Nucleic acid metabolism in yeast II. Metabolism of thymidylate during thymidylate excess death

Summary A discrete class of strains of Saccharomyces cerevisiae, able to utilize, highly efficiently, exogenous deoxythymidine-5-monophosphate (dTMP), was found to be sensitive to concentrations >10 M dTMP in an otherwise complete growth medium. Excess dTMP is cytostatic and cytotoxic: 90% of exponentially growing cells lose colony forming ability within 1 h of exposure to excess dTMP in a growth medium. Uptake of dTMP, adenine, histidine, and leucine does occur during this thymidylate excess death (TED). dTMP is anabolized to higher phosphorylated nucleotides and catabolized to thymidine intracellularly. DNA synthesis is blocked under TED-conditions but not RNA and protein biosynthesis.Abbreviations dTMP deoxythymidine-5-monophosphate - dTDP deoxythymidine-5-diphosphate - dTTP deoxythymidine-5-triphosphate - dThd deoxythymidine - tmp genetic symbol for dTMP-auxotrophy - TMP genetic symbol for dTMP-prototrophy - (tlr) symbol for the phenotype of a yeast strain to efficiently utilize exogenous dTMP     

The identification of a Caenorhabditis elegans homolog of p34cdc2 kinase

We have identified a Caenorhabditis elegans homolog of p34^cdc2 kinase. The C. elegans homolog, ncc-1, is -60% identical to p34^cdc2 of Homo sapiens. When expressed from a constitutive yeast promoter, ncc-1 is capable of complementing a conditional lethal mutation in the CDC28 gene of Saccharomyces cerevisiae, indicating that this C. elegans homolog can properly regulate the cell cycle.     

Cloning of the polyketide synthase gene atX from Aspergillus terreus and its identification as the 6-Methylsalicylic acid synthase gene by heterologous expression

The geneCAL1 (also known asCDC43) ofSaccharomyces cerevisiae encodes the subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant enzymes encoded bycall-1, andcdc43-2 tocdc43-7, expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of thecall/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperature sensitive. Thecall-1 mutation, located most proximal to the C-terminus of the protein, differs from the othercdc43 mutations in several respects. An increase in soluble Rholp was observed in thecall-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype ofcall-1 is most efficiently suppressed by overproduction of Rholp. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious incall-1 cells, but not in othercdc43 mutants or the wild-type strains. Thecdc43-5 mutant cells accumulate Cdc42p in soluble pools andcdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among thecall/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.     

Short nascent DNA pieces, accumulating in Saccharomyces cerevisiae after inhibition of DNA chain elongation, hybridize to specific chromosomal sites

Summary In temperature-sensitive DNA chain elongation mutants (cdc8 ^ts) of Saccharomyces cerevisiae even at the restrictive temperature a small portion of DNA is synthesized, which can be labeled by radioactivity. Under denaturing conditions this product sediments in alkaline sucrose gradients with about 4S. It is probable that these short nascent DNA pieces are derived predominantly from newly activated origins of replication. An alternative and more direct method of limiting the elongation of DNA chains uses araCMP as an inhibitor in nucleoside monophosphate-incorporating yeast strains. As with the cdc8 ^ts mutants the only radioactive products in labeling experiments with [³²P]dTMP are 4S pieces. Potential sites of their formation are small replication bubbles and terminal doublestranded loops observed in electron micrographs of the DNA from araCMP-inhibited cells. The pieces hybridize specifically with the replication origin of the 2 m-plasmid, the chromosome telomeres and a group of chromosomal genes. Other genes and the centromere of chromosome 11 (CEN11) do not react. The 4S pieces hybridize with only three of nine cloned autonomously replicating sequences (ARS). It is concluded that ARS sequences, at least in the presence of araCMP, are not always used as replication origins within their normal environment on the chromosome.     

Nucleic acid metabolism in yeast VI. Utilisation of exogenous dAMP

Summary It is shown that mutants of Saccharomyces cerevisiae able to efficiently utilise exogenous dTMP can also utilise exogenous dAMP. Under extracellular conditions permissive for dTMP uptake label stemming from offered [8-³H]dAMP is incorporated preferentially into alkali-resistant, high molecular weight material (putative DNA): only about 30% of high molecular weight cell-bound dAMP label was found to be sensitive towards mild alkali hydrolysis. This putative RNA label can be minimised to practically zero when mM Ade is employed in a dAMP labelling assay. Exogenous dAMP at 10 M was found to be cytostatic similarly to M dTMP and similarly to inhibit effectively import of exogenous P_i. We conclude from our results that there exists a yeast cytoplasmic membrane permease able to import dAMP. A model of this hypothetical permease system is presented.Abbreviations S. cerevisiae Saccharomyces cerevisiae - TIP yeast cytoplasmic membrane permease importing dTMP under permisive conditions - AIP hypothetical yeast cytoplasmic membrane permease importing dAMP under permissive conditions - tlr symbol for the recessive genetic trait to utilise effeciently exogenous dTMP - tmp symbol for the recessive genetic trait leading to dTMP auxotrophy - dTMP 2-deoxythymidine 5-monophosphate - dAMP 2-deoxyadenosine 5-monophosphate - (d)NMP (deoxy)ribonucleoside 5-monophosphate - dThd 2-deoxythymidine - Thy thymine - dAdo 2-deoxyadenosine - Ado adenosine - Ade adenine - Ura uracil - P_i inorganic phosphate Apart from discrete abbreviations we followed the rules of nomenclature as recommended by the IUPAC-IUB commission of biochemical nomenclature (CBN)     

A study of barley stripe mosaic virus (BSMV) genome

Summary The cell cycle mutant, cdc9, in the yeast Saccharomyces cerevisiae is defective in DNA ligase be deficient in the repair of DNA damaged by methyl methane sulphonate. On the other hand survival of cdc9 after irradiation by -rays is little diferent from that of the wild-type, even after a period of stress at the restrictive temperature. The mutant cdc9 is not allelic with any known rad or mms mutants.     

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.     

Ung1p-mediated uracil-base excision repair in mitochondria is responsible for the petite formation in thymidylate deficient yeast

The budding yeast CDC21 gene, which encodes thymidylate synthase, is crucial in the thymidylate biosynthetic pathway. Early studies revealed that high frequency of petites were formed in heat-sensitive cdc21 mutants grown at the permissive temperature. However, the molecular mechanism involved in such petite formation is largely unknown. Here we used a yeast cdc21-1 mutant to demonstrate that the mutant cells accumulated dUMP in the mitochondrial genome. When UNG1 (encoding uracil-DNA glycosylase) was deleted from cdc21-1, we found that the ung1Δ cdc21-1 double mutant reduced frequency of petite formation to the level found in wild-type cells. We propose that the initiation of Ung1p-mediated base excision repair in the uracil-laden mitochondrial genome in a cdc21-1 mutant is responsible for the mitochondrial petite mutations.     

Defective DNA Synthesis in Permeabilized Yeast Mutants

THE simple eukaryote, Saccharomyces cerevisiae, is suitable for combined genetic and biochemical analysis of the cell division cycle. More than forty temperature-sensitive mutants of S. cerevisiae defective in fifteen genes that control various steps of the yeast cell cycle have been detected by screening a collection of mutants with time-lapse photomicroscopy¹. Mutations in two genes, cdc4 and cdc8, result in defective DNA synthesis at the restrictive temperature². The product of cdc8 is apparently required throughout the period of DNA synthesis, because if a strain defective in this gene is shifted to 36° C within the S period, DNA replication ceases. In contrast, the product of cdc4 is apparently required only at the initiation of DNA synthesis because when a strain carrying a defect in this gene is shifted to 36° C, DNA replication already in progress is not impaired. Cells defective in cdc4, however, fail to initiate new rounds of DNA synthesis at the restrictive temperature. Based on these observations the DNA mutants have been tentatively classified as defective in DNA replication (cdc8) and in the initiation of DNA synthesis (cdc4).     

The fission yeast cell cycle control gene cdc2: isolation of a sequence suc1 that suppresses cdc2 mutant function

Summary A DNA fragment called suc1 has been found to rescue cells mutated in the cell cycle control gene cdc2 of the fission yeast Schizosaccharomyces pombe. The suppressing activity of suc1 is observed when it is present on a multicopy number plasmid. The gene does not hybridise to cdc2 and maps elsewhere in the genome. Its effect is cdc2 allele specific suggesting that it interacts directly with the cdc2 gene function.     

Fission Yeast Cdc24 Is a Replication Factor C- and Proliferating Cell Nuclear Antigen-Interacting Factor Essential for S-Phase Completion

At the nonpermissive temperature the fission yeast cdc24-M38 mutant arrests in the cell cycle with incomplete DNA replication as indicated by pulsed-field gel electrophoresis. The cdc24⁺ gene encodes a 501-amino-acid protein with no significant homology to any known proteins. The temperature-sensitive cdc24 mutant is effectively rescued by pcn1⁺, rfc1⁺ (a fission yeast homologue of RFC1), and hhp1⁺, which encode the proliferating cell nuclear antigen (PCNA), the large subunit of replication factor C (RFC), and a casein kinase I involved in DNA damage repair, respectively. The Cdc24 protein binds PCNA and RFC1 in vivo, and the domains essential for Cdc24 function and for RFC1 and PCNA binding colocalize in the N-terminal two-thirds of the molecule. In addition, cdc24⁺ genetically interacts with the gene encoding the catalytic subunit of DNA polymerase , which is stimulated by PCNA and RFC, and with those encoding the fission yeast counterparts of Mcm2, Mcm4, and Mcm10. These results indicate that Cdc24 is an RFC- and PCNA-interacting factor required for DNA replication and might serve as a target for regulation.     

Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase

In excised pith parenchyma from Nicotiana tabacum L. cv. Wisconsin Havana 38, auxin (naphthalene-1-acetic acid) together with cytokinin (6-benzylaminopurine) induced a greater than 40-fold increase in a p34^cdc2-like protein, recoverable in the p13^suc1-binding fraction, that had high H1 histone kinase activity, but enzyme induced without cytokinin was inactive. In suspension-cultured N. plumbaginifolia Viv., cytokinin (kinetin) was stringently required only in late G2 phase of the cell division cycle (cdc) and cells lacking kinetin arrested in G2 phase with inactive p34^cdc2-like H1 histone kinase. Control of the Cdc2 kinase by inhibitory tyrosine phosphorylation was indicated by high phosphotyrosine in the inactive enzyme of arrested pith and suspension cells. Yeast cdc25 phosphatase, which is specific for removal of phosphate from tyrosine at the active site of p34^cdc2 enzyme, was expressed in bacteria and caused extensive in-vitro activation of p13^suc1-purified enzyme from pith and suspension cells cultured without cytokinin. Cytokinin stimulated the removal of phosphate, activation of the enzyme and rapid synchronous entry into mitosis. Therefore, plants can control cell division by tyrosine phosphorylation of Cdc2 but differ from somatic animal cells in coupling this mitotic control to hormonal signals.Abbreviations BAP 6-benzylaminopurine - BrdUrd 5-bromo-2-deoxyuridine - cdc cell division cycle - Cdc25 cdc phospho-protein phosphatase - CKI cyclin dependent kinase inhibitor - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6 diamidino-2-phenylindole - GST-cdc25 glutathione sulfur transferase-truncated cdc25 fusion - MS Murashige and Skoog (1962) - NAA naphthalene-1-acetic acid - p34^cdc2 34-kDa product of the cdc2 gene     

The Saccharomyces cerevisiae SOC8-1 gene and its relationship to a nucleotide kinase

The yeast SOC8-1 gene was originally identified by partial complementation of cdc8 mutant strains. We have carried out Bal31 deletion analysis of the SOC8-1 gene to define the minimal size which is required for the complementation of the cdc8 mutation. When the SOC8-1 gene is cloned in a multicopy plasmid, it enables temperature-resistant growth in the cdc8 mutant strain, while the SOC8-1 gene in a single copy plasmid does not. Thus, its suppression of the cdc8 mutant is dosage dependent. The high copy number vector carrying the SOC8-1 gene can complement five different cdc8 alleles, indicating that the suppression is not allele specific. Since CDC8 encodes thymidylate kinase, cells bearing a high copy number plasmid containing SOC8-1 gene were tested for the ability to phosphorylate several nucleoside monophosphates, including UMP, GMP and dTMP. Significantly increased phosphorylation activity was observed, suggesting that SOC8-1 encodes a nucleotide kinase. Both restriction enzyme analysis of the SOC8-1 gene and partial purification of the overproduced kinase in SOC8-1 overproducing strains suggest that SOC8-1 may be allelic with URA6. Consistent with these results, both SOC8-1 and URA6 are located on chromosome XI. Thus, one possible suppression mechanism is that SOC8-1 may provide a trans-acting dTMP kinase activity, bypassing the cdc8 gene defect.     

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.     

Control of Herpes simplex virus thymidine kinase gene expression in Saccharomyces cerevisiae by a yeast promoter sequence

Summary This study presents the first evidence that the 5 promoter region of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene (G-3-PD) promoter will permit expression of an adjacent foreign gene. The S. cerevisiae G-3-PD promoter was linked to the herpes simplex virus — thymidine kinase (HSV-TK) gene in a shuttle plasmid capable of autonomous replication in both yeast and Escherichia coli. Since the HSV-TK gene promoter is not functional in yeast, yeast cells containing these plasmids will express the HSV-TK gene and synthesize thymidine kinase only if the yeast promoter fragment is fused to the HSV-TK gene in the proper orientation. The 5 flanking sequences necessary for the expression of heterologous eukaryotic genes in S. cerevisiae are discussed.