Biomedical subjects

R K Storms

Publications and source records attributed to R K Storms.

29 records · Page 2Linked to original sources

PubMed1991

The periodically expressed TMP1 gene of Saccharomyces cerevisiae is subject to START-dependent and START-independent regulation.

The rate of transcription of the Saccharomyces cerevisiae gene encoding thymidylate synthase (TMP1) fluctuates periodically during the cell cycle. The simplest explanation for this pattern of expression is that transcription occurs during the late G1 and early S phases and does not occur during other stages of the cell cycle. In this report, however, we show that TMP1 is subject to regulation that results in at least three different levels of expression: essentially nondetectable expression during stationary phase (G0), moderate level expression in START-arrested growing cells (START-independent), and a high level of expression in proliferating cells (START-dependent). Our analysis also shows that upstream elements important for START-independent expression and required for START-dependent expression are located within a 37-base region.

Base Sequence↗

PubMed1991

Ubiquitin gene expression: response to environmental changes.

It has previously been shown that the yeast ubiquitin genes UBI1, 2 and 3 are strongly expressed during the log-phase of batch culture growth, whereas the UBI4 gene is weakly expressed. We found that heat shock, treatment with DNA-damaging agents, starvation, and the feeding of starved cells all transiently induced UBI4. These results suggest that UBI4 is induced whenever a change in culture conditions dictates a dramatic shift in cellular metabolism, and that UBI4 expression returns to lower levels once cellular metabolism has adapted to the new conditions. In contrast, all of the treatments tested, except starvation, transiently repressed the UBI1, 2 and 3 genes. Although starvation also repressed UBI1, 2 and 3 its effect was not transient, and expression only recovered upon the addition of fresh media. These results, together with others presented here, suggest that high levels of UBI1, 2 and 3 expression are dependant upon ongoing cell growth, and that treatments which slow or stop growth repress their expression.

4-Nitroquinoline-1-oxide↗

PubMed1991

Characterization of a short, cis-acting DNA sequence which conveys cell cycle stage-dependent transcription in Saccharomyces cerevisiae.

Comparison of the 5'-flanking regions of several cell cycle-regulated DNA replication genes of Saccharomyces cerevisiae has revealed the presence of a common sequence, 5'-ACGCGT-3', which is upstream and proximal to mapped transcription initiation sites. This sequence, which is the cleavage site for the restriction endonuclease MluI, is present twice in the upstream region of the yeast thymidylate synthase gene TMP1. Previous studies have implicated these MluI sites as critical components in the cell cycle-dependent transcription of TMP1. In this study, we examined more closely the importance of the ACGCGT sequences for the transcription of this gene. Using site-directed mutagenesis in combination with deletion analysis and subcloning experiments, we found that (i) while both of the TMP1 MluI sites contribute to the total transcription of this gene, the distal site is predominant and (ii) the 9-bp sequence ACGCGTTAA encompassing the distal MluI site exhibits properties of a cell cycle-stage dependent upstream activation sequence element. The results of this study support the notion that the ACGCGT sequence is an integral component of a transcription system which coordinates the cell cycle-dependent expression of DNA replication genes in S. cerevisiae.

Base Sequence↗

PubMed1988

Transcriptional regulation of the cell cycle-dependent thymidylate synthase gene of Saccharomyces cerevisiae.

We have previously shown that transcript levels expressed from the yeast TMP1 gene fluctuate periodically during the yeast cell cycle. However, it was not known whether periodic expression resulted from a regulatory mechanism acting at the level of transcription or a regulatory mechanism acting at the level of cell cycle stage-dependent changes in the stability of the TMP1 transcript. In this report we now show that the periodic expression of TMP1 transcript is primarily controlled at the level of its transcription by sequences which are upstream of its transcription initiation sites. We also localized the upstream sequences necessary for periodic transcription to a 150-base-pair region and show that this region encodes an element(s) with the properties of a periodic upstream activating sequence. The regulatory region defined in this study apparently does not contain consensus sequences similar to those reported for the cell cycle-regulated HO endonuclease or for the histone H2A and H2B genes of Saccharomyces cerevisiae.

Base Sequence↗

PubMed1987

Molecular characterization of the cell cycle-regulated thymidylate synthase gene of Saccharomyces cerevisiae.

The complete nucleotide sequence of a 1.8-kilobase DNA fragment containing the cell cycle-regulated thymidylate synthase gene (TMP 1) of the yeast Saccharomyces cerevisiae is presented. This analysis has revealed a 912-base pair open reading frame which encodes a 304-amino acid residue protein with a calculated Mr of 35,007. The tmp1-6 and cdc21-1 mutant alleles of this gene also have been sequenced, and both show single base pair changes which would result in different amino acid substitutions. The amino acid sequence of the yeast thymidylate synthase gene derived from the DNA sequence shows considerable homology when compared with the human, mouse, Herpesvirus saimiri, Leishmania major, Leishmania tropica, Escherichia coli, Lactobacillus casei, bacteriophage T4, and Bacillus subtilis phage phi 3T enzymes. Northern blot hybridization reveals that the TMP 1 mRNA is a 1.15-kilobase polyadenylated transcript. A set of consensus yeast mRNA splice sequences appears within the open reading frame of TMP 1, but S1 nuclease protection experiments reveal that splicing of the mRNA does not occur. Disruption of the gene by the introduction of a large insertion did not produce any defect besides the expected dependence on dTMP for growth. Specifically, the viability of the mutants in the presence of dTMP indicates that the protein does not play a significant structural role in a complex of replication enzymes.

Alleles↗

PubMed1986

Growth-rate-dependent regulation of the expression and inactivation of thymidylate synthase in Saccharomyces cerevisiae.

Thymidylate synthase activity fluctuated dramatically as cultures of Saccharomyces cerevisiae progressed through the different stages of batch culture growth. During logarithmic growth these yeast cultures each contained about 40 microU (1 microU is 1 pmol of 3H released per min) of thymidylate synthase activity per 10(8) haploid cells, but as cultures entered the stationary phase and during the stationary phase, activity dropped dramatically, eventually reaching undetectable levels. Stimulation of stationary-phase cells with fresh medium resulted in rapid reestablishment of log phase levels. Two mechanisms, the regulation of thymidylate synthase-specific transcripts and the irreversible inactivation of thymidylate synthase activity, acted in concert to regulate activity levels. These results suggested that thymidylate synthase represents a special subset of yeast proteins whose levels per cell vary quickly and dramatically in response to changes in proliferation rates.

Cell Cycle↗

PubMed1985

Efficient expression of the Escherichia coli leuB gene in yeast.

Efficient expression of the Escherichia coli leuB (beta-isopropylmalate dehydrogenase) gene occurred in yeast after in vitro DNase digestion and religation of plasmid bound leuB and the yeast HIS3 DNA which placed the 5' end of the yeast HIS3 gene immediately adjacent to the coding region of the E. coli leuB gene. Two structurally distinct classes of gene fusions were constructed, each involved portions of the yeast HIS3 gene which contributed DNA sequences responsible for leuB expression in yeast. The first class involved fusion of the HIS3 coding region to bacterial DNA resulting in the production of a fusion protein with beta-isopropylmalate dehydrogenase activity. The second class consisted of bacterial DNA, including the leuB coding region, fused to the HIS3 promotor region with the absence of any portion of the HIS3 coding region. In both constructions the HIS3 promotor region is required for transcription, however, translation of the class two fusion is initiated at a bacterial DNA coded AUG, and the 5' end of the mRNA coded by the leuB gene mapped predominantly at bacterial DNA sequences. The DNA sequence responsible for the 5' end of the HIS3 mRNAs remain in the class two gene fusions but this did not preclude the initiation of transcription at bacterial DNA sequences. The pattern of mRNA initiation at bacterial DNA suggests that DNA sequences at, or adjacent to, the site of transcription initiation are involved in the determination of the sites of initiation, and perhaps the frequency at which initiation occurs.

3-Isopropylmalate Dehydrogenase↗

PubMed1984

Cell cycle-dependent expression of thymidylate synthase in Saccharomyces cerevisiae.

Synchronous populations of Saccharomyces cerevisiae cells, generated by two independent methods, have been used to show that thymidylate synthase, in contrast to the vast majority of cellular proteins thus far examined, fluctuates periodically during the S. cerevisiae cell cycle. The enzyme, as assayed by two different methods, accumulated during S period and peaked in mid to late S phase, and then its level dropped. These observations suggest that both periodic synthesis and the instability of the enzyme contribute to the activity profile seen during the cell cycle. Accumulation of thymidylate synthase is determined at the level of its transcript, with synthase-specific mRNA levels increasing at least 10-fold to peak near the beginning of S period and then falling dramatically to basal levels after the onset of DNA synthesis. This mRNA peak coincided with the time during the cell cycle when thymidylate synthase levels were increasing maximally and immediately preceded the peak of DNA synthesis, for which the enzyme provides precursor dTMP.

Cell Cycle↗

PubMed1982

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.

DNA Restriction Enzymes↗

PubMed1981

Genetic complementation of the Saccharomyces cerevisiae leu2 gene by the Escherichia coli leuB gene.

The leucine operon of Escherichia coli was cloned on a plasmid possessing both E. coli and Saccharomyces cerevisiae replication origins. This plasmid, pEH25, transformed leuA, leuB, and leuD auxotrophs of E. coli to prototrophy; it also transformed leu2 auxotrophs of S. cerevisiae to prototrophy. beta-Isopropylmalate dehydrogenase was encoded by the leuB gene of E. coli and the leu2 gene of yeast. Verification that the leuB gene present on pEH26 was responsible for complementing yeast leu2 was obtained by isolating in E. coli several leuB mutations that resided on the plasmid. These mutant leuB- plasmids were no longer capable of complementing leu2 in S. cerevisiae. We conclude that S. cerevisiae is capable of transcribing at least a portion of the polycistronic leu operon of E. coli and can translate a functional protein from at least the second gene of this operon. The yeast Leu+ transformants obtained with pEH25, when cultured in minimal medium lacking leucine, grew with a doubling time three to four times longer than when cultured in medium supplemented with leucine.

3-Isopropylmalate Dehydrogenase↗

PubMed1979

Chimeric plasmids for cloning of deoxyribonucleic acid sequences in Saccharomyces cerevisiae.

Two sets of plasmids, each carrying a Saccharomyces cerevisiae gene and a portion or all of the yeast 2-micron circle linked to the Escherichia coli plasmid pBR322, have been constructed. One of these sets contains a BamHI fragment of S. cerevisiae deoxyribonucleic acid that includes the yeast his3 gene, whereas the other set contains a BamHI fragment of S. cerevisiae that includes the yeast leu2 gene. All plasmids transform S. cerevisiae and E. coli with a high frequency, possess unique restriction endonuclease sites, and are retrievable from both host organisms. Plasmids carrying the 2.4-megadalton EcoRI fragment of the 2-micron circle transform yeast with 2- to 10-fold greater frequency than those carrying the 1.5-megadalton EcoRI fragment of the 2-micron circle. Restriction endonuclease analysis of plasmics retrieved from S. cerevisiae transformed with plasmics carrying the 2.4-megadalton EcoRI fragment showed that in 13 of 96 cases the original plasmic has acquired an additional copy of the 2-mcron circle. These altered plasmids appear to have arisen by means of an interplasmid recombination event while in S. cerevisiae. A clone bank of S. cerevisiae genes based upon one of these composite plasmids has been constructed. By using this bank and selecting directly in S. cerevisiae, the ura3, tyr1, and met2 genes have been cloned.

Cloning, Molecular↗