Physical map of a 1.5 mb region on 12p11.2 harbouring a synpolydactyly associated chromosomal breakpoint.
Synpolydactyly (SPD) is a rare malformation of the distal limbs known to be caused by mutations in HOXD13. We have previously described a complex form of SPD associated with synostoses in three members of a Belgian family, which co-segregates with a t(12;22)(p11.2;q13.3) chromosomal translocation. The chromosome 12 breakpoint of this translocation maps to 12p11.2 between markers D12S1034 and D12S1596. Here we show that a mutation in the HOXD13 gene is not responsible for the phenotype, and present a physical map of the region around the 12p11.2 breakpoint. Starting from D12S1034 and D12S1596, we have established a contig approximately 1.5 Mb in length, containing 13 YAC clones, 16 BAC clones, and 11 cosmid clones. FISH analysis shows that cosmid LL12NCO1-149H4 maps across the breakpoint, and Southern blot experiments using fragments of this cosmid as probes identify a rearranged BamHI fragment in the patients carrying the translocation. A search for expressed sequences within the contig have so far revealed one CpG island, seven anonymous ESTs and three previously characterised genes, DAD-R, KRAG and HT21, all of which were found not to be directly disrupted by the translocation. The gene represented by EST R72964 was found to be disrupted by the translocation. These findings lay the groundwork for further efforts to characterise a gene critical for normal distal limb development that is perturbed by this translocation. (+info)
Granulation rescue and developmental marking of juxtaglomerular cells using "piggy-BAC" recombination of the mouse ren locus.
Mice lacking a functional Ren-1(d) gene exhibit a complete lack of renal juxtaglomerular cell granulation and atypical macula densa morphology. Transgenic mice carrying a 145-kilobase BAC clone encompassing the Ren-1(d) and Ren-2 loci were generated, characterized, and backcrossed with Ren-1(d-/-) mice. Homozygous Ren-1(d)-null mice expressing the BAC clone exhibited complete restoration of normal renal structure. Homologous recombination in Escherichia coli was used to generate a modified version of the BAC clone, in which an IRESbeta-geo cassette was inserted specifically into the Ren-1(d) gene. When introduced into the germline, the modified clone provided a marker for juxtaglomerular cell differentiation and beta-geo was expressed appropriately in juxtaglomerular cells throughout development. Parallel backcross experiments onto the Ren-1(d)-null background demonstrated that the juxtaglomerular cells expressed the modified Ren-1(d) locus in the absence of regranulation. These data demonstrate that the nongranulated cells constitute bona fide juxtaglomerular cells despite their altered morphology, that overexpression of renin-2 cannot compensate for the loss of renin-1(d), and that primary structural differences between the two isoforms are responsible for the differences in granulation. The use of BAC modification as part of functional complementation studies illustrates the potential for in vivo molecular dissection of key physiological mechanisms. (+info)
Effects of 2-G exposure on temperature regulation, circadian rhythms, and adiposity in UCP2/3 transgenic mice.
Altered ambient force environments affect energy expenditure via changes in thermoregulation, metabolism, and body composition. Uncoupling proteins (UCPs) have been implicated as potential enhancers of energy expenditure and may participate in some of the adaptations to a hyperdynamic environment. To test this hypothesis, this study examined the homeostatic and circadian profiles of body temperature (T(b)) and activity and adiposity in wild-type and UCP2/3 transgenic mice exposed to 1 and 2 G. There were no significant differences between the groups in the means, amplitudes, or phases of T(b) and activity rhythms at either the 1- or 2-G level. Percent body fat was significantly lower in transgenic (5.2 +/- 0. 2%) relative to the wild-type mice (6.2 +/- 0.1%) after 2-G exposure; mass-adjusted mesenteric and epididymal fat pads in transgenic mice were also significantly lower (P < 0.05). The data suggest that 1) the actions of two UCPs (UCP2 and UCP3) do not contribute to an altered energy balance at 2 G, although 2) UCP2 and UCP3 do contribute to the utilization of lipids as a fuel substrate at 2 G. (+info)
Comparative fluorescence in situ hybridization mapping of a 431-kb Arabidopsis thaliana bacterial artificial chromosome contig reveals the role of chromosomal duplications in the expansion of the Brassica rapa genome.
Comparative genome studies are important contributors to our understanding of genome evolution. Most comparative genome studies in plants have been based on genetic mapping of homologous DNA loci in different genomes. Large-scale comparative physical mapping has been hindered by the lack of efficient and affordable techniques. We report here the adaptation of fluorescence in situ hybridization (FISH) techniques for comparative physical mapping between Arabidopsis thaliana and Brassica rapa. A set of six bacterial artificial chromosomes (BACs) representing a 431-kb contiguous region of chromosome 2 of A. thaliana was mapped on both chromosomes and DNA fibers of B. rapa. This DNA fragment has a single location in the A. thaliana genome, but hybridized to four to six B. rapa chromosomes, indicating multiple duplications in the B. rapa genome. The sizes of the fiber-FISH signals from the same BACs were not longer in B. rapa than those in A. thaliana, suggesting that this genomic region is duplicated but not expanded in the B. rapa genome. The comparative fiber-FISH mapping results support that chromosomal duplications, rather than regional expansion due to accumulation of repetitive sequences in the intergenic regions, played the major role in the evolution of the B. rapa genome. (+info)
Computational and experimental characterization of physically clustered simple sequence repeats in plants.
The type and frequency of simple sequence repeats (SSRs) in plant genomes was investigated using the expanding quantity of DNA sequence data deposited in public databases. In Arabidopsis, 306 genomic DNA sequences longer than 10 kb and 36,199 EST sequences were searched for all possible mono- to pentanucleotide repeats. The average frequency of SSRs was one every 6.04 kb in genomic DNA, decreasing to one every 14 kb in ESTs. SSR frequency and type differed between coding, intronic, and intergenic DNA. Similar frequencies were found in other plant species. On the basis of these findings, an approach is proposed and demonstrated for the targeted isolation of single or multiple, physically clustered SSRs linked to any gene that has been mapped using low-copy DNA-based markers. The approach involves sample sequencing a small number of subclones of selected randomly sheared large insert DNA clones (e.g., BACs). It is shown to be both feasible and practicable, given the probability of fortuitously sequencing through an SSR. The approach is demonstrated in barley where sample sequencing 34 subclones of a single BAC selected by hybridization to the Big1 gene revealed three SSRs. These allowed Big1 to be located at the top of barley linkage group 6HS. (+info)
Two translocations of chromosome 15q associated with dyslexia.
Developmental dyslexia is characterised by difficulties in learning to read. As reading is a complex cognitive process, multiple genes are expected to contribute to the pathogenesis of dyslexia. The genetics of dyslexia has been a target of molecular studies during recent years, but so far no genes have been identified. However, a locus for dyslexia on chromosome 15q21 (DYX1) has been established in previous linkage studies. We have identified two families with balanced translocations involving the 15q21-q22 region. In one family, the translocation segregates with specific dyslexia in three family members. In the other family, the translocation is associated with dyslexia in one family member. We have performed fluorescence in situ hybridisation (FISH) studies to refine the position of the putative dyslexia locus further. Our results indicate that both translocation breakpoints on 15q map within an interval of approximately 6-8 Mb between markers D15S143 and D15S1029, further supporting the presence of a locus for specific dyslexia on 15q21. (+info)
Cloning of a type I cytokine receptor most related to the IL-2 receptor beta chain.
We have identified a type I cytokine receptor, which we have termed novel interleukin receptor (NILR), that is most related to the IL-2 receptor beta chain (IL-2Rbeta) and physically adjacent to the IL-4 receptor alpha chain gene on chromosome 16. NILR mRNA is most highly expressed in thymus and spleen, and is induced by phytohemagglutinin in human peripheral blood mononuclear cells. NILR protein was detected on human T cell lymphotropic virus type I-transformed T cell lines, Raji B cells, and YT natural killer-like cells. Artificial homodimerization of the NILR cytoplasmic domain confers proliferation to Ba/F3 murine pro-B cells but not to 32D myeloid progenitor cells or CTLL-2 murine helper T cells. In these latter cells, heterodimerization of IL-2Rbeta and the common cytokine receptor gamma chain (gamma(c)) cytoplasmic domains allows potent proliferation, whereas such heterodimerization of NILR with gamma(c) does not. This finding suggests that NILR has signaling potential but that a full understanding of its signaling partner(s) is not yet clear. Like IL-2Rbeta, NILR associates with Jak1 and mediates Stat5 activation. (+info)
MSK1 is required for CREB phosphorylation in response to mitogens in mouse embryonic stem cells.
Mouse embryonic stem (ES) cells homozygous for disruption of the MSK1 gene had no detectable MSK1 activity. However, their activators (extracellular signal related kinase (ERK)1/ERK2) were stimulated normally in mitogen- and stress-activated protein kinase (MSK)1-/- and wild type cells in response to tetradecanoylphorbol acetate (TPA) and epidermal growth factor (EGF). TPA and EGF induced the phosphorylation of cyclic AMP-responsive element binding protein (CREB) at Ser-133 and ATF1 at Ser-63 in wild type cells and this was abolished by inhibition of the mitogen-activated protein kinase cascade. In contrast, the TPA- and EGF-induced phosphorylation of CREB/ATF1 was barely detectable in MSK1-/- cells. However, basal and forskolin-induced phosphorylation was similar, indicating that the MSK1 'knockout' did not prevent CREB phosphorylation by cyclic AMP-dependent protein kinase. Thus MSK1 is required for CREB and ATF1 phosphorylation after mitogenic stimulation of ES cells. (+info)