Fine mapping a major obesity locus (jObes1) using a Berlin Fat Mouse × B6N advanced intercross population.

BACKGROUND/OBJECTIVES: The Berlin Fat Mouse Inbred line 860 is a model for juvenile obesity. Previously, a recessive major effect locus (jObes1) on chromosome 3 between 34 and 44 Mb has been found to be responsible for 39% of the variance of total fat mass at 10 weeks in a (BFMI860 x C57BL/6NCrl) F2 population. The aim of this study was fine mapping of the jObes1 locus. SUBJECTS/METHODS: An advanced intercross line (AIL) was generated from the initial F2 mapping population. Three hundred and forty-four male mice of generation 28 were excessively phenotyped and genotyped using the MegaMuga mouse chip containing 22 164 informative single-nucleotide polymorphisms. Expression of candidate genes was investigated in gonadal adipose tissue, liver and whole brain from mice of different genotype classes. Classical genetic complementation tests were performed to test candidate genes. RESULTS: The high mapping resolution of the AIL reduced the confidence interval for jObes1 from 10 to 0.37 Mb between 36.48 and 36.85 Mb. This region was highly significantly (logarithm (base 10) of odds (LOD) score after Benjamini and Hochberg correction (LOD(BH))>50) associated with total fat mass starting at puberty (6 weeks). Male homozygous carriers of the jObese1 BFMI allele had 3 g more fat than the other genotypes. Surprisingly, this genotype class showed lower body mass until weaning at 3 weeks (LOD(BH)=3.2). The mapped interval contains four genes. Bbs7, the most likely candidate gene that also caused obesity in the complementation test was differentially expressed in all tissues examined, whereas the neighboring cyclin A2 (Ccna2) gene showed differential expression in gonadal adipose tissue. CONCLUSIONS: Using an AIL, the confidence interval for jObes1 could be 27-fold reduced by finding chromosomal recombinations. Although Bbs7 is the most likely obesity gene in the jObes1 region, neighboring genes cannot be entirely excluded. Further examinations are needed to enlighten the mechanism leading to physiological consequences on body mass and fat mass in juvenile animals.

Norovirus antagonism of B-cell antigen presentation results in impaired control of acute infection.

Human noroviruses are a leading cause of gastroenteritis, and so, vaccine development is desperately needed. Elucidating viral mechanisms of immune antagonism can provide key insight into designing effective immunization platforms. We recently revealed that B cells are targets of norovirus infection. Because noroviruses can regulate antigen presentation by infected macrophages and B cells can function as antigen-presenting cells, we tested whether noroviruses regulate B-cell-mediated antigen presentation and the biological consequence of such regulation. Indeed, murine noroviruses could prevent B-cell expression of antigen presentation molecules and this directly correlated with impaired control of acute infection. In addition to B cells, acute control required MHC class I molecules, CD8+ T cells, and granzymes, supporting a model whereby B cells act as antigen presenting cells to activate cytotoxic CD8+ T cells. This immune pathway was active prior to the induction of antiviral antibody responses. As in macrophages, the minor structural protein VP2 regulated B-cell antigen presentation in a virus-specific manner. Commensal bacteria were not required for the activation of this pathway and ultimately only B cells were required for the clearance of viral infection. These findings provide new insight into the role of B cells in stimulating antiviral CD8+ T-cell responses.

High-fat diet leads to a decreased methylation of the Mc4r gene in the obese BFMI and the lean B6 mouse lines.

The melanocortin-4 receptor (Mc4r) plays an important role in body-weight regulation. This study examines the methylation status and expression levels of the Mc4r gene in response to a standard and a high-fat diet in the obese Berlin fat mouse inbred (BFMI) line and the lean C57BL/6NCrl (B6) line of Mus musculus. The methylation status of CpG sites located within the Mc4r exon was analyzed by bisulfite genomic sequencing of genomic DNA of brain tissues, and gene expression analysis was performed by real-time PCR. In both lines, the methylation of CpGs 1-8 (near the transcription start) was lower than methylation of CpGs 9-16 (located towards the end of the selected amplicon). On the standard diet, the methylation status did not differ between the lines. In response to high-fat diet, methylation of the CpGs near the transcription start was decreased in both lines. The Mc4r gene expression, however, was only marginally increased in BMFI mice, whereas there was no change in B6 mice. The results suggest that a long-term high-fat diet might have an effect on the methylation status of the Mc4r gene. However, the effect of methylation on Mc4r expression seems to be a variable compensated by other regulating factors in a line-specific manner.

The yeast retrotransposons Ty1 and Ty3 require the RNA Lariat debranching enzyme, Dbr1p, for efficient accumulation of reverse transcripts.

A mutant screen has been initiated to identify host genes important for the replication of retrotransposons in Saccharomyces cerevisiae. Two mutants were identified that undergo Ty1 and Ty3 transposition at <10% of the wild-type frequency. Both these mutants have deficiencies in the accumulation of full-length Ty1 and Ty3 cDNAs, although Ty proteins (including reverse transcriptase) accumulate at wild-type levels. The DBR1 gene, encoding the yeast debranching enzyme, complements both mutants. This suggests that Dbr1p is important for either reverse transcription or the stability of Ty cDNA, roles that have not been previously reported for this protein. The deficiency in accumulation of Ty cDNAs in dbr1 mutants is apparent when engineered Ty elements are expressed for short time periods (6-10 h) but is not apparent following long expression periods (>24 h).

Cell cycle control of reverse transcriptase activity for the yeast retrotransposon Ty3.

Retroviruses and retrotransposons depend on their host cells to complete their replication cycles. In many cases, the viral step of reverse transcription is blocked when host cells are arrested in their cell cycle and this block is only released when the host cell resumes division. It has previously been shown that a retrotransposon found in Saccharomyces cerevisiae, Ty3, is unable to produce full-length double-stranded DNA, the product of reverse transcription, when the host cells are arrested in G1. In this study we show that, although Ty3 viruslike particles are produced at equivalent levels in arrested and nonarrested cells, the reverse transcriptase enzyme is inactive in arrested cells. The enzyme activity is restored when the arrested cells are permitted to resume cell division. These data suggest that a host factor regulates the activity of Ty3 reverse transcriptase in the cell cycle, which represents a novel cellular control of retroelements.