix of all four nucleotides during synthesis.

The purification of oligonucleotides, annealing, extension and amplification by PCR is essentially as described in Example 3.

2. Generation of Random-Sequence Containing Libraries

Vector Construction pET23d, purchased from Novagen, is the backbone for the construction of pET23d:HSVTK-Dummy. pET23d:HSVTK-Dummy is used in place of pMDC (described in Example 1 and 3) for insertion of random sequences. Briefly, a 1.7kb NcollH i lll fragment is purified from a restriction digest of pT7:HSVTKII (Example 3) and cloned into pET23d restricted with the same enzymes to generate pET23d:HSVTK. The dummy vector is constructed by replacing the tk sequences between the Kpnl and Sacl sites with the KpnVSacl fragment from pMDC (Example 3). Library Construction

Qiagen column purified pET23d:HSVTK-Dummy DNA is restricted with Kpnl and S cl and the vector gel isolated using GenCleanll (BiolOl, La Jolla, CA) to remove the small insert fragment. This vector is ligated with the gel isolated PCR- amplified random fragment overnight at 16°C with T4 DNA ligase. 3. Selection of TK Mutants

The ligated mixture is then used to transform BL21(DE3) tdk ^' cells by electroporation as described in Example 3. The transformants are plated directly onto TK selection plates (Example 3) with a small fraction plated on 2 x YT (16g tryptone/lOg yeast extract/5g NaCl/15g BactoAgar per liter) + carbenicillin at 50μg/ml (carb ⁵⁰ ) to determine the total number of transformants. The plates are incubated at 37°C overnight and scored for growth on TK selection plates and the transformation frequency determined. Colonies that grew on the TK selection plates are picked and restreaked on fresh TK selection plates and 2 X YT + carb ⁵⁰ plates. Approximately 426 positive clones are identified from a library of 1.1 x 10 ⁶ transformants or 0.039% of all transformant conferred TK activity to E. coli BL21(DE3) tdk ^' (Figure 14).

B. Analysis of Mutants

7. Sequence of Selected and Unselected Clones

Seventeen clones that demonstrated TK activity (selected) or are taken from 2 x YT + carb ⁵⁰ plates (unselected) are successfully sequenced. DNA is isolated using Qiagen miniprep kits and subjected to double strand sequencing as described in Example 3. Figure 15 shows the sequences from each group and demonstrates that the initial random oligonucleotides are randomized. In both selected and unselected tk genes, the introduction of secondary mutations at sites distal to the randomized region are observed. However, the mutations are primarily confined to two codons, 155 and 156. These mutations are most likely introduced by contamination during the synthesis of the original random oligonucleotides. All changes at codon 155 are silent. Changes

at codon 156 resulted in alanine to valine, serine or proline alterations. Alignment studies indicate that position 156 is not conserved either for alanine nor for the type of amino acid at that position. Therefore, it is unlikely that these secondary mutations result in any real effect on the enzyme activity of the mutants. All selected mutants contained at least two amino acid changes.

2. Secondary Screening for GCV and ACV Sensitivity

Each of the 426 mutants is picked and used to inoculate 200μl of TK selection medium (Example 3) in a 96 well microtiter plate format. All 426 clones are then serially diluted 10 ⁴ in 0.9% NaCI with a 48-prong replicator (Sigma, St. Louis, MO). 30μl of the last dilution is spread onto TK selection plates containing 1 μg/ml thymidine plus varying concentrations of ganciclovir or acyclovir. Initially 2μg/ml GCV is used and the clones unable to grow are scored as positives since any mutant with increased conversion of a pro-drug to an active toxin results in lethality. On 2μg/ml GCV 197 clones are identified. Sequential plating on 1 μg/ml and 0.5μg/ml GCV lead to the identification of 47 mutants. Plating on ACV plates (1 μg/ml) gave 1 16 ACV sensitive clones. To ensure that the clones are truly sensitive to the nucleoside analog and not simply scored because of the inability to grow on the lower thymidine concentrations used, the 47 GCV and 1 16 ACV clones are plated on TK selection plates containing thymidine at 1 μg/ml (no nucleoside analog). Almost half of the clones are unable to grow on low thymidine for a total of 26 GCV sensitive mutants and 54 ACV sensitive mutants. Results are shown in Figure 16.

C. In Vitro Analysis 7. In Vitro Transcription and Translation.

Plasmid DNA is purified by Qiagen column ehromatography. Transcription and translation of the 80 selected mutants is done as in Example 3 except that the isolated plasmids are not linearized prior to transcription. In vitro translation products are assayed in duplicate for thymidine, ganciclovir and acyclovir phosphorylation and compared to pET23d:HSVTK mRNA translation product assays (see Example 3).

2. Measurement of Enzyme Activity

Radiolabelled nucleosides are present in each assay at l μM, 7.5μM and 7.5μM for thymidine, ganciclovir and acyclovir, respectively. The level of activity is adjusted to reflect the level of protein synthesis as determined from the TCA precipitable counts from a duplicated translation with ³⁵ S methionine. For the majority of the 80

mutant enzymes, the level of thymidine, ganciclovir and acyclovir is less that 1% that of the wild-type TK Ten mutant enzymes displayed greater that 10% phosphorylation with at least one of the nucleosides assayed The nucleotide sequences are shown in Figure 17 Several of the clones contained mutations outside the randomized region Two clones, 30 and 84, have mutations that result in amino acid changes, A152V and A156S, respectively Four clones contain in-frame deletions, three (226, 340 and 411) with -3 deletions and one (197) with a -6 deletion All these mutations are centered around a GC-rich region which encodes for the peptide A P P P A This proline rich peptide is likely to comprise a turn at the tip of a loop section The loss of one or two amino acids may simply result in shortening of the loop All of these mutants contain three to six amino acid alterations within the randomized region as shown in Figure 18 with the respective levels of activity determined //; vitro

D Effect of GCV and ACV on Mammalian Cells Expressing Mutant Thymidine Kinases

7. Subcloning mlo a Mammalian Expression Vector

Three mutant thymidine kinases are selected to evaluate for cell toxicity //; vivo in the presence of ganciclovir or acyclovir Mutant clones number 30, 75 and 132 and the wild-type thymidine kinase genes are restricted with Ncol and blunt-ended with Klenow The gel isolated fragments (Ncol-blunt) are ligated to pCMV restricted with Notl and transformed into E. coli strain NM522 The wild-type TK gene in the wrong orientation relative to the CMV promoter is also used as a control Qiagen column purified clones are sequenced to confirm orientation, sequence and the 5' junction region The clones are designated pCMV, pCMV TK-wrong, pCMV TK, pCMV 30, pCMV 75 and pCMV 132

2. Transfedions

As an initial step to evaluate these mutants, the pCMV clones are introduced in the presence of a neomycin resistant marker plasmid (pSV2neo) into TS13 BHK tk ^" cells (baby hamster kidney cells) by calcium phosphate precipitation using a modified version of Chen and Okayama (Molec. Cell. Biol. 7 2745-2752, 1987)

Briefly, the cell transfections are performed as follows Approximately 5 x 10 ⁵ ts!3 BHK tk ^" cells (ATCC CRL-1632) are plated on 100mm dishes in DMEM + 10% calf serum For each transfection l μg of pSV2neo and lOμg of a pCMV construct (pCMV, pCMV TK-wrong (HSVTK in the wrong orientation relative to the promoter), pCMV HSVTK, pCMV 30, pCMV 75 or pCMV 132 DNA) in 0 25M CaCl ₂ are mixed with 0 5ml 2 x BBS (see Chen and Okayama) and preincubated at 37°C at 2 5% CO ₂ for

24 hours. The CaCl ₂ /DNA mix is added dropwise to the plates and mixed in well. After a 24 hour incubation at 37°C in a 2.5% C0 ₂ wet incubator, the cells are rinsed twice with Dulbecco PBS minus Ca/Mg and fed with fresh DMEM + 10% calf serum. Plates are incubated at 37°C with 6% CO ₂ . After 72 hours post-transfection the cells are split 1 :3 and plated in DMEM + 10% calf serum containing G418 at 600μg/ml.

3. Selection and ED ₅ 0 Determinations

The cells are selected on G418 (600μg/ml) at 37°C for 17 days. During this time the plates are pooled (for each DNA transfection) and split three times at a ratio of 1 :3. Approximately 30-40 clones are selected in this manner for each transfected DNA containing a tk gene in the correct orientation. The pCMV and pCMV:TK-wrong transfections yielded between 130 and 140 clones each. G418 resistant clones are harvested, pooled and plated at a density of 2000 cells/well in lOOμl DMEM + 10% calf serum and 200μg/rnl G418 + 6% CO ₂ in 96 well microtiter plates. A concentration range of either ganciclovir (0.125, 0.25, 0.5, 1, 2.5, 5, 7.5, 10 and 20μM) or acyclovir (0.5, 1, 2.5, 5, 10, 25, 50, 75 and lOOμM) is added to each plate with 8 repeats of each concentration for each transfectant population (the no nucleoside analog controls each had 16 repeats). After three days in the presence of the nucleoside analog, Alamar Blue is added and 6 hours later the plates are scanned using a fluorometer as according to the manufacturer's protocol (Alamar Biosciences, Inc., Sacramento, CA). The plates are incubated a further 24 hours at 37°C and scanned again.

Determination of the fluorescence level of cells incubated in the presence of Alamar Blue directly relates to cell viability. Subtraction of the background fluorescence allows one to plot the cell survival versus the nucleoside analog concentration to determine to effective dose for killing 50% of the cells (ED ₅₀ ). The survival curves are plotted with data from the second scan and are shown in Figures 19 (GCV) and 20 (ACV).

After 4 days on nucleoside analog the effective doses for 50% cell killing with GCV and ACV are determined from Figures 19 and 20 (see Table IV).

TABLE IV

GCV fold over WT ACV fold over WT

WT 20μM 1 25μM 1 30 5μM 4 18μM 1.4 75 0.4μM 50 1.25μM 20 132 20μM 1 25μM 1

4. Enzyme A ssay.s and Immunob/ots

Cell extracts from 2.4 x 10 ⁶ pooled transfectants are assayed for thymidine, ganciclovir and acyclovir activity. The levels of phosphorylation corresponded very well with the activities determined in vitro (rabbit reticulocyte lysate translation products) and the amount of protein expression as determined by western blot analyses. No immunoreactive band is seen in the lanes corresponding to pCMV or pCMV:TK-wrong (TK gene in the wrong orientation). Both the wild-type TK (pCMV:HSVTK) and pCMV: 132 transfected cell lysates exhibited roughly equivalent band intensities. The immunoreactive band for pCMV:30 cell lysates is substantially more intense (5-10 fold) and that of pCMV:75 is approximately half the pCMV:HSVTK band intensity for the equivalent cell number.

5. Testing Mutants in Gliohlastoma Cell Lines Blunt-ended Ncol fragments isolated from pET23d:HSVTK, pET23d:30 and pET23d:75 are cloned into the Hpal site of pLXSN (Miller and Rosman BioTechniques 7:980, 1989). Plasmid purification is done by Qiagen ehromatography and the isolated DNA sequenced to confirm orientation and 5' junction regions. Stable transfectants of rat C6 glioblastomas (ATCC CCL- 107) and a human glioblastoma cell line (SF767) are made as described above with the exception that pSV2-neo is not co¬ transfected since the neomycin phosphotransferase gene is encoded by pLXSN. Selection and analysis is essentially as described above.

E. Kinetic Analysis of Mutant Thymidine Kinases 7. Overexpression of Mutant and Wild-Type Enzymes

A single colony of pET23d:HSVTK, pET23d:30, pET23d:75 and pET23d: 132 in BL21 (DE3)tk ^" cells is used to inoculate 5ml of M9ZB medium (1% tryptone, 0.5% NaCI, 1 x M9 salts, I mM MgS0 ₄ , 100μM CaCl ₂ and 0.2% glucose)

containing cabenicillin at 20μg/ml. The culture is incubated at 37°C overnight. The following day the 5ml culture is used to inoculate 1L M9ZB + cabenicillin at 20μg/ml and the culture allowed to grow at 37°C to OD600 0.1. At that point IPTG is added to 0.4mM and the culture incubated a further 3 hours. The cells are chilled on ice, pelleted by centrifugation and the pellets washed once in cell wash buffer (50mM Tris, pH 7.5, 5mM EDTA, 10% sucrose) prior to freezing the pellets at -70°C. The next day the cells are resuspended in 12ml Buffer 1 ( 50mM Tris, pH 7.5, 10% sucrose, 2mM DTT, 5mM EDTA, ImM PMSF) and the volume split into two 13ml Oakridge ultracentrifuge tubes, lml Buffer 1 containing 3mg lysozyme is added to each tube and the tubes left on ice for 1 hr. An additional l ml Buffer 1 + protease inhibitor mix is added and the tube spun at 35krpm in a Sorvall T-1250 rotor at 4°C. The cleared supernatant is then aliquoted and frozen at -70°C.

2. Affinity Purification A thymidylyl-sepharose column is used for a one step purification procedure (see Example 2). The lml bed volume column is prepared by passing 10ml Buffer 1 followed by 10ml Absorption Buffer (50mM Tris, pH 7.5, 10% sucrose, 2mM DTT, 25mM MgAc ₂ , 1 OmM ATP) over the column. Two ml of the cleared lysate is mixed with 2 ml of Absorption Buffer and passed through a 0.2μm filter. This mix is passed over the column 3 times. The column is washed with 5ml Absorption buffer three times and the 5ml fractions collected. To elute the enzyme, 3 - lml fractions of Thymidine Buffer (300mM Tris, pH 7.5, 10% sucrose, 2mM DTT, 50mM KCl, 600μM thymidine) is passed over the column and each lml fraction collected. The column is reactivated by loading on 10ml High Salt Buffer (50mM Tris, pH 7.5, 10% sucrose, 2mM DTT, 0.5M KCl) and 10ml 50mM Tris, pH 7.5. The column is stored in 50mM Tris pH 7.5 + 0.004% sodium azide. The extent of purification is monitored by Coomassie stained SDS :P AGE analysis and the concentration of purified protein determined using the BioRad Reagent (Bradford Reagent). The fraction containing TK protein is dialyzed against several liters of 50mM Tris, pH 7.5 10% sucrose, 2mM DTT at 4°C to remove thymidine.

3. Enzyme Kinetics

Enzyme assays described above are used to determine the K _m , V _max and Kca, of the purified TK enzymes for the substrates thymidine, ganciclovir and acyclovir.

EXAMPLE 9

ISOLATION OF HUMAN AND MOUSE GUANYLATE KINASES AND

CONSTRUCTION OF HSV-1 THYMIDINE KINASE AND GUANYLATE

KINASE DUAL EXPRESSION VECTORS

This example describes the isolation of the human and mouse guanylate kinase genes and the vector construction for dual expression of herpes thymidine kinase and guanylate kinase.

A. Isolation of the Human Guanylate Kinase Gene

7. Isolation of the Human Guanylate Kinase Gene

Two oligonucleotides are designed to amplify the entire human guanylate kinase open reading frame. The following two oligonucleotides are synthesized by GenSet (La Jolla, CA): 5'-ACTACTGGAT[CCATGGlCGGGCCCCAGGCCTGTG- 3', a 33-mer (SEQUENCE ID. NO. 26) and 5'-

TACTACGGATCCTCAGGCGGCGGTCCTTTGAGC-3', a 33-mer (SEQUENCE ID. NO. 27). The BamHl sites at each end are underlined and the Ncol site at the initiating methionine codon is shown in brackets. The bold nucleotide denotes a nucleotide alteration from the original sequence (GenBank accession number Al 1042). The human guanylate kinase gene is amplified from a cDNA library of human proliferating B lymphocytes stimulated with alpha-CD3. The resulting single band (~600bp) is restricted with BamHl and cloned into pUCl 18 (BamRl) to yield pUCl 18:Hugmk. The insert is sequenced in entirety (both strands) using the following set of oligonucleotides: 5'-CTGCTGAAGAGGCTGCTC-3' (18mer) (DMO 512) (SEQUENCE ID. NO. 28), 5'-ACACAGATGCGGTTTCATG-3' (19mer) (DMO 513) (SEQUENCE ID. NO. 29), 5'-CTGGACGTGGACCTGCAG-3' (18mer) (DMO 514) (SEQUENCE ID. NO. 30), 5'-GTTAATGATGACCACATC-3' (18mer) (DMO 515) (SEQUENCE ID. NO. 31), 5'-TGTAAAACGACGGCCAGT-3' (18mer) (Ml 3 forward primer purchased from ABI) (SEQUENCE ID. NO. 32) and 5'-CAGGAAACAGCTATGACC-3' (18mer) (Ml 3 reverse primer from ABI) (SEQUENCE ID. NO. 33). Sequence analysis revealed identity with the GenBank sequence except for the anticipated alteration at the Ncol site which results in a serine to alanine change (S2A) (Figure 21).

2. Nor/hern Blot 8μg of total RΝA from SP2/0 murine B lymphoma cells is prepared in 1 x MOPS buffer/75% formamide and heat denatured for 10 min at 55°C and loaded on a

1.2% agarose gel in 1 x MOPS buffer. After transfer to nitrocellulose the blot is probed with the human gmk gene.

The 600bp BamHl fragment is gel isolated from pUCl 18:Hugmk and is labeled using the random primer labeling kit from Amersham according to the manufacturer's instructions. The free radiolabel is removed by size exclusion ehromatography. Following hybridization and washes the blot is exposed to X-ray film at -70°C for two days. Autoradiography of the northern blot reveals a single ~750nt RNA species. In a similar experiment using human poly A+ RNA from proliferating B lymphocytes, a single ~750nt band is also observed.

B. Isolation of Mouse Guanylate Kinase Gene 7. Screening a Mouse cDNA Library

A lambda gt lO cDNA library of mouse 702/3 cells (B lymphomas) is probed using the human gene (same probe as used for northern blot analysis). The total number of plaques screened is 2 x 10 ⁵ pfu. Nine independent lambda clones hybridized to the human probe and are plaque purified.

2. Subcloning and Sequence Analysis of Positive Clones

The EcoRl fragments from eight phage DNA preparations are gel isolated and subcloned into pUC1 18 restricted with EcoKl and dephosphorylated. The DNA insert sizes ranged from ~300bp to 1.2kb. Preliminary sequence analysis with primer (Ml 3 forward primer) reveals that all clones began approximately 60bp 5' to the putative ATG start codon as determined by sequence alignment with the human and bovine guanylate kinase sequences and varied at their respective 3' ends. One representative clone (both strands) is completely sequenced using the following oligonucleotides: 5'-TGTGTCCCATACTACTACAAG-3' (21mer) (DMO 592) (SEQUENCE ID. NO. 34), 5'-TGAGAACTCAGCAGCATGCTC-3' (21mer) (DMO 594) (SEQUENCE ID. NO. 35), 5' GTGCTAGATGTCGACCTA-3' (18mer) (DMO 595) (SEQUENCE ID. NO. 36), 5'-ACCTGGATAAAGCCTATG-3' (18mer) (DMO 674) (SEQUENCE ID. NO. 37), 5'-AAGCAGGCGCTCTCTCTGA-3' (19mer) (DM0 675) (SEQUENCE ID. NO. 38), 5'- CTATTTCTCATATGATGT-3' (18mer) (DMO 731) (SEQUENCE ID. NO. 39) and 5'-GTTACAGTGTCTCTAGAG-3' (18mer) (DMO 732) (SEQUENCE ID. NO. 40), 5'-TCCCCCACCTCCAGGC-3' (lόmer) (DMO 748) (SEQUENCE ID. NO. 52), 5'-CTCAGTGTTGCCCAGTCG-3' (18mer) (DMO 749) SEQUENCE ID. NO. 53) and 5'-GCCGAAGATGCTGCTGTG- 3' (18mer) (DMO 750) SEQUENCE ID. NO. 54). The final murine guanylate kinase gene sequence is shown in Figure 22 with the deduced amino acids.

3. Introduction of a New Restriction Site

A novel Ncol restriction site is introduced at the start codon of the mouse guanylate kinase open reading frame as described in Black, M E and Hruby, D E (J. Biol. Chem. 265 17584-17592, 1990) The mutagenic oligonucleotide used is" 5'- CTAGGTCCTG[CCATGG]CGTCCGCG-3' (24mer) (DMO 676) (SEQUENCE ID NO 41) with the Ncol site shown in brackets and the bold nucleotide denoting a C to G change The resulting clone, pUCl 18 Mug k-Ncol, is sequenced to confirm orientation and the 5' junction region

C Construction of Vectors for /// Vitro Transcription and Translation Analysis

Both the human and murine guanylate kinase genes are subcloned into pET23d (see Example 8) The 600bp Nco /Ba Hl fragment from pUC1 18 Hugmk is gel isolated and directionally subcloned into pET23d (see Example 8) restricted with Ncol and BamHl The murine guanylate kinase gene is gel isolated as a ~800bp NcoI/EcoRI fragment using the introduced Ncol site at the ATG and the EcoRl site from the pUC1 18 3' polylinker region, and cloned into pΕT23d (see Example 8) restricted with Ncol and EcoRl The resulting plasmids, pET23d Hgmk and pET23d Mg k, are then used as templates for /// vitro transcription and, the mRΝAs produced, are used in a rabbit reticulocyte lysate cell free translation system as described in Examples 3 and 8 Enzyme assays to confirm full-length protein production and activity are as described in Agarwal et al (Methods in Enzymol. 51 483-490, 1978) with bovine guanylate kinase purchased from Sigma as a positive control

D Purification and Characterization of the Human and Mouse Guanylate Kinases 7. Expression Vector Construction

The pET23d vector (Νovagen, Madison, WI) is used as the vector backbone for the construction of pET HT This vector contains a 6 histidine residue peptide followed by a thrombin cleavage site to allow for the expression of a removable histidine tag fused to the Ν terminus of the target gene product Synthesis of the 6 his- thrombin fusion encoding region is done by PCR amplification of the promoter region of pET23d and extension using the following primers in three sequential PCR amplification steps 5 '-ACT ACT ACT A GATCTCGATC CCGCGAA-3' (27mer) (DMO 604) (SEQUENCE ID NO 42) 5'-ATGATGATGA TGATGGCTGC TAGCCATAGT ATATCTCCTT C-3' (41mer) (DMO 605) (SEQUENCE ID NO 43) 5'- CGGCACCAGG CCGCTGCTGT GATGATGATG ATGATGGCT-3' (39mer) (DMO606) (SEQUENCE ID NO 44), 5- AGTAGTAT[CC ATGG]AGCTGC

CGCGCGGCAC CAGGCCGCTG CT-3' (42mer) (DMO 607) (SEQUENCE ID. NO. 45). Sequence DMO 604 is annealed to the Bghλ region of pET23d in all PCR amplification steps. Sequence DMO 605 is annealed to the region corresponding to the Ncol site in a 3' to 5' orientation and results in the loss of the Ncol site due to a nucleotide mutation shown in bold in the sequence above. Subsequent amplifications with sequence DMO 606 or DMO 607 in the 3' to 5' orientation are paired with sequence DMO 604 to extend the sequence for the addition of 6 histidine codons and a thrombin cleavage site. A new Ncol site is also introduced with sequence DMO 607 as shown in brackets above. The final Bgfll/Nco fragment is cloned into pET23d at the corresponding sites to create pET:HT. pET:HT is sequenced to confirm correct synthesis and insertion. The amino acid sequence of the new vector fusion peptide is: M A S S H H H H H H S S G L V P R G S S M (Ncol site) (SEQUENCE ID. NO. 46) with the thrombin cleavage recognition site underlined. Cleavage with thrombin is between the arginine and glycine residues.

2. Overexpression in E. coli and Affinity Purification Methods for overexpression and analysis are as in Example 8.

Affinity purification using His-Bind Resin (Novagen, Madison WI) is performed according to the manufacturer's instruction. Thrombin is used to cleave off the terminal 17 amino acids to leave three amino acids N-terminal to the guanylate kinase initiating methionine. The leader peptide is then removed by passing the cleavage mix over the His-Bind column a second time.

3. Enzyme Kinetics The K _nι , V _max and K _ca ι values for guanylate, GCV-monophosphate and acyclovir-monophosphate are determined using purified human and mouse guanylate kinases. In addition to using the assay protocol described in Agarwal et al. (Methods in Enzymol. 57:483-490, 1978), the nucleotide products generated from assays performed with radionucleotide substrates are analyzed by thin layer ehromatography and scintillation counting.

E. Construction and Analysis of Dual Expression Vectors in Vivo

The HSV1 tk gene is cloned into the Hpal site of pLXSN (Miller and

Rosman, BioTechniques 7:980-990, 1989) as a Ncol (blunt-ended) fragment and the orientation determined by restriction mapping. This places the ΗSV-1 tk gene behind the MoMLV LTR promoter. The neomycin phosphotransferase gene is replaced by the guanylate kinase gene (human or mouse) as a BamHl (blunt-ended) fragment such that

guanylate kinase gene expression is driven off the SV40 promoter. In addition, vectors are constructed where the tk and gmk gene order is reversed such that the tk gene is expressed from the SV promoter and gmk is expressed from the LTR promoter. Vector constructs with individual genes (tk or gmk) are also constructed. Furthermore, expression vectors containing HSV-1 tk mutants in place of the wild-type HSV-1 tk genes are also constructed.

As in Example 8, plasmid DNA from the constructs described above are used to transfect tsl3 BHK tk- cells, SF767 human glioblastoma cells, and rat C6 glioblastoma cells in the presence of a marker plasmid (pSV2-neo) to enable the selection of transfectants on G418.

Selection of stable transfectants and assays for increased sensitivity to ACV and GCV are as described in Example 8.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(l) APPLICANTS: Loeb. Lawrence A. Black, Margaret

(n) TITLE OF INVENTION: THYMIDINE KINASE MUTANTS

(m) NUMBER OF SEQUENCES: 54

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Seed and Berry

(B) STREET: 6300 Columbia Center, 701 Fifth Avenue

(C) CITY: Seattle

(D) STATE: Washington

(E) COUNTRY: US

(F) ZIP: 98104-7092

(v) COMPUTER READABLE FORM-

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compati le

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 02-MAY-1995

(C) CLASSIFICATION:

(vin) ATTORNEY/AGENT INFORMATION:

(A) NAME: McMasters, David D.

(B) REGISTRATION NUMBER: 33,963

(C) REFERENCE/DOCKET NUMBER: 240052.409C1

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (206) 622-4900

(B) TELEFAX: (206) 682-6031

(C) TELEX: 3723836

(2) INFORMATION FOR SEQ ID NO:l:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1131 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATGGCTTCGT ACCCCGGCCA TCAACACGCG TCTGCGTTCG ACCAGGCTGC GCGTTCTCGC 60

GGCCATAGCA ACCGACGTAC GGCGTTGCGC CCTCGCCGGC AGCAAGAAGC CACGGAAGTC 120

CGCCTGGAGC AGAAAATGCC CACGCTACTG CGGGTTTATA TAGACGGTCC TCACGGGATG 180

GGGAAAACCA CCACCACGCA ACTGCTGGTG GCCCTGGGTT CGCGCGACGA TATCGTCTAC 240

GTACCCGAGC CGATGACTTA CTGGCAGGTG CTGGGGGCTT CCGAGACAAT CGCGAACATC 300

TACACCACAC AACACCGCCT CGACCAGGGT GAGATATCGG CCGGGGACGC GGCGGTGGTA 360

ATGACAAGCG CCCAGATAAC AATGGGCATG CCTTATGCCG TGACCGACGC CGTTCTGGCT 420

CCTCATATCG GGGGGGAGGC TGGGAGCTCA CATGCCCCGC CCCCGGCCCT CACCCTCATC 480

TTCGACCGCC ATCCCATCGC CGCCCTCCTG TGCTACCCGG CCGCGCGGTA CCTTATGGGC 540

AGCATGACCC CCCAGGCCGT GCTGGCGTTC GTGGCCCTCA TCCCGCCGAC CTTGCCCGGC 600

ACCAACATCG TGCTTGGGGC CCTTCCGGAG GACAGACACA TCGACCGCCT GGCCAAACGC 660

CAGCGCCCCG GCGAGCGGCT GGACCTGGCT ATGCTGGCTG CGATTCGCCG CGTTTACGGG 720

CTACTTGCCA ATACGGTGCG GTATCTGCAG TGCGGCGGGT CGTGGCGGGA GGACTGGGGA 780

CAGCTTTCGG GGACGGCCGT GCCGCCCCAG GGTGCCGAGC CCCAGAGCAA CGCGGGCCCA 840

CGACCCCATA TCGGGGACAC GTTATTTACC CTGTTTCGGG CCCCCGAGTT GCTGGCCCCC 900

AACGGCGACC TGTATAACGT GTTTGCCTGG GCCTTGGACG TCTTGGCCAA ACGCCTCCGT 960

TCCATGCACG TCTTTATCCT GGATTACGAC CAATCGCCCG CCGGCTGCCG GGACGCCCTG 1020

CTGCAACTTA CCTCCGGGAT GGTCCAGACC CACGTCACCA CCCCCGGCTC CATACCGACG 1080

ATATGCGACC TGGCGCGCAC GTTTGCCCGG GAGATGGGGG AGGCTAACTG A 1131

(2) INFORMATION FOR SEQ ID N0:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 52 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2: TGGGAGCTCA CATGCCCCGC CCCCGGCCCT CACCCTCATC TTCGATCGCC AT 52

(2) INFORMATION FOR SEQ ID N0:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 56 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: ATGAGGTACC GNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNATGGCG ATCGAA 56

(2) INFORMATION FOR SEQ ID N0:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4: CCCCTCCAGC GCGGTAC 17

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5: CGCGCTCGAG GGGAGCT 17

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION- SEQ ID NO:6 ^• TGGGAGCTCA CATGCCCCGC C 21

(2) INFORMATION FOR SEQ ID NO:7:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7: ATGAGGTACC G 11

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 52 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: l near

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8: TGGGAGCTCA CATGCCCCGC CCCCGGCCCT CACCCTCATC TTCGATCGCC AT 52

(2) INFORMATION FOR SEQ ID N0:9:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 70 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: misc feature

(B) LOCATION: 23..25

(D) OTHER INFORMATION - /note= "80*; Wi l d-Type Nucl eoti de, and 20% Other Three Nucl eoti des "

(ix) FEATURE:

(A) NAME/KEY: misc feature

(B) LOCATION: 41..55

(D) OTHER INFORMATION : /note= "80S. Wi ld-Type Nucl eotide, and 203. Other Three Nucl eoti des"

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9: TGGGAGCTCA CATGCCCCGC CCCCGGCCCT CACCCTCATC TTCGACCGCC ATCCCATCGC 60 CGCCCTCCTG 70

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10: ATGAGGTACC GCGCAGCTGG GTAGCACAGG AGGGCGGC 38

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11: CATGCCTTAT GCCGTGA 17

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..33

(xi) SEQUENCE DESCRIPTION. SEQ ID N0:12:

CCC ATC GCC GCC CTC CTG TGC TAC CCG GCC GCG 33

Pro He Ala Ala Leu Leu Cys Tyr Pro Ala Ala 1 5 10

(2) INFORMATION FOR SEQ ID NO:13:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(n) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:

Pro H e Al a Al a Leu Leu Cys Tyr Pro Al a Al a 1 5 10

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

( B) LOCATION : joi nd . .33 )

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

CCC ATC GCC TCC CTC CTG TGC TAC CCG GCC GCG 33

Pro He Ala Ser Leu Leu Cys Tyr Pro Ala Ala 1 5 10

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15

Pro H e Al a Ser Leu Leu Cys Tyr Pro Al a Al a 1 5 10

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..33

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16.

TCC ATC GGC GCC CTA CAG TGC TAC CCG GTC GCG 33

Ser He Gly Ala Leu Gin Cys Tyr Pro Val Ala 1 5 10

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(n) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION- SEQ ID NO:17:

Ser H e Gly Al a Leu Gi n Cys Tyr Pro Val Al a 1 5 10

(2) INFORMATION FOR SEQ ID NO:18-

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..33

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:

CCC ATC GCC ACC CTG CTG TGC TAC CCG GCC GCG 33

Pro He Ala Thr Leu Leu Cys Tyr Pro Ala Ala 1 5 10

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Pro H e Al a Thr Leu Leu Cys Tyr Pro Al a Al a 1 5 10

(2) INFORMATION FOR SEQ ID N0:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..33

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:

CCC ATC GCC GCC TTA CTG TTA TAC CCG ACC GCG 33

Pro He Ala Ala Leu Leu Leu Tyr Pro Thr Ala 1 5 10

(2) INFORMATION FOR SEQ ID N0:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:

Pro H e Al a Al a Leu Leu Leu Tyr Pro Thr Al a 1 5 10

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..33

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:

CCC ATC GCC GCC CTC GTG TGC TAC CCG GCC GCG 33

Pro He Ala Ala Leu Val Cys Tyr Pro Ala Ala 1 5 10

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:

Pro H e Al a Al a Leu Val Cys Tyr Pro Al a Al a 1 5 10

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 58 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:

TGGGAGCTCA CATGCCCCGC CCCCGGCCCT CACCNNNNNN NNNGACCGCC ATCCCATC 58

(2) INFORMATION FOR SEQ ID N0:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:

ATAAGGTACC GCGCGGCCGG GTAGCANNNN NNNNNGGCGA TGGGATGGCG G 51

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:

ACTACTGGAT CCATGGCGGG CCCCAGGCCT GTG 33

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:

TACTACGGAT CCTCAGGCGG CGGTCCTTTG AGC 33

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:

CTGCTGAAGA GGCTGCTC 18

(2) INFORMATION FOR SEQ ID N0:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:

ACACAGATGC GGTTTCATG 19

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:

CTGGACGTGG ACCTGCAG 18

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:

GTTAATGATG ACCACATC 18

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:

TGTAAAACGA CGGCCAGT 18

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:

CAGGAAACAG CTATGACC 18

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

( B) TYPE : nucl ei c aci d

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:

TGTGTCCCAT ACTACTACAA G 21

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

( B ) TYPE : nucl ei c aci d

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:

TGAGAACTCA GCAGCATGCT C 21

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

( B) TYPE : nucl ei c aci d

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:

GTGCTAGATG TCGACCTA 18

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:

ACCTGGATAA AGCCTATG 18

(2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:

AAGCAGGCGC TCTCTCTGA 19

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:

CTATTTCTCA TATGATGT 18

(2) INFORMATION FOR SEQ ID N0:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:

GTTACAGTGT CTCTAGAG 18

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:

CTAGGTCCTG CCATGGCGTC CGCG 24

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:

ACTACTACTA GATCTCGATC CCGCGAA 27

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:

ATGATGATGA TGATGGCTGC TAGCCATAGT ATATCTCCTT C 41

(2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:

CGGCACCAGG CCGCTGCTGT GATGATGATG ATGATGGCT 39

(2) INFORMATION FOR SEQ ID N0:45"

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:

AGTAGTATCC ATGGAGCTGC CGCGCGGCAC CAGGCCGCTG CT 42

(2) INFORMATION FOR SEQ ID N0:46:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:

Met Ala Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15

Arg Gly Ser Ser Met 20

(2) INFORMATION FOR SEQ ID N0:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:

Al a Leu Thr Leu H e Phe Asp Arg Hi s Pro H e Al a Al a Leu Leu Cys 1 5 10 15

Tyr Pro H e

(2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 606 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 7..600

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:

GGATCC ATG GCG GGC CCC AGG CCT GTG GTG CTG AGC GGG CCT TCG GGA 48 Met Ala Gly Pro Arg Pro Val Val Leu Ser Gly Pro Ser Gly 1 5 10

GCT GGG AAG AGC ACC CTG CTG AAG AGG CTG CTC CAG GAG CAC AGC GGC 96 Ala Gly Lys Ser Thr Leu Leu Lys Arg Leu Leu Gin Glu His Ser Gly 15 20 25 30

ATC TTT GGC TTC AGC GTG TCC CAT ACC ACG AGG AAC CCG AGG CCC GGC 144 He Phe Gly Phe Ser Val Ser His Thr Thr Arg Asn Pro Arg Pro Gly 35 40 45

GAG GAG AAC GGC AAA GAT TAC TAC TTT GTA ACC AGG GAG GTG ATG CAG 192 Glu Glu Asn Gly Lys Asp Tyr Tyr Phe Val Thr Arg Glu Val Met Gin 50 55 60

CGT GAC ATA GCA GCC GGC GAC TTC ATC GAG CAT GCC GAG TTC TCG GGG 240 Arg Asp He Ala Ala Gly Asp Phe He Glu His Ala Glu Phe Ser Gly 65 70 75

AAC CTG TAT GGC ACG AGC AAG GTG GCG GTG CAG GCC GTG CAG GCC ATG 288 Asn Leu Tyr Gly Thr Ser Lys Val Ala Val Gin Ala Val Gin Ala Met 80 85 90

AAC CGC ATC TGT GTG CTG GAC GTG GAC CTG CAG GGT GTG CGG AAC ATC 336

Asn Arg He Cys Val Leu Asp Val Asp Leu Gin Gly Val Arg Asn He

95 100 105 110

AAG GCC ACC GAT CTG CGG CCC ATC TAC ATC TCT GTG CAG CCG CCT TCA 384

Lys Ala Thr Asp Leu Arg Pro He Tyr He Ser Val Gin Pro Pro Ser

115 120 125

CTG CAC GTG CTG GAG CAG CGG CTG CGG CAG CGC AAC ACT GAA ACC GAG 432

Leu His Val Leu Glu Gin Arg Leu Arg Gin Arg Asn Thr Glu Thr Glu

130 135 140

GAG AGC CTG GTG AAG CGG CTG GCT GCT GCC CAG GCC GAC ATG GAG AGC 480

Glu Ser Leu Val Lys Arg Leu Ala Ala Ala Gin Ala Asp Met Glu Ser 145 150 155

AGC AAG GAG CCC GGC CTG TTT GAT GTG GTC ATC ATT AAC GAC AGC CTG 528

Ser Lys Glu Pro Gly Leu Phe Asp Val Val He He Asn Asp Ser Leu 160 165 170

GAC CAG GCC TAC GCA GAG CTG AAG GAG GCG CTC TCT GAG GAA ATC AAG 576

Asp Gin Ala Tyr Ala Glu Leu Lys Glu Ala Leu Ser Glu Glu He Lys

175 180 185 190

AAA GCT CAA AGG ACC GGC GCC TGA GGATCC 606

Lys Ala Gin Arg Thr Gly Ala * 195

(2) INFORMATION FOR SEQ ID N0:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 198 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:

Met Ala Gly Pro Arg Pro Val Val Leu Ser Gly Pro Ser Gly Ala Gly 1 5 10 15

Lys Ser Thr Leu Leu Lys Arg Leu Leu Gin Ala His Ser Gly He Phe 20 25 30

Gly Phe Ser Val Ser His Thr Thr Arg Asn Pro Arg Pro Gly Glu Glu 35 40 45

Asn Gly Lys Asp Tyr Tyr Phe Val Thr Arg Glu Val Met Gin Arg Asp 50 55 60

He Ala Ala Gly Asp Phe He Glu His Ala Glu Phe Ser Gly Asn Leu 65 70 75 80

Tyr Gly Thr Ser Lys Val Ala Val Gin Ala Val Gin Ala Met Asn Arg 85 90 95

He Cys Val Leu Asp Val Asp Leu Gin Gly Val Arg Asn He Lys Ala 100 105 110

Thr Asp Leu Arg Pro He Tyr He Ser Val Gin Pro Pro Ser Leu His 115 120 125

Val Leu Glu Gin Arg Leu Arg Gin Arg Asn Thr Glu Thr Glu Glu Ser 130 135 140

Leu Val Lys Arg Leu Ala Ala Ala Gin Ala Asp Met Glu Ser Ser Lys 145 150 155 160

Glu Pro Gly Leu Phe Asp Val Val He He Asn Asp Ser Leu Asp Gin 165 170 175

Ala Tyr Ala Glu Leu Lys Glu Ala Leu Ser Glu Glu He Lys Lys Ala 180 185 190

Gin Arg Thr Gly Ala * 195

(2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 660 base pairs

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 25..621

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:

CTGGGTCGGG TCCCCGCGGA CGGC ATG GCA GGA CCT AGG CCA GTA GTG CTG 51

Met Ala Gly Pro Arg Pro Val Val Leu 1 5

AGC GGG CCG TCA GGG GCA GGG AAG AGC ACT CTG CTC AAG AAG CTG TTC 99 Ser Gly Pro Ser Gly Ala Gly Lys Ser Thr Leu Leu Lys Lys Leu Phe 10 15 20 25

CAG GAG CAC AGC AGC ATC TTC GGC TTC AGT GTG TCC CAT ACT ACA AGG 147 Gin Glu His Ser Ser He Phe Gly Phe Ser Val Ser His Thr Thr Arg 30 35 40

AAC CCA CGA CCT GGT GAA GAA GAT GGC AAA GAT TAC TAC TTT GTG ACC 195 Asn Pro Arg Pro Gly Glu Glu Asp Gly Lys Asp Tyr Tyr Phe Val Thr 45 50 55

AGG GAG ATG ATG CAG CGT GAT ATT GCA GCA GGG GAC TTC ATT GAG CAT 243 Arg Glu Met Met Gin Arg Asp He Ala Ala Gly Asp Phe He Glu His 60 65 70

GCT GAG TTC TCA GGG AAC CTG TAC GGG ACA AGC AAG GAA GCT GTT CGG 291 Ala Glu Phe Ser Gly Asn Leu Tyr Gly Thr Ser Lys Glu Ala Val Arg 75 80 85

GCT GTG CAG GCC ATG AAC CGC ATC TGC GTG CTA GAT GTC GAC CTA CAA 339

Al a Val Gi n Al a Met Asn Arg H e Cys Val Leu Asp Val Asp Leu Gi n

90 95 100 105

GGT GTG CGC AGC ATC AAG AAG ACT GAT CTG TGT CCC ATC TAC ATC TTT 387 Gly Val Arg Ser He Lys Lys Thr Asp Leu Cys Pro He Tyr He Phe 110 115 120

GTG CAG CCT CCC TCG CTG GAC GTG CTG GAG CAA CGA CTG CGA CTG CGC 435 Val Gin Pro Pro Ser Leu Asp Val Leu Glu Gin Arg Leu Arg Leu Arg 125 130 135

AAC ACT GAG ACT GAG GAG AGT CTG GCA AAG CGG CTG GCA GCT GCA CGG 483 Asn Thr Glu Thr Glu Glu Ser Leu Ala Lys Arg Leu Ala Ala Ala Arg 140 145 150

ACA GAC ATG GAG AGC AGC AAG GAG CCT GGC TTG TTT GAC CTG GTG ATC 531 Thr Asp Met Glu Ser Ser Lys Glu Pro Gly Leu Phe Asp Leu Val He 155 160 165

ATC AAT GAC GAC CTG GAT AAA GCC TAT GCA ACC CTG AAG CAG GCG CTC 579 He Asn Asp Asp Leu Asp Lys Ala Tyr Ala Thr Leu Lys Gin Ala Leu 170 175 180 185

TCT GAG GAA ATC AAG AAA GCA CAG GGA ACT GGC CAC GCC TGA 621

Ser Glu Glu He Lys Lys Ala Gin Gly Thr Gly His Ala * 190 195

AGGCCTGCTT CATTCCACAG AGTGATGTCT GTGGTCTAA 660

(2) INFORMATION FOR SEQ ID NO:51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 199 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:

Met Ala Gly Pro Arg Pro Val Val Leu Ser Gly Pro Ser Gly Ala Gly 1 5 10 15

Lys Ser Thr Leu Leu Lys Lys Leu Phe Gin Glu His Ser Ser He Phe 20 25 30

Gly Phe Ser Val Ser His Thr Thr Arg Asn Pro Arg Pro Gly Glu Glu 35 40 45

Asp Gly Lys Asp Tyr Tyr Phe Val Thr Arg Glu Met Met Gin Arg Asp 50 55 60

He Ala Ala Gly Asp Phe He Glu His Ala Glu Phe Ser Gly Asn Leu 65 70 75 80

Tyr Gly Thr Ser Lys Glu Ala Val Arg Ala Val Gin Ala Met Asn Arg 85 90 95

He Cys Val Leu Asp Val Asp Leu Gin Ala Val Arg Ser He Lys Lys

100 105 110

Thr Asp Leu Cys Pro He Tyr He Phe Val Gin Pro Pro Ser Leu Asp 115 120 125

Val Leu Glu Gin Pro Leu Arg Leu Arg Asn Thr Glu Thr Glu Glu Ser 130 135 140

Leu Ala Lys Arg Leu Pro Ala Ala Arg Thr Asp Met Glu Ser Ser Lys 145 150 155 160

Glu Pro Gly Leu Phe Asp Leu Val He He Asn Asp Asp Leu Asp Lys 165 170 175

Ala Tyr Ala Thr Leu Lys Gin Ala Leu Ser Glu Glu He Lys Lys Ala

180 185 190

Gin Gly Thr Gly His Ala 195

(2) INFORMATION FOR SEQ ID NO:52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:

TCCCCCACCT CCAGGC 16

(2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:

CTCAGTGTTG CGCAGTCG 18

(2) INFORMATION FOR SEQ ID NO:54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:

GCCGAAGATG CTGCTGTC 18