Isolation and amino acid sequence of bovine platelet factor 4

Bovine platelet factor 4 was isolated by affinity chromatography using dextran sulfate Sepharose and purified by subsequent gel filtration. The complete amino acid sequence of this 88-residue, 9505-Da protein was determined by isolation and analysis of the overlapping peptides from tryptic and Staphylococcus aureus hydrolysates of reduced, carboxymethylated, and reductive methylated protein. Primary structure comparison was made between bovine platelet factor 4, human platelet factor 4, and human beta-thromboglobulin. The bovine platelet factor 4 amino-terminal region, which contains two unique phenylalanine residues, is extended by 15 residues relative to human platelet factor 4. The bovine carboxy-terminal region is extended by three residues relative to human platelet factor 4 and differed from beta-thromboglobulin in the absence of two additional terminal residues. Bovine platelet factor 4 shares sequence similarities proportionately with both human platelet factor 4 and beta-thromboglobulin. The sequences of the lysine-rich carboxy-terminal putative heparin binding domains are essentially identical for all three proteins. The heparin neutralizing potencies of bovine and human platelet factor 4 are similar: 40 USP units of heparin neutralized per milligram protein, as measured by a modified chromogenic substrate assay. Heparin neutralization was lost by reduction of the disulfide bonds, but only attenuated by tryptic digestion of the intact protein.     

Structure and analysis of the bovine atrial natriuretic peptide precursor gene

The isolation and sequence analysis of the gene encoding the bovine atrial natriuretic peptide (ANP) precursor is described. The bovine-ANP coding sequences are located on three exons which are interrupted by two intervening sequences. Comparison of the bovine, human, rat and mouse ANP gene sequences reveals a common organization of introns and exons and a high degree of sequence homology in the 5'-flanking and coding regions. Examination of the pre-proANP amino acid sequence derived from the bovine gene with those from rat, mouse and human, indicates a high degree of sequence homology in both the amino-terminal and biologically-active carboxy-terminal ANP region. The latter region in the bovine sequence resembles its human counterpart except for a carboxy-terminal Arg-Arg dipeptide.     

Human Microtubule-Associated Protein 1a (MAP1A) Gene: Genomic Organization, cDNA Sequence, and Developmental- and Tissue-Specific Expression

Microtubule-associated proteins (MAPs) regulate microtubule stability and play critical roles in neuronal development and the balance between neuronal plasticity and rigidity. MAP1a (HGMW-approved symbol MAP1A) stabilizes microtubules in postnatal axons. We describe human MAP1a's genomic organization and deduced cDNA and amino acid sequences. MAP1a is a single-copy gene spanning 10.5 kb. MAP1a coding sequence is contained in five exons. Translation begins in exon 3. Human MAP1a contains 2805 amino acids (predicted molecular weight 306.5 kDa) and is slightly larger than rat MAP1a (2774 amino acids). Like rat and bovine MAP1a, human MAP1a contains conserved tubulin binding motifs in the amino-terminal region. The carboxy-terminal portion contains a conserved pentadecapeptide that is present in the light chain portion of rat and bovine MAP1a/LC2 polyprotein. We show that human MAP1a gene expression occurs almost exclusively in the brain and that there is approximately 10-fold greater gene expression in adult brain compared to fetal brain. Strong, interspecies conservation between human and rat MAP1a cDNA and amino acid sequences indicates important relationships between MAP1a's function and its primary amino acid sequence.     

Mosaic evolution of prepropancreatic polypeptide. II. Structural conservation and divergence in pancreatic polypeptide gene

We demonstrated that nucleotide and amino acid sequences in the carboxyl-terminal regions of rat, mouse, and human prepropancreatic polypeptide exhibit a high degree of divergence, whereas the amino-terminal domains are highly conserved. To understand the molecular basis of this divergence and conservation, we determined the nucleotide sequence of the rat pancreatic polypeptide gene from an islet genomic library and compared it with that of the human gene. Exon 2 of the rat gene encodes the signal peptide and pancreatic polypeptide, exon 3 encodes the carboxyl-terminal region, and exons 1 and 4 encode the 5'- and 3'- untranslated regions of the mRNA, respectively. Exons 1 and 2 of rat and human genes are well conserved. The rat and human genes, however, have exons 3 and 4 of different lengths and heterologous nucleotide sequences. Mutational accumulation in exons 3 and 4 and intron 3 of the rat gene appears to have caused splice junction sliding and translational frameshift, resulting in a structural divergence in the carboxyl-terminal region. Available evidence indicates that the mosaicism of structural conservation and divergence in pancreatic polypeptide genes may have been caused by a difference in the evolutionary rates of the genomic regions.     

Structural features of B family chorion sequences in the silkmoth Bombyx mori, and their evolutionary implications.

Partial protein sequences, and DNA sequences of corresponding cDNA and genomic clones were obtained and analyzed to reveal the primary structural features of major, developmentally middle or late components of the B chorion multigene family in Bombyx mori. Comparisons with other types of sequences confirm and clarify the tripartite domain structure of chorion proteins. Glycine-, leucine- and tyrosine-containing, tandemly repetitive peptides form the bulk of the amino-terminal and carboxy-terminal domains ('arms'). Extensive sequence homologies suggest a common evolutionary origin for the amino-terminal arms of some B. mori B sequences and the corresponding portions of members of a different (A) chorion multigene family in Antheraea polyphemus, a distantly related silkmoth.     

Derivation of the sequence of the signal peptide in human C4b-binding protein and interspecies cross-hybridisation of the C4bp cDNA sequence

A 5' cDNA clone coding for human C4b-binding protein (C4bp) was isolated, characterised and sequenced to complete the cDNA sequence coding for residues 1-32 thus confirming the protein sequence data of Chung et al. [(1985) Biochem. J. 230, 133-141]. The sequence extended to allow derivation of the putative leader peptide sequence which was 32 residues in length and showed a high of hydrophobicity typical of other documented leader sequences. Cross hybridisation was detected between the human C4bp cDNA probes and genomic DNA isolated from various species on Southern blots suggesting that genomic sequence homologous to that coding for C4bp has been conserved during evolution.     

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana.

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.     

Structure and organization of the gene coding for the DNA binding protein of adenovirus type 5

We have determined the nucleotide sequence of a region of adenovirus type 5 (Ad5) DNA located between map positions 61.7 and 71.4, which covers the gene form the 72 kD DNA binding protein (DBP) and the sequence encoding the amino-terminal part of the 100 kD protein. Sequence analysis of cDNA copies of DBP mRNA revealed the existence of two abundant species of spliced mRNA molecules. One species consists of two short leader sequences from positions 75.2 (67 and 68 nucleotides long) and 68.8 (77 nucleotides long), respectively, and the main body of the RNA molecules. The other species contains only the leader sequence from position 75.2 and the main body. The amino acid sequence of DBP is encoded entirely by a long open reading frame of 1587 nucleotides in the main body of DBP mRNA. From the nucleotide sequence of the DBP gene it can be derived that DBP contains 529 amino acid residues and has an actual molecular weight of 59,049 daltons. The sites of mutation in the mutants H5hr404 and H5ts125 were determined at the nucleotide level. Single nucleotide alterations were detected in H5hr404 and H5ts125 in the sequences corresponding to the amino-terminal part and the carboxy-terminal part of DBP, respectively. The implications of these mutations are discussed.     

Sequencing of the nuclear gene for the yeast cytochrome c1 precursor reveals an unusually complex amino-terminal presequence.

Cytochrome c1 is a component of the mitochondrial respiratory chain in most eukaryotes. The protein is coded by nuclear DNA, synthesized as a larger precursor outside the mitochondria and then cleaved to the mature form in two successive steps during its import into the mitochondria. We have cloned the structural gene for yeast cytochrome c1 by functional complementation of a cytochrome c1-deficient yeast mutant with a yeast genomic library in the yeast-Escherichia coli 'shuttle' vector YEp 13. The complete nucleotide sequence of the gene and of its 5'- and 3'-flanking regions was determined. The deduced amino acid sequence of the yeast cytochrome c1 precursor reveals an unusually long transient amino-terminal presequence of 61 amino acids. This presequence consists of a strongly basic amino-terminal region of 35 amino acids, a central region of 19 uncharged amino acids and an acidic carboxy-terminal region of seven amino acids. This tripartite structure of the presequence resembles that of the precursor of cytochrome c peroxidase and supports a previous suggestion on the import pathways of these two precursors.     

Structure of the human genomic region homologous to the bovine prochymosin-encoding gene

Two human genomic libraries were probed with bovine prochymosin (bPC) cDNA. Recombinant clones covering a genomic region homologous to the entire coding region and flanking sequences of the bPC gene were isolated. Human sequences homologous to exons of the bPC gene are distributed in a DNA fragment of 10 kb. Alignment of the human sequences and the exons of bPC reveals that the human 'exons' 1-3, 5 and 7-9 have sizes identical to the corresponding bovine exons, but a nucleotide (nt) has been deleted in the human exon 4 and two nt in the human exon 6. The aligned human sequence and the coding part of bPC gene share 82% nt homology, the value ranging, in separate exons, from 76 (exon 1) to 84% (exons 5 and 6). 150 bp of 5'-flanking sequence of the human gene has 75% homology to the corresponding region of bPC gene and contains a TATA-box in a similar position. A 1-nt deletion in the human exon 4 would shift the translational reading frame of a putative human PC mRNA relative to bPC mRNA, and result in an in-phase terminator spanning codons 163 and 164 in bPC mRNA. Another terminator in-phase with the amino-acid sequence encoded by the bPC gene occurs in the human exon 5 and the second frameshift mutation in exon 6. Thus, the nt sequence analysis of the human genomic region has revealed the presence of mutations that have rendered it unable to produce a full-length protein homologous to bPC and, therefore, we refer to this gene as a human prochymosin pseudogene (hPC psi). Blot-hybridization analysis of human genomic DNA indicates that hPC psi is a single gene in the human genome.     

A cDNA clone for the precursor of rat mitochondrial ornithine transcarbamylase: comparison of rat and human leader sequences and conservation of catalytic sites.

We have cloned a DNA complementary to the messenger RNA encoding the precursor of ornithine transcarbamylase from rat liver. This complementary DNA contains the entire protein coding region of 1062 nucleotides and 86 nucleotides of 5'- and 298 nucleotides of 3'-untranslated sequences. The predicted amino acid sequence has been confirmed by extensive protein sequence data. The mature rat enzyme contains the same number of amino acid residues (322) as the human enzyme and their amino acid sequences are 93% homologous. The rat and human amino-terminal leader sequences of 32 amino acids, on the other hand, are only 69% homologous. The rat leader contains no acidic and seven basic residues compared to four basic residues found in the human leader. There is complete sequence homology (residues 58-62) among the ornithine and aspartate transcarbamylases from E. coli and the rat and human ornithine transcarbamylases at the carbamyl phosphate binding site. Finally, a cysteine containing hexapeptide (residues 268-273), the putative ornithine binding site in Streptococcus faecalis, Streptococcus faecium, and bovine transcarbamylases, is completely conserved among the two E. coli and the two mammalian transcarbamylases.     

Nucleotide sequence of cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones

Recombinant DNA plasmids containing sequences coding for the alpha subunit of the bovine pituitary glycoprotein hormones have been isolated. The nucleotide sequences of three different cDNA clones have been determined. The largest alpha-subunit cDNA clone was found to contain 713 bases including 77 nucleotides from the 5'-untranslated region, 72 nucleotides coding for a precursor segment, 288 nucleotides coding for the mature alpha subunit, and 276 nucleotides from the 3'-untranslated region of the mRNA followed by a poly(A) segment. This cDNA likely represents most of the bovine alpha-subunit mRNA sequence. Nucleotide sequences were obtained from the cDNA inserts of two other alpha-subunit clones, and several differences among the three cDNA sequences have been detected. These differences in nucleotide sequence may represent either individual variation in genomic sequence or cloning artifacts. Comparison of the bovine alpha-subunit cDNA sequence to the sequences of human, rat, and mouse alpha-subunit cDNAs reveals that the bovine sequence has greater than 70% homology with the other cDNAs. The cloned alpha-subunit cDNA should provide a useful probe for further studies of the structure and expression of this interesting gene.     

Cloning and characterization of a novel upstream untranslated exon of the mouse Fgf-1 gene

Fibroblast growth factor 1 (FGF-1 or aFGF), is the prototype member of the heparin-binding growth factors which are capable of angiogenesis in vivo. FGF-1 has been implicated in atherosclerosis, cancer, wound repair and inflammatory autoimmune diseases. As part of an effort to understand the role of FGF-1 in the etiopathogenesis of inflammation and cancer, we have undertaken steps to isolate and characterize the mouse Fgf-1 gene. Southern blotting and sequence analysis displayed considerable conservation within the coding and upstream untranslated regions of Fgf-1 in human, mouse, hamster, rat and bovine. By using primers derived from the 5′-untranslated exon of a rat prostate-specific Fgf-1 cDNA, a 220-bp product was amplified from mouse genomic DNA via PCR. Sequence analysis of this amplicon showed that there was 80% similarity with the corresponding region of the rat FGF-cDNA sequence. Primers designed from this amplicon and the Fgf-1 coding region were used to isolate multiple overlapping genomic clones spanning the entire mouse Fgf-1 gene. Sequencing analysis of the genomic sequence upstream from this novel 5'-untranslated exon did not reveal typical TATA, CCAAT sequences. It appears that the occurrence of multiple untranslated exons for FGF-1 is a highly conserved theme for this gene across species.     

Sequence heterogeneity of murine complementary DNA clones related to the C4 and C4-Slp isoforms of the fourth complement component

Two classes of mRNA encoding the murine C4 protein were identified by sequence analysis of clones isolated from a liver complementary DNA library. The divergence found within a 357 base pair sequence available for comparison is limited to five nucleotide replacements located in the region corresponding to the carboxy-terminal end of the C4d peptide fragment. One of the nucleotide substitutions influences the presence of a site for the Hind III restriction endonuclease. That this restriction site indeed discriminates the two non-allelic genes encoding the mouse C4 and C4-Slp isoforms has been demonstrated by Southern blot analysis and nucleotide sequencing at the genomic level. Circumstantial evidence supports the identification of the gene lacking the Hind III site in the region corresponding to the carboxy-terminal end of the C4d fragment as the one encoding the C4-Slp isotype.     

Isolation of the osteonectin gene: evidence that a variable region of the osteonectin molecule is encoded within one exon

A complementary DNA clone for bovine osteonectin was used to isolate the osteonectin gene from two libraries of bovine genomic DNA fragments. Two overlapping clones were obtained whose relationship was determined by restriction mapping and sequence analysis. The two clones contain the entire osteonectin coding region spanning approximately 11 kilobases of genomic DNA. The coding region of the gene was determined, by electron microscopy and DNA sequencing, to reside in nine exons. In addition, there is at least one 5' exon interrupted by an intron in the 5'-nontranslated sequence of the gene. Excluding this 5' exon and the 3'-terminal exon, the exons are small and approximately uniform in size, averaging 130 +/- 17 base pairs. Three of the exons at the 5' end of the gene were sequenced and appear to encode discrete protein domains. For example, the putative exon 2 contains the coding region for the leader peptide of the molecule. The amino-terminal protein sequence was determined for osteonectin extracted from human, rabbit, and chicken bone and compared with those for bovine, mouse, and pig osteonectin. These data suggest that osteonectin is highly conserved between species, interspecies changes being seen primarily at the amino terminus of the protein and specifically in the region encoded by putative exon 3 in the bovine gene.