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.     

A single gene encodes the catalytic "A" subunit of the bovine vacuolar H(+)-ATPase.

We have previously demonstrated that the 73-kDa (A) subunit of the bovine coated vesicle (H+)-ATPase possesses a nucleotide binding site required for catalytic activity (Arai, H., Berne, M., Terres, G., Terres, H., Puopolo, K., and Forgac, M. (1987) Biochemistry 26, 6632-6638). Here we report the cDNA sequence of the coding region of the bovine brain A subunit. Comparison of the deduced amino acid sequence with those previously reported for the A subunits of vacuolar ATPases from lower eukaryotes, plants, and archaebacteria reveals significant homology, especially in sequences implicated in nucleotide binding. The message encoding the bovine brain A subunit is relatively large, approximately 4.6 kilobases; Northern blotting of RNA isolated from rat brain and human brain tumor cells reveals a message of similar size. Northern analysis of several bovine tissues indicates that only one message for this subunit is expressed. Southern blot analysis of bovine genomic DNA indicates that the bovine A subunit is encoded by a single gene.     

Human liver cDNA clones encoding proteolipid subunit 9 of the mitochondrial ATPase complex

Clones encoding the proteolipid subunit 9 of the mitochondrial ATPase complex have been isolated from a lambda gt10 library of human liver cDNA sequences, using a probe of Neurospora crassa cDNA encoding subunit 9. From nucleotide sequence analysis it is concluded that the amino acid sequence of mature human subunit 9 is identical to that of its bovine counterpart. By comparing the sequence of two cDNA clones (denoted human 1 and 2) to those of two bovine cDNA clones (denoted P1 and P2) recently described by Gay and Walker (EMBO J. 4, 3519-3524, 1985) it is evident that there are close sequence relationships between human 1 and bovine P1, and between human 2 and bovine P2, although both human clones are truncated at their 5'-ends. Thus, as in bovine cells there appears to be at least two human genes encoding subunit 9.     

Molecular cloning and cDNA structure of the regulatory subunit of type I cAMP-dependent protein kinase from rat brain

Complementary DNA (cDNA) clones encoding the regulatory subunit of the type I cAMP-dependent protein kinase (R-I) were isolated by screening of rat brain cDNA libraries. A 1.5-kilobase (kb) cDNA insert containing the entire coding region was sequenced and full amino acid sequence has been deduced from the nucleotide sequence. The clone encodes for a protein of 380 amino acids that shows 97% homology to the bovine R-I subunit. Northern blot analysis demonstrated two major mRNA species (2.8 and 4.4 kb in size) in rat brain and liver.     

Beta subunit of rat liver mitochondrial ATP synthase: cDNA cloning, amino acid sequence, expression in Escherichia coli, and structural relationship to adenylate kinase

The amino acid sequence of all but a few N-terminal residues of the beta subunit of rat liver ATP synthase has been determined from cDNA clones. Rat liver F1-beta is shown to contain 17 amino acid differences from that reported for F1-beta of bovine heart, 2 differences of which involve differences in charge. This may account in part for the observation that bovine heart F1 binds nucleotides with much greater affinity than the rat liver enzyme. Rat liver F1-beta also contains homologous regions with another nucleotide binding protein, adenylate kinase, for which high-resolution structural studies are available. Adjacent to one of these homologous regions is an eight amino acid stretch which bears striking homology to the phosphorylation region of the (Na+,K+)-ATPase. The combination of these two homology regions may constitute at least part of a nucleotide binding domain in F1-beta. Significantly, both rat liver and bovine heart beta contain these regions of homology, whereas the 17 amino acid differences between the two enzymes lie outside this region. The possibility of a second nucleotide binding domain which differs between the two enzymes is discussed. A cDNA clone containing all the regions of homology as well as 11 of the 17 amino acid differences between the bovine heart and rat liver beta subunits has been ligated into the bacterial expression vector pKK223-3. After transformation of a protease-deficient strain of Escherichia coli, this cDNA clone is expressed as a 36-kilodalton protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of two cDNA clones for pyruvate dehydrogenase E1 beta subunit and its regulation in tricarboxylic acid cycle-deficient fibroblast

Two distinct types of cDNA clones encoding for the pyruvate dehydrogenase (PDH) E1 beta subunit were isolated from a human liver lambda gt11 cDNA library and characterized. These cDNA clones have identical nucleotide sequences for PDH E1 beta protein coding region but differ in their lengths and in the sequences of their 3'-untranslated regions. The smaller cDNA had an unusual polyadenylation signal within its protein coding region. The cDNA-deduced protein of PDH E1 beta subunit revealed a precursor protein of 359 amino acid residues (Mr 39,223) and a mature protein of 329 residues (Mr 35,894), respectively. Both cDNAs shared high amino acid sequence similarity with that isolated from human foreskin (Koike, K.K., Ohta, S., Urata, Y., Kagawa, Y., and Koike, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 41-45) except for three regions of frameshift mutation. These changes led to dramatic alterations in the local net charges and predicted protein conformation. One of the different sequences in the protein coding region of liver cDNA (nucleotide position 452-752) reported here was confirmed by sequencing the region after amplification of cDNA prepared from human skin fibroblasts by the polymerase chain reaction. Southern blot analysis verified simple patterns of hybridization with E1 beta cDNA, indicating that the PDH E1 beta subunit gene is not a member of a multigene family. The mechanisms of differential expression of the PDH E1 alpha and E1 beta subunits were also studied in established fibroblast cell lines obtained from patients with Leigh's syndrome and other forms of congenital lactic acidosis. In Northern blot analyses for PDH E1 alpha and E1 beta subunits, no apparent differences were observed between two Leigh's syndrome and the control fibroblasts studied: one species of PDH E1 alpha mRNA and three species of E1 beta mRNA were observed in all the cell lines examined. However, in one tricarboxylic acid cycle deficient fibroblast cell line, which has one-tenth of the normal enzyme activity, the levels of immunoreactive PDH E1 alpha and E1 beta subunits were markedly decreased as assessed by immunoblot analyses. These data indicated a regulatory mutation caused by either inefficient translation of E1 alpha and E1 beta mRNAs into protein or rapid degradation of both subunits upon translation. In contrast, the PDH E1 alpha and E1 beta subunits in two fibroblast cell lines from Leigh's syndrome patients appeared to be normal as judged by 1) enzyme activity, 2) mRNA Northern blot, 3) genomic DNA Southern blot, and 4) immunoblot analyses indicating that the lactic acidosis seen in these patients did not result from a single defect in either of these E1 alpha and E1 beta subunits of the PDH complex.     

cDNA sequence of a Drosophila melanogaster gene, Dfur1, encoding a protein structurally related to the subtilisin-like proprotein processing enzyme furin

Screening a genomic library of Drosophila melanogaster DNA with a human fur cDNA probe resulted in the isolation of DNA clones that apparently belonged to two different DNA regions of the Drosophila genome. Subsequently, corresponding Drosophila cDNA clones were isolated. Nucleotide sequence analysis indicated that these cDNA clones originated from two different genes, which were called Dfur1 and Dfur2. From overlapping Dfur1 cDNA clones, a composite cDNA could be constructed and analysis of its nucleotide sequence revealed the coding sequence for a protein of 899 amino acid residues. This protein, designated Dfurin1, exhibited striking sequence homology to human furin and contained the same protein domains except for the cysteine-rich region. Furthermore, unlike human furin, Dfurin1 possessed an extended amino-terminal region in which a potential transmembrane anchor was present.     

Identification of three human pseudogenes for subunit VIb of cytochrome c oxidase: a molecular record of gene evolution.

Three pseudogenes for the nuclear-encoded subunit VIb of cytochrome c oxidase (COX) were isolated by screening a human genomic library with cloned human cDNA coding for COX subunit VIb. The nucleotide sequences of the pseudogenes, designated psi COX6b-1, psi COX6b-2 and psi COX6b-3, were determined. Pseudogene psi COX6b-1 bears all the hallmarks of a processed pseudogene and diverged from the parental gene after the divergence of man and cow. Alu repetitive elements were integrated into the structural sequences of the other two pseudogenes. Comparison with the human and bovine cDNA sequences encoding COX subunit VIb suggests that psi COX6b-2 and psi COX6b-3 were formed earlier in evolution than psi COX6b-1. Genomic Southern analysis indicated that a few more pseudogenes for COX subunit VIb are likely to be present in the human genome. Identical nt differences with respect to the human cDNA sequence in the pseudogenes provide some clues on the evolution of the ancestral gene coding for COX subunit VIb.     

The gene for the beta subunit of bovine luteinizing hormone encodes a gonadotropin mRNA with an unusually short 5'-untranslated region

Both cDNA and genomic clones encoding the beta subunit of bovine luteinizing hormone (LH) have been isolated and characterized. The nucleotide sequence was determined for the entire gene and 776 base pairs of 5'-flanking sequence. The mRNA cap site and polyadenylation site were mapped by primer extension and S1 nuclease protection, respectively. The bovine LH beta spans less than 1.1 kilobase pairs and has three exons encoding a 550 nucleotide mRNA (excluding the poly(A) tail). Bovine LH beta is a single-copy gene, in contrast to human LH beta, which is a member of the LH/chorionic gonadotropin beta subunit multigene family. Comparison of the bovine LH beta gene with the human LH beta/chorionic gonadotropin gene family reveals a high degree of nucleotide sequence homology, both within the genes and in the 5'-flanking sequences. Despite this extensive sequence conservation, there is a major difference between the two species in the selection of a promoter site. As a result, the bovine LH beta gene produces an mRNA with an usually short 5'-untranslated region of only 6-11 nucleotides.     

Molecular cloning and sequencing of cDNAs encoding the proteolipid subunit of the vacuolar H(+)-ATPase from a higher plant.

To understand the molecular structure of the vacuolar H(+)-translocating ATPase from plants, cDNAs encoding the N,N'-dicyclohexylcarbodiimide-binding 16-kDa proteolipid from oat (Avena sativa L. var. Lang) have been obtained. A synthetic oligonucleotide corresponding to a region of the bovine proteolipid cDNA (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-C.E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5521-5524) was used to screen an oat cDNA library constructed in lambda gt11. The nucleotide sequences of several positive clones (VATP-P1, clones 12, 54, 93) demonstrated the presence of a small multigene family. The four clones showed extensive divergence in their codon usage and their 3'-untranslated regions; however, the deduced amino acid sequences of the proteins were 97-99% identical. These clones encoded the proteolipid subunit as one of them (clone 12) expressed a fusion protein that reacted with an antibody to the 16-kDa proteolipid. The open reading frame of one cDNA clone (VATP-P1) predicted a polypeptide of 165 amino acids with a molecular mass of 16,641. Based on hydropathy plots, a molecule with four membrane-spanning domains was predicted, in which domain IV was especially conserved among different species. This domain showed 80% identity in nucleotide or amino acid sequences between the oat and the bovine proteolipids and contained a glutamate residue that is the putative N,N'-dicyclohexylcarbodiimide-binding residue. The presence of a small multigene family of the 16-kDa proteolipid was confirmed by Southern blot analysis showing that several distinct restriction fragments of oat nuclear DNA hybridized with the VATP-P1 cDNA.     

Cloning and characterization of a cDNA encoding bovine mannan-binding protein

To identify the bovine mannan-binding protein (MBP), a search for the cDNA homologue of human MBP was carried out. cDNA clones encoding bovine MBP were isolated from a bovine liver cDNA library using a cDNA fragment encoding a short collagen region, neck domain and carbohydrate recognition domain of human MBP. The cDNA carried an insert of 747 bp encoding a protein of 249 amino acid (aa) residues with a signal peptide of 19 aa. The mannan-binding protein fraction of bovine serum that eluted with 100 mM mannose from a mannan-Sepharose column was analyzed under reducing conditions by SDS-PAGE. The major band of 33 kDa obtained reacted with anti-human MBP rabbit serum. The partial aa sequence of the purified 33-kDa protein was identical to the aa sequence deduced from the obtained cDNA. Results of the passive hemolysis experiment using sheep erythrocytes coated with yeast mannan suggest that this MBP has the ability to activate complement. Northern blot analysis showed a 1.8-kb mRNA that was expressed only in the liver. Based on results of genomic analysis, this bovine MBP is likely to be a homologue of human MBP and to also have homology to rat and mouse MBP-C which are localized in liver cells rather than to rat and mouse MBP-A found in serum. Alignments of bovine collectins show that bovine MBP cannot be included among the other bovine collectins, such as bovine SP-D, conglutinin and CL-43. Finally, these genomic and biological analyses indicate that the cDNA obtained here encoded a bovine serum MBP.     

Rabbit C-reactive protein. Biosynthesis and characterization of cDNA clones

To study the biosynthesis of rabbit C-reactive protein (CRP), a cDNA library was constructed from CRP mRNA-enriched polysomal poly(A) RNA. Four recombinant plasmids, designated pCX9, pCX23, pCX28, and pCX39, from 39 positive clones were sequenced and found to represent overlapping clones. DNA sequencing of CRP cDNA and primer extension of the 5'-end of CRP mRNA have demonstrated that the complete length of rabbit CRP mRNA consists of 2331 nucleotides and a terminal poly(A) segment. Analysis of the resulting sequence indicated that rabbit CRP mRNA contained a 5'-noncoding region of 107 nucleotides, a leader sequence encoding 20 amino acids, a coding region covering 205 amino acids, and a 3'-noncoding region of 1549 nucleotides. The 3'-noncoding region contained a consensus AAUAAA sequence that is 105 nucleotides upstream from the 3'-terminal poly(A) segment. Using an in vitro translation system, we have confirmed that CRP is synthesized as a precursor polypeptide (Mr approximately equal to 26,000) which undergoes processing to form the mature polypeptide (Mr approximately equal to 23,500). The CRP precursor failed to display a calcium-dependent affinity for phosphorylcholine ligand as demonstrated by mature CRP, suggesting that the phosphorylcholine-binding site of CRP only formed after processing. Northern blot analysis suggested that following induction with turpentine, liver was the only site where CRP mRNA synthesis could be demonstrated and that the change in mRNA concentration correlated with the course of CRP production. Southern blot analysis of liver genomic DNA indicated a single gene copy for CRP.     

Characterization of two different genes (cDNA) for cytochrome c oxidase subunit VIa from heart and liver of the rat.

By antibody screening of a rat liver and a rat heart cDNA library in lambda gt11 two clones coding for the liver- and heart-specific subunit VIa of rat cytochrome c oxidase were isolated. In the heart cDNA sequence a TAA stop codon was found in frame 18 bp 5' upstream of the first methionine codon, thus excluding a leader sequence for this protein. The two cDNAs contain the full-length coding region of two subunits. The amino acid sequences of the two subunits show only 50% homology, whereas 74% homology was found between rat heart and bovine heart subunit VIa. By Northern blot analysis it is shown that the gene for subunit VIa from heart is only expressed in heart and skeletal muscle, whereas that from liver is also expressed in kidney, brain, heart and weakly in muscle.     

The amino acid sequence of the 24-kDa subunit, an iron-sulfur protein, of rat liver mitochondrial NADH dehydrogenase deduced from cDNA sequence

Antiserum directed against bovine heart mitochondrial NADH dehydrogenase has been used to screen a rat liver cDNA expression library in lambda gt11. The insert cDNA of a positive clone was found to represent the 24-kDa subunit of NADH dehydrogenase by epitope selection using nitrocellulose filter containing the expressed proteins. The amino acid sequence deduced from the nucleotide sequence of the cloned cDNA indicated that the 24-kDa subunit is produced as a precursor with an amino-terminal extension, and that its mature form consists of 217 amino acid residues with a molecular weight of 23,933.     

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.