Structural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein pref-1.
Preadipocyte factor 1 (pref-1), a member of the EGF-like protein family, is a transmembrane protein with six tandem EGF-like repeats in the putative extracellular domain. Expression of pref-1 is abolished during the in vitro differentiation of 3T3-L1 preadipocytes to adipocytes, and constitutive expression of pref-1 in preadipocytes inhibits their differentiation [Smas, C.M., & Sul, H.S. (1993) Cell 73, 725-734]. In the present studies, we have isolated and characterized genomic clones for pref-1 and have identified multiple pref-1 transcripts generated by alternate splicing. The pref-1 gene consists of five exons and four introns spanning approximately 7.3 kb. By primer extension analysis, the transcription start site was determined to be 169 bp upstream from the translation initiation codon. We have identified functional promoter sequences by transient transfection using a 2.1 kb fragment of the pref-1 5' flanking region linked to a luciferase gene; the pref-1-luciferase fusion gene construct gave 20-fold higher promoter activity as compared to the promoterless vector. Analysis of exon-intron junctions reveals that unlike the majority of the mammalian EGF-like genes, EGF-like repeats of pref-1 are not encoded by discrete exons. Through RT-PCR and the isolation and analysis of multiple pref-1 cDNA clones, we have identified, in addition to full-length pref-1, five alternately spliced forms with various in-frame deletions of all or a part of the sixth EGF-like repeat, juxta-membrane, and predicted transmembrane domains.(ABSTRACT TRUNCATED AT 250 WORDS)
Smas
C M
CM
Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115.
Green
D
D
Sul
H S
HS
D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002135:Calcium-Binding Proteins; D002454:Cell Differentiation; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004815:Epidermal Growth Factor / Q000096:biosynthesis; D005091:Exons; D015870:Gene Expression; D015698:Genomic Library; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000096:biosynthesis*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D012097:Repressor Proteins; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection
D002135:Calcium-Binding Proteins; D017931:DNA Primers; C512925:Dlk1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D012097:Repressor Proteins; D004815:Epidermal Growth Factor; D012313:RNA; D004247:DNA