Cloning and expression of a cDNA coding for a rat liver plasma membrane ecto-ATPase. The primary structure of the ecto-ATPase is similar to that of the human biliary glycoprotein I

The amino acid sequence of the ecto-ATPase from rat liver was deduced from analysis of cDNA clones and a genomic clone. Immunoblots with antibodies raised against a peptide sequence deduced from the cDNA sequence indicated that the determined amino acid sequence is that of the ecto-ATPase. The deduced sequence predicts a 519-amino acid protein with a calculated molecular mass of 57,388 daltons. There are 16 potential asparagine-linked glycosylation sites in the protein. Hydropathy analysis of the deduced amino acid sequence indicates that the protein has two hydrophobic stretches. One is located at the N-terminal and the other is near the C-terminal end. A full-length clone encoding the ecto-ATPase was expressed transiently in mouse L cells and human HeLa cells. The cell lysate from the transfected cells contained immunoreactive ecto-ATPase and Ca2+-stimulated ATPase activities. The expressed protein is glycosylated and has an apparent molecular weight (100,000) similar to that of the rat liver plasma membrane ecto-ATPase.     

Cloning, expression, and chromosomal localization of the 140-kilodalton subunit of replication factor C from mice and humans.

We have isolated a full-length mouse cDNA encoding a lysine-rich protein of 1,131 amino acids with a calculated molecular mass of 126 kDa. The protein binds in a sequence-unspecific manner to DNA, is localized exclusively in the nucleus, and contains a putative ATP binding site and a stretch of 80 amino acids with homology to the carboxy terminus of prokaryotic DNA ligases. On the basis of the following facts, we conclude that the isolated cDNA encodes the 140-kDa subunit of mouse replication factor C (mRFC140). (i) The sequence around the ATP binding site shows significant homology to three small subunits of human replication factor C. (ii) Polyclonal antibodies raised against the protein encoded by this cDNA cross-react with the 140-kDa subunit of purified human replication factor C (hRFC140) and recognize in mouse cell extracts an authentic protein with an apparent molecular mass of 130 kDa. (iii) Sequence comparison with a human cDNA isolated by using tryptic peptide sequence information from purified hRFC140 revealed 83% identity of the encoded proteins. The mRFC140 gene is ubiquitously expressed, and two mRNAs approximately 5.0 and 4.5 kb long have been detected. The gene was mapped by in situ hybridization to mouse chromosome 5, and its human homolog was mapped to chromosome 4 (p13-p14).     

Isolation and sequence analysis of a cDNA clone encoding a type-1 protein phosphatase catalytic subunit: homology with protein phosphatase 2A

A 1.5 kb clone containing the full-length coding sequence of a type-1 protein phosphatase catalytic subunit has been isolated from a rabbit skeletal muscle cDNA library constructed in lambda gt10. The protein sequence deduced from the cDNA contains 311 residues and has a molecular mass of 35.4 kDa. A single mRNA species at 1.6 kb was visualized by Northern blotting. The type-1 protein phosphatase was strikingly homologous to protein phosphatase 2A, 49% of the amino acids between residues 11 and 280 being identical. The first 10 and last 31 residues were dissimilar. Residues 1-101 of the type-1 protein phosphatase also showed 21% sequence identity with a region of mammalian alkaline phosphatases.     

Isolation and sequencing of cDNAs for the XL-I and XL-III forms of bovine liver xenobiotic-metabolizing medium-chain fatty acid:CoA ligase

The XL-I form of xenobiotic-metabolizing medium-chain fatty acid:CoA ligase was previously purified to apparent homogeneity from bovine liver mitochondria, and the amino acid sequence of a short segment of the enzyme was determined. This sequence was used to develop a probe for screening a bovine cDNA library from which a 1.6 kb cDNA was isolated. This cDNA was sequenced and found to contain the code for the known amino acid sequence. The complete open reading frame was not present in this cDNA, but it was estimated to code for approximately 75% of the XL-I sequence. The XL-III ligase was purified to apparent homogeneity from bovine liver mitochondria. The enzyme eluted from a gel filtration column as a single peak with an apparent molecular weight of ca. 55,000. It ran as a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of 62 kDa. N-Terminal sequence analysis of the enzyme gave no sequence, which indicates a blocked N-terminus. The enzyme was chemically cleaved using CNBr. The resulting peptides were separated by SDS-PAGE. The cleavage pattern revealed two large peptides of ca. 21 and 25 kDa, plus several smaller peptides including a prominent 6 kDa peptide. The N-terminus of the 6, 21, and 25 kDa peptides was sequenced and the 21 and 25 kDa sequences were identical indicating incomplete cleavage. The sequences were used to design probes for screening a bovine liver cDNA library. This resulted in the isolation of a 2,065 bp cDNA. This cDNA was sequenced and found to contain the initiation and termination codons, as well as the requisite amino acid sequences. The open reading frame coded for a 64,922 Da protein. The sequence of XL-III cDNA was markedly different from that of XL-I, indicating the genetic uniqueness of the two ligases. They are, however, 64% homologous, which suggests a common evolutionary origin.     

Cloning of leptin cDNA and assignment to the long arm of chromosome 5 in the marsupial Sminthopsis crassicaudata

We have isolated and sequenced full-length cDNA clones for leptin in the dasyurid marsupial Sminthopsis crassicaudata (fat-tailed dunnart). Southern and in situ hybridisation data indicated a single leptin gene in the S. crassicauda- ta genome, localised to arbitrary chromosome bands 5q24--> q31 on the long arm of chromosome 5, the short-arm terminus of which bears the only nucleolar organising region. The nucleotide sequence of the cDNAs revealed that the primary translation product of S. crassicaudata leptin is composed of 167 amino acid residues, with a potential signal peptide of 21 residues. The mature protein of 146 amino acids is 82% similar to both the mouse and human proteins and is predicted to have a molecular weight of 16.26 kDa. Northern blot analysis revealed that the corresponding mRNA is approximately 3.9 kb in size and is expressed only in white adipose tissue of this marsupial species. Evolutionary analyses indicate that S. crassicaudata leptin cDNA has evolved at a significantly faster rate than cDNAs from eutherian mammals.     

Molecular isolation and sequence determination of the cDNA for the mouse sperm-specific lactate dehydrogenase-X gene

Several cDNA clones for the mouse lactate dehydrogenase-X (LDH-X), a sperm-specific glycolytic enzyme, were isolated from mouse testicular cDNA libraries constructed in the bacteriophage vectors, lambda gt11 and gt10. The largest cDNA clone contains an insert of 1135 base pairs in length and an open reading frame that encodes a 332 amino acid polypeptide with a molecular weight of 35.89 kD. The deduced amino acid sequence of this protein is in close agreement with the published sequence of mouse LDH-X obtained by direct protein sequencing. Northern analysis of RNA isolated from different tissues detected a single size mRNA of 1.5 kilobases in mouse testis but not in brain or liver. The Ldh-x structural gene was estimated to be about 12 kb in size as demonstrated by Southern hybridization analysis of mouse genomic DNA using the full-length cDNA as a probe.     

Molecular cloning of Na(+)-ATPase cDNA from a marine alga, Heterosigma akashiwo

We cloned novel Na(+)-ATPase (HANA) cDNA from marine alga Heterosigma akashiwo. The full-length HANA cDNA was 4467 bp long and coded for a 1330 amino acid protein with a molecular weight of 146,306. The deduced product exhibited around 40% identity in amino acids with Na(+)/K(+)-ATPase alpha-subunits. A hydrophilic sequence of 285 amino acid residues that showed no homology with any sequence listed in databases existed in the M7--M8 junction of HANA. This is the first report on the primary structure of putative Na(+)-transporting ATPase from plant cells.     

The extrinsic 33 kDa polypeptide of the oxygen-evolving complex of photosystem II is a putative calcium-binding protein and is encoded by a multi-gene family in pea

The extrinsic 33 kDa polypeptide of the water-oxidizing complex has been extracted from pea photosystem II particles by washing with alkaline-Tris and purified by ion-exchange chromatography. The N-terminal amino acid sequence has been determined, and specific antisera have been raised in rabbits and used to screen a pea leaf cDNA library in gt11. Determination of the nucleotide sequence of positive clones revealed an essentially full-length cDNA for the 33 kDa polypeptide, the deduced amino acid sequence showing it to code for a mature protein of 248 amino acids with an N-terminal transit peptide of 81 amino acids. The protein showed a high degree of conservation with previously reported sequences for the 33 kDa protein from other species and the sequence contained a putative Ca²⁺-binding site with homology to mammalian intestinal calcium-binding proteins. Northern analysis of total pea RNA indicated a message of approximately 1.4 kb, in good agreement with the size of the cDNA obtained at 1.3 kbp. Southern blots of genomic DNA probed with the labelled cDNA give rise to several bands suggesting that the 33 kDa polypeptide is coded by a multi-gene family.Abbreviations ATZ - anilinothiazolinone - DITC - p-phenylenediisothiocyanate - PTH - phenylthiohydantoin - TFA - trifluoroacetic acid - Tris - tris (hydroxymethyl) aminomethane - bis-Tris - bis (2-hydroxyethyl) imino-tris (hydroxymethyl)-methane - p.f.u. - plaque-forming units     

The b-32 protein from maize endosperm, an albumin regulated by the O2 locus: Nucleic acid (cDNA) and amino acid sequences

Summary The cDNA coding for the b-32 protein, an albumin expressed in maize endosperm cells under the control of the O2 and O6 loci, has been cloned and the complete amino acid sequence of the protein derived. A lambda gt11 cDNA library from mRNA of immature maize endosperm was screened for the expression of the b-32 protein using antibodies against the purified protein. One of the positive clones obtained was used to isolate a full-length cDNA clone. By Northern analysis, the size of the b-32 mRNA was estimated to be 1.2 kb. Hybrid-selected translation assays show that the message codes for a protein with an apparent molecular weight of 30–35 kDa. The nucleotide sequence shows that several internal repeats are present. The protein has a length of 303 amino acid residues (mol. wt. 32430 dalton) and its sequence shows the following features: no signal peptide is observable; it contains seven tryptophan residues, an amino acid absent in maize storage proteins; polar and hydrophobic residues are spread along the sequence; several pairs of basic residues are present in the N-terminal region; the secondary structure allows the prediction of two structural domains for the b-32 protein that would fold up giving rise to a globular shape. The cloning of this gene may help in understanding the role of the O2 and O6 loci in regulating the deposition of zein, the major storage protein of maize endosperm.     

Cloning and nucleotide sequence of a full-length cDNA for human liver gamma-glutamylcysteine synthetase.

We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.