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Bovine mastitis caused by Streptococcus agalactiaeis 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. agalactiaein raw milk. The specificity of the PCR reaction is based on unique S. agalactiaeDNA 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. agalactiaein 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 1ml 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.

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.

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 interspered 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. The largest fragments would be those that contained one or more long minisatellite tracts. Some of these large fragments would be highly polymorphic by virtue of the included minisatellite sequences; by virtue of their complex structure, all would be capable of hybridizing to a wide variety of probes, uncovering a DNA fingerprint pattern.

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.

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction-fragment-length polymorphism allele (RFLP) of the telomeric alpha-globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t-haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.

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 sequencing 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.

AbstractMilk contains a number of micro-components (“functional foods”) having specific health promoting activities. Important among these compounds are the conjugated linoleic acids (CLAs) consistingof a mixture of isomers of C18:2 fatty acid with conjugated doublebonds. The predominant form in milk fat is the cis-9,trans-11isomer, accounting for 75 to 90% of the total CLA content. Biomedical studies with animal models have demonstrated a variety of preventive health effects from CLA including anticarcinogenic, antiatherogenic, antiobesity, immune system enhancement and antidiabetic benefits. Linolenic acid does not form CLA as an intermediate in rumen biohydrogenation, but it does form vaccenic acid (C18:1,t11) that enters the bloodstream from the rumen, through the abomasum and intestine, and is transported to the mammary gland. Several investigations using direct and indirect approaches established that endogenous synthesis of CLA via ∆9-desaturase action on vaccenic acid (VA, C18:1,t11) is the predominant source of the CLA found in milk fat across a range of diets. Thus, rumen VA production and mammary tissue ∆9-desaturase (D9D) are of key importance in determining the CLA content of milk fat. Mammary tissue D9D activity is measured indirectly as the milk desaturase index (DI), calculated as the ratio of milk CLA to the sum of milk CLA and VA. The study is centered on the Italian Brown Swiss and Israeli Holstein dairy cattle populations. Objectives of the project are: i) to identify environmental and physiological factors (e.g., herd, season, parity and stage of lactation) affecting milk CLA content and DI, and determine appropriate correction factors, if needed; ii) to estimate heritability of milk CLA content and DI in the study populations; iii) to implement a total genome scan for QTL affecting milk CLA VA content and DI in the two study populations. using a selective DNA pooling approach based on milk samples for the initial scan, followed by individual genotyping to confirm suggestive QTL regions. For each of the studied populations,, the research includes determination of CLA, VA and DI for more than 2500 individuals of five large sire half-sib families (for the genome scan) and an additional 400 individuals of 20 small sire half-sib families for the heritability analysis. Milk fat is extracted and transmethylated according to Chouinard et al.(1999). Fatty acid methyl esters are analyzed by GC-FID with a highly polar 100 m SP-2560 column, using GLC-60 supplemented with CLA and VA as a standard. Based on the results obtained to date for the Italian Brown Swiss population, the mean (fatty acid composition over the total fatty acid) ±SD( minimum and maximum) are respectively: VA, 1.97±0.77 (0.63 - 5.40); CLA, 0.63±.0.47 (0.35 - 5.31); DI, 0.25±0.097 (0.063 - 0.64). The means for VA and CLA content found are similar to those reported by other studies (control treatment) and reviewed by Collomb et al(2006). The slightly larger variability here reported, anyhow within the limit of values found in literature, is possibly affected by the wide farming conditions of the individuals sampled.

Southern blots of genomic DNA from a variety of species digested by restriction endonucleases having a four-bp specificity, were probed with a bovine genomic clone consisting of seven tandem poly-TG stretches separated by a 29bp linker sequence. Highly variable DNA ‘fingerprint’ patterns were obtained in chicken, sheep, and horse, moderately variable DNA ‘fingerprints’ in mouse and man, and a monomorphic pattern in Drosophila. In chicken, horse and man a (TG)10 synthetic oligonucleotide probe gave results identical to those given by the bovine probe. Furthermore, in chicken the DNA fingerprint variation showed typical Mendelian inheritance and differed from the fingerprints obtained with Jeffreys 33.6 and M13 minisatellite probes. Thus, for a variety of vertebrate species, poty-TG-containing probes can uncover useful genetic variation.