Genetic basis for the biosynthesis of methylglucose lipopolysaccharides in Mycobacterium tuberculosis

Mycobacteria produce two unusual polymethylated polysaccharides, the 6-O-methylglucosyl-containing lipopolysaccharides (MGLP) and the 3-O-methylmannose polysaccharides, which have been shown to regulate fatty acid biosynthesis in vitro. A cluster of genes dedicated to the synthesis of MGLP was identified in Mycobacterium tuberculosis and Mycobacterium smegmatis. Overexpression of the putative glycosyltransferase gene Rv3032 in M. smegmatis greatly stimulated MGLP production, whereas the targeted disruption of Rv3032 in M. tuberculosis and that of the putative methyltransferase gene MSMEG2349 in M. smegmatis resulted in a dramatic reduction in the amounts of MGLP synthesized and in the accumulation of precursors of these molecules. Disruption of Rv3032 also led to a significant decrease in the glycogen content of the tubercle bacillus, indicating that the product of this gene is likely to be involved in the elongation of more than one alpha-(1-->4)-glucan in this bacterium. Results thus suggest that Rv3032 encodes the alpha-(1-->4)-glucosyltransferase responsible for the elongation of MGLP, whereas MSMEG2349 encodes the O-methyltransferase required for the 6-O-methylation of these compounds.     

Impact of bovine somatotropin on ranking for genetic value of dairy sires for milk yield traits and somatic cell score

Records of Holstein cows were used to examine how different models account for the effect of bovine somatotropin (bST) treatment on genetic evaluation of dairy sires for yield traits and somatic cell score. Data set 1 included 65,720 first-lactation records. Set 2 included 50,644 second-lactation records. Set 3 included 45,505 records for lactations three, four and five. Estimated breeding values (EBV) of sires were with three different animal models. With Model 1, bST administration was ignored. With Model 2, bST administration was used as a fixed effect. With Model 3, administration of bST was used to define the contemporary group (herd-year-month of calving-bST). Correlations for EBV of 1,366 sires with treated daughters between pairs of the three models were calculated for milk, fat and protein yields and somatic cell score for the three data sets. Correlations for EBV of sires between pairs of models for all traits ranged from 0.971 to 0.999. Fractions of sires with bST-treated progeny selected in common (top 10 to 15%) were 0.94 and usually greater for all pairs of models for all traits and parities. For this study, the method of statistical adjustment for bST treatment resulted in a negligible effect on genetic evaluations of sires when some daughters were treated with bST and suggests that selection of sires to produce the next generation of sires and cows might not be significantly affected by how the effect of bST is modeled for prediction of breeding values for milk, fat and protein yields and somatic cell score.     

Longitudinal glia in the fly CNS: pushing the envelope on glial diversity and neuron-glial interactions

Interactions between neurons and glial cells are crucial for nervous system development and function in all complex organisms, and many functional, morphological and molecular features of glia are well conserved among species. Here we review studies of the longitudinal glia (LG) in the Drosophila CNS. The LG envelop the neuropil in a membrane sheath, and have features resembling both oligodendrocytes and astrocytes. Because of their unique lineage, morphology and molecular features, the LG provide an excellent model to study the genetic mechanisms underlying glial subtype differentiation and diversity, glial morphogenesis and neuron-glial interactions during development. In addition, they are proving useful in understanding how glial cells maintain ion and neurotransmitter homeostasis and protect neurons from environmental insult.     

Computing numerator relationships between any pair of animals

We describe a simple method to compute the numerator relationship between any or all pairs of animals in the numerator relationship matrix. The method depends on output of the MTDFNRM program from the MTDFREML set of programs. An option of the MTDFNRM program creates a file that includes the inbreeding coefficient for each animal. The method also makes use of how the inbreeding coefficient is traditionally calculated: one-half of the relationship between the animal's parents. To obtain the numerator relationship between any pair of animals, the original pedigree file is augmented with a dummy animal with an identification number (ID) greater than for any animal in the original pedigree file. The ID of the pair of animals for which the relationship is wanted is included as parents. MTDFNRM is then run with the option to create a file of ordered and original IDs for animals and their parents along with the inbreeding coefficients. We then multiply the inbreeding coefficient for a dummy animal by two to obtain the numerator relationship between the two animals designated as parents.     

Production of replication-defective retrovirus by transient transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFRalpha, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80 degrees C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.     

Development of a sensitive liquid chromatography method coupled with a tandem mass spectrometric detection for the clinical analysis of vinflunine and 4-O-deacetyl vinflunine in blood, urine and faeces

A sensitive and specific liquid chromatographic method coupled with tandem mass spectrometric detection was set up and fully validated for the simultaneous quantification of vinflunine (VFL) and its pharmacologically active metabolite, 4-O-deacetyl vinflunine (DVFL). The two compounds, as well as vinblastine (used as internal standard), were deproteinised from blood and faeces, analysed on a cyano type column and detected on a Micromass Quattro II system in the positive ion mode after ionisation using an electrospray ion source. In blood, linearity was assessed up to 200 ng/ml for vinflunine and 100 ng/ml for 4-O-deacetyl vinflunine. The lower limit of quantification was validated at 250 pg/ml for both compounds. In other biological media, the linearity was assessed within the same range; the limit of quantification was adjusted according to the expected concentration levels of each compound. This method was first developed in order to identify the structures and to elucidate the metabolic pathway of vinflunine. Thanks to its high sensitivity and specificity, the method has enabled the quantification of vinflunine and 4-O-deacetyl vinflunine in blood at trace levels, and has contributed to the knowledge of vinflunine metabolism by monitoring up to 10 metabolites.     

Mrc1, a non-essential DNA replication protein,is required for telomere end protection following loss of capping by Cdc13, Yku or telomerase

Proteins involved in telomere end protection have previously been identified. In Saccharomyces cerevisiae, Cdc13, Yku and telomerase, mainly, prevent telomere uncapping, thus providing telomere stability and avoiding degradation and death by senescence. Here, we report that in the absence of Mrc1, a component of the replication forks, telomeres of cdc13 or yku70 mutants exhibited increased degradation, while telomerase-negative cells displayed accelerated senescence. Moreover, deletion of MRC1 increased the single-strandedness of the telomeres in cdc13-1 and yku70Δ mutant strains. An mrc1 deletion strain also exhibited slight but stable telomere shortening compared to a wild-type strain. Loss of Mrc1’s checkpoint function alone did not provoke synthetic growth defects in combination with the cdc13-1 mutation. Combinations between the cdc13-1 mutation and deletion of either TOF1 or PSY2, coding for proteins physically interacting with Mrc1, also resulted in a synthetic growth defect. Thus, the present data suggest that non-essential components of the DNA replication machinery, such as Mrc1 and Tof1, may have a role in telomere stability in addition to their role in fork progression.