Spliced segments at the 5'' terminus of adenovirus 2 late mRNA.

An mRNA fraction coding for hexon polypeptide, the major virion structural protein, was purified by gel electrophoresis from extracts of adenovirus 2-infected cells late in the lytic cycle. The mRNA sequences in this fraction were mapped between 51.7 and 61.3 units on the genome by visualizing RNA-DNA hybrids in the electron microscope. When hybrids of hexon mRNA and single-stranded restriction endonuclease cleavage fragments of viral DNA were visualized in the electron microscope,branched forms were observed in which 160 nucleotides of RNA from the 5' terminus were not hydrogen bonded to the single-stranded DNA. DNA sequences complementary to the RNA sequences in each 5' tail were found by electron microscopy to be located at 17, 20, and 27 units on the same strand as that coding for the body of the hexon mRNA. Thus, four segments of viral RNA may be joined together during the synthesis of mature hexon mRNA. A model is presented for adenovirus late mRNA synthesis that involves multiple splicing during maturation of a larger precursor nuclear RNA.     

Covalent protein-DNA complexes at the 5'' strand termini of meiosis-specific double-strand breaks in yeast.

During meiosis in Saccharomyces cerevisiae, the first chemical step in homologous recombination is the occurrence of site-specific DNA double-strand breaks (DSBs). In wild-type cells, these breaks undergo resection of their 5' strand termini to yield molecules with 3' single-stranded tails. We have further characterized the breaks that accumulate in rad50S mutant stains defective in DSB resection. We find that these DSBs are tightly associated with protein via what appears to be a covalent linkage. When genomic DNA is prepared from meiotic rad50S cultures without protease treatment steps, the restriction fragments diagnostic of DSBs selectively partition to the organic-aqueous interphase in phenol extractions and band at lower than normal density in CsCl density gradients. Selective partitioning and decreased buoyant density are abolished if the DNA is treated with proteinase K prior to analysis. Similar results are obtained with sae2-1 mutant strains, which have phenotypes identical to rad50S mutants. The protein is bound specifically to the 5' strand termini of DSBs and is present at both 5' ends in at least a fraction of breaks. The stability of the complex to various protein denaturants and the strand specificity of the attachment are most consistent with a covalent linkage to DSB termini. We propose that the DSB-associated protein is the catalytic subunit of the meiotic recombination initiation nuclease and that it cleaves DNA via a covalent protein-DNA intermediate.     

Peripheral metabolism of bovine parathyroid hormone in the dog

Four dogs were infused with highly purified bovine parathyroid hormone until constant levels of immunoreactive hormone were attained in the circulation. Simultaneous samples of plasma were then obtained from the aorta, from hepatic, renal, and femoral veins, and later from a pulmonary artery and the left ventricle. Radioimmunoassay of these samples revealed mean arteriovenous differences of ?23% across the liver and ?19% across the kidney. No significant differences were found across the lung or lower extremity. After termination of the infusion the disappearance rate of immunoreactive hormone in external jugular-venous blood was multiexponential: the predominant initial $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was 4, 6, and 8 min, and the terminal component was 60, 54, and 99 min, respectively, in 3 dogs.     

Inhibition of parathyroid hormone release from dispersed bovine cells by ouabain

Ouabain, at concentrations of 3 X 10(-4)-10(-3) M, inhibited secretion of PTH approximately 50% in a freshly dispersed bovine parathyroid cell preparation. This inhibition was found with both unstimulated and secretagogue-stimulated PTH release. Reductions in PTH secretion were found at all concentrations of Ca++ tested between 0.3 mM and 2.0 mM and in the presence of the divalent cation chelators EDTA and EGTA, indicating that extracellular Ca++ is not an absolute requirement for the inhibition. The ouabain inhibition did not appear to be mediated through changes in either adenylate cyclase activity or total cellular cAMP, implying a locus distal to the generation of this cyclic nucleotide. The data suggest that transmembrane potential and/or distribution of monovalent cations across the plasma membrane is important in the maintenance of PTH secretion. The mechanisms involved in this control do not appear to involve extracellular Ca++ directly.     

Unusual nucleotide sequences at the 5'' end of actin genes in Dictyostelium discoideum.

The nucleotide sequences at the 5' end of one actin cDNA and six actin genomic clones from Dictyostelium have been determined. The amino acid sequences derived from the nucleotide sequences show strong conservation for six of the seven genes relative to the NH2-terminal region of Physarum actin. The region 5' to the AUG initiating codon is greater than 90% A+T residues in all of the genes.     

Metabolism of radioiodinated bovine parathyroid hormone in the rat.

Metabolism of bovine 125I-labeled parathyroid hormone was studied in the rat by gel filtration and by sequence analysis of the iodinated fragments. Analysis of the kinetics of hormone metabolism shows that iodinated intact hormone has a multiexponential disappearance curve with a rapid (3 min) initial and a slower (48 min) second component. Iodinated fragments, which rapidly increase during the first 12 min after injection of the intact hormone, subsequently disappear from the circulation with a t1/2 of no greater than 48 min. Plasma samples collected at various time-intervals after intravenous injection of bovine 125I-labeled parathyroid hormone were gel filtered on Bio-gel P-100. Four radioactive peaks were seen. The first and second peaks eluted, respectively, at the void volume of the column and at the position of intact hormone. The third peak consisted of iodinated fragments, and the last peak eluted at the salt volume of the column. Sequence analysis of the iodinated fragments in the third peak showed that it was heterogeneous, containing several different, but closely related, polypeptides. Before 48 min after injection, the most-abundant fragment is one whose amino-terminal amino acid is residue 34. The amino-terminal residue of the next most-common fragment is the amino acid at position 37. No fragments representing cleavages closer to the amino-terminus than residue 34 were seen. The results of these studies are virtually identical with those previously obtained in the dog. The similarities found in the sites of hormone proteolysis and in the kinetics of hormone metabolism in the rat and dog, coupled with the less direct evidence indicating that similar cleavages are also present in man and bovine, are consistent with the view that proteolysis of parathyroid hormone is peripheral tissues is specific, at least in mammalian species, and may be a critical step in controlling the availability of biologically active hormone.     

alpha-Adrenergic inhibition of adenosine 3',5'-monophosphate accumulation and parathyroid hormone release from dispersed bovine parathyroid cells.

The possibility of alpha-adrenergic modulation of cAMP accumulation and parathyroid hormone (PTH) release was investigated in dispersed bovine parathyroid cells. cAMP accumulation due to the mixed alpha- and beta-adrenergic agonists, (-)epinephrine and (-)norepinephrine, was significantly enhanced by the alpha-adrenergic inhibitor phentolamine; that due to the "pure" beta-adrenergic agonist, (-)isoproterenol, was not altered significantly. Direct inhibition of agonist-stimulated cAMP accumulation was effected by adding increasing concentrations of (-)epinephrine to concentrations of (-)isoproterenol maximally stimulating cAMP accumulation. A 50-75% inhibition of cAMP was observed which was specifically blocked by phentolamine. This inhibition was not specific for beta-adrenergic stimulation, as (-)epinephrine also inhibited dopamine-stimulated cAMP accumulation. The inhibition of (-)isoproterenol-stimulated cAMP accumulation by (-)epinephrine was unaffected by ambient calcium concentration. Stimulation of PTH release by (-)epinephrine and (-)norepinephrine was potentiated by phentolamine and inhibited by the beta-adrenergic blocker, (-)propranolol, demonstrating alpha-adrenergic modulation of hormone release and confirming the close relationship between cAMP accumulation and PTH release previously shown in this system. These results demonstrate the presence of an alpha-adrenergic receptor in dispersed bovine parathyroid cells which inhibits agonist-stimulated cAMP accumulation and PTH release by a mechanism independent of extracellular calcium.     

Loss of infectivity of brome mosaic virus RNA after chemical modification of the 3'' or 5'' terminus.

Brome mosaic virus (BMV) RNA that had both termini chemically modified by periodate oxidation and aniline-catalyzed cleavage of the terminal nucleotide had drastically reduced infectivity. BMV RNA that was first enzymatically tyrosylated to protect the 3' terminus from modification, and then modified at the 5' terminus by periodate oxidation and aniline cleavage, had a similar reduction in infectivity. Tyrosylation followed by acetylation modifies only the 3' terminus. Nevertheless, acetylated tyrosyl-BMV RNA was less than one-fourth as infectious as a control sample subjected to procedures that differed only by the presence of tyrosinol (which prevents aminoacylation and subsequent acetylation). For each modified form of viral RNA, care was taken to test the infectivity of appropriate control samples. The integrity of the modified RNAs was examined by gel electrophoresis and by biological translation and aminoacylation assays. We conclude that, in different ways, both the 5'- and 3'-terminal structures of BMV RNA play important roles during infection of the host.     

Vasoactive intestinal polypeptide stimulates parathyroid hormone release by interaction with cyclic adenosine monophosphate production of bovine parathyroid cells.

Influence of vasoactive intestinal polypeptide, neuropeptide Y, calcitonin gene-related peptide, and substance P was investigated on dispersed parathyroid cells of adult cattle. At a physiological concentration of extracellular calcium, vasoactive intestinal polypeptide stimulated the parathyroid hormone release in a dose-dependent manner, whereas no effects were noted for the other peptides. The dependency of PTH secretion upon extracellular calcium was shifted to the right by vasoactive intestinal polypeptide at 10(-6) mol/l, with a tendency for greater effects at low (0.5 mmol/l) than high concentrations (2.0-3.0 mmol/l) of the cation. Vasoactive intestinal polypeptide significantly enhanced cAMP release of the parathyroid cells, whereas no influence was noted on cytoplasmic calcium or pH within the cells. The results suggest that vasoactive intestinal polypeptide stimulates the PTH release by interaction with cAMP production of the parathyroid cells. This effect may contribute to the development of hypercalcemia in patients with neuroendocrine tumours secreting vasoactive intestinal polypeptide.     

Binding of the receptor for 1,25-dihydroxyvitamin D3 to the 5'-flanking region of the bovine parathyroid hormone gene

Receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) were prepared from bovine parathyroid glands and incubated with fragments of DNA of the 5'-flanking region of the bovine parathyroid hormone (PTH) gene covering 1700 base pairs (bp) upstream of the initiation site. In filter binding assays, incubation of the DNA fragment spanning -700 to +50 bp with 200 micrograms cytosolic protein gave 288 +/- 63% (mean +/- S.D.) of binding in the absence of protein. In contrast, there was no significant reaction with the -1350 to -700 bp fragment, nor was there binding of the receptor to a fragment of DNA covering the coding region of the PTH gene. Substitution of bovine serum albumin for the receptor preparation did not induce binding to the -700 to +50 bp fragment. The receptor-binding site was further defined to -700 to -100 bp as deletion of the -100 to +50 bp did not reduce receptor binding. Reaction of receptors further purified by sucrose density ultracentrifugation with a monoclonal antibody in immunoblots revealed a single species with a molecular mass of approximately 50,000 Da, which was absent in preparations of cos-1 cells. Autoradiography following incubation of receptors immobilized on nitrocellulose filters with the -700 to +50 bp fragment indicated a single reactive band coincident with the band in the immunoblot. The DNA fragment did not bind to filters containing preparations of cos-1 cells. Extraction of the receptors in the presence or absence of 1,25-(OH)2D3 (4 nmol/l) or the presence of KCl (150 mmol/l) in the incubation medium had no significant effect on DNA binding to the protein in this assay. (ABSTRACT TRUNCATED AT 250 WORDS)