Expression of somatotropin receptor messenger ribonucleic acid in bovine tissues

The somatotropin receptor mRNA is controlled by at least two different gene promoters that generate two variants with different exon 1 sequences (1A and 1B). The location of 1A and 1B somatotropin receptor mRNA within cattle tissues and, hence, the tissue specificity of the 1A and 1B promoters are unknown. In addition, the cDNA sequence of the 1B somatotropin receptor has not been determined. Our objective, therefore, was to sequence a cDNA for the 1B somatotropin receptor and to analyze bovine tissues for expression of 1A and 1B somatotropin receptor mRNA. Twenty adult tissues and six fetal tissues were collected at slaughter from each of four cows and two fetuses. Messenger RNA was analyzed using ribonuclease protection assays. The adult liver expressed both 1A and 1B mRNA. All other adult tissues expressed 1B mRNA but not 1A mRNA. The greatest amount of 1B mRNA was detected in liver and adipose (abdominal and subcutaneous) tissues. Other tissues had approximately one-half to one-tenth of the amount of 1B mRNA in the liver or adipose tissue. Fetal tissues (including fetal liver) expressed 1B mRNA and not 1A mRNA. Based on cDNA sequencing, the protein encoded by the 1A and 1B mRNA was nearly identical. We concluded that 1A somatotropin receptor mRNA is specific to adult bovine liver. Other adult and fetal bovine tissues expressed 1B somatotropin receptor mRNA with a predicted protein sequence that was similar to the 1A somatotropin receptor.     

Functional neuroimaging of hallucinations in schizophrenia: toward an integration of bottom-up and top-down approaches

Phenol sulfotransferases catalyze the transfer of a sulfonate moiety from 3'-phosphoadenosyl 5'-phosphosulfate to a phenolic group of lipophylic substrates to generate soluble sulfate esters. Using a phenol sulfotransferase cDNA as probe to screen a human leukocyte genomic DNA library constructed in lambda EMBL3, we obtained a clone containing a complete gene sequence. Comparison of the gene sequence with that of the corresponding cDNAs, namely phenol-sulfating phenol sulfotransferase (P-PST) or thermostable sulfotransferase (TS-PST), and human aryl sulfotransferase 1 and 2 (HAST1 and HAST2) indicates that the gene possesses eight short exons separated by seven introns included in approximately 5 kb. HAST2 has a different 5' untranslated sequence, and thus is encoded by a different mRNA species. While the nucleotide sequence corresponding to the 5' noncoding region of P-PST (TS-PST and HAST1) is included in the exon I, the 5' untranslated sequence of HAST2 is located in the beginning of exon IIa. The remaining sequence in exon II that is identical to both P-PST and HAST2 was termed exon IIb. Exons III to VIII, which cover the coding region and the 3' untranslated region, are almost identical in all types of PST or AST cDNAs. These results suggest that the phenol sulfotransferase gene possesses two alternate promoters that drive the expression of the two different mRNA species in a tissue-specific manner. Transfection of chloramphenicol acetyl transferase (CAT) reporter gene vectors containing the 5'-flanking sequence upstream from exon I and exon II, respectively, in transformed human embryonal kidney (293) cells indicate that both sequences possess promoter activity with higher activity for promoter 1. RNA blot analysis indicates that human phenol sulfotransferase gene is expressed in kidney, liver, lung, leukocyte, colon, small intestine, and spleen.     

Molecular cloning of a bovine ornithine decarboxylase cDNA and its use in the detection of restriction fragment length polymorphisms in Holsteins

A cDNA coding for ornithine decarboxylase (ODC) was isolated from a bovine liver cDNA library. The clone (1758 base pairs) consisted of 5'- and 3'-untranslated regions of 185 and 187 nucleotides, respectively, and an open reading frame of 1383 nucleotides encoding an ODC protein (M(r) 51,342 daltons) of 461 amino acids. Comparison of the nucleotide and the predicted amino acid of the cDNA with other mammalian ODCs showed a very high degree of homology both at the DNA and protein levels. The bovine ODC mRNA was identified by northern blot to be a single species with a molecular size of 2.35 kilobase pairs. Primer extension analysis indicated that the 5'-untranslated region of the bovine ODC mRNA was 312 nucleotides long. Southern blot analysis of bovine genomic DNA revealed restriction fragment length polymorphisms when cleaved with restriction enzymes PstI, MspI, TaqI, and Bg/I.     

"Miguel F. Jiménez lecture. The paradigm of modern medicine: molecular genetics

The sequence for bovine link protein cDNA, including 108 bases of the 5' untranslated region (UTR) and 768 nucleotides of the 3' UTR, was determined from polymerase chain reaction products and bovine articular chondrocyte cDNA clones. The deduced primary structure for bovine link protein predicts a protein 354 amino acid residues in length. Comparative analysis with link protein sequence from several other species revealed overall high conservation of protein coding sequence. High nucleotide sequence conservation was observed within the extensive 5' and 3' UTRs of bovine, human, pig, chick and rat link protein mRNA. As evidence that the UTRs might play a role in regulation of link protein mRNA turnover, multiple occurrences of the adenosine-uridine binding factor motif A(Ua)A were found to be conserved between species within 3' UTRs. A polyadenylation signal was conserved between the bovine and chicken sequence, use of which would result in the smallest of multiple bovine link protein mRNA species observed by Northern blot analysis.     

Cloning, characterization, and tissue expression pattern of mouse tuftelin cDNA

Tuftelin is a protein that has been suggested to function during enamel crystal nucleation. Published sequences for bovine tuftelin cDNA and genomic clones proposed different reading frames that radically affected the derived amino acid sequence of the tuftelin carboxyl-terminus. We have isolated and characterized a full-length mouse cDNA clone and a partial porcine cDNA clone that include the region of the proposed frame-shift. The mouse tuftelin clone is 2572 nucleotides in length, exclusive of the poly(A+) tail. Translation from the 5'-most ATG yields a protein of 390 amino acids with an isotope-averaged molecular mass of 44.6 kDa and an isoelectric point of 5.9. Comparison of the bovine, mouse, and porcine cDNAs supports the revised bovine tuftelin amino acid sequence and suggests that the bovine tuftelin translation initiation codon be re-assigned to a more 5' ATG. Re-assigning the translation initiation codon lengthens the tuftelin protein by 52 amino acids, 51 of which are identical between bovine and mouse. At the carboxyl-terminus, the revised bovine and the mouse sequences match at 39 of the final 42 amino acid positions, compared with 2 identities with the originally published bovine reading frame. Northern blot analysis reveals that tuftelin is not ameloblast-specific but is expressed in multiple tissues, including kidney, lung, liver, and testis. Two tuftelin RNA messages, of 2.6 and 3.2 kb, were detected. DNA sequence characterization of an RT-PCR amplification product confirmed expression of tuftelin in kidney, and identified an alternatively spliced mouse tuftelin mRNA lacking exon 2.     

PA26, a novel target of the p53 tumor suppressor and member of the GADD family of DNA damage and growth arrest inducible genes

We report the cystathionine-beta synthase (CBS) gene expression pattern during early human embryogenesis (3 to 6 weeks post conception) by in situ hybridization and in fetal and adult tissue by Northern Blot analysis. Probes were chosen to recognize either the common sequence to all known CBS mRNAs or the sequences of two different major exons 1 issued of we have previously identified. We demonstrate by in situ hybridization that CBS is continuously expressed from the earliest stages studied (22 days post conception) during embryogenesis in the tissues of developing embryos which will after birth present clinical abnormalities in homocystinuria patients. It is expressed at an especially high level in the neural and cardiac systems until the liver primordium appears. In embryonic central nervous system, the whole neural tube and primary brain vesicles are labeled. Secondary brain vesicles labeling are dependent on the neuroepithelium differentiation. The ventricular layer of the rhombencephalon, cranial nerve nuclei and then after cerebellar cortex derived from rhombencephalon ventricular layer are strongly labeled. Thalamus and other derivatives of the diencephalon plate, the neuroblastic layer of the retina, lens and dorsal root ganglia are labeled. After 35 days post conception, CBS mRNAs was detected in endocardial cells and in cells derived from the neural crest of the heart and in particular developing mesodermic regions such as the primitive hepatocytes of the liver, mesonephros vesicles, various endocrine glands and developing bones. We could not detect tissue specificity of different probes at this embryonic stage. Northern blot analysis consistently detected mRNA species in fetal 25 weeks post conception brain, liver and kidney. The common cDNA probe revealed the 2.5 and 3.7 kb mRNA species from brain, liver and kidney. The exon 1b probe detected only the 2.5 kb mRNA and the exon 1c probe the 3.7 kb mRNA in these three tissues. In adult tissue, the 1b probe detected only the 2.5 kb mRNA and the 1c probe only the 3.7 kb mRNA in the liver.     

Identification of a novel cysteine string protein variant and expression of cysteine string proteins in non-neuronal cells

Cysteine string proteins (Csps) are synaptic vesicle proteins thought to be involved in calcium-dependent neurotransmitter release at nerve endings. Here, we report the cloning of two Csp variants, termed Csp1 and Csp2, from bovine adrenal medullary chromaffin cells. The bovine Csp1 appears to be the homologue of rat brain Csp, sharing 95% identity at the amino acid level. The nucleotide sequence of csp2 is identical with that of csp1 except for a 72-base insert which introduces a stop codon into the coding sequence, which would be predicted to result in a truncated protein 3.3 kDa smaller than Csp1. Furthermore, polymerase chain reaction analysis detected homologues of Csp1 and Csp2 in rat kidney, liver, pancreas, spleen, lung, and adrenal gland. Expression of Csps in non-neuronal tissues was confirmed by Northern blotting and by immunoblotting with anti-Csp1 antiserum which also demonstrated expression of both full-length and truncated Csps in spleen. The widespread tissue distribution is inconsistent with a role of Csps as specific regulators of presynaptic calcium channels as previously proposed. We suggest that Csps may have a more general role in membrane traffic in non-neuronal as well as neuronal cells.     

Cloning and sequencing of the URA3 gene of Kluyveromyces marxianus CBS 6556

More than 250 mutations have been detected in the cystic fibrosis (CF) transmembrane regulator (CFTR) gene, most of which are single point mutations or small deletions or insertions of a few nucleotides. Here we report the first large deletion identified in the CFTR gene, which involves 50 kb in two stretches of DNA: one of 10 kb from exon 4 to exon 7, and another of 40 kb, spanning exons 11 to 18. The deletion has been detected via uniparental inheritance of CFTR microsatellite alleles (IVS17BTA and IVS17BCA) in 3 independent CF families. Clinical status of the 3 CF patients, of which two have the delta F508 mutation as the other CF allele, suggests that this mutation is responsible for a severe clinical phenotype, indistinguishable from homozygous delta F508 patients. The deletion detected here suggests that other large, but less complex molecular defects could also exist in the CFTR gene.