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C K Raymond

Publications and source records attributed to C K Raymond.

15 recordsLinked to original sources
PubMed2004

Hypervariability, suppressed recombination and the genetics of individuality.

We define 'genetic individuality' as intraspecies variation that has substantial heritability and involves traits that are sufficiently common that they can be observed in any modest-sized sampling of individuals. We propose that genetic individuality is largely shaped by the combinatory shuffling of a modest number of genes, each of which exists as a family of functionally and structurally diverged alleles. Unequivocal examples of such allele families are found at the O-antigen-biosynthetic locus in Pseudomonas aeruginosa and the human leucocyte antigen locus in humans. We examine characteristic features of these allele families and explore the possibility that genetic loci with similar characteristics can be recognized in a whole-genome scan of human genetic variation.

Alleles↗
PubMed1999

General method for plasmid construction using homologous recombination.

We describe a general method for plasmid assembly that uses yeast and extends beyond yeast-specific research applications. This technology exploits the homologous recombination, double-stranded break repair pathway in Saccharomyces cerevisiae to join DNA fragments. Synthetic, double-stranded "recombination linkers" were used to "subclone" a DNA fragment into a plasmid with > 80% efficiency. Quantitative data on the influence of DNA concentration and overlap length on the efficiency of recombination are presented. Using a simple procedure, plasmids were shuttled from yeast into E. coli for subsequent screening and large-scale plasmid preps. This simple method for plasmid construction has several advantages. (i) It bypasses the need for extensive PCR amplification and for purification, modification and/or ligation techniques routinely used for plasmid constructions. (ii) The method does not rely on available restriction sites, thus fragment and vector DNA can be joined within any DNA sequence. This enables the use of multifunctional cloning vectors for protein expression in mammalian cells, other yeast species, E. coli and other expression systems as discussed. (iii) Finally, the technology exploits yeast strains, plasmids and microbial techniques that are inexpensive and readily available.

Cloning, Molecular↗
PubMed1998

Development of the methylotrophic yeast Pichia methanolica for the expression of the 65 kilodalton isoform of human glutamate decarboxylase.

We describe a protein expression system in the methylotrophic yeast, Pichia methanolica. Methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild-type ADE2 gene. A vacuolar protease-deficient strain was constructed. Two genes encoding alcohol oxidases were found, yet a single isoform of alcohol oxidase was produced during methanol-fed fermentations. The promoter from this gene was used to drive expression. An integrating plasmid for the cytoplasmic expression of the 65 kDa isoform of human glutamate decarboxylase (human GAD65) was assembled. A strain harboring eight copies of this plasmid expressed enzymatically active human GAD65 at levels approaching 0.5 g/l. Identical amounts were made in Pichia pastoris. The recombinant GAD65 was purified to greater than 90% purity.

Alcohol Oxidoreductases↗
PubMed1993

The VPS1 protein is a dynamin-like GTPase required for sorting proteins to the yeast vacuole.

VPS1 encodes a 79 kDa protein required for the proper sorting of soluble vacuolar proteins in Saccharomyces cerevisiae. The N-terminal half of Vps1p, which contains a consensus GTP-binding motif, shares extensive homology with a growing family of high molecular mass GTP-binding proteins. Members of this family have been implicated in a number of cellular processes. Vps1p most closely resembles the microtubule-associated protein dynamin. As predicted from the sequence, Vps1p binds and hydrolyses GTP. However, no requirement for microtubules was found for Vps1p function in protein sorting. In subcellular fractionation experiments Vps1p associates with the membrane fraction; the C-terminal half of Vps1p is important for this association. Mutational analysis of VPS1 generated two classes of mutations, dominant negative and recessive. The dominant mutations all mapped to the N-terminal half of the protein. Recessive mutations gave rise to either truncated or unstable proteins. A potential Vps1p-interacting protein (Mvp1p) has been isolated by screening for suppressors of the dominant alleles of VPS1. Taken together these results suggest that Vps1p is a two-domain protein that is part of a multi-subunit protein complex involved in vacuolar protein sorting.

Biological Transport↗
PubMed1992

The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains.

The product of the VPS1 gene, Vps1p, is required for the sorting of soluble vacuolar proteins in the yeast Saccharomyces cerevisiae. We demonstrate here that Vps1p, which contains a consensus tripartite motif for guanine nucleotide binding, is capable of binding and hydrolyzing GTP. Vps1p is a member of a subfamily of large GTP-binding proteins whose members include the vertebrate Mx proteins, the yeast MGM1 protein, the Drosophila melanogaster shibire protein, and dynamin, a bovine brain protein that bundles microtubules in vitro. Disruption of microtubules did not affect the fidelity or kinetics of vacuolar protein sorting, indicating that Vps1p function is not dependent on microtubules. Based on mutational analyses, we propose a two-domain model for Vps1p function. When VPS1 was treated with hydroxylamine, half of all mutations isolated were found to be dominant negative with respect to vacuolar protein sorting. All of the dominant-negative mutations analyzed further mapped to the amino-terminal half of Vps1p and gave rise to full-length protein products. In contrast, recessive mutations gave rise to truncated or unstable protein products. Two large deletion mutations in VPS1 were created to further investigate Vps1p function. A mutant form of Vps1p lacking the carboxy-terminal half of the protein retained the capacity to bind GTP and did not interfere with sorting in a wild-type background. A mutant form of Vps1p lacking the entire GTP-binding domain interfered with vacuolar protein sorting in wild-type cells. We suggest that the amino-terminal domain of Vps1p provides a GTP-binding and hydrolyzing activity required for vacuolar protein sorting, and the carboxy-terminal domain mediates Vps1p association with an as yet unidentified component of the sorting apparatus.

Ca(2+) Mg(2+)-ATPase↗
PubMed1992

Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants.

The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H(+)-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.

Carboxypeptidases↗
PubMed1990

A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting.

Members of the Mx protein family promote interferon-inducible resistance to viral infection in mammals and act by unknown mechanisms. We identified an Mx-like protein in yeast and present genetic evidence for its cellular function. This protein, the VPS1 product, is essential for vacuolar protein sorting, normal organization of intracellular membranes, and growth at high temperature, implying that Mx-like proteins are engaged in fundamental cellular processes in eukaryotes. Vps1p contains a tripartite GTP binding motif, which suggests that binding to GTP is essential to its role in protein sorting. Vps1p-specific antibody labels punctate cytoplasmic structures that condense to larger structures in a Golgi-accumulating sec7 mutant; thus, Vps1p may associate with an intermediate organelle of the secretory pathway.

Amino Acid Sequence↗
PubMed1990

Molecular analysis of the yeast VPS3 gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle.

vps3 mutants of the yeast Saccharomyces cerevisiae are impaired in the sorting of newly synthesized soluble vacuolar proteins and in the acidification of the vacuole (Rothman, J. H., and T. H. Stevens. Cell. 47:1041-1051; Rothman, J. H., C. T. Yamashiro, C. K. Raymond, P. M. Kane, and T. H. Stevens. 1989. J. Cell Biol. 109:93-100). The VPS3 gene, which was cloned using a novel selection procedure, encodes a low abundance, hydrophilic protein of 117 kD that most likely resides in the cytoplasm. Yeast strains bearing a deletion of the VPS3 gene (vps3-delta 1) are viable, yet their growth rate is significantly reduced relative to wild-type cells. Temperature shift experiments with strains carrying a temperature conditional vps3 allele demonstrate that cells rapidly lose the capacity to sort the vacuolar protein carboxypeptidase Y upon loss of VPS3 function. Vacuolar morphology was examined in wild-type and vps3-delta 1 yeast strains by fluorescence microscopy. The vacuoles in wild-type yeast cells are morphologically complex, and they appear to be actively partitioned between mother cells and buds during an early phase of bud growth. Vacuolar morphology in vps3-delta 1 mutants is significantly altered from the wild-type pattern, and the vacuolar segregation process seen in wild-type strains is defective in these mutants. With the exception of a vacuolar acidification defect, the phenotypes of vps3-delta 1 strains are significantly different from those of mutants lacking the vacuolar proton-translocating ATPase. These data demonstrate that the acidification defect in vps3-delta 1 cells is not the primary cause of the pleiotropic defects in vacuolar function observed in these mutants.

Amino Acid Sequence↗
PubMed1989

Acidification of the lysosome-like vacuole and the vacuolar H+-ATPase are deficient in two yeast mutants that fail to sort vacuolar proteins.

Organelle acidification plays a demonstrable role in intracellular protein processing, transport, and sorting in animal cells. We investigated the relationship between acidification and protein sorting in yeast by treating yeast cells with ammonium chloride and found that this lysosomotropic agent caused the mislocalization of a substantial fraction of the newly synthesized vacuolar (lysosomal) enzyme proteinase A (PrA) to the cell surface. We have also determined that a subset of the vpl mutants, which are deficient in sorting of vacuolar proteins (Rothman, J. H., and T. H. Stevens. 1986. Cell. 47:1041-1051; Rothman, J. H., I. Howald, and T. H. Stevens. EMBO [Eur. Mol. Biol. Organ.] J. In press), failed to accumulate the lysosomotropic fluorescent dye quinacrine within their vacuoles, mimicking the phenotype of wild-type cells treated with ammonium. The acidification defect of vpl3 and vpl6 mutants correlated with a marked deficiency in vacuolar ATPase activity, diminished levels of two immunoreactive subunits of the protontranslocating ATPase (H+-ATPase) in purified vacuolar membranes, and accumulation of the intracellular portion of PrA as the precursor species. Therefore, some of the VPL genes are required for the normal function of the yeast vacuolar H+-ATPase complex and may encode either subunits of the enzyme or components required for its assembly and targeting. Collectively, these findings implicate a critical role for acidification in vacuolar protein sorting and zymogen activation in yeast, and suggest that components of the yeast vacuolar acidification system may be identified by examining mutants defective in sorting of vacuolar proteins.

Adenosine Triphosphatases↗
PubMed1986

Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes.

Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome. The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in lambda EMBL3. Genomic maps were drawn for three ORS571 Nif gene loci. Forty-five Nif::Vi mutants in genomic Nif locus 1 defined two gene clusters separated by 8 kilobase pairs (kb) of DNA. In the first cluster, 36 Nif::Vi mutants mapped to a 7-kb DNA segment that showed DNA homology with Klebsiella pneumoniae nifHDKE and encoded at least two Nif operons. In the other cluster, nine Nif::Vi mutants mapped to a 1.5-kb DNA segment that showed homology with K. pneumoniae and Rhizobium meliloti nifA; this DNA segment encoded a separate Nif operon. Fifteen Nif::Vi mutants mapped to a 3.5-kb DNA segment defined as Nif locus 2 and showed DNA homology with the R. meliloti P2 fixABC operon. Nif locus 2 carries a second nifH (nifH2) gene. Four Nif::Vi mutants mapped to a 2-kb DNA segment defined as Nif locus 3 and showed DNA homology with K. pneumoniae nifB. DNA from lambda Nif phages comprising all three genomic Nif loci was subcloned in plasmid vectors able to stably replicate in ORS571. These plasmid subclones were introduced into ORS571 strains carrying physically mapped Nif::Vi insertions, and genetic complementations were conducted. With the exception of certain mutants mapping to the nifDK genes, all mutants could be complemented to Nif+ when they carried plasmid subclones of defined genomic DNA regions. Conversely, most nifDK mutants behaved as pseudodominant alleles.

Alleles↗
PubMed1985

Vector insertion mutagenesis of Rhizobium sp. strain ORS571: direct cloning of mutagenized DNA sequences.

When the limited-host-range plasmid pVP2021 carrying Tn5 was mobilized into Rhizobium sp. strain ORS571 and stable acquisition of Tn5 was selected, ORS571 plasmid-genome cointegrates were exclusively obtained; direct Tn5 transposition was never observed. In every case, genomic cointegrates exhibited an additional (third) IS50 element that bordered VP2021 DNA sequences but maintained a single Tn5 element. Genomic cointegrates containing IS50 triplications were stable; neither phenotypic reversion nor resolution was detectable. Auxotrophic mutant strains (vector insertion mutants) were identified at expected frequencies among derivatives carrying ostensibly random genomic pVP2021 insertions; N2 fixation (Nif)-defective vector insertion mutants were observed among these derivatives at a frequency of 10(-3). The presence of integrated pVP2021 in ORS571 nif::VP2021 mutant genomes enabled VP2021 to constitute an endogenous cloning vector. After EcoRI or KpnI digestions, genomic nif::pVP2021 DNA sequences contiguous with integrated pVP2021 were directly cloned as new replicons without addition of an exogenous vector. Recombinant plasmids derived from two such nif::pVP2021 mutants hybridized to previously analyzed ORS571 Nif DNA sequences. Recombinant plasmid DNA and ORS571 Nif region DNA were found to be colinear; pVP2021 insertions could be accurately mapped. pVP2021 insertion-mutagenesis thus allows the direct cloning of ORS571 gene sequences for which mutant phenotypes can be selected or screened.

Base Sequence↗
PubMed1984

Extension of bacteriophage lambda host range: selection, cloning, and characterization of a constitutive lambda receptor gene.

A set of plasmids has been constructed that carry a constitutive lamB gene (LamBc phenotype) from Escherichia coli and that confer functional phage lambda receptors to bacteria other than E. coli. This E. coli LamBc strain has been selected to escape both maltose-inducible and glucose-repressible control. Constitutivity results from an IS-3 insertion, carrying a mobile promoter, proximal to lamB. The LamBc DNA has been cloned into both broad and narrow host-range plasmids, and the resulting pTROY plasmids have been transferred to diverse bacteria. Both Salmonella typhimurium/pTROY and Klebsiella pneumoniae/pTROY strains efficiently adsorb phage lambda; Pseudomonas aeruginosa/pTROY strains do not. Introduction of a functional E. coli LamB protein into foreign bacterial will allow these bacteria carrying pTROY plasmids to be infected by phage lambda recombinant DNA libraries, phage lambda::Tn insertion mutagenesis vectors, and in vivo lambda-packaged cosmids.

Bacterial Outer Membrane Proteins↗