Maize rough sheath2 and its Arabidopsis orthologue ASYMMETRIC LEAVES1 interact with HIRA, a predicted histone chaperone, to maintain knox gene silencing and determinacy during organogenesis

Plant shoots are characterized by indeterminate growth resulting from the action of a population of stem cells in the shoot apical meristem (SAM). Indeterminacy within the SAM is specified in part by the class I knox homeobox genes. The myb domain proteins rough sheath2 (RS2) and ASYMMETRIC LEAVES1 (AS1) from maize (Zea mays) and Arabidopsis thaliana, respectively, are required to establish determinacy during leaf development. These proteins are part of a cellular memory system that in response to a stem cell-derived signal keeps knox genes in an off state during organogenesis. Here, we show that RS2/AS1 can form conserved protein complexes through interaction with the DNA binding factor ASYMMETRIC LEAVES2, a predicted RNA binding protein (RIK, for RS2-Interacting KH protein), and a homologue of the chromatin-remodeling protein HIRA. Partial loss of HIRA function in Arabidopsis results in developmental defects comparable to those of as1 and causes reactivation of knox genes in developing leaves, demonstrating a direct role for HIRA in knox gene repression and the establishment of determinacy during leaf formation. Our data suggest that RS2/AS1 and HIRA mediate the epigenetic silencing of knox genes, possibly by modulating chromatin structure. Components of this process are conserved in animals, suggesting the possibility that a similar epigenetic mechanism maintains determinacy during both plant and animal development.     

Evidence for the presence of a CmuA methyltransferase pathway in novel marine methyl halide-oxidizing bacteria

Marine bacteria that oxidized methyl bromide and methyl chloride were enriched and isolated from seawater samples. Six methyl halide-oxidizing enrichments were established from which 13 isolates that grew on methyl bromide and methyl chloride as sole sources of carbon and energy were isolated and maintained. All isolates belonged to three different clades in the Roseobacter group of the alpha subdivision of the Proteobacteria and were distinct from Leisingera methylohalidivorans, the only other identified marine bacterium that grows on methyl bromide as sole source of carbon and energy. Genes encoding the methyltransferase/corrinoid-binding protein CmuA, which is responsible for the initial step of methyl chloride oxidation in terrestrial methyl halide-oxidizing bacteria, were detected in enrichments and some of the novel marine strains. Gene clusters containing cmuA and other genes implicated in the metabolism of methyl halides were cloned from two of the isolates. Expression of CmuA during growth on methyl halides was demonstrated by analysis of polypeptides expressed during growth on methyl halides by SDS-PAGE and mass spectrometry in two isolates representing two of the three clades. These findings indicate that certain marine methyl halide degrading bacteria from the Roseobacter group contain a methyltransferase pathway for oxidation of methyl bromide that may be similar to that responsible for methyl chloride oxidation in Methylobacterium chloromethanicum. This pathway therefore potentially contributes to cycling of methyl halides in both terrestrial and marine environments.     

Summary of the animal homologue section of HLDA8

Development of reagents against leukocyte differentiation antigens in veterinary immunology is slower compared to humans and mice. Cross-reactivity studies with monoclonal antibodies (mAb) generated against human molecules represent an excellent approach for the detection of new reagents for the minor characterised species. Three hundred seventy-seven commercially available mAb from different companies were tested for their reactivity with cells from 17 species--including non-human primates, ruminants, swine, horse, carnivores, rabbit, guinea pig, chicken and fish. In a first round of testing by flow cytometry (FCM) 182 mAb showed reactivity with atleast one of the species described above. Most of the cross-reactivity was found against non-human primate leukocytes, but also species in evolutionarily more distant from humans showed in some cases a clear staining pattern in flow cytometry (FCM). In a second round these FCM-results were confirmed by molecular analyses, by immunoprecipitation studies and analyses on transfectants. Interesting was the broad species-overlapping reactivity of mAb directed against CD9 (11 out of 17 species), CD11a (11/17), CD14 (11/17), CD18 (13/17), CD21 (7/17), CD29 (10/17), CD44 (13/17), CD45 (9/17), CD47 (10/17), and CD49d (13/17), CD61 (6/17), CD86 (7/17), CD91 (5/17), and CD172a (10/17), indicating evolutionary highly conserved epitopes on these surface molecules. Our results suggest the suitability of crossreactive mAb for the animal model studies. Moreover, these findings contribute to our understanding of the evolution of the immune system.     

Numbers of antral follicles during follicular waves in cattle: evidence for high variation among animals, very high repeatability in individuals, and an inverse association with serum follicle-stimulating hormone concentrations

The extent, causes, and physiological significance of the variation in number of follicles growing during ovarian follicular waves in human beings and cattle are unknown. Therefore, the present study examined the variability and repeatability in numbers of follicles 3 mm or greater in diameter during the follicular waves in bovine estrous cycles, and we determined if the variation in number of follicles during waves was associated with alterations in secretion of FSH, estradiol, inhibin, and insulin-like growth factor I (IGF-I). Dairy cattle were subjected to twice-daily ultrasound analysis to count total number of antral follicles 3 mm or greater in diameter throughout 138 different follicular waves. In another study, blood samples were taken at frequent intervals from cows that consistently had low or very high numbers of follicles during waves and were subjected to immunoassays. Results indicate the following: First, despite an approximately sevenfold variation in number of follicles during waves among animals and marked differences in age, stage of lactation, and season of the year, a very highly repeatable (0.95) number of follicles 3 mm or greater in diameter is maintained during the ovulatory and nonovulatory follicular waves of individuals. Second, variation in number of follicles 3 mm or greater in diameter during waves and the inverse association of number of follicles during waves with FSH are not directly explained by alterations in the patterns of secretion of estradiol, inhibin, or IGF-I. Third, ovarian ultrasound analysis can be used reliably by investigators to identify cattle that consistently have low or high numbers of follicles during waves, thus providing a novel experimental model to determine the causes and physiological significance of the high variation in antral follicle number during follicular waves among single-ovulating species, such as cattle or humans.     

Apolipoprotein AI could be a significant determinant of epithelial integrity in rainbow trout gill cell cultures: a study in functional proteomics

The freshwater fish gill forms a barrier against an external hypotonic environment. By culturing rainbow trout gill cells on permeable supports, as intact epithelia, this study investigates barrier property mechanisms. Under symmetrical conditions the apical and basolateral epithelial surfaces contact cell culture media. Replacing apical media with water, to generate asymmetrical conditions (i.e. the situation encountered by the freshwater gill), rapidly increases transepithelial resistance (TER). Proteomic analysis revealed that this is associated with enhanced expression of pre-apolipoprotein AI (pre-apoAI). To test the physiological relevance, gill cells were treated with a dose of 50 microg ml(-1) human apolipoprotein (apoAI). This was found to elevate TER in those epithelia which displayed a lower TER prior to apoAI treatment. These results demonstrate the action of apoAI and provide evidence that the rainbow trout gill may be a site of apoAI synthesis. TER does not differentiate between the trans-cellular (via the cell membrane) and para-cellular (via intercellular tight junctions) pathways. However, despite the apoAI-induced changes in TER, para-cellular permeability (measured by polyethylene glycol efflux) remained unaltered suggesting apoAI specifically reduces trans-cellular permeability. This investigation combines proteomics with functional measurements to show how a proteome change may be associated with freshwater gill function.     

Towards a mutant map of the mouse – new models of neurological, behavioural, deafness, bone, renal and blood disorders

With the completion of the first draft of the human genome sequence, the next major challenge is assigning function to genes. One approach is genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes of interest and subsequent mapping and identification of the mutated genes in question. We (a consortium made up of GlaxoSmithKline, the MRC Mammalian Genetics Unit and Mouse Genome Centre, Harwell, Imperial College, London, and the Royal London Hospital) have used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for use as animal models of human disease and for gene function assignment (Nolan et al., 2000). As of 2003, 35,000 mice have been produced to date in a genome-wide screen for dominant mutations and screened using a variety of screening protocols. Nearly 200 mutants have been confirmed as heritable and added to the mouse mutant catalogue and, overall, we can extrapolate that we have recovered over 700 mutants from the screening programme. For further information on the project and details of the data, see http://www.mgu.har.mrc.ac.uk/mutabase.     

Prevalence of<Emphasis Type="Italic"> BRCA1</Emphasis> and<Emphasis Type="Italic"> BRCA2</Emphasis> mutations among clinic-based African American families with breast cancer

To define the prevalence and relative contributions of BRCA1 and BRCA2 mutations among African American families with breast cancer, we analyzed 28 DNA samples from patients identified through two oncology clinics. The entire coding regions of BRCA1 and BRCA2 were screened by protein truncation test, heteroduplex analysis, or single-stranded conformation polymorphism followed by DNA sequencing of variant bands. Deleterious protein-truncating BRCA1 and BRCA2 mutations were identified in five patients or 18% of the entire cohort. Only 8% (1 of 13) of women with a family history of breast cancer, but no ovarian cancer, had mutations. The mutation rates were higher for women from families with a history of breast cancer and at least one ovarian cancer (three of six, 50%). One woman with a family history of undocumented cancers was also found to carry a deleterious mutation in BRCA2. The spectrum of mutations was unique in that one novel BRCA1 mutation (1625del5) and three novel BRCA2 mutations (1536del4, 6696delTC, and 7795delCT) were identified. No recurrent mutations were identified in this cohort, although one BRCA2 (2816insA) mutation had been previously reported. In addition, two BRCA1 and four BRCA2 missense mutations of unknown significance were identified, one of which was novel. Taken together with our previous report on recurrent mutations seen in unrelated families, we conclude that African Americans have a unique mutation spectrum in BRCA1 and BRCA2 genes, but recurrent mutations are likely to be more widely dispersed and therefore not readily identifiable in this population.