Primary structure of cDNA for bovine beta-casein

In this work the complete sequence of 1097 nucleotide bovine beta-casein cDNA has been determined. Sequencing of end labeled fragments was performed using the method of Maxam and Gilbert. Bovine beta-casein cDNA consist of a 672 nucleotide coding region, flanked by 60 nucleotide 5' and 358 nucleotide 3'-noncoding region. The restriction map of beta-casein cDNA was constructed. The computer analysis was done and the comparisons of the complete sequence of bovine beta-casein cDNA with other sequences, coding caseins in bovine, rat and guinea-pig was performed. It was determined, that cloned cDNA is related to genetic variant A1.     

Restriction analysis of cDNA for cow alpha s1-, beta- and kappa-caseins

By means of in situ hybridization to cloned cDNA fragments coding for cow alpha s1-, beta- and kappa-caseins, screening of the library of clones containing the cDNA complementary to mRNA of lactating cow mammary gland was carried out. The clones containing the sequences of alpha s1-, beta- and kappa-casein cDNAs were shown to constitute 4.0, 3.2 and 0.7% of all the colonies, respectively. The analysis of the data on cross-hybridization points to the absence of extensive regions of homology between the above-mentioned cDNAs. The restriction analysis of cDNAs of the selected clones was carried out and the restriction maps of cDNAs of these three caseins were constructed. The restriction analysis data and determination of the nucleotide sequence of 5'-termini of the studied cDNAs indicated that the cloned sequences were the full-length mRNA copies of alpha s1-, beta- and kappa-caseins. The data obtained on restriction analysis are utilized in mapping the corresponding natural genes of cow caseins.     

cDNA of cattle alpha S1-casein: cloning and nucleotide sequence

The nucleotide sequence of recombinant plasmids representing a full-size cDNA of cow alpha s1-casein was investigated. The corresponding mRNA consists of 1133 nucleotides except for poly(A) and includes 642 nucleotides of the coding region, 63 nucleotides of 5'- and 428 nucleotides of the 3'-noncoding regions. A comparative analysis of nucleotide sequences of cow alpha s1-casein and guinea pig B-casein showed that the homology in the 5'-nontranslatable region is 90.5%, that of a precasein single peptide is 82.22%, while that of the major polypeptide in the coding region is 64% without taking into account the blank spaces. The homology is higher in the 3'-noncoding region than in the coding region and makes up to 72%. The data obtained testify to the high degree of conservatism of sequences in casein mRNA noncoding regions as well as to functional and regulatory role of these sequences in gene expression of caseins.     

Structure and restriction fragment length polymorphism of genes for human liver arylamine N-acetyltransferases.

Genomic DNA clones coding for polymorphic and monomorphic arylamine N-acetyltransferases (NAT) of human liver were isolated from a genomic DNA library, and their restriction maps and partial nucleotide sequences were determined. Messenger RNA for monomorphic NAT was coded in one exon, while mRNA for polymorphic NAT was coded in two exons; the 5'-noncoding region was located in one exon 8 kb upstream from another exon containing the coding and 3'-noncoding regions. Recently, we have shown that there are three types of polymorphic NAT gene; one of the genes corresponds to a high NAT activity, while the other two genes give rise to a low NAT activity. The restriction fragment length polymorphism (RFLP) was analyzed by Southern blot hybridization of genomic DNAs from homozygotes of the three polymorphic NAT genes using various fragments of the cloned NAT gene. RFLPs of polymorphic NAT gene were observed in coding and 3'-flanking region upon digestion with BamHI and KpnI.     

The Bos taurus casein genes. Isolation and characterization of the kappa-casein gene

A region spanning 25 kb of genomic DNA containing the kappa-casein gene, has been isolated from two genomic libraries in EMBL3 and EMBL4 phage vectors. Five phage clones containing kappa-casein gene have been found. Gene organisation has been determined using restriction mapping and a partial sequencing the 5' and 3' flanking regions. The kappa-casein gene includes 5 exons, the first of them coding for 64 nucleotides from the 5' untranslated mRNA zone. The gene is 12.5 kb long, which is almost 16 times longer than the corresponding mRNA. The first intron spans 2.5 kb, the second is the largest one and spans 5.5 kb. The 5' flanking region sequence has been analysed; it contains a TATA box from -30 to -25 bp, somewhat different from the canonic sequence, and a CAAT box at -80 bp.     

The Saccharomyces cerevisiae ADE1 gene: structure, overexpression and possible regulation by general amino acid control.

The ADE1 gene of the yeast Saccharomyces cerevisiae has been cloned by complementation of the ade1 mutation. The nucleotide sequence has been determined for the 918-bp coding region, 240-bp 5'-noncoding region and 292-bp 3'-noncoding region. The sequenced region includes a single large open reading frame coding for a protein of 306 amino acid (aa) residues. The promoter of the ADE1 gene contains a copy of the 5'-TGACTC hexanucleotide, a feature characteristic of promoters under general aa control. Subsequent search of other published purine biosynthesis gene sequences revealed that all of them also contain general aa control signals in their promoter regions. An expression plasmid containing the ADE1 coding region under control of the PHO5 promoter produced N-succinyl-5-aminoimidazole-4-carboxamide ribotide (SAICAR) synthetase in yeast cells at a level of 40% of total cellular protein. One-step purification resulted in an almost homogeneous preparation of SAICAR synthetase.     

Molecular cloning and the nucleotide sequence of cDNA to mRNA for non-neuronal enolase (alpha alpha enolase) of rat brain and liver.

The nucleotide sequence for alpha alpha enolase (non-neuronal enolase: NNE) of rat brain and liver was determined from recombinant cDNA clones. The sequence was composed of 1722 bp which included the 1299 bp of the complete coding region, the 108 bp of the 5'-noncoding region and the 312 bp of the 3'-noncoding region containing a polyadenylation signal. In addition, the poly(A) tail was also found. A potential ribosome-binding site was located 30 nucleotides upstream to the initiation codon in the 5'-noncoding region. The amino acid sequence deduced from the nucleotide sequence was 433 amino acids in length and showed very high homology (82%) to the amino acid sequence of gamma gamma enolase (neuron-specific enolase: NSE), although the nucleotide sequence showed slightly lower homology (75%). The size of NNE mRNA was approximately 1800 bases by Northern transfer analysis and much shorter than that of NSE mRNA (2400 bases) indicating a short 3'-noncoding region. A dot-blot hybridization and Northern transfer analysis of cytoplasmic RNA from the developing rat brains using a labeled 3'-noncoding region of cDNA (no homology between NSE and NNE) showed a decrease of NNE mRNA at around 10 postnatal days and then a gradual increase to adult age without changes of mRNA size. Liver mRNA did not show any significant change during development.     

Cloning, expression and sequence homologies of cDNA for human gamma enolase

The nucleotide sequence of the human gamma-enolase mRNA was determined from recombinant cDNA clones. The sequence spans 2273 bp and includes the complete coding region of 1299 bp, a 5'-noncoding region of 74 bp and a 897-bp-long 3'-noncoding region containing a variant polyadenylation signal (ATTAAA). The deduced amino acid (aa) sequence is 433 aa long and shows a 97% similarity with rat gamma-enolase. Both the 5'- and 3'-untranslated regions are similar (82% and 68%, respectively) to the analogous regions of the rat gamma-enolase gene, suggesting that a strong selective pressure operates on noncoding segments of gamma-enolase mRNAs. The size of the gamma-enolase mRNA expressed in human brain is 2.4 kb. A crosshybridizing 1.5-kb message is detected in human skeletal muscle which may be derived from the beta-enolase-coding gene.     

Molecular cloning of cDNA of S100 alpha subunit mRNA

The primary structure of the bovine S-100 alpha mRNA on the basis of molecular cloning and sequence analysis of the cDNA are described. The sequence is composed of 532 bp which include the 282 bp of the complete coding region, 89 bp at the 5'-noncoding region, 161 bp at the 3'-noncoding region, polyadenylation signal, ATTAAA and poly(A) tail. Northern blot analysis shows that the size of S-100 alpha mRNA is about 700-800 bases long and a single mRNA occurs in bovine brain. Bovine brain contains both S100 alpha and beta subunits and their mRNAs. In contrast, the rat brain contains only S100 beta subunit and its mRNA.     

Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses inducible bile acid 7-dehydroxylation activity. Several new polypeptides are produced in this strain following induction with cholic acid. Genes coding for two copies of a bile acid-inducible 27,000-dalton polypeptide (baiA1 and baiA2) have been previously cloned and sequenced. We now report on a gene coding for a third copy of this 27,000-dalton polypeptide (baiA3). The baiA3 gene has been cloned in lambda DASH on an 11.2-kilobase DNA fragment from a partial Sau3A digest of the Eubacterium DNA. DNA sequence analysis of the baiA3 gene revealed 100% homology with the baiA1 gene within the coding region of the 27,000-dalton polypeptides. The baiA2 gene shares 81% sequence identity with the other two genes at the nucleotide level. The flanking nucleotide sequences associated with the baiA1 and baiA3 genes are identical for 930 bases in the 5' direction from the initiation codon and for at least 325 bases in the 3' direction from the stop codon, including the putative promoter regions for the genes. An additional open reading frame (occupying from 621 to 648 bases, depending on the correct start codon) was found in the identical 5' regions associated with the baiA1 and baiA3 clones. The 5' sequence 930 bases upstream from the baiA1 and baiA3 genes was totally divergent. The baiA2 gene, which is part of a large bile acid-inducible operon, showed no homology with the other two genes either in the 5' or 3' direction from the polypeptide coding region, except for a 15-base-pair presumed ribosome-binding site in the 5' region. These studies strongly suggest that a gene duplication (baiA1 and baiA3) has occurred and is stably maintained in this bacterium.     

The structure and evolution of a 461 amino acid human protein C precursor and its messenger RNA, based upon the DNA sequence of cloned human liver cDNAs.

Human liver cDNA coding for protein C has been synthesized, cloned and sequenced. The abundance of protein C message is approximately 0.02% of total mRNA. Three overlapping clones contain 1,798 nucleotides of contiguous sequence, which approximates the size of the protein's mRNA, based upon Northern hybridization. The cDNA sequence consists of 73 5'-noncoding bases, coding sequence for a 461 amino acid nascent polypeptide precursor, a TAA termination codon, 296 3'-noncoding bases, and a 38 base polyadenylation segment. The nascent protein consists of a 33 amino acid "signal", a 9 amino acid propeptide, a 155 amino acid "light" chain, a Lys-Arg connecting dipeptide, and a 262 amino acid "heavy" chain. Human protein C and Factor IX and X precursors possess about one third identical amino acids (59% in the gamma-carboxyglutamate domain), including two forty-six amino acid segments homologous to epidermal growth factor. Human protein C also has similar homology with prothrombin in the "leader", gamma-carboxyglutamate and serine protease domains, but lacks the two "kringle" domains found in prothrombin.     

Cloning and nucleotide sequence of the simian rotavirus gene 6 that codes for the major inner capsid protein.

The nucleotide sequence of the gene that codes for the major inner capsid protein of the simian rotavirus SA11 has been determined. A DNA copy of mRNA from gene 6 was cloned in the E. coli plasmid pBR322. The full-length gene is 1357 nucleotides long with a 5'-noncoding region of 23 nucleotides and a 3'-noncoding region of 140 nucleotides. The gene contains a single, long, open reading-frame of 1194 nucleotides capable of coding for a protein of 397 amino acids with a molecular weight of 44,816. The predicted protein product is relatively proline-rich with a net charge at neutral pH of -3.5. One stretch of 53 amino acids (encoded by nucleotides 327-485) is basic.     

Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor

Human cDNAs coding for angiogenin, a human tumor derived angiogenesis factor, were isolated from a cDNA library prepared from human liver poly(A) mRNA employing a synthetic oligonucleotide as a hybridization probe. The largest cDNA insert (697 base pairs) contained a short 5'-noncoding sequence followed by a sequence coding for a signal peptide of 24 (or 22) amino acids, 369 nucleotides coding for the mature protein of 123 amino acids, a stop codon, a 3'-noncoding sequence of 175 nucleotides, and a poly(A) tail. The gene coding for human angiogenin was then isolated from a genomic lambda Charon 4A bacteriophage library employing the cDNA as a probe. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (4688 base pairs) was then determined. The coding and 3'-noncoding regions of the gene for human angiogenin were found to be free of introns, and the DNA sequence for the gene agreed well with that of the cDNA. The gene contained a potential TATA box in the 5' end in addition to two Alu repetitive sequences immediately flanking the 5' and 3' ends of the gene. The third Alu sequence was also found about 500 nucleotides downstream from the Alu sequence at the 3' end of the gene. The amino acid sequence of human angiogenin as predicted from the gene sequence was in complete agreement with that determined by amino acid sequence analysis. It is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonuclease are also conserved in angiogenin. This provocative finding is thought to have important physiological implications.     

Human aldolase isozyme gene: the structure of multispecies aldolase B mRNAs.

A complete nucleotide sequence of human aldolase B mRNA was determined with a recombinant cDNA (pHABL120-3). The cDNA insert was composed of 1,652 bases excluding poly(A) tail and the sequence was consistent with the previous results reported by others. However, S1 nuclease mapping and subsequent genomic analysis allowed us to know that the clone possesses two more sites corresponding to 5'-termini in the 5'-noncoding region and another site of polyadenylation in the 3'-noncoding region. In fact, the major aldolase B mRNA species occupying 90% of the total mRNAs initiated at the predominant position corresponding to the position around -82 of the 5'-noncoding sequence in pHABL120-3 and terminated at the distal polyadenylation site. Second species accounting for 9% of the mRNAs initiated at the same site and terminated at the proximal polyadenylation site. The remainings have a longer 5'-noncoding sequence which starts from further upstream region of the major one and pHABL120-3 corresponds to one of these largest clones.     

Molecular cloning and nucleotide sequence of the cDNA for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme

For the studies on the mechanism of induction of peroxisomal beta-oxidation enzymes and biogenesis of the organelle, we have isolated cDNA clones for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. On blotting experiments with liver RNA, the cDNAs hybridized to a 3.0-kilobase RNA which was increased 5-7-fold by the administration of di-(2-ethylhexyl)phthalate to rats. Nucleotide sequencing was carried out for four cloned cDNAs and one obtained by a primer extension method. By overlapping these sequences with each other, we identified 20 nucleotides of 5'-noncoding, 2,166 nucleotides of coding, and 910 nucleotides of 3'-noncoding regions. The deduced amino acid sequence of the enzyme is composed of 722 residues, and the composition agrees with that of the protein data. The sequence was confirmed by the amino acid compositions and sequence analyses of some of the tryptic peptides. The molecular weight of the mature enzyme is calculated to be 78,511 from the predicted amino acid sequence. The enzyme has no terminal peptide extension as a signal for translocation into peroxisomes.     

Complete nucleotide sequence of alfalfa mosaic virus RNA3.

A full-length cDNA clone of alfalfa mosaic virus (AMV) RNA3 was prepared and sequenced. The 2,037 base sequence contains two open reading frames of 903 and 666 nucleotides that code for a 32,400 dalton protein (32.4K protein) and the 24,380 dalton coat protein, respectively. A 5'-noncoding sequence of 240 bases preceeding the 32.4K protein contains homologous regions that may have a function in its translation. The intercistronic junction is 49 bases long, the last 36 bases representing the 5'-end of the subgenomic RNA4. The remaining 179 bases comprise the 3'-terminal noncoding sequence.