Abstract
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.
Highlights
SMP-2 is a r a t liver proteinwhose synthesisis influ- the male rat,androgenscan inducemale-specific hepatic enced by both androgens and aging
A cDNA libraryintheplasmidpBR322was constructed from the female rat liver which contains a high level of SMP-2 mRNAs
Recombinant plasmids were screened by differential colony hybridization to
Summary
To the estimated sizeof the SMP-2 mRNA. The cDNA Isolation and Translation of mRNA-Total cellular RNAs were sequence providesan open reading framofe282 amino acid residues. The female-specific clones were analyzed for SMP-2 cDNA inserts by hybrid-selected mRNA translation (Fig. 1, A and B). The translation products of Digestionof Plasmid DNA by Restriction Enzymes and Sequencing female mRNAs hybrid selected through pBR322 did not react of the cDNA Insert-The SMP-2 cDNA was excised from the recom- with anti-SMP-2 (Fig. lB, lune 6). From these results it can binant plasmid by PstI digestion, and the insert was recovered by electroelution from the 1%agarose gel. Since the cytoplasmic androgen binding protein of the liver has been implicated in androgen action [27], it will be of interest to examine whether this protein can bind to the SMgPe-n2e
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