Coordinate expression of matrix-degrading proteinases and their activators and inhibitors in bovine skeletal muscle.

Matrix metalloproteinases (MMP) responsible for degradation of connective tissue are found in most tissues. The MMP are regulated at the levels of transcription, zymogen activation by plasmin or membrane-type- (MT) MMP, and control of enzyme activity by tissue inhibitors of metalloproteinases (TIMP). Whole bovine skeletal muscle showed multiple MMP activities on gelatin zymography and also expressed mRNA encoding MMP-1, -2, -9, -14, and -16, tissue inhibitors of metalloproteinase (TIMP)-1, -2, and -3 and plasminogen activator and its receptor. Purified intramuscular fibroblasts and myogenic cell culture derived from satellite cells expressed most or all of these elements. Statistical analysis (n = 35) revealed a strong positive correlation among the mRNA levels of several elements of the MMP system, including MMP-2, MMP-14, TIMP-1, -2, and -3 (r = 0.614 to 0.930, P < 0.0001). Our results provide an extensive profile of an extracellular proteolytic cascade involving MMP in skeletal muscle and suggest that 1) the activation cascades of muscle MMP may be initiated by both plasmin and membrane-type MMP; 2) a group of genes involved in the same "arm" of zymogen activation are coexpressed in this tissue; and 3) skeletal muscle cells, in addition to the intramuscular fibroblasts, express an extensive complement of MMP and related proteins.

Analysis of an enhancer trap expressed in regenerating Drosophila imaginal discs.

In Drosophila, imaginal discs are the undifferentiated larval precursors of the pattern of epidermal and sensory neural cells in each adult segment. Although cell fates are already specified by late third instar, disc fragments can either regenerate or duplicate after growth in culture. The outcome depends on signaling between cells across the healed wound and involves a redeployment of the expression patterns of selector genes and other disc pattern genes. We recently used the enhancer-trap method to screen for such genes that are expressed ectopically at the wound-heal site in imaginal discs undergoing regeneration. Here we report the cloning by plasmid rescue of transcribed sequences adjacent to one such enhancer-trap insertion. Using Northern analysis and in situ hybridization we show that one transcript is expressed in the embryo and in imaginal discs in a pattern similar to that of the enhancer trap. We also, by imprecise excision of the enhancer-trap insertion, generated a series of flanking deletions that were mapped using Southern analysis and complementation.

Growth hormone restriction fragment length polymorphisms that segregate with 42-day live weight of mice.

The known correlation between growth hormone levels and growth rate in a number of species prompted us to examine if polymorphic restriction fragment alleles at the growth hormone locus in mice might be associated with differentiable rates of growth. An F2 population of mice was generated from crosses between a line selected for high 42-day weight and an unselected control line. The original selected and control lines exhibited mean 42-day weights of 30.6 +/- 3.8 and 20.5 +/- 2.6 g, respectively. Since the two lines also differed with respect to the restriction fragments detected by hybridization to a rat growth hormone cDNA probe, an analysis of the F2 generation was carried out to determine whether this polymorphism could be considered a quantitative trait locus for 42-day weight. The results of the analysis indicated that a polymorphic HindIII restriction fragment was correlated (P less than 0.05) with 42-day weight. However, the allele that was positively correlated with weight was the one that was fixed in the original control line, rather than the one from the selected line. While these findings support the potential use of restriction fragment length polymorphisms in quantitative trait evaluation of livestock, they also emphasize the requirement for testing such potential quantitative trait loci in the appropriate genetic background.

Changes in phosphoprotein pattern in Schizosaccharomyces pombe.

A variety of evidence suggests that protein phosphorylation (pp) may be important in cell-cycle control. Phosphorylated proteins from S. pombe have been examined for phosphorylation changes under several conditions: known triggers of the division control (low nitrogen, low phosphate), cell size mutants (WEE1 and CDC2 alleles) and cell cycle mutants (CDC2, CDC10, CDC17, CDC25 alleles). Three major phosphorylated proteins (pp38, pp45 and pp54) showed the greatest response to nutritional shifts. The changes in the phosphorylated states of these proteins correlated with growth rate. Some phosphorylations (e.g. pp53) occurred transiently following a stimulus to cell division suggesting a possible involvement with the division mechanism. An allele-specific alteration of charge was noted for pp45 suggesting that this protein is the product of the CDC2 gene. The wee1-6 phosphoprotein pattern is similar to wild-type indicating that this mutant cell line accurately senses its nutritional environment and that the mutation likely affects the transfer of this information to the division control. Cells blocked by various temperature-sensitive cell cycle mutants did not show an alteration of phosphoprotein pattern.

The relationship of cardiolipin biosynthesis to mitochondrial protein synthesis in Tetrahymena pyriformis.

The synthesis of cardiolipin has been investigated following the inhibition of mitochondrial protein synthesis with chloramphenicol. Quantitative measurements of the amount of cardiolipin in the cell during treatment with chloramphenicol as well as pulse-labelling studies using labelled acetate were carried out. The results show that while whole cell phospholipid biosynthesis is depressed by the treatment (probably a reflection of a general cessation of growth), there is no sign of any preferential effect on cardiolipin synthesis. The data also show that as cells are reduced in size as they approach stationary phase there is a six- to seven-fold loss of total cellular phospholipids; however, the amount of cardiolipin is only reduced by four- to five-fold. There is a preferential conservation of cardiolipin as stationary phase is approached with the mole percent cardiolipin phosphorus in the cell rising from 5-7% to 10-12%.