Isolation of a mRNA encoding a glycine-proline-rich beta-keratin expressed in the regenerating epidermis of lizard.
During scale regeneration in lizard tail, an active differentiation of beta-keratin synthesizing cells occurs. The cDNA and amino acid sequence of a lizard beta-keratin has been obtained from mRNA isolated from regenerating epidermis. Degenerate oligonucleotides, selected from the translated amino acid sequence of a lizard claw protein, were used to amplify a specific lizard keratin cDNA fragment from the mRNA after reverse transcription with poly dT primer and subsequent polymerase chain reaction (3'-rapid amplification of cDNA ends analysis, 3'-RACE). The new sequence was used to design specific primers to obtain the complete cDNA sequence by 5'-RACE. The 835-nucleotide cDNA sequence encodes a glycine-proline-rich protein containing 163 amino acids with a molecular mass of 15.5 kDa; 4.3% of its amino acids is represented by cysteine, 4.9% by tyrosine, 8.0% by proline, and 29.4% by glycine. Tyrosine is linked to glycine, and proline is present mainly in the central region of the protein. Repeated glycine-glycine-X and glycine-X amino acid sequences are localized near the N-amino and C-terminal regions. The protein has the central amino acid region similar to that of claw-feather, whereas the head and tail regions are similar to glycine-tyrosine-rich proteins of mammalian hairs. In situ hybridization analysis at light and electron microscope reveals that the corresponding mRNA is expressed in cells of the differentiating beta-layers of the regenerating scales. The synthesis of beta-keratin from its mRNA occurs among ribosomes or is associated with the surface of beta-keratin filaments.
Dalla Valle
Luisa
L
Department of Biology, University of Padova, Italy.
Toffolo
Vania
V
Belvedere
Paola
P
Alibardi
Lorenzo
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000502:physiology*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D000078404:Epidermal Cells; D004817:Epidermis / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007633:Keratins / Q000235:genetics / Q000378:metabolism*; D008116:Lizards / Q000378:metabolism / Q000502:physiology*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D012333:RNA, Messenger / Q000378:metabolism*; D012038:Regeneration / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013623:Tail / Q000502:physiology*
D017931:DNA Primers; D018076:DNA, Complementary; D009841:Oligonucleotides; D012333:RNA, Messenger; D007633:Keratins