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Biomedical subjects

D Botstein

Publications and source records attributed to D Botstein.

At least 181 records · Page 10Linked to original sources
PubMed1986

A rapid, efficient method for isolating DNA from yeast.

A method is described for the purification of chromosomal and plasmid DNA from the yeast Saccharomyces cerevisiae. This method is rapid, gives 75% of theoretical yield, and produces DNA that can be cut with restriction endonucleases. Yeast cells are treated with zymolyase, and the resulting spheroplasts are lysed in the presence of the chaotropic agent guanidine hydrochloride. After a brief ethanol precipitation, protein is removed by treatment with proteinase K followed by phenol-chloroform extraction. After ethanol precipitation, the DNA is sufficiently pure for restriction analysis or for the transformation of Escherichia coli.

Chromosomes↗
PubMed1986

Strategies for studying heterogeneous genetic traits in humans by using a linkage map of restriction fragment length polymorphisms.

Simple single-gene disorders in humans can be genetically mapped by using traditional methods of linkage analysis and increasingly abundant restriction fragment length polymorphisms (RFLPs). Many human diseases and traits, however, can be expected to be genetically heterogeneous (i.e., caused by any one of several genes), and traditional linkage analysis is much less effective in such circumstances. We present two methods, interval mapping and simultaneous search, designed to exploit the full power of a linkage map of the DNA markers. For the simplest situations, only 1/3 as many affected families are needed to map a heterogeneous trait by using these methods. Only 1/5-1/50 as many are needed to detect that genetic heterogeneity is present.

Chromosome Mapping↗
PubMed1986

Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins.

Two alpha-tubulin genes from the budding yeast Saccharomyces cerevisiae were identified and cloned by cross-species DNA homology. Nucleotide sequencing studies revealed that the two genes, named TUB1 and TUB3, encoded gene products of 447 and 445 amino acids, respectively, that are highly homologous to alpha-tubulins from other species. Comparison of the sequences of the two genes revealed a 19% divergence between the nucleotide sequences and a 10% divergence between the amino acid sequences. Each gene had a single intervening sequence, located at an identical position in codon 9. Cell fractionation studies showed that both gene products were present in yeast microtubules. These two genes, along with the TUB2 beta-tubulin gene, probably encode the entire complement of tubulin in budding yeast cells.

Amino Acid Sequence↗
PubMed1986

Genetically essential and nonessential alpha-tubulin genes specify functionally interchangeable proteins.

Microtubules in yeast are essential components of the mitotic and meiotic spindles and are essential for nuclear movement during cell division and mating. The relative importance in these processes of the two divergent alpha-tubulin genes of the budding yeast Saccharomyces cerevisiae, TUB1 and TUB3, was examined through the construction of null mutations and by increasing their copy number on chromosomes and on plasmids. Experiments with null alleles of TUB3 showed that TUB3 was not essential for mitosis, meiosis, or mating. Null alleles of TUB3, however, did cause several phenotypes, including hypersensitivity to the antimicrotubule drug benomyl and poor spore viability. On the other hand, the TUB1 gene was essential for growth of normal haploid cells. Even in diploids heterozygous for a TUB1 null allele, several dominant phenotypes were evident, including slow growth and poor sporulation. This functional difference between the two genes is apparently due to different levels of expression, because extra copies of either gene could suppress the defects caused by a null mutation in the other. We conclude that in spite of the 10% divergence between the products of the two genes, there is no essential qualitative functional difference between them.

Alleles↗
PubMed1986

Effects of null mutations in the hexokinase genes of Saccharomyces cerevisiae on catabolite repression.

Saccharomyces cerevisiae has two homologous hexokinases, I and II; they are 78% identical at the amino acid level. Either enzyme allows yeast cells to ferment fructose. Mutant strains without any hexokinase can still grow on glucose by using a third enzyme, glucokinase. Hexokinase II has been implicated in the control of catabolite repression in yeasts. We constructed null mutations in both hexokinase genes, HXK1 and HXK2, and studied their effect on the fermentation of fructose and on catabolite repression of three different genes in yeasts: SUC2, CYC1, and GAL10. The results indicate that hxk1 or hxk2 single null mutants can ferment fructose but that hxk1 hxk2 double mutants cannot. The hxk2 single mutant, as well as the double mutant, failed to show catabolite repression in all three systems, while the hxk1 null mutation had little or no effect on catabolite repression.

Enzyme Repression↗
PubMed1986

Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase.

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

Alleles↗
PubMed1986

A deletion map of the human Y chromosome based on DNA hybridization.

The genomes of 27 individuals (19 XX males, two XX hermaphrodites, and six persons with microscopically detectable anomalies of the Y chromosome) were analyzed by hybridization for the presence or absence of 23 Y-specific DNA restriction fragments. Y-specific DNA was detected in 12 of the XX males and in all six individuals with microscopic anomalies. The results are consistent with each of these individuals carrying a single contiguous portion of the Y chromosome; that is, the results suggest a deletion map of the Y chromosome, in which each of the 23 Y-specific restriction fragments tested can be assigned to one of seven intervals. We have established the polarity of this map with respect to the long and short arms of the Y chromosome. On the short arm, there is a large cluster of sequences homologous to the X chromosome. The testis determinant(s) map to one of the intervals on the short arm.

Chromosome Banding↗
PubMed1985

Transformation of yeast with linearized plasmid DNA. Formation of inverted dimers and recombinant plasmid products.

The molecular products of DNA double strand break repair were investigated after transformation of yeast (Saccharomyces cerevisiae) with linearized plasmid DNA. DNA of an autonomous yeast plasmid cleaved to generate free ends lacking homology with the yeast genome, when used in transformation along with sonicated non-homologous carrier DNA, gave rise to transformants with high frequency. Most of these transformants were found to harbor a head-to-head (inverted) dimer of the linearized plasmid. This outcome of transformation contrasts with that observed when the carrier DNA is not present. Transformants occur at a much reduced frequency and harbor either the parent plasmid or a plasmid with deletion at the site of the cleavage. When the linearized plasmid is introduced along with sonicated carrier DNA and a homologous DNA restriction fragment that spans the site of plasmid cleavage, homologous recombination restores the plasmid to its original circular form. Inverted dimer plasmids are not detected. This relationship between homologous recombination and a novel DNA transaction that yields rearrangement could be important to the cell, as the latter could lead to a loss of gene function and lethality.

DNA Repair↗
PubMed1985

Phenotypic analysis of temperature-sensitive yeast actin mutants.

The consequences of two different mutations in the single essential actin structural gene of yeast (Saccharomyces cerevisiae) were studied. Both conditional-lethal actin mutants exhibit six phenotypes at the restrictive temperature: disruption of the asymmetric staining pattern of actin assembly; delocalized deposition of chitin on the cell surface; partial inhibition of secretion of the periplasmic protein, invertase; an intracellular accumulation of secretory vesicles; death of cells in the budded portion of the cell cycle upon prolonged incubation at the restrictive condition; and osmotic sensitivity. These results implicate actin in the organization and polarized growth of the yeast cell surface.

Actins↗
PubMed1985

DNA topoisomerase II is required at the time of mitosis in yeast.

We have constructed five new temperature-sensitive DNA topoisomerase II mutations and have analyzed their physiological consequences in yeast. Several lines of evidence suggest that the activity of topoisomerase II is required specifically at the time of miosis. First, top2 mutations cause dramatic lethality at the restrictive temperature, but only if the mutant cells are actively traversing the cell cycle. Second, temperature-shift experiments with synchronized cultures show that the onset of inviability coincides with the time of mitosis. Third, fluorescence microscopy reveals that the normal progression of mitosis is disturbed in mutant cells at the restrictive temperature. Finally, inviability at the restrictive temperature is prevented by nocodazole, an inhibitor of tubulin polymerization that prevents formation of the mitotic spindle. These results are consistent with the hypothesis that the essential function of topoisomerase II is to allow the separation of intertwined chromosomal DNA molecules during mitosis.

Benzimidazoles↗
PubMed1985

Propagation of some human DNA sequences in bacteriophage lambda vectors requires mutant Escherichia coli hosts.

The growth of clones of human genomic DNA fragments in a bacteriophage lambda vector has been examined in a number of different Escherichia coli hosts. A large proportion (8.9%) of the phages carrying different fragments of the human genome fail to grow on standard rec+ hosts but will grow on hosts carrying mutations in the recB, recC, and sbcB genes. Heteroduplex analysis in the electron microscope of DNA from four of these phages revealed substantial secondary structure, including snap-back regions 200-500 base pairs in length. Such structures were not found in phages from the same DNA library that grow in rec+ hosts. These results are interpreted in the light of prior observations [Leach, D.R.F. & Stahl, F. (1983) Nature (London) 305, 448-451] showing that inverted repetitions cloned in phage lambda can be propagated in recB recC sbcB hosts but not in rec+ hosts.

Bacteriophage lambda↗
PubMed1985

Isolation and characterization of mutations in the beta-tubulin gene of Saccharomyces cerevisiae.

Of 173 mutants of Saccharomyces cerevisiae resistant to the antimitotic drug benomyl (BenR), six also conferred cold-sensitivity for growth and three others conferred temperature-sensitivity for growth in the absence of benomyl. All of the benR mutations tested, including the nine conditional-lethal mutations, were shown to be in the same gene. This gene, TUB2, has previously been molecularly cloned and identified as the yeast structural gene encoding beta-tubulin. Four of the conditional-lethal alleles of TUB2 were mapped to particular restriction fragments within the gene. One of these mutations was cloned and sequenced, revealing a single amino acid change, from arginine to histidine at amino acid position 241, which is responsible for both the BenR and the cold-sensitive lethal phenotypes. The terminal arrest morphology of conditional-lethal alleles of TUB2 at their restrictive temperature showed a characteristic cell-division-cycle defect, suggesting a requirement for tubulin function primarily in mitosis during the vegetative growth cycle. The TUB2 gene was genetically mapped to the distal left arm of chromosome VI, very near the actin gene, ACT1; no CDC (cell-division-cycle) loci have been mapped previously to this location. TUB2 is thus the first cell-division-cycle gene known to encode a cytoskeletal protein that has been identified in S. cerevisiae.

Alleles↗
PubMed1984

Catabolic alanine racemase from Salmonella typhimurium: DNA sequence, enzyme purification, and characterization.

The alanine racemase encoded by the Salmonella typhimurium dadB gene was purified to 90% homogeneity from an overproducing strain. At 37 degrees C the enzyme has a specific activity of 1400 units/mg (V max, L- to D-alanine). Active enzyme molecules are monomers of Mr 39 000 with one molecule of pyridoxal 5'-phosphate bound per subunit. The Km's for L- and D-alanine are 8.2 and 2.1 mM, respectively. Measurement of turnover numbers yielded the expected Keq value of 1.0. Determination of 22 of the 25 N-terminal amino acid residues of the purified polypeptide allowed localization of cloned DNA encoding the structural gene. Sequencing of subcloned DNA revealed that the dadB gene encodes a polypeptide of 356 amino acids whose calculated molecular weight (apoenzyme) was 39 044.

Alanine Racemase↗
PubMed1984

Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene.

Two temperature-sensitive mutations have been constructed in the single actin gene (ACT1) of the yeast Saccharomyces cerevisiae by in vitro mutagenesis of the cloned gene followed by integrative transformation of mutagenized DNA into yeast cells. A strategy of allele replacement was used that allowed recessive mutations to be phenotypically expressed in the initial transformants, thus simplifying the screening of large numbers of independently transformed cells. After confirming that several ts mutations were located within the actin structural gene by genetic methods, these mutant alleles were cloned, and the altered amino acid residues were defined by DNA sequence analysis. The two unique mutations resulted in substitution of proline-32 with leucine and alanine-58 with threonine. In the course of isolating these mutations, the observation was made that a high proportion of yeast cells transformed with exogenous DNA by the spheroplast method are temperature sensitive for growth because of genetic changes unrelated to the transforming DNA.

Actins↗