Biomedical subjects

Moshe Mevarech

Publications and source records attributed to Moshe Mevarech.

7 recordsLinked to original sources

PubMed2005

Archaeal genetics - the third way.

For decades, archaea were misclassified as bacteria because of their prokaryotic morphology. Molecular phylogeny eventually revealed that archaea, like bacteria and eukaryotes, are a fundamentally distinct domain of life. Genome analyses have confirmed that archaea share many features with eukaryotes, particularly in information processing, and therefore can serve as streamlined models for understanding eukaryotic biology. Biochemists and structural biologists have embraced the study of archaea but geneticists have been more wary, despite the fact that genetic techniques for archaea are quite sophisticated. It is time for geneticists to start asking fundamental questions about our distant relatives.

Archaea↗

PubMed2004

An alternative pathway for reduced folate biosynthesis in bacteria and halophilic archaea.

Whereas tetrahydrofolate is an essential cofactor in all bacteria, the gene that encodes the enzyme dihydrofolate reductase (DHFR) could not be identified in many of the bacteria whose genomes have been entirely sequenced. In this communication we show that the halophilic archaea Halobacterium salinarum and Haloarcula marismortui contain genes coding for proteins with an N-terminal domain homologous to dihydrofolate synthase (FolC) and a C-terminal domain homologous to dihydropteroate synthase (FolP). These genes are able to complement a Haloferax volcanii mutant that lacks DHFR. We also show that the Helicobacter pylori dihydropteroate synthase can complement an Escherichia coli mutant that lacks DHFR. Activity resides in an N-terminal segment that is homologous to the polypeptide linker that connects the dihydrofolate synthase and dihydropteroate synthase domains in the haloarchaeal enzymes. The purified recombinant H. pylori dihydropteroate synthase was found to be a flavoprotein.

Amino Acid Sequence↗

PubMed2004

Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes.

Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry. Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. DeltaleuB and DeltatrpA mutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying the leuB or trpA gene under control of a constitutive promoter. The use of these selectable markers in deletion of the lhr gene of H. volcanii is described.

3-Isopropylmalate Dehydrogenase↗

PubMed2003

FolM, a new chromosomally encoded dihydrofolate reductase in Escherichia coli.

Escherichia coli (thyA DeltafolA) mutants are viable and can grow in minimal medium when supplemented with thymidine alone. Here we present evidence from in vivo and in vitro studies that the ydgB gene determines an alternative dihydrofolate reductase that is related to the trypanosomatid pteridine reductases. We propose to rename this gene folM.

Amino Acid Sequence↗

PubMed2003

Development of a gene knockout system for the halophilic archaeon Haloferax volcanii by use of the pyrE gene.

So far, the extremely halophilic archaeon Haloferax volcanii has the best genetic tools among the archaea. However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies. In this paper we describe the development of pyrE-based positive selection and counterselection systems to generate an efficient gene knockout system. The H. volacanii pyrE1 and pyrE2 genes were isolated, and the pyrE2 gene was shown to code for the physiological enzyme orotate phosphoribosyl transferase. A DeltapyrE2 strain was constructed and used to isolate deletion mutants by the following two steps: (i) integration of a nonreplicative plasmid carrying both the pyrE2 wild-type gene, as a selectable marker, and a cloned chromosomal DNA fragment containing a deletion in the desired gene; and (ii) excision of the integrated plasmid after selection with 5-fluoroorotic acid. Application of this gene knockout system is described.

Amino Acid Sequence↗

PubMed2002

Genetic evidence for a novel thymidylate synthase in the halophilic archaeon Halobacterium salinarum and in Campylobacter jejuni.

A search of the complete genome sequence of the halophilic archaeon Halobacterium salinarum failed to identify a gene homologous to the thymidylate synthase (thyA) gene present in the closely related Haloferax volcanii. To understand the source of thymidine synthesis in Hbt. salinarum, a genomic library of Hbt. salinarum was constructed and used to complement a Hfx. volcanii thyA deletion mutation. The Hbt. salinarum ORF that complemented the thyA mutation shares sequence homology with ORFs found in numerous microorganisms that lack a thyA gene, including the recently discovered thyX of Helicobacter pylori. We also show that a homolog of the Hbt. salinarum ORF is present in Campylobacter jejuni and is able to complement an Escherichia coli thyA mutant under oxygen-limiting conditions.

Campylobacter jejuni↗

PubMed1997

A Candida albicans RAS-related gene (CaRSR1) is involved in budding, cell morphogenesis and hypha development.

Candida albicans, the most important human fungal pathogen, is a dimorphic fungus that can grow either as a yeast or as a hyphal form in response to medium conditions. A RAS-related C. albicans gene (CaRSR1) was isolated as a suppressor of a cdc24ts bud-emergence mutation of the baker's yeast, Saccharomyces cerevisiae. The deduced protein encoded by CaRSR1 is 248 amino acids long and 56% identical to that encoded by the S. cerevisiae RSR1 (BUD1) gene. Disruption of CaRSR1 in C. albicans indicated that CaRSR1 is involved in both yeast and hypha development. In the yeast phase, CaRSR1 is required for normal (polar) bud site selection and is involved in cell morphogenesis; in the yeast-mycelial transition it is involved in germ tube emergence; and in the development of the hyphae it is involved in cell elongation. The disruption of CaRSR1 leads to reduced virulence in both heterozygote and homozygote disruptants in a dose-dependent manner. The reduced virulence can be attributed to the reduced germination and shorter hyphae resulting from the disruption of CaRSR1.

Amino Acid Sequence↗