Molecular cloning and characterization of a progesterone-dependent cat endometrial secretory protein complementary deoxyribonucleic acid

In the cat, a group of low molecular weight secretory proteins have previously been shown to appear in the endometrium after progesterone (P) administration to an estrogen (E2)-primed animal. Using a polyclonal antibody to these progesterone-dependent proteins (PDP) we have isolated a recombinant cDNA clone corresponding to the mRNA for PDP from a cDNA library prepared using poly(A+) RNA from the endometrium of P-treated E2-primed cats. Comparison of Western blots using the polyclonal antibody and epitope selected antibody demonstrated that the multiple molecular weight and isoelectric forms of the PDP are immunologically related and potentially products of the same gene. Northern analysis revealed that the mRNA for the PDP in the endometrium of P-treated E2-primed cats was 1.8 kilobases in length. Using slot blot analysis, we found that the PDP mRNA levels were low in the endometrium of ovariectomized animals and undetectable in E2-treated animals. With 1 day of P treatment the PDP mRNA levels were readily detectable and they peaked after 5 to 7 days of P treatment. No PDP mRNA was detectable in myometrium, oviduct, or ovary. Sequence analysis revealed that PDP had significant homology to human, rat, and mouse cathepsin L at the nucleotide (80%, 74%, and 73%, respectively) and amino acid (68%, 65%, and 63%, respectively) level. We suggest that PDP via its collagenolytic and elastolytic activities as a cathepsin L is responsible for preparing the endometrium for blastocyst implantation.     

Molecular cloning and sequencing of a rat preprogastrin complementary deoxyribonucleic acid

Gastrin biosynthesis involves a complex series of posttranslational modifications; their elucidation requires a knowledge of the structure of the gastrin precursor. The complete structure of rat preprogastrin was deduced from the nucleotide sequence of a full length cDNA clone isolated from a rat antral cDNA library. Northern blot hybridization analysis of rat antral RNA together with human antral RNA, reveals a single mRNA species of approximately 670 bases. Comparison of this sequence with those of porcine and human gastrin reveals extensive (73%) homology in the gastrin coding region as well as short regions of conserved nucleotides in the noncoding regions. The rat sequence encodes a preprogastrin of 104 amino acids which consists of a signal peptide, a 37 amino acid prosegment; and the gastrin 34 sequence, followed by a glycine (the amide donor), and flanked by pairs of arginine residues. Cleavage at an internal pair of lysine residues yields gastrin 17. Unlike the human and porcine sequences, rat preprogastrin contains a 9 amino acid carboxy-terminal extension peptide (-Ser-Ala-Glu-Glu-Glu-Asp-Gln-Tyr-Asn) which is homologous to the midportion of gastrin 17 including the site of tyrosine sulfation.     

Molecular cloning and expression of mouse placental lactogen I complementary deoxyribonucleic acid

The mouse midpregnancy lactogen or placental lactogen I (mPL-I) is encoded by a 1.0-kilobase mRNA that appears transiently during gestation, with maximal amounts accumulating in the placenta at day 10 of pregnancy. Several cDNA clones for mPL-I have been isolated from a lambda gt11 expression library constructed from day 10-placental RNA. The cDNA sequence indicates that mPL-I is synthesized as a 224 amino acid precursor, and is secreted as a 194 amino acid glycosylated hormone. The deduced amino acid sequence of mPL-I is highly homologous to the known members of the PRL family in the mouse, and hybridization analysis indicates that the mouse genome contains several mPL-I genes. Introduction of the mPL-I cDNA in an expression vector into cultured mouse cells results in the synthesis and secretion of glycosylated mPL-I protein that is recognized by anti-mPL-I antiserum and is biologically active.     

The androgen-dependent mouse seminal vesicle secretory protein IV: characterization and complementary deoxyribonucleic acid cloning

Seminal vesicle secretory protein IV of a mouse has been isolated, and the cDNA coding for its mRNA has been cloned and sequenced. The 556-nucleotides encode 16 amino acid signal peptides and 92 residues of mature protein. Considerable homology between mouse and rat SVS IV cDNA was found. In the leader peptide and 3'-noncoding region there is 92% and 85% homology, respectively. The other regional homologies are 86% for the first 12, 68.5% for the last 35, and 40% for the middle 44 amino acids. The expression of mouse SVS IV mRNA is under the control of androgen. Administration of testosterone to castrated mice resulted in induction of the mRNA level to 50% of the mature male in 96 h of hormone treatment. Secretion of the protein after testosterone injection follows a similar pattern.     

Molecular cloning of complementary deoxyribonucleic acid for an androgen-regulated epididymal protein: sequence homology with metalloproteins

Acidic epididymal glycoprotein (AEG) is a 31,000 molecular weight secretory protein of the rat epididymis. Screening of a rat epididymal cDNA library with affinity-purified AEG antiserum yielded cDNA for AEG. Identity of the clones was verified by comparison of amino acid sequence of the purified protein with the sequence derived from the nucleotide sequence of the cDNA isolates. Two classes of AEG cDNA, approximately 1500 base pairs (bp) and 950 bp in length, differed by 538 bp in the 3'-untranslated region and by four single nucleotide mismatches, one of which was in the coding region. Northern blot hybridization of epididymal RNA revealed two species of AEG mRNA, corresponding in length to each type of cDNA. Analysis of RNA from individual animals provided evidence that the two mRNA species are the products of allelic genes. In vivo studies demonstrated that the level of total AEG mRNA is regulated by androgen. Amino acid sequence homology of AEG with metal-binding domains of several proteins suggests that AEG is a metalloprotein.     

Molecular cloning and nucleotide sequence analysis of complementary deoxyribonucleic acid for the beta-subunit of rat follicle stimulating hormone

To provide a hybridization probe for analysis of the regulation of rat gonadotropin subunit mRNA levels, an effort was made to isolate a cloned cDNA for the beta-subunit of rat FSH (FSH beta). Using a cloned bovine FSH beta cDNA as a hybridization probe, a rat pituitary lambda gt10 cDNA library was screened and a single, strongly hybridizing clone identified. The 874 base pair cDNA insert from this clone contains the complete sequence of rat FSH beta including an amino-terminal precursor segment. Hybridization of this cloned cDNA to rat pituitary RNA demonstrated the presence of an approximately 2.0 kilobase RNA species containing FSH beta sequences. Cloned rat cDNA was also used to demonstrate that estrogen treatment of ovariectomized female rats results in decreases in mRNA concentrations for FSH beta and the beta-subunit of LH with somewhat smaller decreases in alpha-subunit mRNA concentrations. Little or no change was detected in the mRNA for the beta-subunit of TSH.     

Construction and characterization of type II collagen complementary deoxyribonucleic acid clones

The mRNA for type II collagen was purified from embryonic chick sternum or from purified sternal chondrocytes with guanidine thiocyanate as the extractant. Double-stranded cDNAs to procollagen mRNAs from sternum were synthesized and dC-tailed. After annealing with PstI-cleaved, dG-tailed pBR322, this DNA was used to transform Escherichia coli X1776. Transformed colonies were screened by colony hybridization to type I and II collagen cDNAs. Clones that preferentially hybridized to type II cDNA were characterized further. Four such cDNA clones, pCgII-2, 3, 10 and 12, with inserts of 400, 320, 260 and 750 bp, have been identified as type II collagen cDNA clones by several criteria, including their preference for hybridizing with type II rather than type I collagen mRNAs in hybrid-selected translation experiments.     

Molecular characterization of the interferon-induced 15-kDa protein. Molecular cloning and nucleotide and amino acid sequence

We have isolated a cDNA clone for an interferon-induced 15-kDa protein. The cDNA clone was prepared from mRNA isolated from interferon-beta-treated human Daudi cells. The clone of 635 base pairs contains an open reading frame coding for a protein of 145 amino acids, and suggests for the mRNA a 75-base pair 5' untranslated and a 125-base pair 3' untranslated region. Approximately 85% of the amino acid sequence of the 15-kDa protein has been independently obtained from 2 nmol of material using microsequencing technology on the N terminus of the intact protein and on tryptic and chymotryptic peptides. The amino acid sequence of the isolated protein is identical to the amino acid sequence deduced from the cDNA. Northern blot analysis confirmed that the mRNA for the 15-kDa protein is undetectable in untreated cells, but is greatly induced following interferon treatment.     

Molecular cloning and functional characterization of mouse coactosin-like protein.

Coactosin was first isolated from Dictyostelium discoideum and, as reported, human coactosin-like protein (CLP) was identified in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. A mouse CLP (mCLP) cDNA clone was identified among EMBL/GenBank EST sequences. The derived amino acid sequence (142 residues) was 95.1% identical with human CLP. Here, we also show that mCLP interacts with actin and 5LO in the two-hybrid system. High-speed cosedimentation assays and GST-binding assays confirmed these protein interactions. In chemical cross-linking experiments, one molecule of mCLP was covalently linked to either one subunit of actin or one molecule of 5LO. The mCLP-F-actin and mCLP-5LO associations were pH-insensitive and Ca(2+)-independent. However, association with actin was best observed at low salt concentrations, while association with 5LO was favored by salt, indicating different binding characteristics.     

Molecular cloning, complementary deoxyribonucleic acid structure and predicted full-length amino acid sequence of the hormone-inducible regulatory subunit of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase from human testis

In this study, we report the isolation and characterization of a full-length cDNA clone for the hormone-inducible regulatory subunit RII beta (formerly called RII51) of type II cAMP-dependent protein kinase from a human testis cDNA library. The cloned cDNA demonstrated tissue-specific expression of RII beta mRNA in human tissues, with the highest mRNA levels in testis and ovary. The isolated human cDNA clone was 3.3 kilobases (kb) in length and contained 166 base pairs (bp) of G/C-rich 5'-noncoding sequence, an open reading frame of 1254 bp and an A/T-rich 3'-nontranslated region containing 1836 bp followed by an 89 nucleotide long poly(A)-tail. The predicted protein contains 418 amino acids including the start methionine, and the estimated mol wt of human RII beta is 53,856. The nucleotide sequence within the open reading frame and the predicted amino acid sequence of human RII beta are highly conserved compared with partial rat RII beta sequences, displaying 91% and 97% similarity, respectively. Codon preference analysis of the cloned cDNA sequence indicated that the two cAMP-binding domains and the hinge region are highly conserved through evolution, whereas the dimerization domain displayed a codon preference pattern indicative of appearance at a later stage of evolution. The isolated human cDNA detected an FSH- and cAMP-inducible mRNA of 3.2 kb in rat Sertoli cells, thus confirming that the cloned cDNA represents the hormone-inducible regulatory subunit of cAMP-dependent protein kinase. This is the first report documenting the isolation of a full-length cDNA clone for the RII beta of cAMP-dependent protein kinase.     

Molecular cloning and characterization of oppo 1: a haploid germ cell-specific complementary DNA encoding sperm tail protein

We isolated a cDNA clone specifically expressed during spermatogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1085 nucleotides and had an open reading frame of 870 nucleotides encoding a putative protein of 290 amino acid residues. Northern blot analysis revealed a 1.2-kilobase mRNA exclusively expressed in the testis in adult mice; the mRNA was first detected late pachytene stage, and expression increased as the animals matured. The protein encoded by the mRNA had a molecular weight of approximately 33 kDa by Western blot analysis, and was localized to occupy the flagella from the connecting piece through the principal piece. We named this newly isolated gene oppo 1, and we suggest that it plays an important role in sperm tail structure and/or sperm movement.