Histochemical localization of IGF-I and IGF-II mRNA in the rat between birth and adulthood

We describe the postnatal ontogeny and localization of insulin-like growth factors I and II (IGF-I and -II) in the rat. We have used oligodeoxyribonucleotide probes for in situ hybridization (hybridization histochemistry) and for Northern blotting. IGF-II mRNA is strongly expressed in liver, skeletal muscle, perichondrium, leptomeninges and choroid plexus of the newborn. Demonstrable levels fall dramatically in the liver at 18-20 days postnatally but persist for longer periods in muscle and remain undiminished throughout life in the pia/choroid plexus, indicating that different control mechanisms operate in these tissues. IGF-I mRNA is predominantly found in the liver. Its level in this organ rises well before levels of IGF-II fall. This suggests that distinct factors govern the expression of IGF-I and -II genes.     

Nucleotide sequence encoding the biosynthetic dehydroquinase function of the penta-functional arom locus of Aspergillus nidulans.

The nucleotide sequence of a 1.9 Kb HindIII fragment of DNA derived from the arom locus of A.nidulans and encoding the biosynthetic dehydroquinase activity has been determined. The sequences encoding the biosynthetic and catabolic dehydroquinase enzymes of A.nidulans show no detectable homology, strongly suggesting convergent evolutionary pathways. The messenger RNA specified by the arom locus was detected as a 5.3 Kb RNA species.     

A retroviral provirus closely associated with the Ren-2 gene of DBA/2 mice.

We have determined the entire nucleotide sequence of an intra-cisternal A particle (IAP) genome, associated with the Ren-2 gene of DBA/2 mice. This genome (MIARN) displays features common to other IAP retroviral-like genomes. Long terminal repeats (LTRs) are approximately 430 base pairs (bp) in length and show typical retroviral U3-R-U5 organisation, though the R-region, at 120 bp, is much larger than the average IAP. This difference probably arose by the amplification of a pyrimidine-rich sequence, by a slippage-mispairing mechanism. Flanking the 5' LTR is a sequence complementary to a phenylalanine tRNA, strongly conserved in all rodent IAP genomes and probably required to prime the initiation of (-) strand synthesis. Flanking the 3' LTR, is a purine-rich sequence probably required for (+) strand synthesis. The tRNA binding site (TBS) is flanked by six tandem copies of a sequence homologous to the TBS. The relationship of the MIARN element to other IAP genomes and the significance of its association with the highly expressed Ren-2 is discussed.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Alignment of clonedamiE gene ofPseudomonas aeruginosa with theN-terminal sequence of amidase

A restriction enzyme map was constructed for 5.1-kb fragment of Pseudomonas aeruginosa DNA inserted into plasmid pBR322. Restriction enzyme sites were matched to the N-terminal amino acid sequence of amidase to obtain alignment of the amiE gene within the cloned fragment.     

The construction in vitro of derivatives of bacteriophage lambda carrying the amidase genes of Pseudomonas aeruginosa

Summary The amidase genes of Pseudomonas aeruginosa were inserted into a replacement vector following cleavage with the restriction endonuclease HindIII. The recombinant ami was detected by enhanced growth of Escherichia coli around plaques of the recombinant phage on minimal medium containing acetamide as the nitrogen source. Low levels of amidase activity were detected in E. coli cultures infected with ami and these were sufficient to allow growth with acetamide as nitrogen source. Lysis-defective derivatives of ami were made by introducing Q ^-, S ^- mutations. Cultures of E. coli infected with amiQ ^- S ^- synthesised amidase as the major protein. The amidase produced by these cultures was identical to that produced by PAC strains of P. aeruginosa in substrate specificity, thermal stability and immunological crossreaction.     

Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.