Comparison of the three primary structures of deoxyribonuclease isolated from bovine, ovine, and porcine pancreas. Derivation of the amino acid sequence of ovine DNase and revision of the previously published amino acid sequence of bovine DNase

Based on the published bovine DNase sequence (Liao, T.-H., Salnikow, J., Moore, S., and Stein, W. H. (1973) J. Biol. Chem. 248, 1489-1495), the ovine DNase sequence is derived from the amino acid compositions of isolated short peptides covering all regions of the intact polypeptide. The sequence is substantiated by results of automated Edman degradation of the intact polypeptide and of the two middle CNBr fragments, and by elucidation of the complete sequence of the COOH-terminal CNBr peptide. The 12 changes from bovine to ovine DNase are at residues 22 (Ala to Ser), 29 (Val to Leu), 35 (Val to Ala), 54 (Tyr to Asp), 62 (Thr to Ser), 83 (Leu to Val), 121 (His to Pro), 127 (Glu to Ala), 132 (Ala to Pro), 159 (His to Asp), 163 (Val to Ile), and 231 (Ala to Val). A minor genetic variant form of ovine DNase has Val at residue 163. The data from automated Edman degradation of the largest CNBr peptide of bovine DNase show that the published bovine DNase sequence is in error and that an Ile-Val-Arg tripeptide must be inserted between Arg-27 and Arg-28. The corrected sequence is substantiated by two peptides covering this region each with three amino acids more than the published sequence. Comparison of the bovine, ovine, and porcine DNase sequences reveals the following: with the revised bovine sequence, all three DNase sequences can be aligned without a gap; all three DNases have a carbohydrate side chain at Asn-18, but only porcine DNase has carbohydrate at Asn-106; there are 12 changes between bovine and ovine DNases, 56 between bovine and porcine, and 50 between ovine and porcine; there are six highly variable regions and four invariable ones; bovine and ovine DNases have the same length while porcine DNase is longer by 2 amino acid residues at the COOH terminus; the residues around the nucleotide-binding site, the four pairs of salt bridges, and the essential His-134 groups are not changed.     

Nucleotide sequences of two mRNAs for rat brain myelin proteolipid protein

The 3200 and 1600 nucleotide mRNAs encoding rat brain proteolipid protein (PLP), the major protein component of central nervous system myelin, are heterogeneous at their 5' ends, differ in their 3' polyadenylation sites, and are transcribed from a single gene. The mRNAs, which first appear postnatally, encode identical 277 amino acid proteins that are 99% identical to the bovine protein sequence. Thus, PLP has been highly conserved during mammalian evolution. A single amino-terminal methionine is removed post-translationally, indicating that PLP does not require a signal peptide sequence for insertion into the myelin membrane. Mouse and monkey utilize the 3200 but not the 1600 nucleotide mRNA, suggesting that there is no functional necessity for two sizes of rat PLP mRNAs.     

Isolation,structure and biologic activity of chicken intestinal neurotensin

Using a radioimmunoassay towards bovine neurotensin (NT), chicken NT has been purified to homogeneity from extracts of intestine and its amino acid sequence determined to be: <Glu-Leu-His-Val-Asn-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu-OH. The molecule is identical to the bovine peptide except for the 3 amino acid substitutions located in its NH₂-terminal half and italicized above (His/Tyr; Val/Glu; Ala/Pro). The structure for chicken NT is consistent with earlier immunochemical studies which indicated a COOH-terminal homology with bovine NT [1]. The peptide isolated was shown to be near equipotent with bovine NT in its ability to induce hypotension, hyperglycemia, and cyanosis in the anesthesized rat, underscoring the importance of the COOH-terminal residues in NT for biological activity.     

Nucleotide sequences of bovine alpha S1- and kappa-casein cDNAs

The nucleotide sequences corresponding to bovine alpha S1- and kappa-casein mRNAs are presented. An unusual alpha S1-casein cDNA has been characterised whose 5' end commences upstream from its putative TATA box. The alpha S1-casein mRNA is compared to rat alpha-casein mRNA and two components of divergence are identified. Firstly, the two sequences have diverged at a high point mutation rate and the rate of amino acid replacement by this mechanism is at least as great as the rate of divergence of any other part of the mRNAs. Secondly, the protein coding sequence has been subjected to several insertion/deletion events, one of which may be an example of exon shuffling . The kappa-casein mRNA sequence verifies the proposition that it has arisen from a different ancestral gene to the other caseins.     

Bovine and water buffalo <Emphasis Type="Italic">Mx2</Emphasis> genes: polymorphism and antiviral activity

Millennia-long selective pressure of single-strand RNA viruses on the bovine Mx locus has increased the advantages of using the bovine Mx protein to evaluate the ultimate significance of the antiviral role of Mx proteins. The conclusions of research based only on the bovine Mx1 protein showed the need for comprehensive studies that demonstrate the role of all isoforms, individually or together, especially in the presence of a second isoform, the bovine Mx2 gene. This study provides information about bovine and water buffalo Mx2 genes, as well as their allelic polymorphism and basic antiviral potential. Observation of an Mx2 cDNA sequence (2,381 bp) obtained from 15 animals from 11 breeds using primers based on a previous sequence (NCBI accession no. AF335147) revealed several nucleotide substitutions, with eight different alleles and two amino acid exchanges: Gly to Ser at position 302 and Ile to Val at position 354, though the latter was found only in the NCBI database. A water buffalo Mx2 cDNA sequence was identified for the first time, revealing 46 nucleotide substitutions with 12 amino acid variations, in addition to a 9-bp insertion in the 5′ untranslated region UTR, compared with the bovine Mx2 cDNA. Transfected 3T3 cells expressing bovine Mx2 mRNAs coding Gly or Ser at position 302, water buffalo Mx2 mRNA, positive control bovine Mx1 mRNA-expressing cells, and negative control parental 3T3 were subjected to infection with recombinant vesicular stomatitis virus (VSVΔG*-G), as were empty pCI-neo vector-transfected cells. The positive control and all cells expressing Mx2 mRNAs displayed significantly higher levels of antiviral activity against VSVΔG*-G (P < 0.01) than did the negative controls.     

Complete nucleotide sequence of ovine alpha-lactalbumin mRNA

The nucleotide sequence of ovine alpha-lactalbumin mRNA has been determined by chemical sequencing of two cDNA recombinant plasmids and a primer extension product. Ovine alpha-lactalbumin mRNA contains 723 nucleotides (excluding the poly(A) tail), with a 5' non-coding region of 26 nucleotides, followed by the 426 nucleotides of the coding region which determines a sequence signal of 19 amino acid residues and the 123 amino acid residues of mature alpha-lactalbumin. The coding region is followed by a 3' untranslated sequence of 271 nucleotides. The derived amino acid sequence of ovine pre-alpha-lactalbumin differs from that of its bovine counterpart by 8 amino acid substitutions, all but one originating from single mutations. Comparison of sequences of guinea pig, rat and human alpha-lactalbumin mRNAs with their ovine and bovine counterparts has revealed that these molecules have rapidly evolved. The highest degree of conservation was observed in the region coding for the mature protein and corresponds essentially to sequences which interact with UDP-galactosyltransferase and Ca2+ ions.     

The rat gene encoding neurotensin and neuromedin N. Structure, tissue-specific expression, and evolution of exon sequences

Recombinant DNA clones encoding the neurotensin/neuromedin N precursor protein have been isolated from both bovine hypothalamus cDNA and rat genomic libraries using a heterologous canine cDNA probe. Nucleotide sequence analysis of these clones and comparison with the previously determined canine sequence has revealed that 76% of the amino acid residues are conserved in all three species. The protein precursor sequences predicted from bovine hypothalamus and canine intestine cDNA clones vary at only 9 of 170 amino acid residues suggesting that within a species identical precursors are synthesized in both the central nervous system and intestine. The rat gene spans approximately 10.2 kilobases (kb) and is divided into four exons by three introns. The neurotensin and neuromedin N coding domains are tandemly positioned on exon 4. RNA blot analysis has revealed that the rat gene is transcribed to yield two distinct mRNAs, 1.0 and 1.5 kb in size, in all gastrointestinal and all neural tissues examined except the cerebellum. There is a striking variation in the relative levels of these two mRNAs between brain and intestine. The smaller 1.0-kb mRNA greatly predominates in intestine while both mRNA species are nearly equally abundant in hypothalamus, brain stem, and cortex. Sequence comparisons and RNA blot analysis indicate that these two mRNAs result from the differential utilization of two consensus poly(A) addition signals and differ in the extent of their 3' untranslated regions. The relative combined levels of the mRNAs in various brain and intestine regions correspond roughly with the relative levels of immunologically detectable neurotensin except in the cerebral cortex where mRNA levels are 6 times higher than anticipated.     

The epsilon-subunit of ATP synthase from bovine heart mitochondria. Complementary DNA sequence, expression in bovine tissues and evidence of homologous sequences in man and rat.

The epsilon-subunit of ATP synthase from bovine heart mitochondria is assembled into the extrinsic membrane sector, F1-ATPase. The mature protein is 50 amino acid residues in length and its function is unknown. It is a nuclear gene product that is imported into the organelle. A mixture of 64 oligonucleotides 17 bases long, designed on the basis of the known protein sequence, was synthesized and used as a hybridization probe to isolate a cognate cDNA clone from a bovine library. The DNA sequence of this clone was determined, and the protein sequence of the epsilon-subunit deduced from it agrees exactly with that determined by direct sequence analysis of the protein isolated from bovine hearts. The bovine cDNA was used as a hybridization probe to examine the expression of the epsilon-subunit in various bovine tissues. mRNAs related to the cDNA are found in all of these tissues, and no evidence was obtained of the presence of mRNAs for the epsilon-subunit with similar coding sequences and dissimilar 3' non-coding regions. By hybridization experiments with digests of DNA from cow, man and rat it has been shown that sequences related to the bovine cDNA are present in the genomes of all three species. More than one related sequence was detected in all cases, indicating the presence in all three genomes of more than one gene and/or pseudogenes.     

Complete nucleotide sequences of bovine alpha S2- and beta-casein cDNAs: comparisons with related sequences in other species

The nucleotide sequences corresponding to bovine alpha S2- and beta- casein mRNAs have been determined by cDNA analysis. Both sequences appear to be complete at their 5' ends. The nucleotide sequence of alpha S2-casein, when compared with the corresponding cavine A sequence, helps to define the boundaries of a large amino acid repeat (approximately 80 residues) whereas comparisons with the nucleotide sequences of rat gamma- and mouse epsilon-casein mRNAs also reveal extensive sequence similarities. An alignment of these four sequences shows that the divergence of their translated regions has been characterized by the duplication and deletion of discrete segments of sequence that probably correspond to exons. A high degree of nucleotide substitution is also found when the four sequences are compared, except for well-conserved leader-peptide and phosphorylation-site sequences and, to a lesser extent, the 5'-untranslated regions. Similar comparison of the bovine and rat beta-caseins shows that their divergence has involved a high rate of nucleotide substitution but that no major insertions or deletions of sequence have occurred. The several splice sites that have veen defined in the rat beta-casein gene are likely to have been conserved in the bovine. The contrasting evolutionary histories of the alpha- and beta-casein coding sequences correlate with the distinctive functions of these proteins in the casein micelle system in milk.      

Amino-terminal sequence of a phospholipid transfer protein from rat lung

A phospholipid transfer protein from rat lung has been characterized in terms of the amino-terminal sequence. The sequence is Val-Leu-Leu-Lys-Glu-Tyr-Arg-Val-Ile-Leu-Pro-(Val)-His-Val-Asp-Glu-Tyr-Gln-Val- Gly. Comparison of the amino-terminal sequence of the protein from lung with sequences from phosphatidylcholine transfer protein and non-specific phospholipid transfer protein from bovine liver revealed no apparent sequence homology. The sequence showed no homology with fatty acid binding proteins or cellular retinoid binding proteins.     

A novel expression vector for high-level synthesis and secretion of foreign proteins in Escherichia coli: overproduction of bovine pancreatic phospholipase A2

A novel expression plasmid (pTO-N) has been constructed that allows for the production of large quantities of foreign proteins (or fragments thereof) in an unfused state. The vector has a strong and tightly regulated T7 gene 10 promoter and the ompA Shine-Dalgarno (SD) sequence, followed by the ompA sequence and a cloning linker region. The mRNAs produced by the vector are protected by secondary structures at both ends of the mRNAs. The OmpA signal peptide directed the synthesized proteins into the periplasmic space of Escherichia coli. Phospholipase A2 and prophospholipase A2 from bovine pancreas have been produced to a high level by using this expression vector. One additional feature, which is essential for the stable maintenance of the plasmid in the E. coli expression host, BL21 (DE3)[pLysS], is the shortened distance between the 5' secondary structure sequence (immediately following the gene 10 promoter) and the SD sequence. This vector could be particularly useful for synthesis of toxins in E. coli.     

Relations between divalent cation binding and ATPase activity in coupling factor from chloroplast.

Although 150 individual samples of milk from Italian water buffalo (Bubalus arnee) were examined by acid and alkaline gel electrophoresis, no polymorphism was observed for α-lactalbumin and β-lactoglobulin. After isolation and purification of these two proteins their amino acid compositions were determined and compared with those of the corresponding bovine proteins. The sequence alignments of 36 and 17 amino-acids from the N-terminal ends and 2 amino-acids from the C-terminal ends of buffalo α-lactalbumin and β-lactoglobulin, respectively, have been established. Our results indicate that buffalo α-lactalbumin differs from its cow B counterpart by a substitution Asn/Gly at position 17 and by another substitution, likely Glu/Gln or Asp/Asn, at an unknown position. Buffalo β-lactoglobulin is homologous to the bovine B variant. Three substitutions differentiate the two proteins: Ile/Leu and Val/Ile at positions 1 and 162 respectively; a further one, Gln/Ile, has not yet been located. According to these results the B variant of bovine β-lactoglobulin might be the wild type of the Bos genus.     

Marsupial possum neurotensin: a unique mammalian regulatory peptide exhibiting structural homology to the avian analogue

Neurotensin has been isolated from small intestinal extracts of an Australian marsupial, the brush-tailed possum (Trichosurus vulpecula). The primary structure was determined as: pGlu-Leu-His-Val-Asn-Lys-Ala-Arg-Arg-Val-Tyr-Ile-Leu. When compared with bovine neurotensin, marsupial possum neurotensin exhibits four amino acid substitutions. His for Tyr3, Val for Glu4 and Ala for Pro7 are identical with those found in chicken neurotensin. In addition, substitution of Pro10 with Val is unique among all neurotensins sequenced to date. Marsupial possum neurotensin is therefore of unique primary structure, displaying most sequence homology with its avian counterpart. This neurotensin may thus resemble the phylogenetic precursor present at the time of divergence of primitive mammals and birds.     

Cloning and sequence analysis of cDNAs for human high molecular weight and low molecular weight prekininogens. Primary structures of two human prekininogens

cDNA sequences for both human high molecular weight (HMW) and low molecular weight (LMW) prekininogens have been isolated by molecular cloning and determined by sequence analysis. The sequence determination together with the S1 nuclease mapping and RNA blot-hybridization analyses indicate that human HMW and LMW prekininogen mRNAs share an identical sequence throughout the 5'-untranslated region and the protein-coding region up to the sequence encoding the 12 amino acids distal to the bradykinin sequence, and the two mRNAs then completely diverge from each other. The signal peptide, the heavy chain (H chain), and the bradykinin moiety, which are common between the two prekininogens, consist of 18, 362, and 9 amino acids, respectively, while the light chains (L chains) of the HMW and LMW prekininogens are composed of 255 and 38 amino acids, respectively. All 17 cysteine residues present in the human and bovine H chains are located at exactly equivalent positions, indicating that the human H chain, like the bovine counterpart, can form 8 loop structures, each connected by two adjacent cysteine residues. The L chains of human and bovine kininogens differ in the protein lengths as well as in some amino acids crucial for the processing of the kininogens by kallikrein. Based upon this finding, we have discussed the molecular basis for the different modes of processing of human and bovine HMW kininogens and for the different kinetics of contact activation reactions exhibited by the two HMW kininogens.     

Bovine parathymosin: amino acid sequence and comparison with rat parathymosin

Parathymosin has been purified from calf liver and its primary sequence established, except for a segment containing approximately 11 amino acid residues in the central part of the polypeptide chain. Bovine parathymosin contains approximately 101 amino acid residues and shows 90% identity with rat parathymosin, with substitution of Glu for Asp at positions 21, 57, and 58, Asp for Glu at positions 60 and 63, and Ala for Val at position 77. Three non-conservative substitutions were Ala for Thr at position 81, Leu for Arg at position 78, and Val for Lys at position 79. The replacement at the last two positions of a pair of basic by hydrophobic amino acid residues may account for differences in chromatographic behavior observed for the bovine and rat polypeptides. Analysis of the NH2-terminus employed a new deblocking procedure which was also employed to analyze rat parathymosin, requiring correction of the previously published NH2-terminal sequence for that polypeptide.     

A model for Sec incorporation with the regions upstream of the UGA Sec codon to play a key role

For eukaryotic selenoprotein mRNAs, it has been proposed that the SECIS element in the 3'-UTR is required for recognition of UGA as a Sec codon. Some proteins which bind to SECIS (SBP) have been reported. However, it is not clear how the SECIS element in the 3'-UTR can mediate Sec insertion far at the in-frame UGA Sec codons. The idea that there must be a signal near the UGA Sec codon is still being considered. Therefore, we searched for a protein which binds to an RNA sequence surrounding the UGA Sec codon on human GPx mRNA. We found a protein, prepared from bovine brain microsomes, which strongly bound to the RNA fragment upstream of the UGA Sec codon but not to the RNA sequence downstream of the UGA codon. This protein also bound to the SECIS sequence in the 3'-UTR of human GPx, and this binding to SECIS was competed with the RNA fragment upstream of the UGA Sec codon. We also obtained the similar results with the RNA fragments of type I iodothyronine 5'-deiodinase (5'DI) mRNAs. Comparison of such RNA fragments with SECIS fragments revealed similarities in the region upstream of the in-frame UGA Sec codon of several Se-protein mRNAs. The study thus favors a novel model of Sec incorporation at the UGA Sec codon that involves the regions upstream of the UGA codon of mRNAs of mammalian selenoproteins. This model explains that the stem-loop structure covering the UGA codon is recognized by SBP and how the UGA Sec codon escapes from attack by eRF.     

Sequence analysis of cloned cDNA for rat substance P precursor: existence of a third substance P precursor

The sequence of the mRNA for the rat substance P precursor (preprotachykinin A) has been elucidated by molecular cloning and sequence analysis. The deduced amino acid sequence of rat preprotachykinin A indicates that it contains both substance P and substance K but differs in the sequence organization from either bovine alpha- or beta-preprotachykinin A reported previously. The existence of the bovine mRNA for the third preprotachykinin A has thus been examined and evidenced by the isolation of the corresponding cDNA clone. This mRNA, named gamma-preprotachykinin A mRNA, deletes the sequence precisely corresponding to the exon 4 sequence of the preprotachykinin A gene. Thus, alternative RNA splicing in the expression of the single preprotachykinin A gene results in the generation of three different forms of the preprotachykinin A mRNAs.     

Characterization of two novel forms of cholecystokinin isolated from bovine upper intestine

The primary structures of two novel forms of cholecystokinin, isolated from bovine upper intestine are reported. The two peptides are composed of 33 and 39 amino acid residues, respectively, the larger being an N-terminally extended form of the shorter peptide. The primary structure of the 39 amino acid peptide is: (Formula: see text) This amino acid sequence differs from the porcine hormone at positions 13 and 15, which are Val and Met, respectively, in pig, the same amino acid substitutions have previously been found to occur also in dog.