Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.

Functional and structural conservation of Schizosaccharomyces pombe dTMP kinase gene.

We describe the isolation and identification of the Schizosaccharomyces pombe dTMP kinase gene by the complementation of a Saccharomyces cerevisiae cell cycle mutant cell, cdc8. The isolated cDNA contains an open reading frame which can encode a protein with the molecular weight of 24,151. The deduced protein sequence is highly conserved among known dTMP kinase sequences from different organisms. The isolated gene should facilitate our study of its enzymatic activity, as well as nucleotide metabolism and cell cycle regulation in this organism.

Mutation analysis of Saccharomyces cerevisiae CDC6 promoter: defining its UAS domain and cell cycle regulating element.

Using beta-galactosidase as the reporter gene, we carried out mutagenesis experiments to investigate the 5' promoter region of the CDC6 gene. Our results showed that the DNA element, between -262 and -170, is important for the upstream activating sequence (UAS) activities. On the basis of the DNA sequence, there is a Mlu I (-204) and a Mlu I-like (-216) element located within the middle of the UAS region. Insertion and deletion mutagenesis analysis of the Mlu I sequence has indicated that the internal CGCG sequence of the Mlu I site (ACGCGT) is important for gene expression. Furthermore, when DNA elements containing the Mlu I sites were subcloned into the tester plasmid, periodic expression of a reporter gene throughout the cell cycle was observed, as evidenced by the beta-galactosidase activities and lacZ mRNA. Because the possible transcriptional initiation sites of the CDC6 transcript have been previously defined (Zhou and Jong, 1990, J. Biol. Chem. 264, 9022-9029), we propose a model regarding the construct of the CDC6 promoter region. This 5' promoter construct contains a UAS region and a Mlu I element (MCB box) typical of a family of cell cycle-regulated genes involved in DNA metabolism. Previous genetic studies have not completely defined the CDC6 execution point in the functional yeast cell cycle map. Our results favor the possibility that the CDC6 gene is required, and directly involved, in the initiation of DNA replication.

Amplification of gene ends from gene libraries by polymerase chain reaction with single-sided specificity.

Isolation of a full-length gene on the basis of a limited sequence information is often troublesome and challenging. Tremendous effort is needed to isolate a specific gene by screening cDNA or genomic libraries by oligonucleotide or nucleic acid probes. In those methods, basically nucleic acid probes are used in a screening process to check whether or not a plaque or a colony contains the sequence of interest. There have been attempts to isolate specific DNA fragments using immobilized DNA, in which particular DNA fragments were enriched by hybrid selection and then the concentrated library was screened by a specific DNA probe (1,2). Recently, polymerase chain reaction (PCR) has been applied to the cloning of genes. Friedmann et al. (3) first used PCR to screen λgt11 library with two gene-specific primers. This protocol can be effectively used to isolate a particular DNA fragment between two specific primers or to generate nucleic acid probe from cDNA libraries. The unknown sequences flanking the fragment between the two specific primers cannot be amplified by this method.

Saccharomyces cerevisiae nucleoside-diphosphate kinase: purification, characterization, and substrate specificity.

Nucleoside-diphosphate kinase is an enzyme which catalyzes the phosphorylation of nucleoside diphosphates into the corresponding triphosphates for nucleic acid biosynthesis. In this communication, we describe the purification and characterization of nucleoside-diphosphate kinase from yeast. The purified protein appears to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel analysis, with a molecular weight of about 17,000-18,000. An estimate from the fast protein liquid chromatography Superose 12 gel filtration shows a native molecular weight of about 68,000 to 70,000. The results suggest that yeast nucleoside-diphosphate kinase is composed of four subunits. Substrate specificity studies show that the relative activity of nucleoside diphosphates (NDP) as phosphate acceptors is in the order of dTDP greater than CDP greater than UDP greater than dUDP greater than GDP greater than or equal to dGDP greater than dCDP greater than dADP greater than ADP; and the relative activity of triphosphate donors is in the order of UTP greater than dTTP greater than CTP greater than dCTP greater than dATP greater than ATP greater than or equal to dGTP greater than GTP. The Km and Vm of dTDP, dGDP, dCDP, dUDP, CDP, and UDP have been determined. The rate constant studies indicate that the purified NDP kinase prefers using, to a slight extent, dTDP (approximately 800 min-1) as the substrate rather than other tested deoxyribo- and ribonucleotides (350-450 min-1). The broad substrate specificity and kinetic data suggest that the enzyme is involved in both DNA and RNA metabolism.

DNA synthesis in yeast cell-free extracts dependent on recombinant DNA plasmids purified from Escherichia coli.

In our attempts to establish a cell-free DNA replication system for the yeast Saccharomyces cerevisiae, we have observed that recombinant DNA plasmids purified from Escherichia coli by a common procedure (lysozyme-detergent lysis and equilibrium banding in cesium chloride ethidium bromide gradients) often serve as templates for DNA synthesis by elongation enzymes. The templates could be elongated equally well by enzymes present in the yeast cell-free extracts, by the large proteolytic fragment of E. coli DNA polymerase I or by T4 DNA polymerase. The template activity of the purified plasmids was dependent on the presence of heterologous DNA segments in the bacterial vectors. The template activity could be diminished by treatment with alkali. We propose that the ability of recombinant plasmids isolated from bacterial hosts to serve as elongation templates may lead to erroneous conclusions when these plasmids are used as templates for in vitro replication or transcription reactions.

Isolation of the gene encoding yeast single-stranded nucleic acid binding protein 1.

A yeast gene encoding SSB-1, a single-stranded nucleic acid binding protein, has been isolated by screening a lambda gt11 genomic DNA library. The gene is located on a 1.84-kilobase chromosomal Bgl II-BamHI fragment. Yeast strains carrying the high-copy-number vector YEp24 with an SSB1 gene insert overproduce SSB-1 3-fold and SSB-1 mRNA 10-fold. A typical haploid cell contains about 20,000 molecules of SSB-1; thus, the cells can tolerate up to 60,000 copies. Yeast SSB-1 was expressed in Escherichia coli cells by using a phage T7 expression system. Spores containing the gene disrupted at a point within the coding sequence germinate and grow normally; thus, the gene is not essential. Protein blots show that no SSB-1 or novel immunologically related species that might retain SSB-1 activity are present in cells containing the disrupted SSB1 genes. Southern analysis and protein blots suggest the presence in yeast of a second, related, but nonidentical gene and two immunologically related proteins of 55 kDa and 75 kDa.

Cellular mechanisms of microbial proteins contributing to invasion of the blood-brain barrier.

One of the least understood issues in the pathogenesis and pathophysiology of microbial infection of the central nervous system (CNS) is how microorganisms cross the blood-brain barrier (BBB), which separates brain interstitial space from blood and is formed by the tight junctions of brain microvascular endothelial cells (BMEC). BMEC monolayer and bilayer culture systems have been developed as in vitro models to dissect the mechanisms of adhesion and invasion involved in pathogenesis of CNS infection caused by microbes. Viral, bacterial, fungal and parasitic pathogens may breach the BBB and enter the CNS through paracellular, transcellular and/or Trojan horse mechanisms. Conceivable evidence suggests that microbial proteins are the major genetic determinants mediating penetration across the BBB. Several bacterial proteins including IbeA, IbeB, AslA,YijP, OmpA, PilC and InlB contribute to transcellular invasion of BMEC. Viral proteins such as gp120 of HIV have been shown to play a role in penetration of the BBB. Fungal and parasitic pathothogens may follow similar mechanisms. SAG1 of Toxoplasma gondii has been suggested as a ligand to mediate host-cell invasion. Understanding the fundamental mechanisms of microbial penetration of the BBB may help develop novel approaches to prevent the mortality and morbidity associated with central nervous system (CNS) infectious diseases.

Characterization of Saccharomyces cerevisiae thymidylate kinase, the CDC8 gene product. General properties, kinetic analysis, and subcellular localization.

Thymidylate kinase is the product of the CDC8 gene of Saccharomyces cerevisiae (Jong, A.Y.S., Kuo, C.-L., and Campbell, J.L. (1984) J. Biol. Chem. 259, 11052-11059). In this communication we report the catalytic properties of the enzyme. The enzyme catalyzes the phosphorylation of deoxythymidine monophosphate to form deoxythymidine diphosphate in the presence of phosphate donor. ATP and dATP are the most efficient phosphate donors. In addition to dTMP, the yeast enzyme can use dUMP and 5-iodo-dUMP as phosphate acceptors. Kinetic analysis gives a Km of 0.5 mM for dTMP and 2 mM for dUMP. dTMP has a 7-fold greater rate constant than dUMP. Thymidylate kinase requires a divalent cation and is active over the entire range of pH from 6 to 9. The relative inhibitory effects of related compounds on yeast thymidylate kinase activity are in the order of dTDP greater than thymidine greater than 5-iodo-dUMP greater than ADP greater than or equal to dADP greater than dUMP greater than dTTP greater than dUDP, if dTMP is used as the phosphate acceptor. dTDP is a competitive inhibitor, with a Ki of 0.62 mM. Subcellular fractionation indicates that thymidylate kinase is found in the combined nuclear and cytoplasmic fraction but not in the mitochondria.

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.

Multiple species of single-stranded nucleic acid-binding proteins in Saccharomyces cerevisiae.

DNA affinity chromatography has been used to identify the major single-stranded nucleic acid binding proteins (SSBs) of Saccharomyces cerevisiae. There are five abundant species having molecular masses of 50, 45, 31, 23, and 20 kDa. Four of these proteins are cytoplasmic and one is mitochondrial. To date, three of the proteins have been purified to homogeneity. The purified proteins are designated SSB-m, SSB-1, and SSB-2, with molecular masses of 20, 45, and 50 kDa, respectively. SSB-m is found only in mitochondrial subcellular fractions. SSB-1 stimulates purified yeast DNA polymerase I, while SSB-2 inhibits DNA polymerase I. An antibody against SSB-1 has been prepared in rabbits and purified by SSB-1-Sepharose affinity chromatography. The purified antibody specifically inhibits DNA synthesis in an in vitro replication system, suggesting that SSB-1 may be involved in DNA replication in vivo. SSB-2 has the highest affinity for single-stranded DNA of all three proteins. It may represent a new class of eukaryotic SSB, on the basis of molecular weight, inhibition of DNA polymerase and antigenicity. Antibodies have also been prepared against SSB-2. The immunological reagents have been used to show that SSB-1, SSB-2, and SSB-m are antigenically distinct, as well as to study the relationship of these three SSBs to other proteins in yeast.

Molecular cloning of Saccharomyces cerevisiae CDC6 gene. Isolation, identification, and sequence analysis.

The CDC6 gene product is required for entering the S phase of the cell cycle in Saccharomyces cerevisiae. It has been isolated on recombinant plasmids by selection for complementation of temperature-sensitive alleles with a yeast genomic library. The entire complementing activity is carried on a 1.8-kilobase chromosomal DNA fragment, as revealed by deletion mapping. Northern blotting shows that the size of the CDC6 mRNA is about 1.7 kilobases. A Southern blot of yeast chromosomes which were separated by the field inversion gel electrophoresis method indicates that the isolated DNA fragment is derived from chromosome X. The locus from which the clone was derived was marked by integration with a nutritional marker and found by meiotic mapping to cosegregate with CDC6. Thus, we conclude that we have isolated the authentic CDC6 gene. Nucleotide sequence analysis of the CDC6 gene has revealed an open reading frame that encodes a protein with Mr = 57,969. There are five potential Asn-X-(Ser/Thr) glycosylation sites and a highly conserved nucleotide-binding site in the CDC6 sequence. Although computer surveys indicate overall sequence homology between S. cerevisiae CDC6 protein and Saccharomyces pombe CDC10 START protein, they may not be functionally equivalent as evaluated by the complementation assay.

Temporal and spatial distributions of yeast nucleoside diphosphate kinase activities and its association with the Cdc8p.

Nucleoside diphosphate kinase (E.C. 2.7.4.6.) is a broad substrate-specific enzyme that catalyzes the phosphorylation of nucleoside diphosphates to the corresponding triphosphates in nucleic acid biosynthesis. In this report, we investigate its spatial and temporal distributions in yeast to understand how the enzyme exerts its gene function(s). Our results show that the enzyme is predominantly cytoplasmic. A substantial amount of enzyme activity (40-50%) may be associated with the cell membrane. Less than 1% of total activity was detected in the nuclear fraction. Approximately 3% was found in the mitochondrial fraction. When yeast cultures were synchronized, we found that Saccharomyces cerevisiae nucleoside diphosphate kinase did not show cell cycle periodicity, as Schizosaccharomyces pombe enzyme did. To explore its link with DNA synthesis, we investigated its relationship with the Cdc8p (dTMP kinase). We demonstrated a physical interaction between these proteins in vitro, as evidenced that the GST:Cdc8p protein affinity column could retain a subpopulation of nucleoside diphosphate kinase activity from yeast crude extract. Furthermore, when GST:Cdc8p protein was expressed in yeast, the protein could bind to the glutathione-agarose, along with nucleoside diphosphate kinase, suggesting that there is an interaction between GST:Cdc8p and nucleoside diphosphate kinase in vivo. Our results provide evidence for at least a two-enzyme complex that may well facilitate nucleotide channeling in the cell.

Pulsed field gel electrophoresis labeling method to study the pattern of Saccharomyces cerevisiae chromosomal DNA synthesis during the G1/S phase of the cell cycle.

Yeast Saccharomyces cerevisiae is an excellent model to study eukaryotic DNA replication. Since yeast lacks thymidine kinase, it is difficult to assay DNA synthesis. In this report, a novel approach called the pulsed field gel electrophoresis (PFGE) labeling method is used to investigate yeast chromosomal DNA synthesis. In this method, yeast cells are first labeled by 32P in vivo and chromosomal DNA molecules are then resolved by pulsed field gel electrophoresis. A linear 32P labeling of chromosomal-size DNA molecules can be observed up to 100 min in an asynchronized culture. In an alpha-factor arresting-and-releasing synchronized culture, we observed that 32P can be rapidly taken up in the S phase. Our results show that all of the chromosomes are labeled at approximately the same time (within a 15-min interval), suggesting that the temporal order of all chromosomal DNA synthesis is synchronized in the S phase. Cell cycle blockers were used to arrest the yeast cultures to study DNA synthesis that coincides with cell cycle analysis. Our results show that alpha-factor and hydroxyurea block the cell cycle at the late G1 and S phases, respectively, resulting in the failure of DNA synthesis. Nocodazole blocks the cell cycle after the S phase and thus shows no effect on chromosomal DNA synthesis. Since the PFGE labeling method is highly specific to chromosomal DNA synthesis, we used it to examine the DNA synthesis pattern in various cell cycle mutants. All of the G1/S cdc mutants tested, cdc4, 6, 7, 8, 17, 2, and 9, cannot label the chromosomal size of DNA molecules; but the G2/M cdc mutant cdc13 can.(ABSTRACT TRUNCATED AT 250 WORDS)

The CDC8 gene of yeast encodes thymidylate kinase.

Thymidylate kinase catalyzes the phosphorylation of thymidine 5'-monophosphate to thymidine 5'-diphosphate in the pathway of synthesis of dTTP from dTMP. We have purified the enzyme approximately 5000-fold from a plasmid-bearing strain of the yeast Saccharomyces cerevisiae that over produces the activity 6-fold. The protein appears homogeneous by sodium dodecyl sulfate-polyacrylamide gel analysis and has a molecular weight of 25,000. The amino acid composition and the sequence of amino acids on the NH2 terminus have been determined. Our interest in thymidylate kinase stems from the fact that R. A. Sclafani and W. Fangman (personal communication) recently presented genetic evidence that this enzyme is encoded by the CDC8 gene of yeast. In this paper, we show, by several biochemical criteria, that thymidylate kinase is the product of the CDC8 gene. First, extracts of strains bearing six different alleles of cdc8 show no thymidylate kinase activity. Secondly, strains carrying the CDC8 gene on a high-copy-number plasmid produce 6-fold higher levels of the kinase activity than does wild type. Third, the DNA sequence of the CDC8 gene reveals an open reading frame that encodes a protein with the same amino-terminal sequence as purified thymidylate kinase.

The CDC4 gene product is associated with the yeast nuclear skeleton.

The CDC4 gene product of Saccharomyces cerevisiae is required at the late G1/S phase boundary of the cell cycle. In an attempt to better understand the function of CDC4, we performed experiments to localize this protein in the yeast cell. Using antisera, directed against a TrpE-CDC4 fusion protein, to analyze immuno-blots of different subcellular fractions from yeast, we demonstrated that the CDC4 gene product localizes in the nucleus by two different biochemical preparations of the yeast nucleoskeletal proteins. Immunofluorescence microscopy further confirmed its nuclear localization. These data support a model that includes the CDC4 gene product as a component of the yeast nuclear skeleton. The significance of this association in relationship to the biological role of CDC4 is discussed.

Molecular characterization of Saccharomyces cerevisiae URA6 gene. DNA sequence, mutagenesis analysis, and cell cycle regulation relevant to its suppression mechanism to cdc8 mutation.

Yeast SOC8 DNA fragment was isolated as a wild type dominant suppressor of cdc8 mutation. We have used Bal31 deletion analysis to define the minimal 1 kilobase HpaI-NcoI DNA element required for complementing the cdc8 mutation. The complementing sequence harbored a multicopy plasmid also enhanced by uridine monophosphate kinase in crude extracts. DNA sequence analysis revealed an open reading frame encoding a protein with a molecular weight of 24,949. Since our SOC8 sequence was identical to that of URA6 gene, which encodes uridine monophosphate kinase, we conclude that SOC8 is allelic with URA6, and we use the term URA6 hereafter. Northern blotting experiments showed that the size of mRNA is about 0.9 kibobases. Primer extension experiments showed multiple transcriptional starting sites primarily located at -160 The size and the deduced amino acid composition are consistent with information obtained from purified uridine monophosphate kinase. Thus, both molecular genetic and biochemical evidence supports a notion that the URA6 is SOC8 encoding a yeast uridine monophosphate kinase. Mutagenesis analysis of its putative nucleotide-binding site, altering essential lysine to glutamic acid, resulted in loss of its uridine monophosphate kinase activity. Complementation analysis studies indicated that the mutated ura6 gene abolished its ability to complement ura6 mutant cells; nor could it suppress cdc8 mutation. Unlike CDC8, the mRNA level of the URA6 gene did not fluctuate throughout the cell cycle; presumably, the temporal order of these two enzymatic activities might be different during cell cycle progression. These data may explain an incomplete suppression of cdc8 by URA6, as previously observed. Taken together, the results support our previous speculation that the suppression of the cdc8 mutation mechanisms by URA6 is due to the provision of the trans-acting dTMP kinase activity to complement the cdc8 defect.