Isolation and characterization of the rat chromosomal gene for a polypeptide (pS1) antigenically related to statin.
Increasing evidence shows the existence of nonproliferation-specific gene(s) whose expression is mostly present in growth-arrested cells. One member of this gene family has been identified by previous work as a nuclear protein of 57,000 Da, termed statin. Logical extensions of statin research are to identify the genomic and cDNA clones encoding for statin and to study the regulation of statin gene expression. During the search for the statin gene, we have identified a cDNA clone and a genomic clone named S1 and S10, respectively, by screening a rat brain lambda gt11 expression library with the statin antibody and subsequently using S1 cDNA as a probe to screen a rat genomic cosmid library. Here, we report the cloning and sequencing of the S1 cDNA and S10 genomic clones. Primary sequence analyses indicate that the derived amino acid sequence of S1 shares high homology (greater than 92.6%) with human elongation factor 1 alpha (EF-1 alpha), whereas the 5'- and 3'-untranslated regions are less than 20% homologous. Despite the unusually high degree of similarity between S1 and human EF-1 alpha at the amino acid sequence level, their protein products are different and immunologically distinct. The in vitro transcription and translation product of S1 (pS1), a 49,000-Da polypeptide, reacts only with the monoclonal antibody against statin; this antibody exhibits no antigenic reaction to the EF-1 alpha protein. Northern blot analysis shows that the S1 message is most abundant in G0 phase of 3T3 mouse fibroblasts, but becomes significantly reduced in G1 and S phase cells. EF-1 alpha messages do not show such dramatic changes during cell cycle phase transition. These findings suggest that the expression of the identified S1 cDNA clone is specific for nonproliferating cells and that the in vitro translation product of the S1 cDNA is recognized by the statin antibody. Genomic Southern blots indicate that S1 cDNA is encoded by a single copy gene in the rat genome and is a unique member of the EF-1 alpha/S1 supermultigene family. DNA sequence analysis demonstrates that the rat S1 transcription unit is 12 kilobase pairs in length and contains seven introns. The organization of exons is virtually identical between S1 and human EF-1 alpha. In contrast, neither a TATA box nor a CAAT box is located in the proximal 5'-flanking regions from positions -1 to -1359 of the S1 gene, where we could expect to find the regulatory region containing the elements controlling gene expression; no evident sequence homology to the human EF-1 alpha gene is detected in this region.(ABSTRACT TRUNCATED AT 400 WORDS)
Ann
D K
DK
Department of Pharmacology, Medical School, University of Minnesota, Minneapolis 55455.
Moutsatsos
I K
IK
Nakamura
T
T
Lin
H H
HH
Mao
P L
PL
Lee
M J
MJ
Chin
S
S
Liem
R K
RK
Wang
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D018797:Cell Cycle Proteins; D004247:DNA / Q000235:genetics; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D005786:Gene Expression Regulation; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D009687:Nuclear Proteins; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000235:genetics; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D012313:RNA / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D014158:Transcription, Genetic
D018797:Cell Cycle Proteins; C503234:EEF1A2 protein, human; D000939:Epitopes; D009687:Nuclear Proteins; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D011506:Proteins; D012313:RNA; D004247:DNA