Acridine-linked oligonucleotide probes for the short dAT-rich motifs in the 3'-untranslated region of cytokine genes

We have investigated the use of oligonucleotide probes for identifying cDNA clones containing the short dAT-rich motifs found in the 3'-untranslated region of cytokine genes. To obtain sufficiently stable duplexes between the octameric probes used to identify genes containing the sequence dTATTTATT and its complement, it was necessary to couple an intercalating agent, an acridine derivative (acr), to the 5'-positions of the probes. The resulting octamers 5'-acr-dAATAAATA and, particularly, 5'-acr-dTATTTATT were successfully used to distinguish the complementary sequences in cDNA from internal, single point mismatched sequences. Southern blot analyses of plasmids containing IL-1 beta and IL-8 gave positive results with the 3' degenerate probe, 5'-acr-dTATTTATTN, clearly showing that the very short probe approach can be used in this type of analysis. Subsequently, in slot blot analyses we found that, even without the degenerate nucleotide, N, plasmids bearing cytokine sequences with at least 7 contiguous matched nucleotides could be unambiguously identified with 5'-acr-dTATTTATT. Unfortunately, because of the ubiquity of these dAT-rich sequences in bacterial DNA, it was not possible to use these probes for direct colony screening. In contrast to the results obtained with DNA, at the RNA level, with IL-1 beta mRNA bound to nitrocellulose, the hybrid formed with 5'-acr-dAATAAATA was very unstable, even in 1M LiCl solution at 2 degrees C; however, in the same salt solution the slightly longer acridine-coupled probes 5'-acr-dAATAAATAGGG and 5'-acr-dAAAGAACAA remained hybridized to their complementary sequences up to about 18 degrees C.     

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.     

Regulation of Escherichia coli secA mRNA translation by a secretion-responsive element.

The Escherichia coli secA gene, whose translation is responsive to the proficiency of protein export within the cell, is the second gene in a three-gene operon and is flanked by gene X and mutT. By using gene fusion and oligonucleotide-directed mutagenesis techniques, we have localized this translationally regulated site to a region at the end of gene X and the beginning of secA. This region has been shown to bind SecA protein in vitro. These studies open the way for a direct investigation of the mechanism of secA regulation and its coupling to the protein secretion capability of the cell.     

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).     

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.     

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.     

Phylogenetic relationships among four members of Stizostedion (Percidae) determined by mitochondrial DNA and allozyme analyses

Phylogenetic relationships among four Stizostedion species were examined using mitochondrial DNA (mtDNA) and allozyme analyses. Twenty-six allozyme loci were scored, and mtDNA variation was examined using 24 restriction endonucleases, yielding 48–57 restriction sites among the species. Genetic distance analyses show that the two North American species ( S. canadense and S. vitreum ) cluster in one group, while the two European species ( S. hciopercu and S. vogense ) form a second group. Nei's genetic distance between these two groups was 0.7 ± 0.2 for allozymes, while the corresponding mtDNA sequence divergence was 14.8 ± 2.0%, suggesting that these two groups diverged approximately 10 million years ago. Thus, these data are consistent with the hypothesis that Stizostedion colonized North America during the Pliocene.     

Structure and biological activity of crustacean gastrointestinal peptides identified with antibodies to gastrin/cholecystokinin.

Four gastrin/cholecystokinin-like peptides (G/CCK) which cross-react with a specific C-terminal gastrin/CCK antiserum have been isolated from the stomach of the marine crustacean Nephrops norvegicus. The molecular weight of the four peptides was estimated between 1000 and 2000 Da by molecular sieving. By radioimmunoassay, the cross-reactivity of these peptides with human gastrin 17-I was found to be around 0.03%. Pure peptidic fractions were recovered after four successive steps of HPLC. Amino-acid analysis suggested a similarity between the four peptides identified which may belong to a new family. A limited homology between the C-terminus of one Nephrops peptide and vertebrate G/CCK was found after sequencing. Two of the peptides exhibited secretagogue effects on crustacean isolated midgut glands. The Nephrops peptides, although structurally distinct from the vertebrate G/CCKs, appear to serve similar biological functions in crustaceans.