Yechezkel Kashi

Simple sequence repeats as a source of quantitative genetic variation

Most traits in biological populations appear to be under stabilizing selection, which acts to eliminate quantitative genetic variation. Yet, virtually all measured traits in biological populations continue to show significant quantitative genetic variation. The paradox can be resolved by postulating the existence of an abundant, though unspecified, source of mutations that has quantitative effects on phenotype, but does not reduce fitness. Does such a source actually exist? We propose that it does, in the form of repeat-number variation in SSRs (simple sequence repeats, of which the triplet repeats of human neurodegenerative diseases are a special case). Viewing SSRs as a major source of quantitative mutation has broad implications for understanding molecular processes of evolutionary adaptation, including the evolutionary control of the mutation process itself.

Detection of a chromosomal region with two quantitative trait loci, affecting cold tolerance and fish size, in an F2 tilapia hybrid

Abstract We searched for genetic linkage between microsatellite DNA markers and quantitative trait loci (QTL) for cold tolerance and fish size (body weight and standard length) in two unrelated F 2 families of interspecific tilapia hybrids ( Oreochromis mossambicus × Oreochromis aureus ). The first experiment was based on a family of 60 fish scanned for 20 microsatellites. A second experiment was conducted with a family of 114 fish scanned for 6 microsatellites in one linkage group, in order to test for QTL found in the first experiment. This two-step experimental design was used in order to protect against “false positive” associations. In both families, significant associations were found for two loci within the same linkage group. The two QTL, near UNH879 for cold tolerance, and near UNH130 for body size, were estimated to be 22 cM distant from each other, with no interaction found between the two traits. One of these loci, UNH879 , was also associated with sex determination. Distortion from the expected Mendelian genotypic ratio was observed for three markers: UNH130 , UNH180 and UNH907 , suggesting linkage with a QTL affecting survival. These results identify a chromosomal region in the tilapia genome harboring several QTL affecting fitness traits.

Resistance to Adjuvant Arthritis Is Due to Protective Antibodies Against Heat Shock Protein Surface Epitopes and the Induction of IL-10 Secretion

Adjuvant arthritis (AA) is an experimental model of autoimmune arthritis that can be induced in susceptible strains of rats such as inbred Lewis upon immunization with CFA. AA cannot be induced in resistant strains like Brown-Norway or in Lewis rats after recovery from arthritis. We have previously shown that resistance to AA is due to the presence of natural as well as acquired anti-heat shock protein (HSP) Abs. In this work we have studied the fine specificity of the protective anti-HSP Abs by analysis of their interaction with a panel of overlapping peptides covering the whole HSP molecule. We found that arthritis-susceptible rats lack Abs to a small number of defined epitopes of the mycobacterial HSP65. These Abs are found naturally in resistant strains and are acquired by Lewis rats after recovery from the disease. Active vaccination of Lewis rats with the protective epitopes as well as passive vaccination with these Abs induced suppression of arthritis. Incubation of murine and human mononuclear cells with the protective Abs induced secretion of IL-10. Analysis of the primary and tertiary structure of the whole Mycobacterium tuberculosis HSP65 molecule indicated that the protective epitopes are B cell epitopes with nonconserved amino acid sequences found on the outer surface of the molecule. We conclude that HSP, the Ag that contains the pathogenic T cell epitopes in AA, also contains protective B cell epitopes exposed on its surface, and that natural and acquired resistance to AA is associated with the ability to respond to these epitopes.

Finding approximate tandem repeats in genomic sequences.

An efficient algorithm is presented for detecting approximate tandem repeats in genomic sequences. The algorithm is based on a flexible statistical model which allows a wide range of definitions of approximate tandem repeats. The ideas and methods underlying the algorithm are described and its effectiveness on genomic data is demonstrated.

Molecular-Genetic Biodiversity in a Natural Population of the Yeast Saccharomyces cerevisiae From “Evolution Canyon”: Microsatellite Polymorphism, Ploidy and Controversial Sexual Status

The yeast S. cerevisiae is a central model organism in eukaryotic cell studies and a major component in many food and biotechnological industrial processes. However, the wide knowledge regarding genetics and molecular biology of S. cerevisiae is based on an extremely narrow range of strains. Studies of natural populations of S. cerevisiae, not associated with human activities or industrial fermentation environments, are very few. We isolated a panel of S. cerevisiae strains from a natural microsite, “Evolution Canyon” at Mount Carmel, Israel, and studied their genomic biodiversity. Analysis of 19 microsatellite loci revealed high allelic diversity and variation in ploidy level across the panel, from diploids to tetraploids, confirmed by flow cytometry. No significant differences were found in the level of microsatellite variation between strains derived from the major localities or microniches, whereas strains of different ploidy showed low similarity in allele content. Maximum genetic diversity was observed among diploids and minimum among triploids. Phylogenetic analysis revealed clonal, rather than sexual, structure of the triploid and tetraploid subpopulations. Viability tests in tetrad analysis also suggest that clonal reproduction may predominate in the polyploid subpopulations.

Vibrio cholerae Strain Typing and Phylogeny Study Based on Simple Sequence Repeats

ABSTRACT Vibrio cholerae is the etiological agent of cholera. Its natural reservoir is the aquatic environment. To date, practical typing of V. cholerae is mainly serological and requires about 200 antisera. Simple sequence repeats (SSR), also termed VNTR (for variable number of tandem repeats), provide a source of high genomic polymorphism used in bacterial typing. Here we describe an SSR-based typing method that combines the variation in highly mutable SSR loci, with that of shorter, relatively more stable mononucleotide repeat (MNR) loci, for accurate and rapid typing of V. cholerae. In silico screening of the V. cholerae genome revealed thousands of perfect SSR tracts with an average frequency of one SSR every 152 bp. A panel of 32 V. cholerae strains, representing both clinical and environmental isolates, was tested for polymorphism in SSR loci. Two strategies were applied to identify SSR variation: polymorphism of SSR tracts longer than 12 bp (L-SSR) assessed by capillary fragment-size analysis and MNR polymorphism assessed by sequencing. The nine L-SSR loci tested were all polymorphic, displaying 2 to 13 alleles per locus. Sequence analysis of eight MNR-containing loci (MNR-multilocus sequence typing [MLST]) provided information on both variations in the MNR tract itself, and single nucleotide polymorphism (SNP) in their flanking sequences. Phylogenetic analysis of the combined SSR data showed a clear discrimination between the clinical strains belonging to O1 and O139 serogroups, and the environmental isolates. Furthermore, discrimination between 27 strains of the 32 strains was achieved. SSR-based typing methods combining L-SSR and MNR-MLST were found to be efficient for V. cholerae typing.

Vibrio cholerae Hemagglutinin/Protease Degrades Chironomid Egg Masses

ABSTRACT Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. The disease does not persist in a chronic state in humans or animals. The pathogen is naturally present as a free-living organism in the environment. Recently, it was suggested that egg masses of the nonbiting midge Chironomus sp. (Diptera) harbor and serve as a nutritive source for V. cholerae, thereby providing a natural reservoir for the organism. Here we report that V. cholerae O9, O1, and O139 supernatants lysed the gelatinous matrix of the chironomid egg mass and inhibited eggs from hatching. The extracellular factor responsible for the degradation of chironomid egg masses (egg mass degrading factor) was purified from V. cholerae O9 and O139 and was identified as the major secreted hemagglutinin/protease (HA/P) of V. cholerae. The substrate in the egg mass was characterized as a glycoprotein. These findings show that HA/P plays an important role in the interaction of V. cholerae and chironomid egg masses.

A PCR method based on 16S rRNA sequence for simultaneous detection of the genus Listeria and the species Listeria monocytogenes in food products

The genus Listeria comprises six closely related species, of which only Listeria monocytogenes is a human pathogen. The rapid and sensitive detection of L. monocytogenes is important in the food industry as well as in medical diagnosis. In this study, a PCR-based method for the rapid, specific, and sensitive detection of L. monocytogenes in food products was developed. The PCR is based on DNA sequences and primer pairs that are found within the 16S subunit of the rRNA gene and are specific to the Listeria genus and to L. monocytogenes within the Listeria genus. The primers for the Listeria genus and for L. monocytogenes were used in the same reaction mix for their simultaneous detection. In addition, a pair of bacterial primers universal to any bacterial DNA at the 16S subunit of the rRNA gene were developed as a positive control. For the detection of Listeria and L. monocytogenes in food products, the method includes selective enrichment for Listeria followed by DNA extraction and a specific PCR reaction. The method detects 1 to 5 CFU in a 25-g sample in < or = 24 h. It can be easily incorporated into the routine screening of diverse food products and readily adapted for clinical use.

Production of 2-phenylethanol from L-phenylalanine by a stress tolerant Saccharomyces cerevisiae strain.

Aims:  Screening for a robust, stress tolerant Saccharomyces cerevisiae strain for production of 2‐phenylethanol (PEA) from l‐phenylalanine.

Heat acclimation prolongs the time to central nervous system oxygen toxicity in the rat: Possible involvement of HSP72

Oxygen toxicity of the central nervous system (CNS-OT) can occur during diving with oxygen-enriched gas mixtures, or during hyperbaric medical treatment. CNS-OT is characterised by convulsions and sudden loss of consciousness, which may be fatal in diving. Heat acclimation is known to provide cross-tolerance to various forms of stress in different organs, including the brain. We hypothesised that heat acclimation may delay the onset of CNS-OT in the rat. Male Sprague-Dawley rats were acclimated to an ambient temperature of 32 degrees C for 4 weeks. Rats in the control group were kept at 24 degrees C. Both groups were exposed to oxygen at 608 kPa. EEG was recorded continuously until the appearance of the first electrical discharge preceding clinical convulsions. CO(2) production was measured simultaneously with the EEG. Latency to CNS-OT was measured and brain samples were taken for evaluation of heat shock protein 72 (HSP72) levels by Western blot analysis at the end of the acclimation period and during 4 weeks of deacclimation. Latency to CNS-OT was twice as long in the heat-acclimated rat, with insignificant changes in CO(2) production. This prolongation continued for 2 weeks during deacclimation. There was a significant increase in the level of HSP72 following heat acclimation, with a subsequent decrease during deacclimation. We conclude that heat acclimation prolongs latency to CNS-OT in a way that does not involve changes in metabolic rate. During deacclimation there was a linear relationship between latency to CNS oxygen toxicity and the level of HSP72. A possible beneficial effect of HSP72 is discussed.