Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae.

Genes encoding tuna, pigeon, and horse cytochromes c were constructed with synthetic oligodeoxyribonucleotides having preferred codons and portions of the iso-1-cytochrome c-encoding gene from the yeast Saccharomyces cerevisiae. The genes were ligated into an expression vector, which contains the normal 5'- and 3'-untranslated regions of the yeast iso-1-cytochrome c gene, and were integrated in single copy into the chromosome. Yeast strains were also constructed with multiple integrated copies of the pigeon gene. The heterologous and normal mRNA levels of the single-copy strains were equivalent. Although the N-terminal methionines were completely cleaved in the heterospecific proteins, the levels of trimethylation of Lys72 and acetylation of N-terminal glycines ranged from 39-78% and 10-70%, respectively. Horse cytochrome c was produced at a nearly normal level, whereas the pigeon and tuna cytochromes c were produced at approx. 40% of the normal levels. The levels of the cytochromes c and growth of the mutant yeast strains indicated that the heterospecific cytochromes c had approx. 50% specific activity in vivo.     

The role of the epidermal growth factor-1 and hydrophobic stack domains of human factor IX in binding to endothelial cells.

To determine the function and specificity in factor IX of the first epidermal growth factor (EGF)-like domain and the eight-amino acid hydrophobic stack encoded by exon C (residues 39-46), these domains were replaced by the corresponding polypeptide regions of factor X and chimeric proteins were produced in human embryo kidney cells. Both chimeras were activated by factor XIa at a rate similar to plasma factor IX and exhibited calcium-dependent fluorescence quenching similar to plasma factor IX. Both chimeras competed equally for binding to the endothelial cell receptor. Our findings make it unlikely that the first EGF-like domain or the hydrophobic stack of factor IX are responsible for the specific binding of factor IX to its endothelial cell receptor.     

Efficient expression of a Trypanosoma cruzi antigen in Escherichia coli and Staphylococcus aureus and its rapid purification

A Trypanosoma cruzi antigen gene was closed into a fusion vector based on the IgG binding domain of Staphylococcus aureus protein A. This vector transformed into Escherichia coli or Staphylococcus aureus and produced about 12 mg fusion protein/l culture. In E. coli, the product remained intracellular while in S. aureus it was excreted into the growth medium. The hybrid protein was purified by IgG Sepharose affinity chromatography. The presence of a cleavage site for enterokinase between protein A and the T. cruzi antigen in the fusion protein allowed the efficient release of the unfused antigen by enzymatic treatment. Further affinity chromatography through IgG Sepharose resulted in the production of the T. cruzi antigen free of protein A.The authors are with the Department of Molecular Genetics, BioSidus S.A., Constitución 4234, 1254, Buenos Aires, Argentina.     

Depalmitylation with hydroxylamine alters the functional properties of rhodopsin

Rhodopsin, the photosensitive protein found in rod photoreceptors, has two covalently attached palmitates that are thought to anchor a portion of the C terminus to the disc membrane, forming a fourth cytoplasmic loop. Using hydroxylamine (NH2OH) to cleave the thioester linkage, we have characterized the effect of depalmitylation on certain functional properties of rhodopsin. Treatment of rod outer segment membranes (prepared from rat retinas previously labeled in vivo with [3H]palmitate) with 1 M NH2OH typically removed greater than or equal to 75% of the [3H]palmitate initially bound to rhodopsin. Spectrophotometry of rod outer segment membranes that had been treated with 1 M NH2OH indicated preservation of 85% of the native rhodopsin and no effect on the shape of the absorbance spectrum of rhodopsin. In vivo labeled rhodopsin that had been treated with 1 M NH2OH did not reincorporate free endogenous [3H] palmitate over a 2-h incubation period. Both NH2OH-treated and untreated rhodopsin incorporated [14C]palmitate from exogenously added [14C]palmitoyl-CoA. This incorporation was substantially greater in the NH2OH-treated sample. The removal of palmitate by NH2OH inhibited rhodopsin regeneration by 44% and increased the ability of rhodopsin to activate transducin's light-dependent GTPase activity by 61%. However, the removal of palmitate from rhodopsin did not affect the light-dependent binding of transducin (T alpha and T beta gamma).     

Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.     

Molecular mapping of the mouse ob mutation.

The mouse ob mutation has been mapped relative to a series of RFLPs among the progeny of three separate mouse crosses: an intraspecific backcross, an intraspecific intercross, and an interspecific intercross. Genotypic assignment at the ob locus was made by making use of measurements of body mass index and the plasma concentrations of glucose and insulin. These data have suggested that the development of diabetes in these animals is a consequence of unlinked polygenes. There was also evidence that unlinked Mus spretus alleles can diminish the obesity of ob/ob mice. From these data we have mapped several markers on chromosome 6 with the following order: cen-Cola-2-Met-ob-Cpa-Tcrb. The homologs of markers that flank ob map to human chromosome 7q, suggesting that if there is a human homologue of ob, it maps to 7q31.     

Genes newly identified as regulated by glucocorticoids in murine thymocytes

Glucocorticoids induce dramatic biochemical and morphological changes in lymphocytes through an unknown process that requires RNA and protein synthesis. In order to identify genes involved in this response, we previously isolated 11 cDNA clones from the murine WEHI-7TG thymoma cell line that correspond to mRNAs induced by glucocorticoids. We now report the isolation of two new cDNA clones whose gene expression is regulated by glucocorticoids in WEHI-7TG cells. We further characterize the two new cDNA clones, as well as those described previously, by examining the response of each of the corresponding mRNAs to glucocorticoids in murine thymocytes. With the exception of two, all cDNAs correspond to genes that are induced by glucocorticoids in murine thymocytes within 4 h of treatment. We previously identified two of the cDNAs as the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein. We have now identified four additional cDNA clones that correspond to the genes for calmodulin, mitochondrial phosphate carrier protein, immunoglobulin (Ig)-related glycoprotein (GP-70), and the 70 kilodalton autoantigen for Lupus and Graves diseases. Two other cDNA clones represent previously undescribed genes: one shares a high similarity to known sequences for the family of G-protein-coupled receptors and the other to a human placental-specific protein, PP11. Another cDNA appears to contain sequences for an unknown gene and the remnants of a mouse transposon. ETn. The remaining clones represent new, unidentified genes induced by glucocorticoids in murine thymocytes and in the WEHI-7TG cell line.     

The reproductive behavior of six species of Namib desert tenebrionid beetles (Coleoptera: Tenebrionidae)

The reproductive behavior of six species of tenebrionid beetles (Coleoptera: Tenebrionidae) was studied in the Namib Desert of southern Africa. In three species, males follow closely behind females (following behavior), while in the other three species, males mount females and remain clasped to them for extended periods (riding behavior). Following behavior occurs before and sometimes after copulation, while riding behavior occurs primarily after copulation. Males of all six species guard females from contesting males, although the effectiveness of guarding is greater in riding species. The evolution of the two male mating strategies does not appear to be related to operational sex ratio differences but, rather, to differential tendencies of females to remate. Variation in total pair duration within following and riding species may be attributed partly to species differences in operational sex ratio. However, pair durations are not affected by experimental manipulations of sex ratio in each species.     

Developmental expression of syndecan, an integral membrane proteoglycan, correlates with cell differentiation

Syndecan is an integral membrane proteoglycan that behaves as a matrix receptor by binding cells to interstitial matrix and associating intracellularly with the actin cytoskeleton. Using immunohistology, we have now localized this proteoglycan during the morphogenesis of various derivatives of the surface ectoderm in mouse embryos. Syndecan is expressed on ectodermal epithelia, but is selectively lost from the cells that differentiate into the localized placodes that initiate lens, nasal, otic and vibrissal development. The loss is transient on presumptive ear, nasal and vibrissal epithelia; the derivatives of the differentiating ectodermal cells that have lost syndecan subsequently re-express syndecan. In contrast, syndecan is initially absent from the mesenchyme underlying the surface ectoderm, and is transiently expressed when the surface ectoderm loses syndecan. These results demonstrate that expression of syndecan is developmentally regulated in a distinct spatiotemporal pattern. On epithelia, syndecan is lost at a time and, location that correlates with epithelial cell differentiation and, on mesenchyme, syndecan is acquired when the cells aggregate in proximity to the epithelium. This pattern of change with morphogenetic events is unique and not duplicated by other matrix molecules or adhesion receptors.