Search PubMed⌕ Search

Biomedical subjects

J R Broach

Publications and source records attributed to J R Broach.

At least 73 records · Page 4Linked to original sources
PubMed1987

A yeast plasmid partitioning protein is a karyoskeletal component.

The Saccharomyces yeast plasmid, 2-micron circle, encodes a partitioning system that ensures equidistribution of plasmid molecules to both progeny following cell division. This system consists of two proteins encoded in plasmid genes REP1 and REP2 and a cis-active noncoding locus, designated REP3. We have raised antibodies against a REP1 beta-galactosidase fusion protein and used them to identify the authentic REP1 protein in plasmid-bearing yeast cells. We find that REP1 protein is located exclusively in the nucleus and co-purifies with a karyoskeletal protein subfraction operationally and morphologically equivalent to the nuclear matrix-pore complex-lamina fraction of higher cells. The carboxyl half of the REP1 protein exhibits strong sequence homology to myosin heavy chain, vimentin, and nuclear lamins A and C, indicating a fibrous structure for the protein. From these observations, we suggest that REP1 protein may promote plasmid partitioning by intercalating into the nuclear lamina of the host cell to provide dispersed anchorage sites for attachment of plasmid molecules.

Amino Acid Sequence↗
PubMed1987

Functional domains of SIR4, a gene required for position effect regulation in Saccharomyces cerevisiae.

The product of the Saccharomyces cerevisiae SIR4 gene, in conjunction with at least three other gene products, prevents expression of mating-type genes resident at loci at either end of chromosome III, but not of the same genes resident at the MAT locus in the middle of the chromosome. To address the mechanism of this novel position effect regulation, we have conducted a structural and genetic analysis of the SIR4 gene. We have determined the nucleotide sequence of the gene and found that it encodes a lysine-rich, serine-rich protein of 152 kilodaltons. Expression of the carboxy half of the protein complements a chromosomal nonsense mutation of sir4 but not a complete deletion of the gene. These results suggest that SIR4 protein activity resides in two portions of the molecule, but that these domains need not be covalently linked to execute their biological function. We also found that high-level expression of the carboxy domain of the protein yields dominant derepression of the silent loci. This anti-Sir activity can be reversed by increased expression of the SIR3 gene, whose product is normally also required for maintaining repression of the silent loci. These results are consistent with the hypothesis that SIR3 and SIR4 proteins physically associate to form a multicomponent complex required for repression of the silent mating-type loci.

Amino Acid Sequence↗
PubMed1987

Expression of Rous sarcoma virus transforming protein pp60v-src in Saccharomyces cerevisiae cells.

The Rous sarcoma virus (RSV) pp60v-src protein was expressed in Saccharomyces cerevisiae cells either from a plasmid vector carrying the v-src gene or in yeast cells containing a single-copy v-src gene chromosomally integrated. In both yeast strains, v-src gene transcription is regulated by the galactose-inducible GAL10 promoter. Growth in galactose-containing medium resulted in constitutive expression of pp60v-src in the integrated strain and transient expression of higher levels of pp60v-src in the plasmid-bearing strain. The concentration of pp60v-src in the plasmid-bearing strain at its peak of expression was approximately threefold lower than that found in RSV-transformed mammalian cells. pp60v-src synthesized in yeast cells was phosphorylated in vivo on sites within the amino and carboxyl halves of the molecule. In immune complex kinase assays, the yeast pp60v-src was autophosphorylated on tyrosine and was able to phosphorylate exogenous substrates such as casein and enolase. The specific activity of pp60v-src synthesized in yeast cells was approximately 5- to 10-fold higher than that made in mammalian cells. Induction of pp60v-src caused the death of the plasmid-bearing yeast strain and transient inhibition of growth of the single-copy strain. Concomitantly, this induction resulted in high levels of tyrosine phosphorylation of yeast cell proteins. This indicates that pp60v-src functions as a tyrosine-specific phosphotransferase in yeast cells and suggests that hyperphosphorylation of yeast proteins is inimical to cell growth.

Avian Sarcoma Viruses↗
PubMed1987

Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function.

Two proteins in the yeast Saccharomyces cerevisiae that are encoded by the genes RAS1 and RAS2 are structurally and functionally homologous to proteins of the mammalian ras oncogene family. We examined the role of fatty acylation in the maturation of yeast RAS2 protein by creating mutants in the putative palmitate addition site located at the carboxyl terminus of the protein. Two mutations, Cys-318 to an opal termination codon and Cys-319 to Ser-319, were created in vitro and substituted in the chromosome in place of the normal RAS2 allele. These changes resulted in a failure of RAS2 protein to be acylated with palmitate and a failure of RAS2 protein to be localized to a membrane fraction. The mutations yielded a Ras2- phenotype with respect to the ability of the resultant mutants to grow on nonfermentable carbon sources and to complement ras1- mutants. However, overexpression of the ras2Ser-319 product yielded a Ras+ phenotype without a corresponding association of the mutant protein with the membrane fraction. We conclude that the presence of a fatty acyl moiety is important for localizing RAS2 protein to the membrane where it is active but that the fatty acyl group is not an absolute requirement of RAS2 protein function.

Acylation↗
PubMed1986

Site-specific recombination promotes plasmid amplification in yeast.

All stable, naturally occurring circular yeast DNA plasmids contain a pair of long, nontandem inverted repeats that undergo frequent reciprocal recombination. This yields two plasmid inversion isomers that exist in the cell in equal numbers. In the 2 mu circle plasmid of S. cerevisiae such inversion is catalyzed by a plasmid-encoded site-specific recombinase, FLP. We show that the site-specific recombination system of 2 mu circle enables the plasmid to increase its mean intracellular copy number in yeast cells growing under nonselective conditions. This apparently occurs by a FLP-induced transient shift in the mode of replication from theta to double rolling circle as initially proposed by Futcher. This capability may ensure stable maintenance of the plasmid by enabling it to correct downward deviations in copy number that result from imprecision of the plasmid-encoded partitioning system.

Chromosome Inversion↗
PubMed1986

Survival strategies of the yeast plasmid two-micron circle.

The multicopy yeast plasmid 2-micron circle uses a number of strategies to insure its persistence in its host. The plasmid confers no selective phenotype to the cell in which it is resident. Nonetheless, the plasmid is lost at less than 1 per 10(5) cell divisions during continuous exponential growth. We have determined that the plasmid persists at least in part due to the ability of the plasmid to amplify its mean copy number when its cellular copy level is low and to distribute plasmid molecules equally between mother and daughter cells at mitosis. We have found that amplification of plasmid copy number occurs by a novel mechanism in which site-specific recombination induces a transient shift in the mode of replication from theta to rolling circle. Equitable partitioning of plasmid molecules requires plasmid-encoded proteins and a centromere-like segment on the plasmid. We have accumulated evidence consistent with a model of partitioning in which the partitioning proteins form a transnuclear structure that is responsible for distributing plasmid molecules throughout the nucleus prior to cell division. In this chapter we describe evidence supporting the existence and mode of action of these two plasmid strategies and discuss the extent to which these strategies may be a pervasive facet of the biology of eukaryotic extrachromosomal elements.

Amino Acid Sequence↗
PubMed1986

Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle.

The FLP protein of the Saccharomyces cerevisiae plasmid 2 microns circle catalyzes site-specific recombination between two repeated segments present on the plasmid. In this paper we present results of experiments we performed to define more precisely the features of the FLP recognition target site, which we propose to designate FRT, and to determine the actual recombination crossover point in vivo. We found that essential sequences for the recombination event are limited to an 8-base-pair core sequence and two 13-base-pair repeated units immediately flanking it. This is the region identified as the FLP binding site in vitro and at which FLP protein promotes specific single-strand cleavages (B. J. Andrews, G. A. Proteau, L. G. Beatty, and P. D. Sadowski, Cell 40:795-803, 1985; J. F. Senecoff, R. C. Bruckner, and M. M. Cox, Proc. Natl. Acad. Sci. USA 82:7270-7274, 1985). Mutations within the core domain can be suppressed by the presence of the identical mutation in the chromatid with which it recombines. However, mutations outside the core are not similarly suppressed. We found that strand exchange during FLP recombination occurs most of the time within the core region, proceeding through a heteroduplex intermediate. Finally, we found that most FLP-mediated events are reciprocal exchanges and that FLP-catalyzed gene conversions occur at low frequency. The low level of gene conversion associated with FLP recombination suggests that it proceeds by a breakage-joining reaction and that the two events are concerted.

Base Sequence↗
PubMed1985

Signals for transcription initiation and termination in the Saccharomyces cerevisiae plasmid 2 micron circle.

By S1 nuclease protection experiments and primer extension analysis, we determined precisely the cap and polyadenylation sites of transcripts from the four genes of the yeast 2 micron circle plasmid, as well as those of other plasmid transcripts of unknown function. In addition, we used deletion analysis to identify sequences necessary for polyadenylation in plasmid transcripts. Our results indicate that plasmid genes constitute independent transcription units and that plasmid mRNAs are not derived by extensive processing of precursor transcripts. In addition, we found that the D coding region of 2 micron circle is precisely encompassed by a polyadenylated transcript, suggesting that this coding region constitutes a functional plasmid gene. Our identification of the position of plasmid polyadenylation sites and of sequences necessary for polyadenylation provides support for a tripartite signal for polyadenylation as proposed by Zaret and Sherman (K.S. Zaret and F. Sherman, Cell 28:563-573, 1982). Finally, these data highlight salient features of the transcriptional regulatory circuitry that underlies the control of plasmid maintenance in the cell.

Base Sequence↗
PubMed1985

Properties of REP3: a cis-acting locus required for stable propagation of the Saccharomyces cerevisiae plasmid 2 microns circle.

Stable propagation of the yeast plasmid 2 microns requires an origin of replication, a cis-active locus designated REP3, and two plasmid-encoded proteins which are the products of the REP1 and REP2 genes. The three REP loci appear to constitute a partitioning system, ensuring equal distribution of plasmid molecules to mother and daughter cells after mitosis. We have localized the REP3 site completely within a segment of five-and-one-half direct tandem repeats of a 62-base-pair unit, bordered by HpaI and AvaI restriction sites within the large unique region of the 2 microns genome. In addition, we find that the repeated elements are functionally distinct. Only a subset of the repeats is necessary to promote full partitioning activity. The other repeats appear to promote plasmid transcription. These results are discussed in the context of a model of plasmid copy control involving titration of a plasmid-specific protein by the repeated elements within REP3.

Base Sequence↗
PubMed1984

Identification of sites required for repression of a silent mating type locus in yeast.

There are three loci in the yeast Saccharomyces, each containing one of two possible genetic elements that can determine cell type. At one of these loci, MAT, this information is expressed to establish the mating type of the cell. At the other two loci, HML and HMR, this same information is phenotypically and transcriptionally silent, even though a large amount of identical sequence flanks MAT, HML and HMR coding regions. Transcriptional repression of HML and HMR requires the trans active gene products of four loci, designated variously as MAR or SIR, that are unlinked to each other or to MAT, HML or HMR. We have examined the phenotypic expression of a cloned, plasmid-borne copy of HML and of various deletion and insertion derivatives of this plasmid following their reintroduction into Mar+/Sir+ yeast strains. From these data, we have identified two sites flanking the locus, both of which are required for MAR/SIR repression of the locus. In addition, we demonstrate that each of these sites promotes autonomous replication in yeast. Abraham et al. (1984) have presented evidence demonstrating that a similar regulatory structure exists at the other silent locus, HMR. From an analysis of the sequences of these four regulatory sites, we have identified several specific sequences that may be involved in mediating repression of these loci and in promoting replication in yeast. These results are discussed in the context of potential models for the mechanism of regulation of the silent mating type loci.

Base Sequence↗
PubMed1983

The yeast plasmid 2mu circle encodes components required for its high copy propagation.

The yeast plasmid 2mu and certain hybrid plasmids constructed from it are maintained stably and at high copy number in yeast cells. By examining various mutant hybrid 2mu plasmids, we show that these properties require the integrity of four plasmid loci. Two of these, designated REP1 and REP2, are active in trans and correspond to two open coding regions of 2mu. The other two loci are active only in cis and correspond to the origin of replication and to a region, designated REP3, located several hundred bp away from the origin and consisting of direct repeats of a 62 bp sequence. We propose that the REP loci constitute a copy control system that overrides normal cellular restriction on plasmid replication and amplifies the plasmid when copy number is low.

Base Sequence↗
PubMed1983

Yeast plasmid 2-micron circle promotes recombination within bacterial transposon Tn5.

The site-specific recombination system (FLP) encoded by the yeast plasmid 2-micron circle can also act in yeast on the inverted repeats of the bacterial transposon Tn5. The efficiency of this recombination is dependent on the location of Tn5 within the 2-micron circle genome but can be as high as that observed for 2-micron circle itself. Comparison of the DNA sequences between the Tn5 repeat and the 2-micron circle recombination region reveals certain strikingly similar structural features that might be important in the recombination reaction.

Base Sequence↗
PubMed1982

Recombination within the yeast plasmid 2mu circle is site-specific.

The multicopy yeast plasmid, 2mu circle, encodes a specialized recombination system. It contains two regions, each 599 bp in length, that are precise inverted repeats of each other and between which recombination occurs readily. In addition, this recombination requires the product of a 2mu circle gene, designated FLP. By examining the products of FLP-mediated recombination of plasmids containing single insertions within one of the repeated regions, we show that this recombination occurs only at a specific site within the repeat. This result was confirmed from analysis of the ability of plasmids containing various deletions within one of the repeated regions to serve as substrates for FLP-mediated recombination. These experiments limit the recombination site to a sequence of less than 65 bp. In addition, by mutational analysis of the recombination potential of a hybrid plasmid containing the entire 2mu circle genome, we have shown that FLP is only the 2mu circle gene necessary for this site-specific recombination. Finally, we describe a sensitive assay for recombination between the repeated sequences of 2mu circle; using it, we demonstrate that even in the absence of FLP gene product, recombination between the repeats occurs at a low but detectable level during meiosis.

Base Sequence↗
PubMed1982

Genetic properties of chromosomally integrated 2 mu plasmid DNA in yeast.

We obtained strains of yeast with large segments of 2 mu plasmid DNA integrated at several chromosomal locations by selecting genetically for recombination between a chromosomal sequence carried on a 2 mu-circle-containing hybrid plasmid and a homologous sequence on the chromosome. In all diploids examined, the presence of 2 mu circle sequences causes a marked instability of the chromosome into which the 2 mu DNA is inserted. Although in some cases the loss of genetic markers is due to physical loss of the entire chromosome, in most cases the loss of markers appears to be due to a mitotic homozygotization of markers: the allelic information from the homologous chromosome replaces the information distal to the integrated 2 mu DNA. The instability caused by integrated 2 mu DNA sequences requires the activity of the specialized site-specific recombination system encoded by the 2 mu plasmid. We propose that the presence of integrated 2 mu DNA allows efficient integration of additional copies of the intact 2 mu plasmid by the action of the plasmid-coded special recombination system. Unequal sister-strand exchanges within the inverted repetition would result in the formation of dicentric chromosomes whose breakage during mitosis might begin a cycle analogous to the breakage-fusion-bridge cycle described many years ago in maize.

Base Sequence↗