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Complementary DNA and amino acid sequence of rat liver microsomal, xenobiotic epoxide hydrolase.

The coding nucleotide sequence for rat liver microsomal, xenobiotic epoxide hydrolase was determined from two overlapping cDNA clones, which together contain 1750 nucleotides complementary to epoxide hydrolase mRNA. The single open reading frame of 1365 nucleotides codes for a 455 amino acid polypeptide with a molecular weight of 52,581. The deduced amino acid composition agrees well with those determined by direct amino acid analysis of the rat protein, and the amino acid sequence is 81% identical to that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and comparison to codon usage for NADPH-cytochrome P-450 oxidoreductase and cytochromes P-450b, P-450d, and P-450PCN, suggest that epoxide hydrolase is more conserved than cytochromes P-450b and P-450PCN; comparison of the extent of sequence conservation for 12 homologous proteins between the rat and rabbit, including cytochrome P-450b, supports this hypothesis, and indicates that much of epoxide hydrolase is constrained to maintain its hydrophobic character, consistent with its intramembranous location. The predicted membrane topology of epoxide hydrolase delineates 6 membrane-spanning segments, less than the 8 or 10 predicted for two cytochrome P-450 isozymes; the lower number of membrane-spanning segments predicted for epoxide hydrolase correlates with its lesser dependence on the membrane for maintenance of its tertiary structure and catalytic activity.

Amino Acid Sequence↗

Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein.

Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, 32P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1,700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1,500, 1,850, 1,850 and 2,500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

Base Sequence↗

Molecular cloning and nucleotide sequence of DNA complementary to human decidual prolactin mRNA.

Three human decidual prolactin (PRL) cDNA clones (pdPL-1:349 base pairs, pdPL-2:584 base pairs, pdPL-3:807 base pairs without poly(A) tract) were prepared and sequenced. The insert DNA of the largest clone pdPL-3 contained the coding region corresponding to 217 amino acid residues including 18 amino acid residues of the signal peptide, and 157 nucleotides of the 3'-untranslated region. A comparison of the pdPL-3 cDNA sequence with that of human pituitary PRL cDNA revealed 4 silent nucleotide differences. Two of the base changes occurred in the third position of the amino acid codons, and the other two occurred in the 3'-untranslated region. Therefore, the amino acid sequence of decidual PRL deduced from the nucleotide sequence of pdPL-3 was identical with that of pituitary PRL. Minor changes were also observed among the three PRL mRNAs: a silent change in the third codon in the translated region, and another change in the 3'-untranslated region. Southern blot hybridization of human cellular DNA with the pdPL-3 probe indicates that PRL gene may occur once per haploid genome. Therefore, these minor changes may reflect microheterogeneity of PRL gene. The existence of multiple poly(A)-adjacent sequences was shown in the three species of human decidual PRL mRNA and in human pituitary PRL mRNA. This variation may be due to heterogeneity in the processing at the 3' terminus of mRNA or the termination sites of the transcription.

Amino Acid Sequence↗

The structure of cloned DNA complementary to catfish pancreatic somatostatin-14 messenger RNA.

Pancreatic poly(A) RNA isolated from the channel catfish (Ictalurus punctatus) was enriched for sequences corresponding to somatostatin mRNA on isokinetic sucrose gradients. Double-stranded cDNA was synthesized and inserted into the Pst I site pBR322 via the poly(dG) . poly(dC) tailing method. Escherichia coli was transformed with this DNA, and colonies containing somatostatin cDNA sequences were identified by hybridization using a primer-extended somatostatin cDNA. The somatostatin cDNA was obtained by extending a 5'-labeled undecanucleotide primer complementary to somatostatin mRNA with reverse transcriptase using catfish poly(A) RNA as a template. The synthetic primer d(T-T-C-C-A-G-A-A-G-A-A) was deduced from the amino acid sequence Phe-Phe-Trp-Lys present in somatostatin-14. Twenty positive colonies were obtained upon screening 2000 transformants. The restriction maps of the plasmid DNA obtained from the positive colonies were examined. Nineteen of these plasmids contained sequences corresponding to somatostatin-14, while one contained a sequence corresponding to somatostatin-22. The nucleotide sequence of pancreatic somatostatin-14 is reported here. The cDNA contains 350 nucleotides in the 3' noncoding region, 345 nucleotides in the coding region, and 104 nucleotides in the 5'-untranslated region. The mRNA codes for a precursor to somatostatin which is 114 amino acids in length. The preprosomatostatin has a sequence of hydrophobic amino acids at the NH2 terminus, followed by a connecting peptide of approximately 75 amino acids. The sequence Arg-Lys precedes somatostatin-14. Analysis of genomic DNA from the channel catfish reveals that somatostatin-14 and somatostatin-22 are present on different restriction fragments.

Amino Acid Sequence↗

An investigation into the solution structures of two self-complementary DNA oligomers, 5'-d(C-G-T-A-C-G) and 5'-d(A-C-G-C-G-C-G-T), by means of nuclear-Overhauser-enhancement measurements.

A 500 MHz 1H-n.m.r. study on two self-complementary alternating pyrimidine-purine oligodeoxyribonucleotides, 5'-d(C-G-T-A-C-G) and 5'-d(A-C-G-C-G-C-G-T), is presented. By using the proton-proton nuclear Overhauser effect virtually complete assignments are obtained and a large number of interproton distances [113 in the case of 5'-d(C-G-T-A-C-G) and 79 in the case of 5'-d(A-C-G-C-G-C-G-T)], both intra- and inter-nucleotide, are determined. The interproton-distance data are consistent with an overall right-handed B-DNA-type structure for both oligonucleotides, in agreement with their B-type c.d. spectra. However, whereas 5'-d(C-G-T-A-C-G) adopts a conventional B-type structure with a mononucleotide repeating unit, the interproton-distance data provide evidence that 5'-d(A-C-G-C-G-C-G-T) has a dinucleotide repeating unit consisting of alternation in glycosidic bond and sugar pucker conformations.

Base Composition↗