Molecular cloning and characterization of cDNA for androgen-repressible rat liver protein, SMP-2

• Published in: The Journal of biological chemistry Vol. 262; no. 2; pp. 822 - 825
• Main Authors: Chatterjee, B, Majumdar, D, Ozbilen, O, Murty, C V, Roy, A K
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 15.01.1987; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; androgens; Androgens - pharmacology; Animals; Base Sequence; Biological and medical sciences; Biotechnology; Calcium-Binding Proteins; cDNA; Cloning, Molecular; DNA - metabolism; Female; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; liver; Liver - drug effects; Liver - metabolism; Male; Methods. Procedures. Technologies; Molecular cloning; Nucleic Acid Hybridization; nucleotide sequence; Plasmids; Protein Biosynthesis; proteins; Proteins - genetics; Rats; Rats, Inbred Strains; repression; RNA, Messenger - genetics; Sulfotransferases; Transcription, Genetic; Androgen; Rat; Liver; Rodentia; Gene expression; Characterization; Proteins; Vertebrata; Regulation(control); Mammalia; Complementary DNA; Animal; Hormonal regulation; Repression derepression; Molecular cloning; Sex steroid hormone
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)75860-8

SMP-2 is a rat liver protein whose synthesis is influenced by both androgens and aging. The steady-state level of its mRNA is repressed by the androgen. Compared to the adult male, SMP-2 mRNA is found in higher amounts in the prepubertal and senescent male rat livers which show relative androgen insensitivity. A cDNA library in the plasmid pBR322 was constructed from the female rat liver which contains a high level of SMP-2 mRNAs. Recombinant plasmids were screened by differential colony hybridization to 32P-labeled single-stranded cDNAs from adult female and adult male hepatic poly(A)+ RNAs. From a total of 3500 recombinant clones, 11 highly female specific clones were identified. From these female specific colonies the SMP-2 cDNA-containing plasmid (pSP11) was identified by its ability to select an mRNA species whose translation product is immunochemically and electrophoretically indistinguishable from SMP-2. This insert represents a 571-base pair portion of the SMP-2 cDNA. Rescreening of the library at a high colony density using the 32P-labeled cDNA insert of pSP11 identified several positive clones with larger inserts. Hybrid-selected mRNA translation again confirmed these clones to carry SMP-2 cDNA sequences. The plasmid pSP4a containing a 1040-base pair cDNA insert of SMP-2 was characterized by DNA sequence analysis. The size of the cDNA insert of pSP4a is close to the estimated size of the SMP-2 mRNA. The cDNA sequence provides an open reading frame of 282 amino acid residues. A comparison of the translated amino acid sequence with the protein sequences of NBRF-PIR, PSQNEW, and LOSALA data bases did not establish any sequence homology with known proteins. Northern blot analysis using the 32P-labeled cDNA insert of pSP4a confirms the androgenic repression of the SMP-2 mRNA.