Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.     

The sequence of a cloned cDNA for the beta subunit of bovine thyrotropin predicts a protein containing both NH2-and COOH-terminal extensions

Cloned cDNAs containing sequences coding for the beta subunit of bovine thyrotropin have been identified. The complete nucleotide sequence of the largest of the beta subunit cDNA inserts has been determined. This cDNA contains 35 nucleotides from the 5' untranslated region of thyrotropin beta subunit mRNA and 60 nucleotides coding for an NH2-terminal precursor segment. This is followed by 339 nucleotides which code for the published amino acid sequence of the thyrotropin beta subunit. Following the 339 nucleotide beta subunit coding sequence, no termination codon is encountered for another 15 nucleotides. Thus, the cDNA codes for a thyrotropin beta subunit containing an additional 5 amino acids at the COOH terminus. The cDNA also contains 82 nucleotides of 3' untranslated sequence followed by a short poly(A) segment. Comparison of the bovine cDNA sequence to the recently described mouse thyrotropin beta subunit cDNA sequence reveals considerable homology throughout the coding sequence, including the COOH-terminal extension. These findings suggest the possibility that a thyrotropin beta subunit precursor is processed at both the NH2 and COOH termini.     

Molecular structure and flanking nucleotide sequences of the natural chicken ovomucoid gene

Five independent clones containing the natural chicken ovomucoid gene have been isolated from a chicken gene library. One of these clones, CL21, contains the complete ovomucoid gene and includes more than 3 kb of DNA sequences flanking both termini of the gene. Restriction endonuclease mapping, electron microscopy and direct DNA sequencing analyses of this clone have revealed that the ovomucoid gene is 5.6 kb long and codes for a messenger RNA of 821 nucleotides. The structural gene sequence coding Ifor the mature messenger RNA is split into at least eight segments by a minimum of seven intervening sequences of various sizes. The shortest structural gene segment is only 20 nucleotides long. All seven intervening sequences are located within the peptide coding region of the gene, and the sequences at the 5' and 3' untranslated regions of the mRNA are not interrupted by intervening sequences. The DNA sequences of the regions flanking the 5' and 3' termini of the gene have been determined. Thirty nucleotides before the start of the messenger RNA coding sequence is the heptanucleotide TATATAT, which is also present in a similar location relative to the chicken ovalbumin gene and other unique sequence eucaryotic genes. This sequence resembles that of the Pribnow box in procaryotic genes where a promoter function has been implicated. Seven nucleotides past the 3' end of the gene is the tetranucleotide TTGT, a sequence found to be present at identical locations as either TTTT or TTGT in other eucaryotic genes that have been sequenced. These conserved DNA sequences flanking eucaryotic genes may serve some regulator function in the expression of these genes.     

Evolution of the casein multigene family: conserved sequences in the 5'' flanking and exon regions.

The rat alpha- and bovine alpha s1-casein genes have been isolated and their 5' sequences determined. The rat alpha-, beta-, gamma- and bovine alpha s1-casein genes contain similar 5' exon arrangements in which the 5' noncoding, signal peptide and casein kinase phosphorylation sequences are each encoded by separate exons. These findings support the hypothesis that during evolution, the family of casein genes arose by a process involving exon recruitment followed by intragenic and intergenic duplication of a primordial gene. Several highly conserved regions in the first 200 base pairs of the 5' flanking DNA have been identified. Additional sequence homology extending up to 550 base pairs upstream of the CAP site has been found between the rat alpha- and bovine alpha s1-casein sequences. Unexpectedly, the 5' flanking promoter regions are conserved to a greater extent than both the entire mature coding and intron regions of these genes. These conserved 5' flanking sequences may contain potential cis regulatory elements which are responsible for the coordinate expression of the functionally-related casein genes during mammary gland development.     

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.     

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.     

Isolation and characterization of human cDNA clones encoding the alpha and the alpha' subunits of casein kinase II

Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two alpha or alpha' subunits (or one of each) and two beta subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell lambda gt10 library using cDNA clones isolated from Drosophila melanogaster [Saxena et al. (1987) Mol. Cell. Biol. 7, 3409-3417]. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5' untranslated region) and followed by 871 bp (3' untranslated region). The hT4.1 clone was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells [Meisner et al. (1989) Biochemistry 28, 4072-4076]. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 bp (5' untranslated region) and followed by 550 bp (3' untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of alpha and alpha' subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the alpha and alpha' subunits of casein kinase II. Microsequence data determined from separated preparations of bovine casein kinase II alpha subunit and alpha' subunit [Litchfield et al. (1990) J. Biol. Chem. 265, 7638-7644] confirmed that hT4.1 encoded the alpha subunit and hT9.1 encoded the alpha' subunit. These studies show that there are two distinct catalytic subunits for casein kinase II (alpha and alpha') and that the sequence of these subunits is largely conserved between the bovine and the human.     

Sequence homologies in the protamine gene family of rainbow trout

We have sequenced five different rainbow trout protamine genes plus their flanking regions. The genes are not clustered and do not contain intervening sequences. There is an extremely high degree of sequence conservation in the coding and 3' untranslated regions of the gene. Downstream sequences exhibit little homology though conserved regions are found 250 base pairs 3' to the gene. There are four regions upstream of the gene that are highly conserved in the six clones, including the canonical Goldberg - Hogness box which is 45 base pairs 5' to the coding region. A second homologous region is found 90 bases upstream. Although in the same approximate location as the CAAT box found upstream of other genes, it does not contain the canonical CAAT sequence. Further upstream of the protamine genes at -115 there is an A-T rich sequence while a 25 base pair conserved sequence is located 150 bases upstream. In addition we report the presence of a potential Z-DNA region of predominantly A-C repeats approximately one kilobase downstream of one of the genes.     

A short repeat in the genome of the DNA sequences flanking bovine growth hormone genes

The phage clones containing a gene coding for bovine growth hormone were isolated from a bovine genomic library. Comparison of the 5' and 3' regions flanking the bovine growth hormone gene by Southern blot hybridization revealed that they share homology. Screening the bovine genomic library by nick-translated DNA fragment from 5' flanking region leads to conclusion that this sequence is present in 0.1% of clones. Each analysed clone carrying the sequence contains some copies of it.     

The relationship between rat major acute phase protein and the kininogens

The rat major acute phase protein (alpha 1-MAP) is a cysteine protease inhibitor. The stoichiometry of the interaction between the inhibitor and enzyme was shown to be 1:2. A cDNA clone specific for rat alpha 1-MAP was isolated from a cDNA library prepared from an inflamed rat liver RNA template. The 1458-base pair insert was sequenced and positively identified by alignment with a partial amino acid sequence obtained by radiosequence analysis of the primary translation product for alpha 1-MAP. Complete sequence analysis determined the alpha 1-MAP cDNA coded for the entire protein with the exception of the first four amino acids of the signal peptide, all of which were identified by radiosequencing. The coding sequence spans 1282 nucleotides, followed by 115 base pairs of a 3' untranslated region. Two putative active sites, suggested by the enzyme-inhibitor ratio, have been identified by analysis of internal duplications of the alpha 1-MAP sequence and the alignment of these regions with the sequences of several low molecular weight cysteine protease inhibitors. A computer homology analysis of the protein sequence revealed a 59.3% overall identity between rat alpha 1-MAP and bovine low molecular weight (LMW) kininogen. The homology included the signal peptide regions. LMW kininogen is a precursor of bradykinin. alpha 1-MAP does contain a bradykinin sequence; the flanking amino acids are different, however. Evidence for the expression of the LMW and a high molecular weight kininogen from the same gene, and the high degree of homology between these proteins and the rat acute phase protein suggest that all three proteins belong to a precisely regulated gene family.     

Nucleotide sequence analysis of the cloned salmon preproinsulin cDNA

A cDNA library was constructed using polyadenylated RNA from salmon (Oncorhynchus keta) Brockmann bodies, plasmid vector pBR322, and in vitro recombinant DNA techniques. Insulin-related clones were identified with a cDNA probe generated from the same RNA and enriched for insulin sequences. Two recombinants were shown to contain the nucleotide sequence of the entire coding region and parts of the 5' and 3' untranslated regions. The salmon preproinsulin mRNA is about 760 nucleotides long, 315 of which code for the protein, while about 190 and 200 nucleotides belong to the 5' and 3' flanking regions, respectively. Comparison of the nucleotide sequences of salmon insulin mRNA with those from other species reveals that sequence conservation is limited to the regions coding for the B and A peptides and two segments of the signal peptide. The C-peptide region exhibits no significant sequence homology with the C-peptides of other vertebrates. The 5' and 3' untranslated regions of the salmon preproinsulin mRNA are homologous only with the anglerfish mRNA, whereas there is no evident homology with those of birds and mammals. In addition to establishing the sequence of the preproinsulin mRNA, cloned salmon insulin cDNA provides a specific probe for the analysis and isolation of genomic DNA fragments containing insulin genes.     

Isolation and characterization of the rat proenkephalin gene

The rat proenkephalin gene has been isolated by molecular cloning and characterized by DNA-sequence analysis. The gene exhibits a structural organization similar to that of the human gene. The nucleotide sequence encoding the biologically active opioid peptides which are generated from the proenkephalin precursor as well as the 3' untranslated region of the mRNA are found on a large exon at the 3' end of the gene (Exon III). The nucleotide sequence encoding the N terminus of the mature protein and its signal peptide are located on Exon II while Exon I encodes the 5' untranslated region of the mRNA. The nucleotide sequence of these exons and their flanking regions has been determined and compared to the human proenkephalin gene. Analysis of the nucleotide sequence homology between the human and rat proenkephalin gene reveals the presence of highly conserved regions within both the coding and noncoding portions of the genes. Enkephalin-coding sequences as well as 5' flanking sequences appear to be the most highly conserved. The importance and possible function of these sequences are discussed.     

Cloning and characterization of the C. elegans histidyl-tRNA synthetase gene.

In this paper, we report the cloning and sequencing of the C. elegans histidyl-tRNA synthetase gene. The complete genomic sequence, and most of the cDNA sequence, of this gene is now determined. The gene size including flanking and coding regions is 2230 nucleotides long. Three small introns (45-50 bp long) are found to interrupt the open reading frame. The open reading frame translates to 523 amino acids. This putative protein sequence shows extensive homology with the human and yeast histidyl-tRNA the histidyl-tRNA synthetase gene is a single copy gene. Hence, it is very likely that it encodes both the cytoplasmic and the mitochondrial histidyl-tRNA synthetases. It is likely to be trans-spliced since it contains a trans-splice site in its 5' untranslated region.