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D Botstein

Publications and source records attributed to D Botstein.

At least 217 records · Page 12Linked to original sources
PubMed1982

Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase.

The SUC2 gene of yeast (Saccharomyces) encodes two forms of invertase: a secreted, glycosylated form, the synthesis of which is regulated by glucose repression, and an intracellular, nonglycosylated enzyme that is produced constitutively. The SUC2 gene has been cloned and shown to encode two RNAs (1.8 and 1.9 kb) that differ at their 5' ends. The stable level of the larger RNA is regulated by glucose; the level of the smaller RNA is not. A correspondence between the presence of the 1.9 kb RNA and the secreted invertase, and between the 1.8 kb RNA and the intracellular invertase, was observed in glucose-repressed and -derepressed wild-type cells. In addition, cells carrying a mutation at the SNF1 locus fail to derepress synthesis of the secreted invertase and also fail to produce stable 1.9 kb RNA during growth in low glucose. Glucose regulation of invertase synthesis thus is exerted, at least in part, at the RNA level. A naturally silent allele (suc2 degrees) of the SUC2 locus that does not direct the synthesis of active invertase was found to produce both the 1.8 and 1.9 kb RNAs under normal regulation by glucose. A model is proposed to account for the synthesis and regulation of the two forms of invertase: the larger, regulated mRNA contains the initiation codon for the signal sequence required for synthesis of the secreted, glycosylated form of invertase; the smaller, constitutively transcribed mRNA begins within the coding region of the signal sequence, resulting in synthesis of the intracellular enzyme.

Cell Compartmentation↗
PubMed1982

Single-copy sequence hybridizes to polymorphic and homologous loci on human X and Y chromosomes.

Use of a 4.5-kilobase-pair (kb) segment of single-copy DNA from a human genomic library as a hybridization probe of genomic human DNAs revealed allelic Taq I restriction fragments 10.6, 11.8, and 14.6 kb long. Among 12 unrelated individuals, all 6 males exhibited the 14.6-kb fragment in addition to one of the other fragments. Three of the females displayed 10.6- and 11.8-kb fragments, and the other three displayed only one fragment length; none had the 14.6-kb fragment. Hybridization of this probe to Taq I-digested DNAs from human-rodent hybrid cell lines (which have partial complements of human chromosomes) demonstrated segregation of the 14.6-kb fragment with the human Y chromosome and segregation of the 10.6- and 11.8-kb fragments with the human X chromosome. Furthermore, hybridization of this probe to Taq I-digested DNAs from 48 members of a single kindred revealed Y-linked inheritance of the 14.6-kb fragment and X-linked inheritance of the 10.6- and 11.8-kb fragments. These experiments demonstrate homology between single-copy sequences on the human X and Y chromosomes.

Alleles↗
PubMed1982

Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro.

Short single-stranded gaps can be constructed by limited exonuclease action at single-stranded breaks (nicks) placed at predetermined sites on closed circular DNA molecules. As efficient primer-templates for DNA polymerase, single-stranded gaps can be repaired in vitro to regenerate an intact DNA duplex. In this report two in vitro reaction schemes are described that produce a high frequency of errors during repair ("misrepair") of gaps and thereby allow the efficient recovery of mutations limited to the nucleotide sequence at or near the original gap. In the first of these misrepair schemes, nucleotide misincorporations are stimulated by omission of one of the four deoxynucleoside triphosphates; the misincorporations are trapped by the presence of excess DNA ligase in the reaction mixture. The second misrepair scheme involves the misincorporation of an excision-resistant alpha-thiophosphate nucleotide, followed by gap filling in the presence of all four conventional deoxynucleoside triphosphates. When applied to short gaps constructed at one of several unique restriction sites on the small plasmid pBR322, both gap misrepair methods yielded mutations within the targeted restriction site at high frequency (6--42%). A majority of the sequence changes identified were base substitutions; transversions and transitions are approximately equally represented. The remaining sequence changes were an insertion of a single base pair and deletions of one to four base pairs.

DNA Polymerase I↗
PubMed1982

Cold-sensitive cell-division-cycle mutants of yeast: isolation, properties, and pseudoreversion studies.

We isolated 18 independent recessive cold-sensitive cell-division-cycle (cdc) mutants of Saccharomyces cerevisiae, in nine complementation groups. Terminal phenotypes exhibited include medial nuclear division, cytokinesis, and a previously undescribed terminal phenotype consisting of cells with a single small bud and an undivided nucleus. Four of the cold-sensitive mutants proved to be alleles of CDC11, while the remaining mutants defined at least six new cell-division-cycle genes: CDC44, CDC45, CDC48, CDC49, CDC50 and CDC51.--Spontaneous revertants from cold-sensitivity of four of the medial nuclear division cs cdc mutants were screened for simultaneous acquisition of a temperature-sensitive phenotype. The temperature-sensitive revertants of four different cs cdc mutants carried single new mutations, called Sup/Ts to denote their dual phenotype: suppression of the cold-sensitivity and concomitant conditional lethality at 37 degrees. Many of the Sup/Ts mutations exhibited a cell-division-cycle terminal phenotype at the high temperature, and they defined two new cdc genes (CDC46 and CDC47). Two cold-sensitive medial nuclear division cdc mutants representing two different cdc genes were suppressed by different Sup/Ts alleles of another gene which also bears a medial nuclear division function (CDC46). In addition, the cold-sensitive medial nuclear division cdc mutant csH80 was suppressed by a Sup/Ts mutation yielding an unbudded terminal phenotype with an undivided nucleus at the high temperature. This mutation was an allele of CDC32. These results suggest a pattern of interaction among cdc gene products and indicate that cdc gene proteins might act in the cell cycle as complex specific functional assemblies.

Cell Cycle↗
PubMed1982

Determination of the order of gene function in the yeast nuclear division pathway using cs and ts mutants.

Cold-sensitive (cs) and heat-sensitive (ts) conditional-lethal mutations that affect specifically the cell division cycle of budding yeast (Saccharomyces cerevisiae) were used to determine the order of gene function. Reciprocal temperature-shift experiments using cs-ts double mutants revealed a detailed order of function among genes whose execution points and mutant phenotypes are very similar. The data suggest that the nuclear branch of the overall cell-cycle pathway itself contains at least one branch.

Cell Cycle↗
PubMed1982

Genetic mapping of mutations in a highly radiation-resistant mutant of Salmonella typhimurium LT2.

The genes involved in the high radiation resistance of mutant R68 of Salmonella typhimurium LT2 were mapped by conjugation. It was observed that the high radiation resistance involved genes localized in two regions of the chromosome, which have been designated as garA and garB for high gamma resistance. The garA gene mapped near gal and uvrB at about 18 map units, and the garB gene mapped near purC at about 49 map units. The resistance of R68 was reduced to the wild-type level by the acquisition of the two wild-type alleles, garA+ and garB+. Recombinants carrying the garA or garB gene repaired single-strand breaks in their DNA faster than did the wild-type strain. However, only those with the garA mutation showed a marked increase in UV irradiation resistance above the wild-type level, whereas those with garB mutation exhibited an increased rate of spontaneous degradation of DNA beyond the level observed in recA cells.

Chromosome Mapping↗
PubMed1982

Regulation of HIS4-lacZ fusions in Saccharomyces cerevisiae.

The beginning of the Saccharomyces cerevisiae HIS4 gene has been fused to the structural gene for Escherichia coli beta-galactosidase. This construction, which contains HIS4 DNA from -732 to +30 relative to the translation initiation codon, has been integrated into the yeast genome at two chromosomal locations, HIS4 and URA3. At both locations, this 762-base-pair stretch of DNA is sufficient for initiating expression of beta-galactosidase activity in S. cerevisiae and confers upon this activity the regulatory response normally found for HIS4.

Base Sequence↗
PubMed1981

Yeast genes fused to beta-galactosidase in Escherichia coli can be expressed normally in yeast.

A plasmid was constructed that allows the selection in vivo of gene fusions between the Escherichia coli beta-galactosidase gene and the yeast (Saccharomyces cerevisiae) URA3 gene. A large yeast DNA fragment containing the URA3 gene was placed upstream of an amino-terminally deleted version of the lacZ gene. The plasmid vehicle contains sequences that allow selection and maintenance of the plasmid in both yeast and E. coli. Selection for Lac+ in E. coli yielded numerous deletions that fused the lacZ gene to the URA3 gene and flanking yeast sequences, to the bacterial tetracycline-resistance gene from the parent plasmid pBR322, and to the yeast 2-micrometer plasmid DNA. Some of these fusion plasmids produced beta-galactosidase activity when introduced into yeast. One of the fusions to the URA3 gene itself has been shown to place the expression of beta-galactosidase activity under uracil regulation in yeasts.

Escherichia coli↗
PubMed1981

Mutants of yeast defective in sucrose utilization.

Utilization of sucrose as a source of carbon and energy in yeast (Saccharomyces) is controlled by the classical SUC genes, which confer the ability to produce the sucrose-degrading enzyme invertase (Mortimer and Hawthorne 1969). Mutants of S. cerevisiae strain S288C (SUC2+) unable to grow anaerobically on sucrose, but still able to use glucose, were isolated. Two major complementation groups were identified: twenty-four recessive mutations at the SUC2 locus (suc2-); and five recessive mutations defining a new locus, SNF1 (for sucrose nonfermenting), essential for sucrose utilization. Two minor complementation groups, each comprising a single member with a leaky sucrose-nonfermenting phenotype, were also identified. The Suc2 mutations isolated include four suppressible amber mutations and five mutations apparently exhibiting intragenic complementation; complementation analysis and mitotic mapping studies indicated that all of the suc2 mutations are alleles of a single gene. These results suggest that SUC2 encodes a protein, probably a dimer or multimer. No invertase activity was detected in suc2 probably a dimer or multimer. No invertase activity was detected in suc2 mutants,--The SNF1 locus is not tightly linked to SUC2. The snf1 mutations were found to be pleiotropic, preventing sucrose utilization by SUC2+ and SUC7+ strains, and also preventing utilization of galactose, maltose and several nonfermentable carbon sources. Although snf1 mutants thus display a petite phenotype, classic petite mutations do not interfere with utilization of sucrose, galactose or maltose. A common feature of all the carbon utilization systems affected by SNF1 is that all are regulated by glucose repression. The snf1 mutants were found to produce the constitutive nonglycosylated form of invertase, but failed to produce the glucose-repressible, glycosylated, secreted invertase. This failure cannot be attributed to a general defect in production of glycosylated and secreted proteins because synthesis of acid phosphatase, a glycosylated secreted protein not subject to glucose repression, was not affected by snf1 mutations. These findings suggest that the SNF1 locus is involved in the regulation of gene expression by glucose repression.

Genetic Code↗
PubMed1981

Genetic evidence for a silent SUC gene in yeast.

The SUC genes (SUC1-SUC7) of Saccharomyces are a family of genes that are dispersed in the yeast genome. A SUC+ allele at any locus confers the ability to produce the enzyme invertase and, thus, to ferment sucrose. Most yeast strains do not carry SUC+ alleles at all possible SUC loci. We have investigated the naturally occurring negative (suc0) alleles present at SUC loci with the aim of distinguishing between two possible models for the structure of suc0 alleles: (1) suc0 alleles correspond to a simple absence of SUC genetic information; (2) suc0 alleles are "silent" SUC genes that either produce a defective product or are not expressed. To facilitate these studies, sucrose-nonfermenting strains were constructed that are congenic to S. cerevisiae strain S288C (SUC2+), but carry at the SUC2 locus the naturally occurring negative allele, suc2(0), of strain FL100 (Lacroute 1968). These strains were used to study the genetic properties of the suc2(0) allele of FL100 and the suc0 alleles (suc1(0), suc3(0), etc.) of S288C. The suc2(0) allele was shown to revert to an active Suc+ state and to provide functional information at three points in the SUC2 gene in recombination experiments; this suc2(0) gene thus appears to be a "silent" gene. Similar tests for silent SUC genes in S288C (corresponding to loci other than SUC2) failed to reveal any additional silent genes.

Alleles↗
PubMed1980

Secretion of beta-lactamase requires the carboxy end of the protein.

Synthesis and secretion of beta-lactamase were studied in Salmonella typhimurium infected with P22 phage carrying the structural gene for beta-lactamase (the bla gene) in mutant or wild-type form. The wild-type gene was shown to specify two forms of beta-lactamase which differ in molecular weight by about 2500 daltons. This difference is consistent with removal, predicted on other grounds, of 23 amino-terminal residues (the "signal" sequence). All bla- mutants, including chain-terminating mutants lacking as much as 50% or as little as 10% of the protein, were apparently unaffected in this processing step. Pulse-chase experiments showed that more than 85% of the wild-type (as well as mutant) proteins are synthesized as complete overlength precursors before being processed to their mature forms. Virtually all the mature wild-type protein appears in the periplasmic space whereas a large fraction of the precursor appears in the cytoplasm. In contrast, both the precursor and processed forms of beta-lactamase proteins synthesized by chain-terminating mutants (including one which lacks only 10% of its residues from the carboxy end) are not secreted and apparently remain soluble in the cytoplasm. These results show that the carboxy-terminal amino acid sequence (at least) of beta-lactamase is essential to successful transport across the cytoplasmic membrane, and suggest that the presence (and probably also the act of removal) of the signal sequence does not suffice to ensure secretion.

Amino Acid Sequence↗