Molecular cloning of complementary DNA encoding mouse seminal vesicle-secreted protein SVS I and demonstration of homology with copper amine oxidases

The primary structure of mouse SVS I was determined by peptide sequencing and nucleotide sequencing of cloned cDNA. The precursor molecule consists of 820 amino acid residues, including a signal peptide of 24 residues, and the mature polypeptide chain of 91 kDa has one site for potential N-linked glycosylation. The SVS I is homologous with amiloride-binding protein 1 (ABP1), a diamine oxidase. However, it probably lacks enzymatic activity, because the cDNA codes for His instead of Tyr at the position of the active-site topaquinon. The SVS I monomer probably binds one molecule of copper, because the His residues coordinated by Cu(II) are conserved. The SVS I gene consists of five exons and is situated on mouse chromosome 6,B2.3. It is located in a region of 100 kilobases (kb) containing several genes with homology to SVS I, including the gene of ABP1 and two other proteins with homology to diamine oxidase. The locus is conserved on rat chromosome 4q24, but the homologous region on human chromosome 7q34-q36 solely contains ABP1. The other genes with homology to diamine oxidase were probably present in a progenitor of primates and rodents but were lost in the evolutionary lineage leading to humans-presumably during recombination between chromosomes. The estimated molecular mass of rat SVS I is 102 kDa (excluding glycosylation). The species difference in size of SVS I is caused by tandem repeats of 18 amino acid residues in the central part of the molecule: The mouse has seven repeats, and the rat has 12 repeats.     

A new member of the prolactin-growth hormone gene family expressed in mouse placenta.

Mouse placenta has been found to contain an mRNA that encodes a previously unidentified member of the prolactin-growth hormone family. This 1.1-kb mRNA (designated PRP mRNA) was detected as a cDNA clone that hydridized to a cDNA clone of mouse proliferin, a recently described growth-associated placental protein related to prolactin. PRP mRNA levels are highest in the fetal part of the placenta and peak at day 12 of gestation, decreasing gradually until term. The 972-bp sequence of PRP mRNA, determined from two cDNA clones, encodes a protein of 244 amino acid residues that has a hydrophobic leader sequence. The protein encoded by PRP mRNA has significant homology to all of the members of the prolactin family, yet is different from each of them; it also differs from mouse placental lactogen. Nucleotide sequence homology is most extensive between PRP and proliferin mRNAs, particularly at their 5' ends, where they share 92 of the first 97 nucleotides.     

Cloning and sequencing of the rat preproepidermal growth factor cDNA: comparison with mouse and human sequences.

We have cloned and sequenced the cDNA corresponding to the rat preproepidermal growth factor (ppEGF) mRNA. The cDNA contained 4,801 nucleotides, similar to that reported for the mouse (4,749 nucleotides) and the human mRNAs (4,871 nucleotides). The predicted protein sequence would contain 1,133 amino acids, smaller than that reported for the mouse (1,217 amino acids) and the human sequences (1,207 amino acids). The results of the sequencing of several cDNA clones suggested the existence of more than one structural gene for ppEGF. In addition, there was an occurrence of alternative splicing events, resulting in deletions of entire exons from the mature mRNA. These alternative splicing events do not create frameshift mutations but cause a deletion of one or more of the "EGF-like" repeat units from the ppEGF. There is approximately the same homology between the rat and mouse amino acid sequences both in the EGF region and in the other regions of the ppEGF protein. We conclude that, because of this conservation of homology, there may be an important function performed by these other regions of the ppEGF besides their function as a precursor for the EGF protein.     

Molecular cloning and sequence analysis of human placental alkaline phosphatase

The complete amino acid sequence of the precursor and mature forms of human placental alkaline phosphatase have been inferred from analysis of a cDNA. A near full-length PLAP cDNA (2.8 kilobases) was identified upon screening a bacteriophage lambda gt11 placental cDNA library with antibodies against CNBr fragments of the enzyme. The precursor protein (535 amino acids) displays, after the start codon for translation, a hydrophobic signal peptide of 21 amino acids before the amino-terminal sequence of mature placental alkaline phosphatase. The mature protein is 513 amino acids long. The active site serine has been identified at position 92, as well as two putative glycosylation sites at Asn122 and Asn249 and a highly hydrophobic membrane anchoring domain at the carboxyl terminus of the protein. Significant homology exists between placental alkaline phosphatase and Escherichia coli alkaline phosphatase. Placental alkaline phosphatase is the first eukaryotic alkaline phosphatase to be cloned and sequenced.     

Nucleotide sequence of cloned cDNA coding for mouse epsilon casein

We isolated cDNA clones, which correspond to the mRNA coding for the smallest of the seven mouse caseins. From the nucleotide sequence of the cDNA we deduced the amino acid sequence of the protein, which we named epsilon casein. Mouse epsilon casein and cow alpha s2 casein show amino acid sequence homologies in the N-terminal region of the mature protein. The signal peptide of mouse epsilon casein shows in length and sequence remarkable homology to the signal peptides of the calcium-precipitable caseins of other species. In accordance with this group of caseins mouse epsilon casein contains in the sequence -Ser-Ser-Glu-Glu- a site for potential multiple phosphorylation.     

Complete nucleotide and derived amino acid sequences of the fourth component of mouse complement (C4). Evolutionary aspects

The nucleotide sequence coding for the fourth component of mouse complement (C4) has been determined from a cloned genomic DNA fragment and a cloned cDNA fragment. The amino acid sequence of the protein was deduced. The single chain precursor protein (pro-C4) consists of 1719 amino acid residues. The mature beta, alpha, and gamma subunits contain 654, 766, and 291 amino acids, respectively. One potential carbohydrate attachment site is predicted for the beta chain, three for the alpha chain, and none for the gamma chain. From a comparison with human C4 cDNA sequence an extensive overall sequence homology, 79% in nucleotides and 76% in amino acids, is observed. There is conservation in both the position and number of cysteine residues in human and mouse C4. We compared the mouse C4 amino acid sequences with those of mouse C3 and human alpha 2-macroglobulin and the evolutionary relationship among these three proteins is discussed.     

Isolation of recombinant plasmids containing structural gene sequences for rat seminal vesicle secretory proteins IV and V

Double stranded cDNA was made to partially purified mRNA for the small seminal vesicle secretory proteins IV and V. This ds cDNA was then inserted into the Pst 1 site of pBR322 by the (G-C) homopolymer tailing technique. Bacterial transformants harboring plasmids with specific inserts were identified by translation of mRNA that was hybridized to plasmid DNA immobilized on nitrocellulose. Separate plasmids were obtained with cDNA inserts for both SVS IV and V. Neither hybridization results nor preliminary restriction analysis gave any indication for homology between them.     

Androgen regulated expression of a spermine binding protein gene in mouse ventral prostate.

A full length cDNA (MP25) encoding the major mouse prostatic secretory glycoprotein (p25), whose expression is androgen dependent, has been cloned and characterised. Steady-state levels of mRNA are decreased approximately 100-fold after 3 days castration but are restored progressively over 4 days with testosterone treatment. The secreted glycoprotein appears to be a spermine binding protein since the nucleotide and predicted amino acid sequence of MP25 shares extensive homology with a spermine binding protein (SBP) found in rat ventral prostate. Genomic clones indicate that there is a single gene for SBP which consists of 4 exons, the first of which is only 11bp in length. The second exon encodes the signal peptide, the third contains a portion of the spermine binding protein unique to the mouse and the largest exon encodes the bulk of the secreted protein.     

Androgen-dependent protein from mouse vas deferens. cDNA cloning and protein homology with the aldo-keto reductase superfamily

We report the cloning and sequencing of a 1225-base pair cDNA encoding an abundant protein from mouse vas deferens. An open reading frame of 948 nucleotides encodes a protein of 316 amino acids with a calculated mass of 35,965 Da. A high degree of homology was found between this protein and members of the aldo-keto reductase superfamily, especially aldose reductase from human placenta (82%) and from rat lens (80%), suggesting that it could be an aldose reductase. Castration resulted in a marked decrease in the level of the 1.4 kilobase mRNA coding for the 35,965 protein, whereas administration of testosterone to castrated males resulted in a marked increase. Southern hybridization analysis of mouse genomic DNA revealed relatively simple patterns of bands.     

The primary structure of human Rieske iron-sulfur protein of mitochondrial cytochrome bc1 complex deduced from cDNA analysis

We isolated a cDNA encoding human Rieske Fe-S protein of mitochondrial cytochrome bc1 complex from a fibroblast cDNA library by colony hybridization. The cDNA contains the nucleotide sequence encoding all of the amino acids (274 residues) comprising the putative precursor to the protein. Based on the known amino acid sequence of bovine Rieske Fe-S protein, the N-terminal extension sequence is presumed to be composed of 78 amino acids with a molecular weight of 8053. The mature protein consists of the same number of amino acid residues as that of its rat and bovine counterparts, having a homology of about 92% with the latter.     

Molecular cloning of a cDNA that encodes a serine protease with chymotryptic and collagenolytic activities in the hepatopancreas of the shrimp Penaeus vanameii (Crustacea, Decapoda).

Two clones were isolated by screening a shrimp hepatopancreas cDNA library with a DNA fragment obtained by PCR amplification using two oligonucleotides based on the partial protein sequence of Penaeus vanameii chymotrypsin purified earlier. One of these clones, PVC 7 contains a complete cDNA coding for a serine protease. The deduced amino acid sequence shows the existence of a 270 residue-long preproenzyme containing a highly hydrophobic signal peptide of 14 amino acids. This suggests the existence of a putative zymogen form of the enzyme containing a 30 amino acid-long peptide which is cleaved to give a mature protein of 226 residues. A highly preferred codon usage is observed for this protein. The other obtained cDNA was found to encode the less predominant variant of the protein. Sequence alignments show that shrimp chymotrypsin is highly homologous with crab collagenase (77% homology taking into account the same amino acid at the same position, and 83% homology taking into account amino acids with conserved function) and that it is more similar to mouse trypsin (41% homology of strictly conserved amino acids) than to hornet chymotrypsin (35% homology).     

Primary structure of elongation factor 1β from Artemia

cDNA complementary to mRNA coding for the elongation factor EF-1β has been cloned. A γgt 11 cDNA library has been screened with an antiserum against EF-1β which exchanges GDP bound to EF-1α with exogenous GTP during protein synthesis. The derived amino acid sequence corresponds to 208 amino acids including the N-terminal methionine which is absent in the mature protein. About sixty percent of the protein was sequenced by direct protein sequence analysis. Comparison of Artemis salina EF-1β with Escherichia coli EF-Ts shows no evident homology.     

Cloning and sequencing of the 23 kDa mouse photoreceptor cell-specific protein.

The 23 kDa protein was localized by immunocytochemistry to photoreceptor cells of the mouse retina, and bovine and mouse cDNA clones were isolated and sequenced. The deduced amino acid sequences showed that the mouse 23 kDa protein is 91% identical to the bovine protein, and is the same as S-modulin, the CAR (cancer-associated retinopathy) protein and recoverin, the Ca(2+)-dependent activator of photoreceptor guanylate cyclase. The amino acid sequence reveals two Ca2+ binding sites, no internal repeats, 59% homology to the chicken visinin protein and 40% homology to calmodulin while Northern analysis demonstrated a single 1.0 kb mRNA species in bovine and mouse retina.     

Monkey erythropoietin gene: cloning, expression and comparison with the human erythropoietin gene

The erythropoietin (Epo) gene from Cynomolgus monkeys has been isolated from a kidney cDNA library using mixed 20-mer oligodeoxynucleotide probes. The gene encodes a 168 amino acid (aa) mature protein with a calculated Mr of 18,490 and a presumptive signal peptide of 24 aa. The Epo gene, when transfected into Chinese hamster ovary (CHO) cells, produces a glycosylated protein with an apparent Mr of 34,000. The expressed product is biologically active in vivo. The monkey gene exhibits 92% and 94% homology to the human gene at the aa and nucleotide sequence levels, respectively. When compared with the human Epo, monkey Epo has an additional 3-aa residue at the N terminus of the mature protein and a deletion of an internal lysine residue.