Human thymidine kinase gene: molecular cloning and nucleotide sequence of a cDNA expressible in mammalian cells.

A cDNA containing the entire coding region of the human thymidine kinase gene has been molecularly cloned. The cDNA is under the control of a simian virus 40 promoter and is expressible in mammalian cells. The complete nucleotide sequence of the human thymidine kinase cDNA has been determined. The cDNA is 1,421 base pairs in length and has a large open reading frame of 702 base pairs capable of specifying a protein with a molecular weight of 25,504. Genomic Southern blotting experiments show that sequences homologous to the human thymidine kinase cDNA are conserved among many vertebrates, including prosimians (lemur), tree shrews, rats, mice, and chickens. Direct comparison of the nucleotide sequences of the human thymidine kinase cDNA and the chicken thymidine kinase gene reveals ca. 70% overall homology. This homology is extended further at the amino acid sequence level, with greater than 74% amino acid residues matched between the human and chicken thymidine kinase proteins.     

Structure and expression of the Chinese hamster thymidine kinase gene.

My colleagues and I have cloned a nearly full-length Chinese hamster thymidine kinase (TK) cDNA in a lambda gt10 vector and characterized this cDNA by nucleotide sequencing. The hamster TK protein is encoded in this cDNA by a 702-base-pair open reading frame which specifies a 25,625-dalton protein closely homologous to the previously described human and chicken TK proteins. Using cDNA nucleotide sequence data in conjunction with sequence data derived from selected subclones of the hamster TK gene recombinant phage lambda HaTK.5, we have resolved the structure of the TK gene, finding the 1,219 base pairs of the cDNA sequence to be distributed through 11.2 kilobases of genomic DNA in at least seven exon segments. In addition, we have constructed a variety of Chinese hamster TK minigenes and exonuclease III-S1 derivatives of these genes which have permitted us to define the limits of the Chinese hamster TK gene promoter and demonstrate that efficient TK transformation of Ltk- cells by TK minigenes depends on the presence of both TK intervening sequences and sequences 3' to the site of mRNA polyadenylation.     

Nucleotide sequence of the herpes simplex virus type 2 (HSV-2) thymidine kinase gene and predicted amino acid sequence of thymidine kinase polypeptide and its comparison with the HSV-1 thymidine kinase gene

To analyze the boundaries of the functional coding region of the HSV-2(333) thymidine kinase gene (TK gene), deletion mutants of hybrid plasmid pMAR401 H2G, which contains the 17.5 kbp BglII-G fragment of HSV-2 DNA, were prepared and tested for capacity to transform LM(TK-) cells to the thymidine kinase-positive phenotype. These studies showed that hybrid plasmids containing 2.2-2.4 kbp subfragments of HSV-2 BglII-G DNA transformed LM(TK-) cells to the thymidine kinase-positive phenotype and suggested that the region critical for transformation might be less than 2 kbp. That the activity expressed in the transformants was HSV-2 thymidine kinase was shown by experiments with type-specific enzyme-inhibiting rabbit antisera and by disc-polyacrylamide gel electrophoresis analyses. DNA fragments of the HSV-2 TK gene were subcloned in phage M13mp9 and M13mp8. A sequence of 1656 bp containing the entire coding region of the TK gene and the flanking sequences was determined by the dideoxynucleotide chain termination method. Comparisons with the HSV-1(Cl 101) TK gene revealed that PstI, PvuII, and EcoRI cleavage sites had homologous locations as did promoter, translational start and stop, and polyadenylation signals. Extensive homology was observed in the nucleotide sequence preceding the ATG translational start signal and in portions of the coding region of the genes. Comparisons of the predicted amino acid sequences of the HSV-1 and HSV-2 thymidine kinase polypeptides revealed that both were enriched in alanine, arginine, glycine, leucine, and proline residues and that clear, but interrupted homology existed within several regions of the polypeptide chains. Stretches of 15-30 amino acid residues were identical in conserved regions. The possibility is suggested that domains containing some of the conserved amino acid sequences might have a role in substrate binding and as major antigenic determinants.     

Mapping and identification of the vaccinia virus thymidine kinase gene

The thymidine kinase gene of vaccinia virus (VV) was mapped on the viral genome by using cloned fragments of the viral DNA to hybridize to early viral mRNA. Individual DNA fragments that represented about half of the viral genome were assayed, both for their ability to arrest the cell-free synthesis of active VV thymidine kinase and for their ability to select functional mRNA for the viral enzyme. Both activities were located in HindIII fragment J, which maps near the middle of VV DNA and contains about 2.6% of the genome (4,800 base pairs). This DNA fragment encodes four known early polypeptides, and to determine which of these was thymidine kinase, early VV mRNA was fractionated by sucrose gradient centrifugation and used to direct cell-free synthesis of the active enzyme. The thymidine kinase mRNA cosedimented with several species that encoded polypeptides in the molecular weight range 15,000 to 25,000. Hybridization of these mRNAs to HindIII-J DNA selected a message that directed the synthesis of thymidine kinase and a single polypeptide with an apparent molecular weight of 19,000. The native molecular weight of VV thymidine kinase is about 80,000, so these data indicate that, unlike thymidine kinase from several other sources, the active VV enzyme is probably a tetramer of 19,000-molecular-weight subunits.     

Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences.

Two functional cytosolic thymidine kinase (tk) cDNA clones were isolated from a mouse L-cell library. An RNA blot analysis indicated that one of these clones contains a nearly full-length tk sequence and that LTK- cells contain little or no TK message. The nucleotide sequences of both clones were determined, and the functional mouse tk cDNA contains 1,156 base pairs. An analysis of the sequence implied that there is an untranslated 32-nucleotide region at the 5' end of the mRNA, followed by an open reading frame of 699 nucleotides. The 3' untranslated region is 422 nucleotides long. Thus, the gene codes for a protein containing 233 amino acids, with a molecular weight of 25,873. A comparison of the coding sequences of the mouse tk cDNA with the human and chicken tk genes revealed about 86 and 70% homology, respectively. We also isolated the tk gene from a mouse C57BL/10J cosmid library. The structural organization was determined by restriction mapping, Southern blotting, and heteroduplex analysis of the cloned sequences, in combination with a mouse tk cDNA. The tk gene spans approximately 11 kilobases and contains at least five introns. Southern blot analysis revealed that this gene is deleted in mouse LTK- cells, consistent with the inability of these cells to synthesize TK message. This analysis also showed that tk-related sequences are present in the genomes of several mouse strains, as well as in LTK- cells. These segments may represent pseudogenes.     

Nucleotide sequence of the herpes simplex virus type 2 thymidine kinase gene.

We have determined the complete nucleotide sequence of the thymidine kinase gene of herpes simplex virus (HSV) type 2 strain 333. The sequence of the thymidine kinase gene exhibits an open translational reading frame of 1,128 nucleotides encoding a protein of 376 amino acids. The DNA sequence was compared with that of the HSV type 1 thymidine kinase gene from strain MP (S. L. McKnight, Nucleic Acids Res. 8:5949-5964, 1980) and from strain CL 101 (M. J. Wagner, J. A. Sharp, and W. C. Summers, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981) to assess the extent of intra- and intertypic variation for one viral gene. The nucleotides encoding the structural gene varied 1.7% between the two HSV type 1 strains and 19% between HSV type 1 and HSV type 2, which translated to differences in the amino acid sequence of the two proteins of 1.9 and 27%, respectively. The DNA encoding the 5' regulatory sequences appeared to be more conserved than the DNA coding for the structural gene, and the DNA at the 3' end of the gene was the least homologous.     

Cloning and sequence analysis of thymidine kinase from the oral bacterium Streptococcus gordonii

Abstract Thymidine kinase is an important enzyme in the pyrimidine nucleotide salvage pathway and catalyzes the formation of thymidylate from thymidine using ATP as a phosphate donor. The gene encoding thymidine kinase of the oral bacterium Streptococcus gordonii was cloned and the nucleptide sequence determined. The inferred amino acid sequence of thymidine kinase (191 amino acids) exhibited 43% identity with type H thymidine kinase from Escherichia coli . The S. gordonii thymidine kinase expressed in Escherichia coli KY895 ( tdk⁻ ) was inhibited by thymidline triphosphate, a feature typical of type II thymidine kinases. Immediately 3' to the tdk gene, and possibly co-transcribed with it, was the gene encoding release factor 1 ( prfA ).     

Mapping of a vaccinia host range sequence by insertion into the viral thymidine kinase gene.

A vaccinia virus mutant deleted of ca. 18 kilobase pairs at the left-hand end of the genome is unable to multiply on many human cell lines. To determine whether all or some of the deleted sequences were responsible for the host range property, the corresponding region from wild-type DNA was cloned in three pieces into a vaccinia transplacement vector containing the thymidine kinase gene on the HindIII J fragment. The next step was to transfer these pieces to the genome of the host range deletion mutant by in vivo homologous recombination around the thymidine kinase locus. Transfer of one 5.2-kilobase-pair EcoRI fragment was found to restore a wild-type phenotype on the host range mutant, thus demonstrating that only a small portion of the 18-kilobase-pair deletion contains the host range function(s). This result also illustrates that the method initially devised for inserting foreign genes into vaccinia virus DNA is useful for studies of the vaccinia genome.     

Identification and nucleotide sequence of the thymidine kinase gene of Shope fibroma virus.

The thymidine kinase (TK) gene of Shope fibroma virus (SFV), a tumorigenic leporipoxvirus, was localized within the viral genome with degenerate oligonucleotide probes. These probes were constructed to two regions of high sequence conservation between the vaccinia virus TK gene and those of several known eucaryotic cellular TK genes, including human, mouse, hamster, and chicken TK genes. The oligonucleotide probes initially localized the SFV TK gene 50 kilobases (kb) from the right terminus of the 160-kb SFV genome within the 9.5-kb BamHI-HindIII fragment E. Fine-mapping analysis indicated that the TK gene was within a 1.2-kb AvaI-HaeIII fragment, and DNA sequencing of this region revealed an open reading frame capable of encoding a polypeptide of 176 amino acids possessing considerable homology to the TK genes of the vaccinia, variola, and monkeypox orthopoxviruses and also to a variety of cellular TK genes. Homology matrix analysis and homology scores suggest that the SFV TK gene has diverged significantly from its counterpart members in the orthopoxvirus genus. Nevertheless, the presence of conserved upstream open reading frames on the 5' side of all of the poxvirus TK genes indicates a similarity of functional organization between the orthopoxviruses and leporipoxviruses. These data suggest a common ancestral origin for at least some of the unique internal regions of the leporipoxviruses and orthopoxviruses as exemplified by SFV and vaccinia virus, respectively.     

Evolution of the herpes thymidine kinase: identification and comparison of the equine herpesvirus 1 thymidine kinase gene reveals similarity to a cell-encoded thymidylate kinase.

We have identified the equine herpesvirus 1 (EHV-1) thymidine kinase gene (TK) by DNA-mediated transformation and by DNA sequencing. Alignment of the amino acid sequence of the EHV-1 TK with the TKs from 3 other herpesviruses revealed regions of homology, some of which correspond to the previously identified substrate binding sites, while others have as yet, no assigned function. In particular, the strict conservation of an aspartate within the proposed nucleoside binding site suggests a role in ATP binding for this residue. Comparison of 5 herpes TKs with the thymidylate kinase of yeast revealed significant similarity which was strongest in those regions important to catalytic activity of the herpes TKs, and, therefore we propose that the herpes TK may be derived from a cellular thymidylate kinase. The implications for the evolution of enzyme activities within a pathway of nucleotide metabolism are discussed.     

Nucleotide sequence of the genes III, VI and I of bacteriophage M13.

A DNA region of 2750 base pairs encompassing the genes III, VI and I of bacteriophage M13 has been sequenced by the Maxam-Gilbert procedure. By establishing the nucleotide changes introduced by several amber mutations, the coding region and the regulatory signals of each gene have been deduced. The genes appear to span 1275 base pairs (gene III; mol.wt. 44,748) 339 base pairs (gene VI; mol.wt. 12,264) and 1047 base pairs (gene I; mol.wt. 39,500). Their separating non-codogenic regions are extremely short, namely two and one base pair, respectively. The C-terminal end of gene I, however, intrudes 23 nucleotides into gene IV. From the nucleotide sequence it appears that the minor capsid protein of the phage, which is encoded by gene III, is synthesized in a precursor form containing 18 extra amino acids at its N-terminal end. Furthermore, in this capsid protein two clusters of a fourfold repeat of the sequence Glu-Gly-Gly-Gly-Ser are apparent. Gene VI appears to code for a small, extremely hydrophobic polypeptide. Its total hydrophobic amino acids content of 51% suggests that this protein can only function in the host cell membrane.     

Accuracy of intrachromosomal gene conversion in mouse cells.

Results of several recent studies suggest that homologous recombination and related processes in mammalian cells are highly mutagenic. We have examined the products of intrachromosomal gene conversion events that encompassed the last intron of the chicken thymidine kinase gene. Following plasmid rescue and DNA sequencing, we find no mutations associated with twenty conversion events representing 5380 total base pairs of which 2414 base pairs are intron sequence. Based on these studies we conclude that intrachromosomal gene conversion in mouse cells is not a highly mutagenic process but rather it operates with fidelity.     

猪伪狂犬病毒鲁A株TK基因的序列测定及其结构功能与进化分析

对猪伪狂犬病毒鲁A株(PRV LA株)TK基因进行了克隆和序列测定,并分析比较了该序列与PRV NIA-3株、Ea株、SH以及HSV-1和VZV的同源性,结果表明:在全长1048bp的DNA序列中,包括着一个963bp的开放阅读框(ORF),可编码320个氯基酸组成的多肽;在整个TK基因的ORF内,PRV LA株与PRV NIA-3株、PRV Ea株、PRV SH株、HSV-1、VZV的TK基因比较,核苷酸的同源性分别为98.9%、99.5%、99.3%、36.4%、39.1%,氨基酸的同源性分别为98.4%、99.7%、98.7%、36.6%、37.2%.PRV LA株TK具有疱疹病毒胸苷激酶催化结构域的保守氨基酸共有序列和亚结构域特征序列.将PRV-LA TK、人类和小鼠的胸苷酸激酶、人类脱氧胞苷激酶、人类腺苷酸激酶的对应于这两个亚结构域的氨基酸用DNA Star分析的进化树表明,疱疹病毒的TK与人类和小鼠的胸苷酸激酶的亲缘关系比与人类脱氧胞嘧啶激酶的亲缘关系更近,因此疱疹病毒的TK基因在进化上可能起源于宿主细胞的胸苷酸激酶基因.     

Nucleotide sequence of the SV40 DNA restriction fragment Hind C-Hap 2.

We here report the nucleotide sequence of the SV40 DNA fragment Hind C - Hap 2. The fragment was labeled at the 5'-ends by means of polynucleotide kinase and gamma-(32P) ATP and digested with a suitable restriction enzyme. The separated products were then partially degraded with the base-specific reagents dimethyl-sulphate or hydrazine followed by direct analysis on polyacrylamide gel. The Hind C - Hap 2 sequence is 126 base pairs long and one of the three possible reading frames for translation does not contain any termination codon. So, although no protein is known to be encoded by this region, the possibility cannot yet be completely ruled out. The sequence also contains several AT-rich blocks.     

Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells.

To test the validity of various models for recombination between extrachromosomal DNAs in mammalian cells, we measured recombination between a plasmid containing a herpesvirus thymidine kinase (tk) gene with an internal BamHI linker insertion mutation (ptkB8) and a tk gene deleted at both ends (tk delta 3' delta 5'). The two DNAs shared 885 base pairs of perfect tk homology except for the interruption at the linker insertion site. Recombination events that restored the mutated insertion site to wild type were monitored by the generation of hypoxanthine-aminopterine-thymidine-resistant colonies after cotransformation of Ltk- cells with the two DNAs. We found that cleavage of the ptkB8 DNA at the linker insertion site was essential for gene restoration. If the tk delta 3' delta 5' DNA was ligated into mp10 vector DNA, then recombination with the cleaved ptkB8 DNA was inefficient. In contrast, if it was excised from that vector by cleavage at flanking restriction sites, then recombination was stimulated about 150-fold. Using restriction site polymorphisms, we showed that most of the recombination events leading to restoration of the tk gene with the excised tk delta 3' delta 5' fragment involved three double-strand duplexes: two ptkB8 DNAs and one tk delta 3' delta 5' fragment. These results are much more readily explained by the single-strand annealing model of recombination than by the double-strand break repair model, and they suggest that the deficiency of the latter pathway for extrachromosomal mammalian recombination may be due, at least in part, to the obligate tripartite nature of the reaction. Finally, we measured the effect of DNA homology on the efficiency of the ptkB8-tk delta 3' delta 5' reaction. Our results showed a near-linear relationship between the efficiency of recombination and the amount of homology flanking either side of the linker insertion site. Moreover, we could detect thymidine kinase-positive transformants with as little as 10 base pairs of homology.     

Biosynthesis of bacterial glycogen: primary structure of Salmonella typhimurium ADPglucose synthetase as deduced from the nucleotide sequence of the glgC gene.

The nucleotide sequence of a 1.4-kilobase-pair fragment containing the Salmonella typhimurium LT2 glgC gene coding for ADPglucose synthetase was determined. The glgC structural gene contains 1,293 base pairs, having a coding capacity of 431 amino acids. The amino acid sequence deduced from the nucleotide sequence shows that the molecular weight of ADPglucose synthetase is 45,580. Previous results of the total amino acid composition analysis and amino acid sequencing (M. Lehmann and J. Preiss, J. Bacteriol. 143:120-127, 1980) of the first 27 amino acids from the N terminus agree with that deduced from nucleotide sequencing data. Comparison of the Escherichia coli K-12 and S. typhimurium LT2 ADPglucose synthetase shows that there is 80% homology in their nucleotide sequence and 90% homology in their deduced amino acid sequence. Moreover, the amino acid residues of the putative allosteric sites for the physiological activator fructose bisphosphate (amino acid residue 39) and inhibitor AMP (amino acid residue 114) are identical between the two enzymes. There is also extensive homology in the putative ADPglucose binding site. In both E. coli K-12 and S. typhimurium LT2, the first base of the translational start ATG of glgA overlaps with the third base TAA stop codon of the glgC gene.