Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast.

(Deoxy)thymidylate (dTMP) kinase is an enzyme which phosphorylates dTMP to dTDP in the presence of ATP and magnesium. This enzyme is important in cellular DNA synthesis because the synthesis of dTTP, either via the de novo pathway or through the exogenous supply of thymidine, requires the activity of this enzyme. It has been suggested that the activities of the enzymes involved in DNA precursor biosynthesis, such as thymidine kinase, thymidylate synthase, thymidylate kinase, and dihydrofolate reductase, are subjected to cell cycle regulation. Here we describe the cloning of a human dTMP kinase cDNA by functional complementation of a yeast dTMP kinase temperature-sensitive mutant at the non-permissive temperature. The nucleotide sequence of the cloned human cDNA is predicted to encode a 24 KD protein that shows considerable homology with the yeast and vaccinia virus dTMP kinase enzymes. The human enzyme activity has been investigated by expressing it in yeast. In this work, we demonstrate that the cloned human cDNA, when expressed in yeast, produces dTMP kinase activity.     

Genetic and Molecular Analysis of the Soe1 Gene: A Trna(3)(glu) Missense Suppressor of Yeast Cdc8 Mutations

The CDC8 gene of Saccharomyces cerevisiae encodes deoxythymidylate (dTMP) kinase and is required for nuclear and mitochondrial DNA replication in both the mitotic and meiotic cell cycles. All cdc8 temperature-sensitive mutants are partially defective in meiotic and mitochondrial functions at the permissive temperature. In a study of revertants of temperature-sensitive cdc8 mutants, the SOE201 and SOE1 mutants were isolated. The SOE201 mutant is a disome of chromosome X to which the cdc8 gene maps. Using the chromosome X aneuploids to vary cdc8 gene dosage, we demonstrate that different levels of dTMP kinase activity are required for mitotic, meiotic or mitochondrial DNA replication. The SOE1 mutant contains a dominant suppressor that suppresses five different cdc8 alleles but does not suppress a complete cdc8 deletion. The SOE1 gene is located less than 1.5 cM from the CYH2 gene on chromosome VII and is adjacent to the TSM437-CYH2 region, with the gene order being SOE1-TSM437-CYH2. SOE1 is an inefficient suppressor that can neither suppress the cdc8 hypomorphic phenotype nor restore dTMP kinase activity in vitro. SOE1 is a single C to T mutation in the anticodon of a tRNA(3Glu) gene and thereby, produces a missense suppressor tRNA capable of recognizing AAA lysine codons. We propose that the resultant lysine to glutamate change stabilizes thermo-labile dTMP kinase molecules in the cell.     

Characterization of a nucleotide kinase encoded by bacteriophage t7

Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. The phosphate donors are dTTP, dGTP, and ribo-GTP as well as the thymidine and guanosine triphosphate analogs ddTTP, ddGTP, and dITP. The nucleotide kinase is found in solution as a 256-kDa complex consisting of ～12 monomers of the gene 1.7 protein. The two molecular weight forms co-purify as a complex, but each form has nearly identical kinase activity. Although gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg(2+) in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Neither dTDP nor dGDP is a phosphate acceptor of nucleoside triphosphate donors. Gene 1.7 protein exhibits two different equilibrium patterns toward deoxyguanosine and thymidine substrates. The K(m) of 4.4 × 10(-4) m obtained with dTTP for dTMP kinase is ～3-fold higher than that obtained with dGTP for dGMP kinase (1.3 × 10(-4) m), indicating that a higher concentration of dTTP is required to saturate the enzyme. Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction.     

Purification and characterization of Saccharomyces cerevisiae uridine monophosphate kinase

The SOC8 gene was isolated as an extragenic suppressor of cdc8 mutant cells. It has been suggested that SOC8 is allelic with the URA6 gene which was originally identified as a uridine monophosphate kinase. In this article, we describe the purification of the uridine monophosphate kinase from a yeast Saccharomyces cerevisae strain that overproduces the activity 8-fold. The protein was purified through Fast-Flow Q-Separose, phosphocellulose, blue-agarose, and fast protein liquid chromatography Superose 12 columns, and appears homogeneous by sodium dodecyl sulfate-polyacrylamide gel analysis. The uridine monophosphate kinase contains a single polypeptide with a molecular weight of 25,000, as evidence by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analysis. The amino acid composition has also been determined. Substrate specificity studies show that the relative activity of nucleoside monophosphates is in order of UMP greater than dUMP, and to a lesser extent, dTMP, GMP, and dGMP. The Km and Vm of UMP, dUMP, and dTMP have been determined.     

The activities of thymidine metabolising enzymes during the cell cycle of a human lymphocyte cell line LAZ-007 synchronised by centrifugal elutriation

The activities throughout the cell cycle of thymidine kinase (EC 2.7.1.21), dihydrothymine dehydrogenase (EC 1.3.1.2), thymidine phosphorylase (EC 2.4.2.4) and dTMP phosphatase (EC 3.1.3.35) were measured in the Epstein-Barr virally transformed human B lymphocyte line LAZ-007. Cells were synchronised at different stages of the cell cycle using the technique of centrifugal elutriation. The degree of synchrony in each cycle-stage cell population was determined by flow microfluorimetric analysis of DNA content and by measurement of thymidine incorporation into DNA. The activity of the anabolic enzyme thymidine kinase was low in the G₁ phase cells, but increased many-fold during the S and G₂ phases, reaching a maximum after the peak of DNA synthesis, then decreasing in late G₂ + M phase. By contrast, the specific activities of the enzymes involved in thymidine and thymidylate catabolism, dihydrothymine dehydrogenase, thymidine phosphorylase and dTMP phosphatase remained essentially constant throughout the cell cycle, indicating that the fate of thymidine at different stages of the cell cycle is governed primarily by regulation of the level of the anabolic enzyme thymidine kinase and not by regulation of the levels of thymidine catabolising enzymes.     

Escherichia coli thymidylate kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding tmk locus.

Thymidylate kinase (dTMP kinase; EC 2.7.4.9) catalyzes the phosphorylation of dTMP to form dTDP in both de novo and salvage pathways of dTTP synthesis. The nucleotide sequence of the tmk gene encoding this essential Escherichia coli enzyme is the last one among all the E. coli nucleoside and nucleotide kinase genes which has not yet been reported. By subcloning the 24.0-min region where the tmk gene has been previously mapped from the lambda phage 236 (E9G1) of the Kohara E. coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987), we precisely located tmk between acpP and holB genes. Here we report the nucleotide sequence of tmk, including the end portion of an upstream open reading frame (ORF 340) of unknown function that may be cotranscribed with the pabC gene. The tmk gene was located clockwise of and just upstream of the holB gene. Our sequencing data allowed the filling in of the unsequenced gap between the acpP and holB genes within the 24-min region of the E. coli chromosome. Identification of this region as the E. coli tmk gene was confirmed by functional complementation of a yeast dTMP kinase temperature-sensitive mutant and by in vitro enzyme assay of the thymidylate kinase activity in cell extracts of E. coli by use of tmk-overproducing plasmids. The deduced amino acid sequence of the E. coli tmk gene showed significant similarity to the sequences of the thymidylate kinases of vertebrates, yeasts, and viruses as well as two uncharacterized proteins of bacteria belonging to Bacillus and Haemophilus species.     

Isolation and nucleotide sequence of a Saccharomyces cerevisiae protein kinase gene suppressing the cell cycle start mutation cdc25

We have isolated two unlinked yeast genes complementing the cell division cycle mutant cdc25-1, one containing the wild type allele CDC25 and the other acting as an extragenic suppressor of the cdc25-1 lesion if present on a multicopy plasmid. Nucleotide sequence analysis of the suppressor gene has revealed an open reading frame that encodes a 45,000-dalton protein belonging to the protein kinase family. The cdc25-suppressing protein kinase (PK-25) shows 48% sequence similarity to the catalytic subunit (CA) of mammalian cAMP-dependent protein kinase and 27-31% similarity to cyclic nucleotide-independent enzymes, including the yeast CDC28 gene product. The PK-25 gene was targeted by integrative transformation into a chromosomal region unlinked to the CYR2 site, the structural gene of CA. The cdc25-suppressing protein kinase is also functionally different from CA, since cyr2 strains deficient in the free catalytic subunit remain temperature sensitive if transformed with a multicopy plasmid containing the PK-25 gene. Furthermore, a deficiency of the cAMP-binding regulatory subunit (RA) caused by the bcy1 mutation fails to suppress the cdc25 mutation, indicating that PK-25 does not interact with the cAMP receptor protein. Our data suggest that the cdc25 suppressor gene encodes a cAMP-independent protein kinase involved in the control of the cell cycle start.     

小麦蓝矮病植原体胸苷酸激酶基因(tmk)的分离、原核表达及酶活性分析

[目的]克隆、表达小麦蓝矮病(WBD)植原体胸苷酸激酶基因(tmk),并分析酶活性,进一步研究胸苷酸激酶在植原体感染宿主及繁殖过程中的功能和作用机理,更好地防治植原体病害.[方法]PCR方法扩增tmk基因并进行序列分析,连接pET30a( )表达载体后原核表达,经Ni-NTA柱层析纯化后进行酶催化活性分析.[结果]首次从小麦蓝矮病(WBD)植原体基因组中分离出胸苷酸激酶基因(tmk),该基因包含tmk-1和tmk-2两种,大小分别为630 bp和624 bp,其编码的氨基酸序列均包含3个与结合NTP/NMP相关的保守功能区.表达的融合蛋白TMK-1活性极低,酶活仅16.4 U/mg,而 TMK-2酶活高达112.41 U/mg,且其最适催化条件为32℃、pH 7.3、1.5 mmol/L Mg2 和 1 mmol/L ATP.[结论]分析了胸苷酸激酶活性中心的一级结构序列及其催化活性随条件变化而改变的性质,为深入研究小麦蓝矮病植原体胸苷酸激酶在侵染寄主及其在宿主体内增殖的转录性质奠定基础.     

A dominant negative allele of p34cdc2 shows altered phosphoamino acid content and sequesters p56cdc13 cyclin.

The cdc2 gene product, a 34-kDa phosphoprotein with serine/threonine protein kinase activity, has been implicated as the key component in the regulation of the eucaryotic cell cycle. Activation of the cdc2 protein kinase is regulated by its phosphorylation state and by interaction with other proteins. We have mutagenized the fission yeast cdc2 gene to obtain conditionally dominant negative alleles. One of these mutants, named DL2, is characterized in this report. Overexpression of the mutant protein in a wild-type cdc2 background is lethal and leads to arrest in the G2 phase of the cell cycle. The mutant phenotype is the result of a single amino acid change in the GDSEID motif of the protein, a region of identity in all cdc2 homologs, and results in a nonfunctional protein that shows an altered content of phosphothreonine. Multicopy suppressors of the dominant negative phenotype have been isolated, and one of these has been shown to encode the cdc13 cyclin B gene product.     

The FT210 cell line is a mouse G2 phase mutant with a temperature-sensitive CDC2 gene product

The mouse cell FT210 was isolated as a G2 phase mutant with a possible defect in the histone H1 kinase. We determined that a temperature-sensitive lesion in this cell line lies in the CDC2 gene. DNA sequence analysis revealed two point mutations in highly conserved regions of the gene: an isoleucine to valine change in the PSTAIR region, and a proline to serine change at the C-terminal region of the protein p34. These mutations cause the p34 protein kinase to become inactivated and degraded in FT210 cells at the restrictive temperature, 39 degrees C. The consequence of this temperature-induced inactivation of the CDC2 gene product is cell cycle arrest at the mid to late G2 phase, and this arrest can be alleviated by the introduction of the human CDC2 homolog.     

Characterization of the fission yeast mcs2 cyclin and its associated protein kinase activity.

We have previously described the isolation of mcs2-75, a mutation obtained as an allele-specific suppressor of a dominant allele of cdc2. mcs2 was cloned and determined to be an essential gene, the product of which shares homology with the cyclin family of proteins. In contrast to the behavior of some, but not all cyclins, the mcs2 protein is constant in its abundance and localization throughout the cell cycle. A kinase activity that co-precipitates with mcs2 can be detected when myelin basic protein (MBP) is provided as an exogenous substrate. This kinase activity is constant throughout the cell cycle. mcs2 does not appear to associate with the cdc2 protein kinase or an antigenically related kinase. Finally, a protein kinase termed csk1 (cyclin suppressing kinase) was isolated as a high copy suppressor of an mcs2 mutation. csk1 is not essential, however, the level of kinase activity that co-precipitates with mcs2 is reduced approximately 3-fold in strains harboring a csk1 null allele. Therefore, csk1 may encode a protein kinase physically associated with mcs2 or alternatively may function as an upstream activator of the mcs2-associated kinase.     

Isolation and characterization of an Escherichia coli mutant deficient in dTMP kinase activity

Escherichia coli LD0181 is sensitive to 15 micrograms of 2',3'-dideoxythymidine per ml. A derivative that was resistant to 40 micrograms of the same chemical per ml at 30 degrees C and that had lost the ability to grow on enriched medium at 42 degrees C was isolated after nitroso-guanidine mutagenesis. This mutant, TD105, produced a dTMP kinase with 25-fold lower specific activity and a 5-fold higher Km for dTMP than the parental strain. The dTMP pool in TD105 was 4.4-fold higher than in the parent. In addition to temperature sensitivity and resistance to 2',3'-dideoxythymidine, the mutant exhibited a hypersensitivity to 5-bromo-2'-deoxyuridine. All three of these phenotypes are cotransducible. The tmk gene was mapped by cotransduction to approximately 30 min on the E. coli map.     

A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle

Probes derived from cDNAs encoding isozymes of rat protein kinase C (PKC) were used to screen the genome of the budding yeast S. cerevisiae. A single gene (PKC1) was isolated that encodes a putative protein kinase closely related to the alpha, beta, and gamma subspecies of mammalian PKC. Deletion of PKC1 resulted in recessive lethality. Cells depleted of the PKC1 gene product displayed a uniform phenotype, a characteristic of cell division cycle (cdc) mutants, and arrested cell division at a point subsequent to DNA replication, but prior to mitosis. Unlike most cdc mutants, which continue to grow in the absence of cell division, PKC1-depleted cells arrested growth with small buds. PKC1 may regulate a previously unrecognized checkpoint in the cell cycle.     

A cdc2-related kinase oscillates in the cell cycle independently of cyclins G2/M and cdc2.

The Eg1 gene in Xenopus laevis is related in sequence to the cdc2+ gene. We show here that the Eg1 gene product (cdk2) possesses histone H1 protein kinase activity and binds to PSTAIR antibodies as well as to Sepharose beads linked to the 13-kDa product of the suc 1 gene (p13suc1). Eg1 protein kinase is active only in an Mr approximately 200,000 complex with other proteins but is not associated with any of the three known Xenopus mitotic cyclins or with any newly synthesized protein in egg extracts that exhibit cell cycle oscillations in vitro. The protein kinase activity of Eg1 oscillates in the mitotic cell cycle, being high in M-phase and low in interphase. Hyperactivation of cdc2 kinase by the addition of cyclin A has no effect on the activity or oscillatory behavior of Eg1. Inhibition of cdc2 kinase activation by emetine or RNase treatment of oscillating extracts does not inhibit the activation of Eg1 but does block deactivation normally seen during exit from mitosis. These results indicate that Eg1 is regulated by a cell cycle clock independently of cyclin and cdc2 kinase.     

Suppression of a yeast cyclic AMP-dependent protein kinase defect by overexpression of SOK1, a yeast gene exhibiting sequence similarity to a developmentally regulated mouse gene.

Saccharomyces cerevisiae cyclic AMP-dependent protein kinase (A kinase) activity is essential for growth and cell cycle progression. Dependence on A kinase function can be partially relieved by the inactivation of a second kinase encoded by the gene YAK1. We have isolated two new genes, SOK1 and SOK2 (suppressor of kinase), as gene dosage suppressors of the conditional growth defect of several temperature-sensitive A kinase mutants. Overexpression of SOK1, like lesions in YAK1, also restores growth to a strain (tpk1 tpk2 tpk3) lacking all A kinase activity. The SOK1 gene is not essential, but a sok1::HIS3 disruption abrogates suppression of an A kinase defect by yak1. These results suggest that Yak1 and Sok1 define a linear pathway that is partially redundant with that of the A kinase. Activation of Sok1, by SOK1 overexpression or by inactivation of the negative regulator Yak1, renders a cell independent of A kinase function. The implications of such a model are particularly intriguing in light of the nuclear localization pattern of the overexpressed Sok1 protein and the primary sequence homology between SOK1 and a recently described, developmentally regulated mouse gene.     

Cloning and characterization of the Saccharomyces cerevisiae CDC6 gene.

The yeast cell division cycle gene CDC6 was isolated by complementation of a temperature-sensitive cdc6 mutant with a genomic library. The amino acid sequence of the 48 kDalton CDC6 gene product, as deduced from DNA sequence data, includes the three consensus peptide motifs involved in guanine nucleotide binding and GTPase activity, a target site for cAMP-dependent protein kinase and a carboxy-terminal domain related to metallothionein sequences. A plasmid-encoded CDC6-beta-galactosidase hybrid protein was located at the plasma membrane by indirect immunofluorescence. Disruption experiments indicate that the CDC6 gene product is essential for mitotic growth.     

A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle regulators.

In a screen of mouse erythroleukemia cDNA expression libraries with anti-phosphotyrosine antibodies, designed to isolate tyrosine kinase coding sequences, we identified several cDNAs encoding proteins identical or very similar to known protein-tyrosine kinases. However, two frequently isolated cDNAs, clk and nek, encode proteins which are most closely related to protein kinases involved in regulating progression through the cell cycle, and contain motifs generally considered diagnostic of protein-serine/threonine kinases. The clk gene product contains a C-terminal cdc2-like kinase domain, most similar to the FUS3 catalytic domain. The Clk protein, expressed in bacteria, becomes efficiently phosphorylated in vitro on tyrosine as well as serine/threonine, and phosphorylates the exogenous substrate poly(glu, tyr) on tyrosine. Direct biochemical evidence indicates that both protein-tyrosine and protein-serine/threonine kinase activities are intrinsic to the Clk catalytic domain. These results suggest the existence of a novel class of protein kinases, with an unusual substrate specificity, which may be involved in cell cycle control.     

Regulation of cdc2 activity in Schizosaccharomyces pombe: the role of phosphorylation.

The cdc2 protein kinase, first identified as a cell cycle gene required for transition into the S- and M-phases of budding and fission yeast, has been shown to act as a key component in the regulation of the eukaryotic cell cycle. The periodic activation of cdc2 kinase, which is required for entry into M-phase, is regulated by subunit association with cyclin B, the cdc25, wee1, mik1 gene products and differential phosphorylation of the cdc2 protein. Phosphorylation at Tyr 15 inhibits activation of the cdc2/cdc13 complex whereas phosphorylation of Thr 167 is required for kinase activity.     

Substrate specificity of vaccinia virus thymidylate kinase

Anti-poxvirus therapies are currently limited to cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], but drug-resistant strains have already been characterized. In the aim of finding a new target, the thymidylate (TMP) kinase from vaccinia virus, the prototype of Orthopoxvirus, has been overexpressed in Escherichia coli after cloning the gene (A48R). Specific inhibitors and alternative substrates of pox TMP kinase should contribute to virus replication inhibition. Biochemical characterization of the enzyme revealed distinct catalytic features when compared to its human counterpart. Sharing 42% identity with human TMP kinase, the vaccinia virus enzyme was assumed to adopt the common fold of nucleoside monophosphate kinases. The enzyme was purified to homogeneity and behaves as a homodimer, like all known TMP kinases. Initial velocity studies showed that the Km for ATP-Mg2+ and dTMP were 0.15 mm and 20 microM, respectively. Vaccinia virus TMP kinase was found to phosphorylate dTMP, dUMP and also dGMP from any purine and pyrimidine nucleoside triphosphate. 5-Halogenated dUMP such as 5-iodo-2'-deoxyuridine 5'-monophosphate (5I-dUMP) and 5-bromo-2'-deoxyuridine 5'-monophosphate (5Br-dUMP) were also efficient alternative substrates. Using thymidine-5'-(4-N'-methylanthraniloyl-aminobutyl)phosphoramidate as a fluorescent probe of the dTMP binding site, we detected an ADP-induced conformational change enhancing the binding affinity of dTMP and analogues. Several thymidine and dTMP derivatives were found to bind the enzyme with micromolar affinities. The present study provides the basis for the design of specific inhibitors or substrates for poxvirus TMP kinase.