Cloning of full-length elastin cDNAs from a human skin fibroblast recombinant cDNA library: further elucidation of alternative splicing utilizing exon-specific oligonucleotides.
A human cDNA library was constructed utilizing RNA isolated from cultured skin fibroblasts. Recombinant clones containing elastin sequences were identified by plaque hybridizations with previously characterized human placental elastin cDNAs. Seven positive recombinant clones with inserts of approximately 3.2-2.2 kb were isolated. Characterization of the clones by restriction endonuclease analysis and dot-blot hybridizations with exon-specific synthetic oligonucleotides demonstrated considerable variability in the primary nucleotide sequence. Dideoxy nucleotide sequencing confirmed this finding. The variability is most likely a result of alternative splicing of exons from the primary elastin transcripts. The two largest clones contained approximately 1 kb of 3' untranslated sequence and approximately 2.2 kb of translated sequence encoding 730 amino acids. Six amino acids, encoded by exon 12A, have not been previously noted in human elastin cDNAs. In addition, these human skin fibroblast clones contained a 49 bp 5' untranslated sequence. These results demonstrate that there is considerable variability in the processed nucleotide sequence of the elastin mRNAs. These transcripts may code for isoforms of tropoelastin with different biologic properties.
Fazio
M J
MJ
Department of Dermatology H, Jefferson Medical College, Philadelphia, PA 19107.
Olsen
D R
DR
Kauh
E A
EA
Baldwin
C T
CT
Indik
Z
Z
Ornstein-Goldstein
N
N
Yeh
H
H
Rosenbloom
J
J
Uitto
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D004274:DNA, Recombinant; D004549:Elastin / Q000235:genetics* / Q000302:isolation & purification; D005091:Exons; D005347:Fibroblasts / Q000032:analysis*; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012326:RNA Splicing; D012333:RNA, Messenger / Q000302:isolation & purification; D012867:Skin / Q000166:cytology*
D004274:DNA, Recombinant; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D004247:DNA; D004549:Elastin