ABSTRACT
From an expression library in lambda UniZAP, derived from porcine corpus luteum (CL), a clone lambda MCP9 was detected by hybridization with a porcine MCP-1 specific probe. A pBluescriptSK-derivative pMCP9 was generated from lambda MCP9 by in vivo excision and was shown to contain an open reading frame (ORF) encoding a protein highly homologous to bovine monocyte chemoattractant protein-2 (MCP-2). Comparison of amino acid sequences of known MCPs identified the protein encoded by pMCP9 as porcine MPC-2. The 3' untranslated region of pMCP9 was completed by 3' RACE. Northern analysis using RNA from porcine luteal cells and probes specific for porcine MCP-1 and MCP-2 revealed that porcine luteal cells express both MCPs. According to Southern analysis MCP-2, like MCP-1, is specified by a single copy gene.
Subject(s)
Chemotactic Factors/biosynthesis , Corpus Luteum/metabolism , Monocyte Chemoattractant Proteins , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Chemokine CCL8 , Chemotactic Factors/genetics , Cloning, Molecular , DNA, Complementary , Female , Molecular Sequence Data , Sequence Homology, Amino Acid , SwineABSTRACT
A basic protein of apparent molecular weight 15 kD, designated bSVSP15, was purified from bovine seminal vesicle secretion to homogeneity, employing affinity absorption to calmodulin-Sepharose and reverse-phase HPLC. Immunoblotting identified bSVSP15 in bovine seminal plasma and seminal vesicle secretion, but it was not present either in extracts of bovine ampulla, epididymis, and testis or in serum or follicular fluid. When added to cAMP phosphodiesterase, bSVSP15 inhibited the activation of enzymatic activity by calmodulin in a reversible manner. Immunoscreening of a lambda gt11 expression cDNA library from bovine seminal vesicle tissue yielded two positive clones, pSVS4 and pSVS5, which were characterized by sequencing. Both sequences are identical, except for the 3' region. Because the derived amino acid sequence comprises, with an identity of 81%, the amino-terminal 21 residues of bSVSP15, cDNA clones pSVS4 and pSVS5 represent bSVSP15-specific mRNAs. The mature protein bSVSP15 contains 101 residues and is preceded by 25 residues of a signal sequence, characteristic for secretory proteins. Northern analysis identified two bSVSP15-specific mRNAs of 900 bp and 1200 bp, respectively. Sequence comparison yielded high homologies to human C-type natriuretic peptide. We conclude from this result that bSVSP15 is identical with the hitherto unknown bovine C-type natriuretic peptide.
Subject(s)
Calmodulin-Binding Proteins/isolation & purification , Prostatic Secretory Proteins , Proteins/isolation & purification , Semen/chemistry , 3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Amino Acid Sequence , Amino Acids/analysis , Animals , Base Sequence , Calmodulin-Binding Proteins/chemistry , Calmodulin-Binding Proteins/genetics , Cattle , Cloning, Molecular , DNA, Complementary , Humans , Male , Molecular Sequence Data , Proteins/chemistry , Proteins/genetics , Restriction Mapping , Seminal Plasma Proteins , Sequence Homology, Amino AcidABSTRACT
RT PCR employing poly(A+)RNA from porcine luteal cells and a combination of primers designed from the known bovine MCP-1 cDNA identified the luteal cells as a source of MCP-1. This finding is corroborated by results from Northern analysis using total RNA from luteal cells. To characterize the complete porcine MCP-1 cDNA, poly (A+)RNA was isolated from porcine corpus luteum, transcribed into cDNA and the latter cloned into the expression vector lambda Uni-ZapXR. A digoxigenin-labeled DNA probe of 375 bp was obtained by PCR and employed to screen the library. From the positive clones pMCP5, pMCP7 and pMCP10, the clone pMCP5 was selected and both strands of the cDNA insert were sequenced. The cDNA insert was 742 bp long, with an open reading frame (ORF) encoding a protein of 99 amino acid residues which by comparison with known amino acid sequences of MCPs yielded highest identities with MCP-1 sequences. We therefore assume that pMCP5 encodes the amino acid sequence for porcine MCP-1.
