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1.  Mutations in the homologous ZDS1 and ZDS2 genes affect cell cycle progression. 
Molecular and Cellular Biology  1996;16(10):5254-5263.
The Saccharomyces cerevisiae ZDS1 and ZDS2 genes were identified as multicopy suppressors in distinct genetic screens but were found to encode highly similar proteins. We show that at semipermissive temperatures, a yeast strain with a cdc28-1N allele was uniquely deficient in plasmid maintenance in comparison with strains harboring other cdc28 thermolabile alleles. Quantitative analysis of plasmid loss rates in cdc28-1N strains carrying plasmids with multiple replication origins suggests that a defect in initiating DNA replication probably causes this plasmid loss phenotype. The ZDS1 gene was isolated as a multicopy suppressor of the cdc28-1N plasmid loss defect. A zds1 deletion exhibits genetic interactions with cdc28-1N but not with other cdc28 alleles. SIN4 encodes a protein which is part of the RNA polymerase II holoenzyme-mediator complex, and a sin4 null mutation has pleiotropic effects suggesting roles in transcriptional regulation and chromatin structure. The ZDS2 gene was isolated as a multicopy suppressor of the temperature-sensitive growth defect caused by the sin4 null mutation. Disruption of either ZDS1 or ZDS2 causes only modest phenotypes. However, a strain with both ZDS1 and ZDS2 disrupted is extremely slowly growing, has marked defects in bud morphology, and shows defects in completing S phase or entering mitosis.
PMCID: PMC231525  PMID: 8816438
2.  Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. 
Molecular and Cellular Biology  1995;15(5):2612-2624.
Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G1/S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G0. Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G1 inhibited entry into S phase, whereas microinjection performed near the G1/S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G1/S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle.
PMCID: PMC230491  PMID: 7739542
3.  Inactivation of a Cdk2 inhibitor during interleukin 2-induced proliferation of human T lymphocytes. 
Molecular and Cellular Biology  1994;14(7):4889-4901.
Peripheral blood T lymphocytes require two sequential mitogenic signals to reenter the cell cycle from their natural, quiescent state. One signal is provided by stimulation of the T-cell antigen receptor, and this induces the synthesis of both cyclins and cyclin-dependent kinases (CDKs) that are necessary for progression through G1. Antigen receptor stimulation alone, however, is insufficient to promote activation of G1 cyclin-Cdk2 complexes. This is because quiescent lymphocytes contain an inhibitor of Cdk2 that binds directly to this kinase and prevents its activation by cyclins. The second mitogenic signal, which can be provided by the cytokine interleukin 2, leads to inactivation of this inhibitor, thereby allowing Cdk2 activation and progression into S phase. Enrichment of the Cdk2 inhibitor from G1 lymphocytes by cyclin-CDK affinity chromatography indicates that it may be p27Kip1. These observations show how sequentially acting mitogenic signals can combine to promote activation of cell cycle proteins and thereby cause cell proliferation to start. CDK inhibitors have been shown previously to be induced by signals that negatively regulate cell proliferation. Our new observations show that similar proteins are down-regulated by positively acting signals, such as interleukin 2. This finding suggests that both positive and negative growth signals converge on common targets which are regulators of G1 cyclin-CDK complexes. Inactivation of G1 cyclin-CDK inhibitors by mitogenic growth factors may be one biochemical pathway underlying cell cycle commitment at the restriction point in G1.
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PMCID: PMC358861  PMID: 7516474
4.  Simian virus 40 origin auxiliary sequences weakly facilitate T-antigen binding but strongly facilitate DNA unwinding. 
Molecular and Cellular Biology  1990;10(4):1719-1728.
The complete simian virus 40 (SV40) origin of DNA replication (ori) consists of a required core sequence flanked by two auxiliary sequences that together increase the rate of DNA replication in monkey cells about 25-fold. Using an extract of SV40-infected monkey cells that reproduced the effects of ori-auxiliary sequences on DNA replication, we examined the ability of ori-auxiliary sequences to facilitate binding of replication factors and to promote DNA unwinding. Although the replicationally active form of T antigen in these extracts had a strong affinity for ori-core, it had only a weak but specific affinity for ori-auxiliary sequences. Deletion of ori-auxiliary sequences reduced the affinity of ori-core for active T antigen by only 1.6-fold, consistent with the fact that saturating concentrations of T antigen in the cell extract did not reduce the stimulatory role of ori-auxiliary sequences in replication. In contrast, deletion of ori-auxiliary sequences reduced the efficiency of ori-specific, T-antigen-dependent DNA unwinding in cell extracts at least 15-fold. With only purified T antigen in the presence of topoisomerase I to unwind purified DNA, ori-auxiliary sequences strongly facilitated T-antigen-dependent DNA conformational changes consistent with melting the first 50 base pairs. Under these conditions, ori-auxiliary sequences had little effect on the binding of T antigen to DNA. Therefore, a primary role of ori-auxiliary sequences in DNA replication is to facilitate T-antigen-dependent DNA unwinding after the T-antigen preinitiation complex is bound to ori-core.
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PMCID: PMC362278  PMID: 2157141
5.  Upstream regulatory sequences of the yeast RNR2 gene include a repression sequence and an activation site that binds the RAP1 protein. 
Molecular and Cellular Biology  1989;9(12):5359-5372.
The small subunit of ribonucleotide reductase in Saccharomyces cerevisiae (RNR2) was induced 3- to 20-fold by a variety of DNA-damaging agents. Induction of the RNR2 transcript by at least one of these agents, methyl methanesulfonate, did not require protein synthesis. To identify sequences involved in the regulation of RNR2, we introduced deletions upstream of the transcription start site. Sequences required for induction were contained within a 200-base-pair region that could confer methyl methanesulfonate inducibility on the heterologous CYC1 promoter. This region contained a repression sequence and at least two positive activation sites. One of these activation sites bound RAP1, a protein known to associate with mating-type silencers and the upstream activation sequences of a number of genes. The behavior of deletions of the repression sequence suggests that induction of RNR2 may occur, at least in part, through relief of repression.
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PMCID: PMC363704  PMID: 2685560
6.  Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro. 
Molecular and Cellular Biology  1989;9(2):469-476.
We determined the fidelity of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases (RTs) during DNA synthesis in vitro using the M13mp2 lacZ alpha gene as a mutational target. Both RTs commit an error approximately once for every 30,000 nucleotides polymerized. DNA sequence analysis of mutants generated in a forward mutation assay capable of detecting many types of errors demonstrated that avian myeloblastosis virus RT produced a variety of different mutations. The majority (58%) were single-base substitutions; all of which resulted from the misincorporation of either dAMP or dGMP. Minus-one frameshifts were also common, composing about 30% of the mutations. In addition to single-base events, eight mutants contained sequence changes involving from 2 to 59 bases. The frequency of these mutants suggests that, at least during DNA synthesis in vitro, RTs also commit errors by mechanisms other than classical base miscoding and misalignment. We examined the ability of RTs to synthesize DNA from a mismatched primer terminus at a sequence where the mismatched base was complementary to the next base in the template. Unlike cellular DNA polymerases which polymerize from the mismatched template-primer, RTs preferred to polymerize from a rearranged template-primer containing a matched terminal base pair and an unpaired base in the template strand. The unusual preference for this substrate suggests that the interactions between RTs and the template-primer are different from those of cellular DNA polymerases. The overall error rate of RT in vitro is sufficient to account for the estimated mutation rate of these viruses.
PMCID: PMC362622  PMID: 2469002
7.  Identification of the gene for the yeast ribonucleotide reductase small subunit and its inducibility by methyl methanesulfonate. 
Molecular and Cellular Biology  1987;7(10):3673-3677.
We have identified, cloned, and sequenced the gene for the small subunit of ribonucleotide diphosphate reductase of Saccharomyces cerevisiae. The protein and its transcript are induced about 10-fold by the alkylating agent methyl methanesulfonate, a result which suggests that the gene is induced by DNA damage.
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PMCID: PMC368022  PMID: 3316984
8.  Expression of the c-myc proto-oncogene during development of Xenopus laevis. 
Molecular and Cellular Biology  1986;6(12):4499-4508.
We isolated and characterized Xenopus laevis c-myc cDNAs from an oocyte-specific library. These cDNA clones encompass 2.35 kilobases of the X. laevis c-myc RNA and contain the entire coding domain of 1,257 nucleotides of the 419-amino acid-long X. laevis c-myc protein. The 2.7-kilobase X. laevis c-myc mRNA is expressed in the oocyte, maintained in the egg, and is present throughout the early cleavage stages of embryogenesis. At the time of transcriptional activation in the embryo the c-myc RNA levels show a significant decline and then reaccumulate continuously throughout the remainder of premorphogenic development. At the early neurula stage of embryogenesis the pattern of c-myc RNA expression is elevated in the mesoderm with respect to the endoderm and ectoderm. In the adult X. laevis the c-myc mRNA is expressed in some (e.g., skin, muscle) but not all differentiated tissues. The X. laevis c-myc protein migrates as a doublet of 61,000- and 64,000-dalton species. Both species are phosphorylated in oocytes and somatic cells, exhibit extremely short half-lives of less than 30 min, and are localized to the nuclear fraction of somatic cells. By contrast, the oocyte protein shows both cytoplasmic and germinal vesicle distribution and appears to be stable.
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PMCID: PMC367234  PMID: 3540613

Results 1-8 (8)