Characterization of squid crystallin genes. Comparison with mammalian glutathione S-transferase genes.
Previous experiments have indicated that the crystallins of the squid lens (S-crystallins) are evolutionarily related to glutathione S-transferases (GST) (EC 2.5.1.18). Here we confirm by peptide sequencing that the crystallins of the lens of the squid Ommastrephes sloani pacificus comprise a family of GST-like proteins. Squid lens extracts showed 400 times less GST activity than those of liver using 1-chloro-2,4-dinitrobenzene as a substrate, suggesting that the abundant GST-like crystallins lack enzymatic activity. Four different cDNAs (pSL20-1, pSL18, pSL11, and pSL4) showed 20-25% similarity in homologous regions with mammalian GST polypeptides. pSL20-1, pSL18, and pSL4 each encode an S-crystallin with a unique internal peptide that is unrelated to mammalian GSTs or any other sequence in GenBank. The S-crystallin family is encoded in a minimum of 9-10 genes, and the exon-intron structures of at least two of these (SL20-1 and SL11) are similar to those of the mammalian GST genes. The SL20-1 gene has six exons, with the its unique internal peptide encoded precisely in exon 4; the SL11 gene lacks a unique internal peptide and has five exons. Experiments using bacterial chloramphenicol acetyltransferase as a reporter gene showed that at least 84 and 111 base pairs of 5'-flanking sequence are needed for function of the SL20-1 and SL11 promoters, respectively, in a transfected rabbit lens epithelial cell line (N/N1003A). Within these regions each has a putative TATA box and an upstream AP-1 site overlapping with antioxidant responsive-like elements, which are regulatory elements in the rat GST Ya and quinone reductase genes responsive to oxidative stress.
Tomarev
S I
SI
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
Zinovieva
R D
RD
Piatigorsky
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003459:Crystallins / Q000235:genetics*; D004247:DNA / Q000235:genetics; D049832:Decapodiformes; D005982:Glutathione Transferase / Q000235:genetics*; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D014158:Transcription, Genetic
D003459:Crystallins; D012313:RNA; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D005982:Glutathione Transferase