Quantitative trait loci mapping for conjugated linoleic acid, vaccenic acid and ∆(9) -desaturase in Italian Brown Swiss dairy cattle using selective DNA pooling.

A selective DNA pooling approach was applied to identify QTL for conjugated linoleic acid (CLA), vaccenic acid (VA) and Δ(9) -desaturase (D9D) milk content in Italian Brown Swiss dairy cattle. Milk samples from 60 animals with higher values (after correction for environmental factors) and 60 animals with lower values for each of these traits from each of five half-sib families were pooled separately. The pools were genotyped using the Illumina BovineSNP50 BeadChip. Sire allele frequencies were compared between high and low tails at the sire and marker level for SNPs for which the sires were heterozygous. An r procedure was implemented to perform data analysis in a selective DNA pooling design. A correction for multiple tests was applied using the proportion of false positives among all test results. BTA 19 showed the largest number of markers in association with CLA. Associations between SNPs and the VA and Δ(9) -desaturase traits were found on several chromosomes. A bioinformatics survey identified genes with an important role in pathways for milk fat and fatty acids metabolism within 1 Mb of SNP markers associated with fatty acids contents.

Genome-wide analysis of DNA turnover and gene expression in stationary-phase Saccharomyces cerevisiae.

Exponential-phase yeast cells readily enter stationary phase when transferred to fresh, carbon-deficient medium, and can remain fully viable for up to several months. It is known that stationary-phase prokaryotic cells may still synthesize substantial amounts of DNA. Although the basis of this phenomenon remains unclear, this DNA synthesis may be the result of DNA maintenance and repair, recombination, and stress-induced transposition of mobile elements, which may occur in the absence of DNA replication. To the best of our knowledge, the existence of DNA turnover in stationary-phase unicellular eukaryotes remains largely unstudied. By performing cDNA-spotted (i.e. ORF) microarray analysis of stationary cultures of a haploid Saccharomyces cerevisiae strain, we demonstrated on a genomic scale the localization of a DNA-turnover marker [5-bromo-2'-deoxyuridine (BrdU); an analogue of thymidine], indicative of DNA synthesis in discrete, multiple sites across the genome. Exponential-phase cells on the other hand, exhibited a uniform, total genomic DNA synthesis pattern, possibly the result of DNA replication. Interestingly, BrdU-labelled sites exhibited a significant overlap with highly expressed features. We also found that the distribution among chromosomes of BrdU-labelled and expressed features deviates from random distribution; this was also observed for the overlapping set. Ty1 retrotransposon genes were also found to be labelled with BrdU, evidence for transposition during stationary phase; however, they were not significantly expressed. We discuss the relevance and possible connection of these results to DNA repair, mutation and related phenomena in higher eukaryotes.

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. METHODS AND RESULTS: Saccharomyces cerevisiae natural isolates that include thermotolerant and multi-stress resistant strains were screened for production of PEA. The strains were compared by their growth rate, maximum biomass dry weight and production yields, and it was shown that high biomass is required for obtaining high concentrations of PEA. One thermotolerant strain, Ye9-612, was the most efficient, and under optimal conditions produced 0.85 g l(-1) PEA in shaking flasks, and 4.5 g l(-1) in a fed-batch fermentation. Strain Ye9-612 was also found to be tolerant to high concentrations of PEA, and maintained high biomass dry weight in the presence of 2.5 g l(-1) PEA. CONCLUSIONS: We found a highly productive thermotolerant S. cerevisiae strain that is resistant to high concentrations of PEA. SIGNIFICANCE AND IMPACT OF THE STUDY: PEA is inhibitory to growing yeast cells and therefore a robust strain is needed for excessive industrial production. This is the first description of such a stress tolerant strain, and the PEA concentration obtained in the fermentation is the highest reported to date.

A survey of laboratory-confirmed isolates of invasive listeriosis in Israel, 1997-2007.

During the 11-year period from 1997 to 2007, 321 isolates of Listeria monocytogenes from sporadic cases of invasive listeriosis were reported to the national reference laboratory in Israel. Of these isolates, 113 (35%) were identified from perinatal cases, and 208 (65%) from non-perinatal cases. The prevalent serovars were 4b, 1/2b, 1/2a and 4c. Serovar 4b was identified in 80.5% of the perinatal isolates (P=0.0162), while the number of 1/2b and 1/2a strains increased in the > or = 60 years old group (P=0.0285). Resistance to tetracycline was found in eight 4b isolates. The seasonal distribution showed that 206 isolates (64.2%) were submitted during the hot season (May-October). The estimated morbidity for the study period was 4.4 per million. The incidence of invasive listeriosis was higher in the perinatal group (5.6/100 000), than in individuals aged > or = 60 years (1.5/100 000).

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.

A PCR-based method for the detection of Streptococcus agalactiae in milk.

Bovine mastitis caused by Streptococcus agalactiae is mainly subclinical and therefore can be diagnosed only in the laboratory. We developed a polymerase chain reaction (PCR)-based method for specific and sensitive detection of S. agalactiae in raw milk. The specificity of the PCR reaction is based on unique S. agalactiae DNA sequences within the 16S subunit of the rRNA genes. Two pairs of sequences were used as positive controls; general streptococci primers, which anneal to conserved areas within the 16S rRNA subunit gene, and primers, which anneal to sequences within bovine mitochondrial DNA. The method of detection includes selective enrichment of S. agalactiae in the milk sample, followed by DNA extraction using a rapid and simple procedure developed for this purpose, and specific PCR reaction with appropriate controls. The method enables the detection of one bacterium in 1 ml of raw milk. The method developed can be easily incorporated as part of routine screening of bulk milk collection tanks for early detection of infected cows in a herd.

Genome Evolution of the Cyanobacterium Nostoc linckia under Sharp Microclimatic Divergence at "Evolution Canyon," Israel.

We describe the genomic DNA diversity and divergence of the cyanobacterium Nostoc linckia from "Evolution Canyon," a microsite consisting of ecologically contrasting slopes, south-facing slope (SFS) and north-facing slope (NFS), at lower Nahal Oren, Mt. Carmel, Israel. The opposing slopes share their limestone lithology but vary greatly in their ecology, primarily because of different levels of solar radiation (which is six times higher on the SFS than on the NFS). The warm and xeric SFS displays a tropical African savanna, whereas the cool and mesic NFS displays a temperate South European Mediterranean live-oak maquis shrub forest. The cyanobacterium Nostoc linckia tested here is a sessile microorganism, growing as a carpet on rock surfaces and constantly exposed to environmental fluctuations of solar radiation, temperature, and desiccation. We demonstrate remarkable interslope and intraslope genetic divergence of the genome (including both coding and noncoding regions) of Nostoc linckia, by using 211 AFLP (amplified fragment length polymorphism) DNA molecular marker loci. Genetic polymorphism of N. linckia subpopulations on the ecologically harsher SFS was significantly (p <0.05) higher (p = 99.53%) than was that of the subpopulations on the climatically milder nfs (p = 85.78%). genetic polymorphism (p) and gene diversity (he) were significantly correlated with variables influencing aridity stress: solar radiation (sr) (rp = 0.956; p = 0.046), temperature (tm) (rp = 0.993; p = 0.0068), and day-night temperature difference (tdd) (rp = 0.975; p = 0.025). as in other tested organisms from "evolution canyon", but even more exceptionally because of its completely sedentary nature, we suggest that the climatically stressed sfs environment is responsible for this marked increase of genetic polymorphism, which is maintained by the combined evolutionary forces of diversifying and balancing selection. This could highlight the importance of ecological stress and selection in evolution and its remarkable effect on the genetic system across the prokaryotic genome.

Simple sequence repeats in Escherichia coli: abundance, distribution, composition, and polymorphism.

Computer-based genome-wide screening of the DNA sequence of Escherichia coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1 to 6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in noncoding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in noncoding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the noncoding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in noncoding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.

Development of a polymerase chain reaction-based assay for the detection of Alternaria fungal contamination in food products.

Alternaria sp. are important fungal contaminants of vegetable, fruit, and grain products, including Alternaria alternata, a contaminant of tomato products. To date, the Howard method, based on microscopic observation of fungal filaments, has been the standard examination for inspection of tomato products. We report development of a polymerase chain reaction (PCR)-based method for detection of Alternaria DNA. PCR primers were designed to anneal to the internal transcribed regions ITS1 and ITS2 of the 5.8S rRNA gene of Alternaria but not to other microbial or tomato DNA. We demonstrate use of the PCR assay to detect Alternaria DNA in experimentally infested and commercially obtained tomato sauce and tomato powder. Use of the PCR method offers a rapid and sensitive assay for the presence of Alternaria DNA in tomato products. The apparent breakdown of DNA in tomato sauce may limit the utility of the assay to freshly prepared products. The assay for tomato powder is not affected by storage time.

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.

Minisatellite and microsatellite length variation at a complex bovine VNTR locus.

Length variation at the BTGL1 variable number of tandem repeat (VNTR) locus, which includes both minisatellite and microsatellite motifs, was examined in a wide sample of cattle. A total of 22 alleles were uncovered. The distribution of allele size variation implicated mechanisms involving both minisatellite and microsatellite sequences. This was confirmed by direct sequences of two alleles. Differences between the two alleles involved repeat number variation of microsatellite motifs and a complex event involving the minisatellite motif, but point mutations were not observed.

Primary malignant tumors (lymphoma excluded) in patients with chronic lymphocytic leukemia. A series of 22 cases from a retrospective study of 248 patients.

This retrospective study was conducted to evaluate the incidence of primary malignant tumors in patients with chronic lymphocytic leukemia (CLL). Between 1974 and 1994, 313 patients were hospitalized for CLL. Patients lost to follow-up were excluded (n = 65). Among the remaining 248 patients (167 men and 81 women), 22 patients (8.8%) developed primary malignant tumors (17 males, 5 females; age range 48 to 90 years); those who developed Richter's lymphoma (n = 9) or basal cell epithelioma (n = 5) were excluded from the cohort of the cancer patient. There were 18 epithelial tumors (3 lung adenocarcinoma) and 4 parenchymal tumors. We observed only one cancer of the prostate, and no breast cancer. The malignant tumor occurred at the same time as CLL in 8 cases and after a delay of 3 to 11 years in the 14 other cases. There was no particular features in the stage of duration of CLL before diagnosis of cancer. Outcome was particularly unfavorable, 20 deaths, compared with mortality in the other 226 patients (53.5%). These findings, together with the data in the literature, emphasize the importance of careful follow-up in patients with CLL.

Mechanism of GroEL action: productive release of polypeptide from a sequestered position under GroES.

The chaperonin GroEL is a large, double-ring structure that, together with ATP and the cochaperonin GroES, assists protein folding in vivo. GroES forms an asymmetric complex with GroEL in which a single GroES ring binds one end of the GroEL cylinder. Cross-linking studies reveal that polypeptide binding occurs exclusively to the GroEL ring not occupied by GroES (trans). During the folding reaction, however, released GroES can rebind to the GroEL ring containing polypeptide (cis). The polypeptide is held tightly in a proteolytically protected environment in cis complexes, in the presence of ADP. Single turnover experiments with ornithine transcarbamylase reveal that polypeptide is productively released from the cis but not the trans complex. These observations suggest a two-step mechanism for GroEL-mediated folding. First, GroES displaces the polypeptide from its initial binding sites, sequestering it in the GroEL central cavity. Second, ATP hydrolysis induces release of GroES and productive release of polypeptide.

Residues in chaperonin GroEL required for polypeptide binding and release.

Chaperonins are ring-shaped protein complexes that are essential in the cell, mediating ATP-dependent polypeptide folding in a variety of compartments. Recent studies suggest that they function through multiple rounds of binding and release of non-native proteins: with each round of ATP-driven release into the bulk solution, a substrate protein kinetically partitions between folding to the native state or rebinding to another chaperonin molecule. To gain further insight into the mechanism of polypeptide binding and release by the chaperonin GroEL from Escherichia coli, we have undertaken a mutational analysis that relates the functional properties of GroEL to its crystal structure. Our functional tests identify a putative polypeptide-binding site on the inside surface of the apical domain, facing the central channel, consisting of hydrophobic residues. These same residues are essential for binding of the co-chaperonin GroES, which is required for productive polypeptide release. A highly conserved residue, Asp 87, positioned within a putative nucleotide-binding pocket in the top of the equatorial domain, is essential for ATP hydrolysis and polypeptide release.

How is it that microsatellites and random oligonucleotides uncover DNA fingerprint patterns?

Minisatellites, microsatellites, and short random oligonucleotides all uncover highly polymorphic DNA fingerprint patterns in Southern analysis of genomic DNA that has been digested with a restriction enzyme having a 4-bp specificity. The polymorphic nature of the fragments is attributed to tandem repeat number variation of embedded minisatellite sequences. This explains why DNA fingerprint fragments are uncovered by minisatellite probes, but does not explain how it is that they are also uncovered by microsatellite and random oligonucleotide probes. To clarify this phenomenon, we sequenced a large bovine genomic BamHI restriction fragment hybridizing to the Jeffreys 33.6 minisatellite probe and consisting of small and large Sau3A-resistant subfragments. The large Sau3A subfragment was found to have a complex architecture, consisting of two different minisatellites, flanked and separated by stretches of unique DNA. The three unique sequences were characterized by sequence simplicity, that is, a higher than chance occurrence of tandem or dispersed repetition of simple sequence motifs. This complex repetitive structure explains the absence of Sau3A restriction sites in the large Sau3A subfragment, yet provides this subfragment with the ability to hybridize to a variety of probe sequences. It is proposed that a large class of interspersed tracts sharing this complex yet simplified sequence structure is found in the genome. Each such tract would have a broad ability to hybridize to a variety of probes, yet would exhibit a dearth of restriction sites. For each restriction enzyme having 4-bp specificity, a subclass of such tracts, completely lacking the corresponding restriction sites, will be present. On digestion with the given restriction enzyme, each such tract would form a large fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

GroEL-mediated protein folding proceeds by multiple rounds of binding and release of nonnative forms.

The chaperonin GroEL is a ribosome-sized double-ring structure that assists in folding a diverse set of polypeptides. We have examined the fate of a polypeptide during a chaperonin-mediated folding reaction. Strikingly, we find that, upon addition of ATP and the cochaperonin GroES, polypeptide is released rapidly from GroEL in a predominantly nonnative conformation that can be trapped by mutant forms of GroEL that are capable of binding but not releasing substrate. Released polypeptide undergoes kinetic partitioning: a fraction completes folding while the remainder is rebound rapidly by other GroEL molecules. Folding appears to occur in an all-or-none manner, as proteolysis and tryptophan fluorescence indicate that after rebinding, polypeptide has the same structure as in the original complex. These observations suggest that GroEL functions by carrying out multiple rounds of binding aggregation-prone or kinetically trapped intermediates, maintaining them in an unfolded state, and releasing them to attempt to fold in solution.

Primary structure and function of a second essential member of the heterooligomeric TCP1 chaperonin complex of yeast, TCP1 beta.

A role for heterooligomeric TCP1 complex as a chaperonin in the eukaryotic cytosol has recently been suggested both by structural similarities with other chaperonins and by in vitro experiments showing it to mediate ATP-dependent folding of actin, tubulin, and luciferase. Here we present the primary structure of a second subunit of the complex and present genetic and functional analyses. The TCP1 beta amino acid sequence, predicted from the cloned gene, bears 35% identity to TCP1, termed here TCP1 alpha, containing the same highly conserved residues found in the collective sequence of chaperonins. The predicted product was identified as the fastest-migrating species of the TCP1 complex purified from soluble extracts of yeast. The TCP1 beta gene, like TCP1 alpha, is essential. Strains containing lethal disruptions of either gene could not be rescued by additional copies of the other. Spores bearing disruption of either gene germinated as single, large-budded cells. Similarly, large-budded cells were observed following shift to 37 degrees C of strains carrying temperature-sensitive mutations in either TCP1 alpha or TCP1 beta. The arrested cells contained replicated DNA present in single nuclear masses, associated with abnormal tubulin staining patterns, supporting the assertion that mitotic spindle formation and function are impaired. We conclude that TCP1 beta supplies an essential function that partially overlaps with that of TCP1 alpha in acting as a molecular chaperone in tubulin and spindle biogenesis.