Nucleotide sequence of a cDNA clone encoding mouse protamine 1

The nucleotide sequence of a 404-base cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine has been determined. This insert, isolated from a mouse testis cDNA library, encodes a polypeptide of 50 amino acids of which 28 are arginine, 9 are cysteine, and 3 are tyrosine. The insert contains the complete 3' noncoding region of 151 bases and most of the 5' noncoding region. The predicted amino acid sequence of mouse protamine 1 is about 80% homologous to boar protamine and 67% homologous to bull protamine and contains the central, highly basic domain of four arginine clusters found in the trout protamines. The identification of a cDNA clone for a mouse protamine will facilitate studies of the evolution, regulation, and protein-DNA interaction of this nuclear protein unique to haploid spermatogenic cells.     

Nucleotide sequence of a bovine protamine cDNA

The nucleotide sequence of a 441-base cDNA encoding the bovine protamine has been determined. This insert, isolated from a bovine spermatid-specific cDNA library, encodes a polypeptide of 50 amino acids of which 26 are arginine, 7 are cysteine, and 2 are tyrosine. The insert contains the complete 3'-noncoding region of 150 bases and most of the 5'-noncoding region. The predicted amino-acid sequence of bovine protamine is about 96% homologous to ram protamine, 76% to boar protamine, 64% to mouse protamine 1 and 52% to human protamine 1 and contains the central, highly basic domain of four arginine clusters found in the trout protamines. Our results show that bovine protamine is 50 amino-acid residues in length and not 47 residues as previously published (Coelingh, J.P. et al. (1972) Biochim. Biophys. Acta 285, 1-14).     

The nucleotide sequence of boar transition protein 2 (TNP2) cDNA and haploid expression of the gene during spermatogenesis

A cDNA clone coding for boar transition protein 2 (TNP 2) was isolated from a randomly primed cDNA library of boar testis. Sequence analysis revealed an open reading frame of 414 bp (corresponding to 138 amino acids), 33 bp of the 5' untranslated and about 300 bp of the 3' untranslated region. As compared to TNP 2 of mouse and rat, similarity with TNP 2 of the boar is approximately 70% at the nucleotide level and only about 40% on the basis of amino acid sequence. The similarity between boar and bull TNP2 is 77% and 64%, respectively. Northern blot experiments with RNA of different boar tissues and in situ hybridization on mature boar testis sections revealed testis-specific expression of the TNP 2 gene which is restricted to haploid germ cells. Hybridization experiments of boar TNP2 cDNA with testicular RNA of boar, bull, rat and mouse revealed decreasing intensities of the hybridization signals. With human testicular RNA no hybridization could be obtained.     

Cloning of the cDNA encoding rat homologue of the mismatch repair gene MSH2 and its expression during spermatogenesis

A rat cDNA clone encoding the mismatch repair protein MSH2 has been isolated and characterized. The cDNA has an open reading frame of 2802 nucleotides in length coding for a protein of 933 amino acids (100 kDa). It shows significant homology to human and mouse MSH2. Northern blot analysis of rat MSH2 in the testes of rats of different ages showed maximum expression at 20 days, at which time the germ cells are undergoing premeiotic DNA replication. We observed down-regulation in the expression of rat MSH2 beyond 25 days by which time the germ cells have entered meiotic prophase.     

Nucleotide sequence and expression of a cDNA encoding chick brain actin depolymerizing factor

Chick brain actin depolymerizing factor (ADF) is a 19-kDa protein that severs actin filaments and binds actin monomers. We have obtained a cDNA encoding ADF by screening a chick embryo lambda gt11 cDNA library with both a rabbit anti-ADF antiserum and two oligonucleotide probes. Several non-full-length clones of 636 bases and one full-length clone of 1886 bases were isolated and sequenced. The full-length cDNA encodes a protein of 165 amino acids with a calculated molecular weight of 18,520. The deduced amino acid sequence shows 73% identity with the porcine brain actin binding protein cofilin. The coding region of the ADF cDNA has been placed in an expression vector, and the resulting protein shows immunoreactivity with an anti-ADF antiserum but not with an anti-cofilin antibody. The expressed ADF has been purified and has an actin depolymerizing activity identical with that of brain ADF. Like cofilin, ADF contains a sequence similar to the nuclear transport signal sequence of the SV40 large T antigen and a calcium/calmodulin-dependent protein kinase II phosphorylation consensus sequence. Northern blots of both embryonic chick brain and muscle RNA revealed two ADF mRNAs of length 2.1 and 0.9 kilobases. Southern blots suggest that the ADF gene is present in a single copy within the chicken genome. ADF contains regions of homology with other actin binding proteins including tropomyosin, gelsolin, and depactin.     

Nucleotide sequence and expression of a cDNA clone encoding a fetal rat binding protein for insulin-like growth factors

The insulin-like growth factors (IGFs), IGF-I and IGF-II, occur in plasma and tissue fluids complexed to specific binding proteins. Although the role of the binding proteins is not completely defined, they are capable of modulating the biological activity of the IGFs. In order to better understand the function of these proteins, we have isolated a clone from the BRL-3A rat liver cell line that encodes a protein corresponding to the IGF binding protein in fetal rat serum. The cDNA clone encodes a precursor protein of 304 amino acids (32,886 daltons), comprised of a 34-residue hydrophobic prepeptide and a 270-residue mature protein (29,564 daltons). The deduced amino acid sequence agrees with the sequence of 173 amino acid residues determined by Edman degradation. The mature protein contains 18 cysteines and no N-glycosylation sites. It contains an Arg-Gly-Asp (RGD) sequence near the carboxyl terminus. A similar sequence is present on many extracellular matrix proteins and contributes to their recognition by cellular adhesion receptors. The cloned cDNA has been transcribed in vitro and the resulting RNA expressed in Xenopus oocytes. Injected oocytes secrete a 33-kDa protein that is immunoprecipitated by polyclonal antibodies to the BRL-3A binding protein and binds IGF-I and IGF-II with the same affinity and specificity as does purified BRL-3A binding protein. The binding protein cDNA probe hybridizes to an approximately 2-kilobase mRNA in BRL-3A cells and in multiple fetal rat tissues including liver, kidney, intestine, and lung. Levels of this mRNA are greatly reduced in the corresponding adult tissues. The rat IGF binding protein is closely related to the partial amino acid sequences reported for a bovine IGF binding protein and more distantly related to a human IGF binding protein that recently has been cloned. No significant homologies were identified to other proteins. Thus, the rat IGF binding protein that we have cloned appears to be a distinct member of a family of related IGF binding proteins. We postulate that the structurally distinct IGF binding proteins may have different biological functions.