Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.     

Bacteriophage P22-mediated specialized transduction in Salmonella typhimurium: high frequency of aberrant prophage excision.

The temperate bacteriophage P22 mediates both generalized and specialized transduction in Salmonella typhimurium. Specialized transduction by phage P22 is different from, and less restricted than, the well characterized specialized transduction by phage lambda, due to differences in the phage DNA packaging mechanisms. Based on the properties of the DNA packaging mechanism of phage P22 a model for the generation of various types of specialized transducing particles is presented that suggests generation of substantial numbers of specialized transducing genomes which are heterogeneous but only some of which have terminally redundant ends. The primary attachment site, ataA, for phage P22 in S. typhimurium is located between the genes proA,B and supQ newD. (The newD gene is a substitute gene for the leuD gene, restoring leucine prototrophy of leuD mutant strains.) The proA,B and supQ newD genes are very closely linked and thus cotransducible by generalized transducing particles. Specialized transducing particles can carry either proA,B or supQ newD but not both simultaneously, and thus cannot give rise to cotransduction of the proA,B and supQ newD genes. This difference is used to calculate the frequency of generalized and specialized transducing particles from the observed cotransduction frequency in phage lysates. By this method, very high frequencies of supQ newD (10(-2)/PFU)- and proA,B (10(-3)/PFU)-specialized transducing particles were detected in lysates produced by induction of lysogenic strains. These transducing particles most of which would have been produced by independent aberrant excision events (which include in situ packaging), were of various types.     

Copper Toxicosis in Bedlington Terriers

Copper toxicosis of Bedlington Terriers (Chronic progressive hepatitis) is a genetically transmitted disease. The typical feature of this disease is accumulation of copper in the liver tissue. The changes vary from mild hepatitis to chronic progressive hepatitis and cirrhosis. The material of this study consists of 2 cases of copper toxicosis examined at the Department of Pathology in Helsinki in the years 1980–82. Moreover a re-examination of tissue samples was made of all Bedlington Terriers examined during the years 1969–1982 at the same department. Six of the 14 examined dogs showed a positive reaction for copper in their liver tissues. The possible relationship of the examined dogs is not yet known.     

Resolution of Holliday junction analogs by T4 endonuclease VII can be directed by substrate structure

Endonuclease VII is an enzyme from bacteriophage T4 capable of resolving four-arm Holliday junction intermediates in recombination. Since natural Holliday junctions have homologous (2-fold) sequence symmetry, they can branch migrate, creating a population of substrates that have the branch point at different sites. We have explored the substrate requirements of endonuclease VII by using immobile analogs of Holliday junctions that lack this homology, thereby situating the branch point at a fixed site in the molecule. We have found that immobile junctions whose double-helical arms contain fewer than nine nucleotide pairs do not serve as substrates for resolution by endonuclease VII. Scission of substrates with 2-fold symmetrically elongated arms produces resolution products that are a function of the particular arms that are lengthened. We have confirmed that the scission products are those of resolution, rather than nicking of individual strands, by using shamrock junction molecules formed from a single oligonucleotide strand. A combination of end-labeled and internally labeled shamrock molecules has been used to demonstrate that all of the scission is due to coordinated cleavage of DNA on opposite sides of the junction, 3' to the branch point. Endonuclease VII is known to cleave the crossover strands of Holliday junctions in this fashion. The relationship of the long arms to the cleavage direction suggests that the portion of the enzyme which requires the minimum arm length interacts with the pair of arms containing the 3' portion of the crossover strands on the bound surface of the antiparallel junction.     

Pre-proparathyroid hormone: fidelity of the translation of parathyroid messenger RNA by extracts of wheat germ.

Pre-proparathyroid hormone is the major protein synthesized in wheat-germ extracts in response to addition of an 8-15S fraction of parathyroid RNA. The accuracy of the translation of the mRNA from parathyroid tissue was examined by analysis of the carboxyl-terminal tryptic peptide and the amino-terminal amino acid of the protein, by analysis of the size distribution of the mRNA, and by translation of the mRNA in a second cell-free extract. When 8-15S RNA was fractionated on a sucrose gradient containing formamide, RNA that supported the synthesis of pre-proparathyroid hormone was present in a single symmetrical peak, suggesting that it was homogeneous. Analyses by paper chromatography and electrophoresis of the proline-containing tryptic peptides of pre-proparathyroid hormone indicate that they are identical with the corresponding proline-containing peptides of parathyroid hormone. Because the COOH-terminal tryptic peptide of parathyroid hormone contains proline, the data indicate that the COOH termini of pre-proparathyroid hormone and parathyroid hormone are identical. Methionine from initiator [35S]Met-tRNAfMet was rapidly incorporated into pre-proparathyroid hormone by the wheat-germ extract, and a single-step Edman degradation selectively removed almost all of the initiator [35S]methionine present in pre-proparathyroid hormone. Translation of the 8-15S RNA in a cell-free extract from Krebs-II ascites cells resulted in a protein that comigrated with pre-proparathyroid hormone on sodium dodecyl sulfate-acrylamide gel electrophoresis. These data support the conclusion that the wheat-germ system accurately translates the mRNA for parathyroid hormone, and they strengthen the contention that pre-proparathyroid hormone is the initial biosynthetic product.     

Pseudomonas aeruginosa polysaccharide Psl supports airway microbial community development

Pseudomonas aeruginosa dominates the complex polymicrobial cystic fibrosis (CF) airway and is a leading cause of death in persons with CF. Oral streptococcal colonization has been associated with stable CF lung function. However, no studies have demonstrated how Streptococcus salivarius, the most abundant streptococcal species found in individuals with stable CF lung disease, potentially improves lung function or becomes incorporated into the CF airway biofilm. By utilizing a two-species biofilm model to probe interactions between S. salivarius and P. aeruginosa, we discovered that the P. aeruginosa exopolysaccharide Psl promoted S. salivarius biofilm formation. Further, we identified a S. salivarius maltose-binding protein (MalE) that is required for promotion of biofilm formation both in vitro and in a Drosophila melanogaster co-infection model. Finally, we demonstrate that promotion of dual biofilm formation with S. salivarius is common among environmental and clinical P. aeruginosa isolates. Overall, our data supports a model in which S. salivarius uses a sugar-binding protein to interact with P. aeruginosa exopolysaccharide, which may be a strategy by which S. salivarius establishes itself within the CF airway microbial community.Subject terms: Bacteriology, Biofilms, Microbiome, Clinical microbiology