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D Botstein

Publications and source records attributed to D Botstein.

At least 199 records · Page 11Linked to original sources
PubMed1984

Functional interchangeability of DNA replication genes in Salmonella typhimurium and Escherichia coli demonstrated by a general complementation procedure.

Twenty-four genes from Salmonella typhimurium that affect DNA replication were isolated from a lambda-Salmonella genomic library by lysogenic complementation of temperature-sensitive mutants of Salmonella or E. coli, using a new plaque complementation assay. The complementing lambda clones, which make red plaques in this assay, and noncomplementing mutant derivatives, which make uncolored plaques, were used to further characterize the temperature-sensitive Salmonella mutants and to establish the functional similarity of E. coli and Salmonella DNA replication genes. For 17 of 18 E. coli mutants representing distinct loci, a Salmonella gene that complemented the mutant was found. This result indicates that single Salmonella replication proteins are able to function in otherwise all E. coli replication complexes and suggests that the detailed properties of Salmonella and E. coli replication proteins are very similar. The other seven Salmonella genes that were cloned were unrelated functionally to any E. coli genes examined. --As an aid to the derivation of chromosomal mutations affecting some of the cloned genes, a general method was developed for placing a transposon in the Salmonella chromosome in a segment corresponding to cloned DNA. Chromosomal mutations were derived in Salmonella affecting a gene (dnaA) that was cloned by complementation of an E. coli mutant by using the transposon-encoded drug resistance as a selectable marker in local mutagenesis.

Alleles↗
PubMed1984

Genetic analysis of DNA replication in bacteria: dnaB mutations that suppress dnaC mutations and dnaQ mutations that suppress dnaE mutations in Salmonella typhimurium.

We have isolated and characterized extragenic suppressors of mutations in two different target genes that affect DNA replication in Salmonella typhimurium. Both the target and the suppressor genes are functional homologues of known replication genes of E. coli that were identified in intergeneric complementation tests. Our results point to interactions in vivo involving the dnaB and dnaC proteins in one case and the dnaQ and dnaE proteins in the other case. The suppressor mutations, which were isolated as derivatives of lambda-Salmonella in vitro recombinants, were detected by an adaptation of the red plaque complementation assay. This method was applicable even when the locus of suppressor mutations was not chosen in advance.

Alleles↗
PubMed1984

Formation of inverted dimer plasmids after transformation of yeast with linearized plasmid DNA.

The formation of an inverted dimer plasmid on transformation with linear molecules is formally analogous to the fusion of the daughters of a broken chromosome at their broken ends. In the latter case, this leads to the formation of a dicentric chromosome, which could break at anaphase. Hence the process is cyclic. Similarly, when our linear molecules are modified by the addition of a cloned yeast centromere, dicentric inverted dimers are not obtained. Instead, we obtain monocentric plasmids with partial duplication and deletion that apparently derive from a process of fusion, bridge-breakage, and fusion. This is not surprising, since it is known that dicentric plasmids undergo breakage in yeast (Mann and Davis 1983). However, any apparent similarity of this process to that which occurs with a broken chromosome in maize must be tempered by the special nature of the transformation process. Most significantly, inverted dimers are rare when sonicated carrier DNA is not present during the transformation. This requirement is not understood, but it is a condition that may not be met in a yeast cell harboring a broken chromosome. It is possible that carrier DNA induces a repair process that results in fusion. On the other hand, a property of the transformation process that results in an inhibition of fusion may be overcome by the presence of carrier DNA. Most inverted dimers are apparently formed from an interaction between two input linear molecules. We cannot rule out the possibility that a minor fraction derive from a single molecule. Thus, the fusion of two input molecules is a much more efficient process than a replicative process that could occur with single linear molecule. For a similar fusion process to occur with a broken yeast chromosome, replication would be required. We do not know if a broken yeast chromosome can replicate. Evidence consistent with the presence of a breakage-fusion-bridge process in yeast has been obtained through the formation of dicentric chromosomes via meiotic recombination (Haber et al. 1984). Spores from these meioses sometimes give rise to a clone that is mixed for markers of the chromosome that could have been dicentric. A process of fusion-bridge-breakage could account for the formation of some of these mixed clones. However, the dicentric chromosomes apparently often survive meiotic disjunction and break in the spore's first mitotic anaphase or possibly in a later generation. Thus, the interpretation of the origin of these mixed clones is uncertain. Some aspects of the fusion process are especially intriguing.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromosome Mapping↗
PubMed1984

A molecular approach to defining the inherited components in epilepsy and other diseases of uncertain etiology.

A general method has been developed which, in theory, will make it possible to follow the inheritance of virtually all genes in human families. This method, based on genetic linkage, envisions the use of cloned single copy human deoxyribonucleic acid probes to reveal restriction fragment length polymorphisms as genetic markers. Such markers can be assembled into a linkage map which can be applied to analysis of inherited diseases. It is speculated that such a map might help to clarify the role that heredity plays in the etiology of epilepsy.

Alleles↗
PubMed1983

Structure and function of the yeast URA3 gene. Differentially regulated expression of hybrid beta-galactosidase from overlapping coding sequences in yeast.

Expression of the URA3 gene of Saccharomyces cerevisiae was studied by analysis of URA3-lacZ gene fusions constructed in vitro. Synthesis of hybrid beta-galactosidase by fusions in frame with the coding sequence for orotidine-5'-phosphate decarboxylase (OMPdecarboxylase) was found to be normally regulated even when only 11 nucleotides of URA3 coding sequence remained, indicating that all transcription initiation and regulatory sites are present at the beginning of the URA3 gene. An upstream initiator codon that begins a short overlapping coding sequence in another reading frame was also found to be active in producing hybrid beta-galactosidase. However this beta-galactosidase synthesis showed little or no regulation. Nuclease protection experiments revealed numerous species of URA3 mRNA. The regulation of these is consistent with the idea that the URA3 protein and the overlapping peptide are translated from differentially regulated mRNAs of different lengths.

Base Sequence↗
PubMed1983

Isolation of the beta-tubulin gene from yeast and demonstration of its essential function in vivo.

A DNA fragment from yeast (Saccharomyces cerevisiae) was identified by its homology to a chicken beta-tubulin cDNA and cloned. The fragment was shown to be unique in the yeast genome and to contain the gene for yeast beta-tubulin, since it can complement a benomyl-resistant conditional-lethal mutation. A smaller subfragment, when used to direct integration of a plasmid to the benomyl resistance locus in a diploid cell, disrupted one of the beta-tubulin genes and concomitantly created a recessive lethal mutation, indicating that the single beta-tubulin gene of yeast has an essential function. Determination of the nucleotide sequence reveals extensive amino acid sequence homology (more than 70%) between yeast and chicken brain beta-tubulins.

Amino Acid Sequence↗
PubMed1983

A rapid chromosome-mapping method for cloned fragments of yeast DNA.

A rapid and generally applicable method is described for mapping a cloned yeast DNA segment to the chromosome(s) from which it originated. The method is based upon the recent finding that the integration into a yeast chromosome of a segment of the 2 mu plasmid DNA results, in heterozygous diploids, in the specific loss of genetic information from the chromosome into which the 2 mu DNA was integrated (Falco et al. 1982). After verification of the accuracy of the method using several genes whose position was known in advance, the method was used to locate the yeast actin gene, which lies on the left arm of chromosome VI, about 50 cM distal to CDC4.

Actins↗
PubMed1983

Two alanine racemase genes in Salmonella typhimurium that differ in structure and function.

Mutations were isolated in a previously undescribed Salmonella typhimurium gene encoding an alanine racemase essential for utilization of L-alanine as a source of carbon, energy, and nitrogen. This new locus, designated dadB, lies within one kilobase of the D-alanine dehydrogenase locus (dadA), which is also required for alanine catabolism. The dadA and dadB genes are coregulated. Mutants (including insertions) lacking the dadB alanine racemase do not require D-alanine for growth unless a mutation is introduced at a second locus, designated dal. Two genes specifying alanine racemase activity were cloned from S. typhimurium. The two cloned DNA sequences do not cross-hybridize with each other; one was shown to contain the dadB gene.

Alanine Racemase↗
PubMed1983

Organization of the SUC gene family in Saccharomyces.

The SUC gene family of yeast (Saccharomyces) includes six structural genes for invertase (SUC1 through SUC5 and SUC7) found at unlinked chromosomal loci. A given yeast strain does not usually carry SUC+ alleles at all six loci; the natural negative alleles are called suc0 alleles. Cloned SUC2 DNA probes were used to investigate the physical structure of the SUC gene family in laboratory strains, commercial wine strains, and different Saccharomyces species. The active SUC+ genes are homologous. The suc0 allele at the SUC2 locus (suc2(0) in some strains is a silent gene or pseudogene. Other SUC loci carrying suc0 alleles appear to lack SUC DNA sequences. These findings imply that SUC genes have transposed to different chromosomal locations in closely related Saccharomyces strains.

Alleles↗
PubMed1983

The secreted form of invertase in Saccharomyces cerevisiae is synthesized from mRNA encoding a signal sequence.

The SUC2 gene of Saccharomyces cerevisiae encodes two differently regulated mRNAs (1.8 and 1.9 kilobases) that differ at their 5' ends. The larger RNA encodes a secreted, glycosylated form of invertase and the smaller RNA encodes an intracellular, nonglycosylated form. We have determined the nucleotide sequence of the amino-terminal coding region of the SUC2 gene and its upstream flanking region and have mapped the 5' ends of the SUC2 mRNAs relative to the DNA sequence. The 1.9-kilobase RNA contains a signal peptide coding sequence and presumably encodes a precursor to secreted invertase. The 1.8-kilobase RNA does not include the complete coding sequence for the signal peptide. The nucleotide sequence data prove that SUC2 is a structural gene for invertase, and translation of the coding information provides the complete amino acid sequence of an S. cerevisiae signal peptide.

Amino Acid Sequence↗
PubMed1982

Lethal disruption of the yeast actin gene by integrative DNA transformation.

A mutant allele of the chromosomal locus corresponding to the cloned actin gene of the yeast Saccharomyces cerevisiae has been constructed by DNA transformation with a hybrid plasmid which integrates into, and thereby disrupts, the protein-encoding sequences of the gene. In a diploid strain of yeast, disruption of the actin gene on one chromosome results in a mutation that segregates as a recessive lethal tightly linked to a selectable genetic marker on the integrated plasmid. The actin gene, therefore, must encode an essential function for yeast cell growth.

Actins↗
PubMed1982

Single-stranded gaps as localized targets for in vitro mutagenesis.

Short single-stranded gaps in circular DNA molecules can be generated enzymatically, often at predetermined sites. These can serve as targets for in vitro mutagenesis procedures that result in alterations in nucleotide sequence within or very near the gap. Deamination of unpaired cytosine residues with sodium bisulfite has been used to induce mutations in the BglI restriction site of SV40 DNA and within defined regions of the beta-lactamase gene on pBR322. A new method of induction of mutations at gaps, called "gap misrepair," has been developed; it was used to cause changes at the HindIII and C1aI restriction sites on pBR322 DNA. Gap misrepair reactions using DNA polymerase I of Micrococcus luteus in the presence of T4 DNA ligase and three of the four deoxynucleoside triphosphates yielded all three possible substitutions for adenine and cytosine residues in the DNA.

Chemical Phenomena↗
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↗
PubMed1982

Evidence for posttranslational translocation of beta-lactamase across the bacterial inner membrane.

Secretion of beta-lactamase was studied in Salmonella typhimurium infected with P22 phage carrying wild-type and mutant alleles of the structural gene. Cellular location of precursor and mature products of wild-type and temperature-sensitive and chain-terminating mutants was analyzed by cell fractionation and by trypsin accessibility in intact and lysed spheroplasts. The precursors of wild-type and all these mutants (none of which alter the signal peptide) are found sequestered within the cell, while all the mature forms have at least partially been translocated across the inner membrane. Thus most beta-lactamase molecules traverse the membrane after completion of their translation. It seems that the carboxyl terminus of beta-lactamase is not required for translocation across the inner membrane but is required for the protein to appear in the periplasm as a soluble species.

Amino Acid Sequence↗
PubMed1982

Diverse effects of mutations in the signal sequence on the secretion of beta-lactamase in Salmonella typhimurium.

Mutations in the beta-lactamase structural gene that alter the signal peptide were used to study secretion into the periplasm of Salmonella typhimurium. Processing and cellular location of mutant gene products were followed by pulse-chase and cell-fractionation experiments and by trypsin accessibility in intact and lysed spheroplasts. The precursor proteins examined never appear as a free species in the periplasm. Two of the signal-sequence mutants accumulate a precursor form that is trypsin-accessible in intact spheroplasts; the precursors synthesized by the remaining mutants resemble wild-type in that they remain trypsin-inaccessible. One of the latter mutants does produce mature protein, but at a very reduced rate. It thus appears that signal-sequence mutations can affect more than one step in the secretion process, and that processing of the signal peptide is not required for the protein to be translocated (at least partially) across the inner membrane.

Amino Acid Sequence↗