Isolation and characterisation of keratin mRNA from the scale epidermis of the embryonic chick.
The keratin polypeptides of the epidermis from the leg scale region of 17-day-old embryonic chicks were extracted as S-carboxymethylated derivatives and characterised by electrophoresis on SDS and pH 9.5 urea gels including a combination of both in two dimensions. Proteins were isolated that gave X-ray diffraction patterns typical of alpha- and beta- (avian feather) keratins. An mRNA fraction was isolated from 17-day-old scale tissue by guanidinium chloride extraction and sucrose gradient fractionation. The mRNA was translated in the wheat germ system to give a major product indistinguishable from the molecular weight class (Mr 14 500) of scale beta-keratin polypeptides. A cDNA library was constructed in pBR322 from a 15 S mRNA subfraction and two recombinant clones were selected by their strong hybridisation to cDNA prepared from the 15 S mRNA. The sequencing of these has yielded details of the relatedness of two scale keratin genes including their 3' untranslated regions. Almost half of the protein sequences of the two homologous scale keratins has been deduced and a notable feature of the scale keratin structure appears to be the presence of at least two sequence domains consisting of 13 amino acid repeats.
Wilton
S D
SD
Crocker
L A
LA
Rogers
G E
GE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D004247:DNA / Q000302:isolation & purification; D004274:DNA, Recombinant / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004817:Epidermis / Q000032:analysis; D007633:Keratins / Q000235:genetics*; D010957:Plasmids; D012333:RNA, Messenger / Q000302:isolation & purification*
D004274:DNA, Recombinant; D012333:RNA, Messenger; D007633:Keratins; D004247:DNA