cDNA sequence of the human integrin beta 5 subunit

A novel integrin receptor involved in cell adhesion to the matrix protein vitronectin has recently been described from a human lung epithelial-derived cell line (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69). This receptor has an alpha subunit that appears identical to the alpha v of the vitronectin receptor alpha v beta 3 expressed in melanoma and endothelial cells, but is complexed with a distinct beta subunit, beta 5. cDNA clones coding for beta 5 have been isolated and used to determine the mRNA and amino acid sequence of this new subunit. A 3.3-kilobase mRNA was found to code for a mature protein of 775 amino acid residues with a hydrophobic leader sequence of 24 amino acids. A 56% identity was found between the beta 5 and beta 3 protein sequences, making them the most closely related of the integrin beta subunits. Polymerase chain reaction abundance analysis revealed that alpha v and beta 5 mRNAs were found in seven very different cell lines, compared with beta 3 mRNA which was found in only three of the them, indicating that this new integrin receptor may be widely distributed.     

Molecular cloning of the cDNA encoding the third polypeptide (gamma) of brain calmodulin-dependent protein kinase II

cDNA clones for three distinct types of rat brain calmodulin-dependent protein kinase II have been isolated. Two of them were identified as cDNA clones for the alpha and beta subunits of this kinase. The other showed a nucleotide sequence similar but, not identical, to that encoding either the alpha or beta subunit. The cDNA sequence encoded a polypeptide, designated gamma, consisting of 527 amino acid residues with a molecular weight of 59,038. The deduced amino acid sequence of gamma was 84 and 87% homologous to those of alpha and beta, respectively. Higher homologies of the sequences were found in the amino-terminal halves of the three species, alpha, beta, and gamma. RNA blot analysis revealed that the mRNAs for alpha, beta, and gamma were expressed in rat brain with different regional specificities.     

Molecular cloning of three distinct forms of the Na+,K+-ATPase alpha-subunit from rat brain

Rat brain and kidney cDNA libraries were constructed and screened with a cDNA insert corresponding to the mRNA for the sheep kidney Na+,K+-ATPase catalytic subunit. The alpha-subunit cDNAs isolated from the kidney library were derived from a single class of messenger RNA, and the brain cDNAs were derived from three classes of messenger RNA. The most abundant brain cDNA, which spans 5.1 kilobases, encodes the alpha(+) form of the enzyme. The second most abundant brain cDNA, which spans 3.65 kilobases, is identical with that of the kidney form and therefore encodes the alpha isoform. The third class of cDNA, which spans 3.55 kilobases, was present at low abundance and encodes an isoform of the alpha-subunit, designated alpha III, which has not been identified previously. The complete nucleotide sequence and deduced amino acid sequence for each of the brain and kidney cDNAs have been determined. In addition, we have identified a lysine-rich sequence that may function as a movable, ion-selective gate during cation binding and occlusion and have also identified several amino acid sequence variations that appear to explain some of the well-known species and tissue differences in cardiac glycoside sensitivity.     

Identification of a putative isoform of the Na,K-ATPase beta subunit. Primary structure and tissue-specific expression

We have isolated cDNA clones from rat brain and human liver encoding a putative isoform of the Na,K-ATPase beta subunit. The rat brain cDNA contains an open reading frame of 870 nucleotides coding for a protein of 290 amino acids with a calculated molecular weight of 33,412. The corresponding amino acid sequence shows 98% identity with its human liver counterpart. The proteins encoded by the rat and human cDNAs exhibit a high degree of primary sequence and secondary structure similarity with the rat Na,K-ATPase beta subunit. We have therefore termed the polypeptides these cDNAs encode a beta 2 subunit with the previously characterized rat cDNA encoding a beta 1 subunit. Analysis of rat tissue RNA reveals that the beta 2 subunit gene encodes a 3.4-kilobase mRNA which is expressed in a tissue specific fashion distinct from that of rat beta 1 subunit mRNA. Cell lines derived from the rat central nervous system shown to lack beta 1 subunit mRNA sequences were found to express beta 2 subunit mRNA. These results suggest that different members of the Na,K-ATPase beta subunit family may have specialized functions.     

Molecular cloning of cDNA for the import precursor of human subunit B of H(+)-ATP synthase in mitochondria.

The nucleotide sequence of the import precursor of subunit b of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat subunit b as a probe. The sequence was composed of 1,134 nucleotides including a coding region for the import precursor of subunit b and noncoding regions on the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame were found to consist of 256 and 214 amino acid residues with molecular weights of 28,893 and 24,610, respectively. The presequence of 42 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.     

Molecular cloning and sequence analysis of cDNA for the catalytic subunit 1 alpha of rat kidney type 1 protein phosphatase, and detection of the gene expression at high levels in hepatoma cells and regenerating livers as compared to rat livers.

A cDNA clone containing the full coding sequence of a type 1 protein phosphatase catalytic subunit 1 alpha has been isolated from a rat kidney lambda gt 10 library. The protein sequence deduced from the cDNA contains 330 amino acid residues with a molecular mass of 38 kDa. The cDNA clone from rat kidney was 89% identical at the nucleotide level in the coding region to type 1 protein phosphatase 1 alpha from rabbit skeletal muscle. However, the two protein sequences were completely identical. The type 1 alpha protein phosphatase from rat kidney shows 49% homology of amino acid sequence to the rat type 2A alpha protein phosphatase. Thus, the protein sequence of type 1 alpha protein phosphatase was completely conserved between rat and rabbit. The mRNA levels of type 1 protein phosphatase were determined in rat liver, AH13, a strain of rat hepatoma, and regenerating rat liver by Northern blot analysis using the cDNA fragment as a probe, under which conditions a single mRNA of 1.5 kb was detected. The mRNA levels of AH13 were remarkably increased when compared to those of normal ivers, whereas the mRNA levels of regenerating livers were slightly but significantly increased. These results demonstrate a marked increase in gene expression of type 1 protein phosphatase in hepatoma cells, suggesting an important role of the type 1 protein phosphatase in hepatocarcinogenesis.     

Multiple mRNA species code for the catalytic subunit of the cAMP-dependent protein kinase from LLC-PK1 cells. Evidence for two forms of the catalytic subunit

We present evidence for the existence of two forms of the catalytic (C) subunit of the cAMP-dependent protein kinase. A lambda gt-11 cDNA library constructed from poly(A)-rich RNA from the porcine kidney cell line, LLC-PK1, was screened using a 1.5-kb EcoRI fragment from a bovine cDNA for the C subunit. Two independent classes of cDNAs were identified on the basis of partial restriction map and sequence data. These two cDNAs, lambda CAT4 and lambda CAT3, apparently encode two forms of C subunit designated C alpha and C beta, respectively. The nucleotide sequence of the C alpha and C beta cDNAs revealed differences in the coding region and particularly in the 3' untranslated region. However, the deducted amino acid sequences of C alpha and C beta subunits were 96% homologous to the sequences so far determined. Specific probes from the 3' coding region of the two cDNA species were used to investigate C subunit mRNA expression in LLC-PK1 cells. Northern analysis showed a major mRNA species of 2.8 kb with the C alpha probe while the C beta probe detected two mRNA species of 5.0 kb and 3.8 kb. These data were supported by genomic blot analysis which showed distinct hybridization patterns with either the C alpha or C beta probes. All the available evidence suggests that at least two distinct genes encode the C subunit which are expressed in LLC-PK1 cells.     

cDNA cloning and sequencing for the import precursor of subunit B in H(+)-ATP synthase from rat mitochondria

The nucleotide sequence of the import precursor of subunit b of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 1,124 nucleotides including a coding region for the import precursor of subunit b and noncoding regions of both the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame consisted of 256 and 214 amino acid residues with a molecular weight of 28,867 and 24,628, respectively. The presequence of 42 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.     

Identification of a cDNA clone for the beta-subunit of the pyruvate dehydrogenase component of human pyruvate dehydrogenase complex

We report the isolation of a 1.5 kb cDNA clone for the beta subunit of human pyruvate dehydrogenase (E1) from a human liver lambda gt11 cDNA library using anti-E1 serum. We generated a peptide sequence of 24 amino acids starting from the N-terminus of bovine heart mature E1 beta. The identity of the E1 beta cDNA clone was confirmed by the similarity between the amino acid sequence deduced from the cDNA nucleotide sequence and the known amino acid sequence of bovine heart E1 beta. In Northern analysis of total RNA extracted from human heart, the E1 beta cDNA clone hybridized to a major 1.6 kb and a minor 5.2 kb RNA species.     

Identification of a cDNA clone that contains the complete coding sequence for a 140-kD rat NCAM polypeptide

Neural cell adhesion molecules (NCAMs) are cell surface glycoproteins that appear to mediate cell-cell adhesion. In vertebrates NCAMs exist in at least three different polypeptide forms of apparent molecular masses 180, 140, and 120 kD. The 180- and 140-kD forms span the plasma membrane whereas the 120-kD form lacks a transmembrane region. In this study, we report the isolation of NCAM clones from an adult rat brain cDNA library. Sequence analysis indicated that the longest isolate, pR18, contains a 2,574 nucleotide open reading frame flanked by 208 bases of 5' and 409 bases of 3' untranslated sequence. The predicted polypeptide encoded by clone pR18 contains a single membrane-spanning region and a small cytoplasmic domain (120 amino acids), suggesting that it codes for a full-length 140-kD NCAM form. In Northern analysis, probes derived from 5' sequences of pR18, which presumably code for extracellular portions of the molecule hybridized to five discrete mRNA size classes (7.4, 6.7, 5.2, 4.3, and 2.9 kb) in adult rat brain but not to liver or muscle RNA. However, the 5.2- and 2.9-kb mRNA size classes did not hybridize to either a large restriction fragment or three oligonucleotides derived from the putative transmembrane coding region and regions that lie 3' to it. The 3' probes did hybridize to the 7.4-, 6.7-, and 4.3-kb message size classes. These combined results indicate that clone pR18 is derived from either the 7.4-, 6.7-, or 4.3-kb adult rat brain RNA size class. Comparison with chicken and mouse NCAM cDNA sequences suggests that pR18 represents the amino acid coding region of the 6.7- or 4.3-kb mRNA. The isolation of pR18, the first cDNA that contains the complete coding sequence of an NCAM polypeptide, unambiguously demonstrates the predicted linear amino acid sequence of this probable rat 140-kD polypeptide. This cDNA also contains a 30-base pair segment not found in NCAM cDNAs isolated from other species. The significance of this segment and other structural features of the 140-kD form of NCAM can now be studied.     

Cloning and sequence analysis of cDNA clones for bovine aortic-endothelial-cell transglutaminase.

A cDNA clone encoding transglutaminase was isolated from a bovine-endothelial-cell cDNA library using oligonucleotide probes designed based on partial amino acid sequences of the purified protein. Sequencing of the cDNA insert revealed an open reading frame of 2061 bp coding for a protein of 687 amino acids. The sequence of bovine endothelial-cell transglutaminase was 88, 82, 80, 37, 37 and 37% identical with that of human endothelial, rat macrophage, guinea-pig liver, human and rat keratinocyte transglutaminases, and the human blood-coagulation factor XIIIa subunit, respectively. The cDNA clone was hybridized to a single mRNA species of 3.9 kb in the liver, lung, spleen and heart but not hybridized to RNA from the brain. Northern-blot analysis of mRNA from retinoid-treated cultured vascular endothelial cells revealed that retinoids were able to induce a large increase in the transglutaminase mRNA levels.     

Developmentally regulated plant genes: the nucleotide sequence of a wheat gliadin genomic clone

Gliadins, the major wheat seed storage proteins, are encoded by a multigene family. Northern blot analysis shows that gliadin genes are transcribed in endosperm tissue into two classes of poly(A)+ mRNA, 1400 bases (class I) and 1600 bases (class II) in length. Using poly(A)+ RNA from developing wheat endosperm we constructed a cDNA library from which a number of clones coding for alpha/beta and gamma gliadins were identified by hybrid-selected mRNA translation and DNA sequencing. These cDNA clones were used as probes for the isolation of genomic gliadin clones from a wheat genomic library. One such genomic clone was characterized in detail and its DNA sequence determined. It contains a gene for a 33-kd alpha/beta gliadin protein (a 20 amino acid signal peptide and a 266 amino acid mature protein) which is very rich in glutamine (33.8%) and proline (15.4%). The gene sequence does not contain introns. A typical eukaryotic promoter sequence is present at -104 (relative to the translation initiation codon) and there are two normal polyadenylation signals 77 and 134 bases downstream from the translation termination codon. The coding sequence contains some internal sequence repetition, and is highly homologous to several alpha/beta gliadin cDNA clones. Homology to a gamma-gliadin cDNA clone is low, and there is no homology with known glutenin or zein cDNA sequences.     

alpha Subunit of rat pituitary glycoprotein hormones. Primary structure of the precursor determined from the nucleotide sequence of cloned cDNAs

Double-stranded complementary DNAs were constructed enzymatically from polyadenylated RNA extracted from pituitary glands of ovariectomized rats, were inserted into the Pst I site of plasmid pBR322 and were cloned in Escherichia coli chi 1776. Cloned cDNAs encoding the precursor to the alpha subunit (pre-alpha) of the glycoprotein hormones were identified by hybridization with a restriction fragment of a previously cloned and sequenced cDNA encoding the precursor to the alpha subunit of mouse thyrotropin (Chin, W. W., Kronenberg, H. M., Dee, P. C., Maloof, F., and Habener, J. F. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 5329-5333). The DNA sequences of the two largest rat cDNA inserts (591 and 554 base pairs) were determined and the amino acid sequence of the rat pre-alpha subunit was deduced from these sequences. The composite sequence determined from these cDNAs spans 610 base pairs, or almost the entire length of the messenger RNA (mRNA) of 800 bases, when account is taken of the 3' poly(A) tract. The rat alpha precursor consists of a 24 amino acid leader sequence and a 96 amino acid alpha subunit apoprotein. The amino acid homologies between the rat and mouse, and between the rat and human sequences are 95% and 74%, respectively. Nucleotide homologies between the rat and mouse cDNAs in the coding and untranslated regions are 94% and 80%, respectively. This cloned cDNA will be applied to analysis of the structure of the rat alpha subunit gene(s) and of the regulation of alpha subunit gene expression.     

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.     

The alpha subunit of meprin A. Molecular cloning and sequencing, differential expression in inbred mouse strains, and evidence for divergent evolution of the alpha and beta subunits.

Meprin A, a membrane-bound oligomeric metalloendopeptidase, contains two different subunits, alpha and beta. We report here the cloning and sequencing of the alpha subunit cDNA. The translated polypeptide consists of 760 amino acids, including a preprosequence (77 amino acids) that precedes the NH2 terminus of the purified enzyme. The next 198 amino acids constitute the "astacin family" protease domain, which includes the astacin family signature sequence, HE(L,I)XHXXGFXHE(Q,H)XRXDRDX(Y,H)(V,I)X(I,V). An immunoglobulin/major histocompatibility complex protein signature was found at the end of the protease domain. At the COOH terminus of the alpha subunit, there is an epidermal growth factor-like domain, followed by a transmembrane domain, and six additional amino acids. Ten potential glycosylation sites have been identified, and at least three of those sites are glycosylated. Northern blot analyses of kidney tissue from C57BL/6 and C3H/He mice indicate that variations in meprin A activity in these strains reflect differences in the levels of the alpha subunit mRNA. Several internal peptide sequences obtained from the beta subunit indicate that it is approximately 50% identical to the alpha subunit. Furthermore, NH2-terminal sequence analyses (39 residues) indicate that rat and mouse alpha are 79% identical, rat and mouse beta are 74% identical, and that alpha and beta subunits for both species are 47% identical. These data indicate that alpha and beta are closely related products of divergent evolution.     

A novel low molecular weight ribonucleic acid (RNA) related to transforming growth factor alpha messenger RNA

Human transforming growth factor alpha (TGF alpha) is coded for by an mRNA of about 4800 nucleotides. The cDNA sequence demonstrates that the 50 amino acid TGF alpha is embedded in a larger 160 amino acid precursor protein. We report here that in addition to the 4800 nucleotide TGF alpha mRNA, there is a novel second RNA species of about 350 nucleotides that hybridizes to a human TGF alpha cDNA probe. This small RNA species has been found in the RNA of several human tumor cells including HT1080, A549, A431, A2058, and A673. We have demonstrated an inverse relationship between the amounts of the 4800 nucleotide TGF alpha mRNA and the 350 nucleotide novel RNA in these human cells. Restriction enzyme cleavage of a human TGF alpha cDNA probe into three separate domains consisting of a processed coding region and 5'- and 3'-preprocessed coding and untranslated regions showed that only the 3'-untranslated region hybridized to the 350 nucleotide RNA. Using sense and anti-sense single-stranded 3'-untranslated region probes, we determined that the 350 nucleotide RNA band may be composed of multiple species of RNA which are related to the anti-sense DNA strand that is opposite to the strand that codes for the 4800 nucleotide TGF alpha mRNA.     

Cloning of cDNA encoding a 32-kDa protein. An accessory polypeptide of the H+-ATPase from chromaffin granules

The purified H+-ATPase from chromaffin granules is composed of several polypeptides, one of which has an apparent molecular weight of 39,000. Immunoblots with the antibody against this protein and various membrane preparations showed that similar or even identical polypeptides may be associated with the H+-ATPases from synaptic vesicle, kidney microsomes, and lysosomes. A cDNA library was constructed from bovine adrenal medulla, and the cDNA encoding the polypeptide was isolated and sequenced. Search in DNA and protein data banks revealed no significant homology to known genes. Hydrophobicity plot revealed no obvious transmembrane segments with the exception of one stretch of hydrophobic and neutral amino acid starting at leucine 16. The cDNA was shown to encode the entire polypeptide by the virtue of an amino acid sequence corresponding to the N terminus of the open reading frame and by subunit and site-specific antibodies. The cDNA was cloned into an expression vector, transcribed by T7 polymerase, and translated by reticulocyte lysate. Even though the cDNA encodes a protein with a molecular weight of 31,495, the translation product comigrated on sodium dodecyl sulfate gels with the subunit of the purified H+-ATPase. In line with several other subunits of vacuolar H+-ATPases, no signal sequence was detected in the translated gene. Northern blots revealed the presence of a single mRNA of about 1.6 kb in bovine adrenal medulla. However, liver, lung, and kidney may contain additional mRNA of about 1.7 kb.