👤 Mathew B Dobbs

While initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3' tag digital gene expression (3'DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance. Using 3'DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (P<0.05) and tended to increase expression of HSP90AA1 (P<0.07) but had no effect on expression of SOD1 or CAT. Changes in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene networks involved in embryonic development. It is likely that the increased resistance of morula-stage embryos to heat shock as compared to embryos at earlier stages of development is due in part to developmental acquisition of mechanisms to prevent accumulation of denatured proteins and free radical damage. Show less

Distal arthrogryposis type I (DA1) is a disorder characterized by congenital contractures of the hands and feet for which few genes have been identified. Here we describe a five-generation family with DA1 segregating as an autosomal dominant disorder with complete penetrance. Genome-wide linkage analysis using Affymetrix GeneChip Mapping 10K data from 12 affected members of this family revealed a multipoint LOD(max) of 3.27 on chromosome 12q. Sequencing of the slow-twitch skeletal muscle myosin binding protein C1 (MYBPC1), located within the linkage interval, revealed a missense mutation (c.706T>C) that segregated with disease in this family and causes a W236R amino acid substitution. A second MYBPC1 missense mutation was identified (c.2566T>C)(Y856H) in another family with DA1, accounting for an MYBPC1 mutation frequency of 13% (two of 15). Skeletal muscle biopsies from affected patients showed type I (slow-twitch) fibers were smaller than type II fibers. Expression of a green fluorescent protein (GFP)-tagged MYBPC1 construct containing WT and DA1 mutations in mouse skeletal muscle revealed robust sarcomeric localization. In contrast, a more diffuse localization was seen when non-fused GFP and MYBPC1 proteins containing corresponding MYBPC3 amino acid substitutions (R326Q, E334K) that cause hypertrophic cardiomyopathy were expressed. These findings reveal that the MYBPC1 is a novel gene responsible for DA1, though the mechanism of disease may differ from how some cardiac MYBPC3 mutations cause hypertrophic cardiomyopathy. Show less