The nucleotide sequence of a complete chicken delta-crystallin cDNA

The nucleotide sequence of a full length cDNA of delta-crystallin mRNA from chicken lens has been determined using a delta-crystallin cDNA clone (pB delta 11), which represents the mRNA sequence of 1530 nucleotides from the poly(A) junction but does not contain the 5'-terminal sequence of 44 nucleotides of the mRNA. The 5'-terminal sequence of the mRNA, absent in the cDNA clone, has been determined with a stretch of cDNA sequence by the primer extension procedure. The amino acid sequence deduced from the nucleotide sequence is consistent with the amino acid sequences of several tryptic peptides, the total amino acid composition, and the mol. wt. of delta-crystallin estimated by SDS-polyacrylamide gel electrophoresis. The computer-assisted analysis predicts high alpha-helical content throughout the polypeptide. Sequence analyses have revealed that gene 1 encodes the mRNA from which the cDNA clone was derived.     

The chicken delta-crystallin gene family. Two genes of similar structure in close chromosomal approximation

delta-Crystallin is a major protein product of the differentiated chicken lens. We have isolated two, non-allelic delta-crystallin genes using a recombinant bacteriophage/chicken genomic DNA library. There appear to be only these two delta-crystallin genes in the haploid chicken genome. Southern hybridization and R-loop analyses indicate that the two genes are oriented on the chromosome with similar 5'-3' polarity. delta 1, arbitrarily designated as the directionally 5' of the two genes, is 6.7 kilobases in length, while delta 2 is 9.2 kilobases. The two delta-crystallin genes are about 4.2 kilobases apart. Structurally, both genes are arranged in a similar and characteristic pattern of 17 exons/16 introns, as judged by electron microscopy. The delta-crystallin gene locus represents a simple model for the study of structural co-evolution and/or functional co-expression of two related genes within a developmentally modulated region of the genome.     

Sequence analysis of pigeon delta-crystallin gene and its deduced primary structure. Comparison of avian delta-crystallins with and without endogenous argininosuccinate lyase activity.

delta-Crystallin is a major lens protein present in the avian and reptilian lenses. To facilitate the cloning of the delta-crystallin gene, cDNA was constructed from the poly(A)+ RNA of pigeon lenses, amplified by the polymerase chain reaction (PCR). The PCR product was then subcloned into pUC19 vector and transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing two clones, containing 1.4 kb DNA inserts coding for delta-crystallin allowed the construction of a complete, full-length reading frame of 1,417 bp covering a deduced protein sequence of 466 amino acids, including the universal translation-initiating methionine. The pigeon delta-crystallin shows 88, 83 and 69% sequence identity to duck delta 2, chicken delta 1 crystallins and human argininosuccinate lyase respectively. It is also shown that, in contrast to duck delta 2 crystallin which has a high argininosuccinate lyase activity, pigeon delta-crystallin appears to contain very low activity of this enzyme, despite the fact that they share a highly homologous structure. A structural comparison of delta-crystallins with or without enzymatic activity suggested several amino acid replacements which may account for the loss of argininosuccinate lyase activity in the lenses of certain avian species.     

Nucleotide sequence of a chicken delta-crystallin gene.

We have determined the complete nucleotide sequence of one of the two non-allelic delta-crystallin genes in the chicken, arbitrarily designated delta-gene 1, using a genomic clone (lambda g delta 106) containing the entire gene sequence. By comparison of the genomic sequence and the delta-crystallin cDNA sequence previously determined, we have identified exon sequences in the genomic sequence. Thus, the presence of 17 exons and 16 introns in the gene has been clarified. The delta-crystallin polypeptide deduced from the exon sequences consists of 465 amino acids which is larger, by 19 amino acid residues, than the polypeptide deduced from the cDNA sequence previously reported. Re-examination of the cDNA sequence using the same cDNA clone previously used shows that the present exon sequences are correct and the molecular weight of the deduced delta-crystallin polypeptide is 50,615 daltons instead of the previously reported value of 48,447 daltons. In addition, some structural features of the delta-crystallin gene including putative expression signals are discussed.     

Sequence of the chicken delta 2 crystallin gene and its intergenic spacer. Extreme homology with the delta 1 crystallin gene

The chicken delta-crystallin locus consists of 2 nonallelic, tandemly arranged genes (5'-delta 1-delta 2-3'). Only the delta 1 gene is known to be expressed. The nucleotide sequence for the delta 1 gene has been reported recently (Nickerson, J.M., Wawrousek, E.F., Hawkins, J.W., Wakil, A.S., Wistow, G.J., Thomas, G., Norman, B.L., and Piatigorsky, J. (1985) J. Biol. Chem. 260, 9100-9105; Ohno, M., Sakamoto, H., Yasuda, K., Okada, T.S., and Shimura, Y. (1985) Nucleic Acids Res. 13, 1593-1606). We now report the sequence for the delta 2 gene and the 4-kilobase intergenic spacer between the two delta-crystallin genes. The delta 2 gene, like the delta 1 gene, has 17 exons and 16 introns. The homologous exons are remarkably similar: exons 3-17 are identical in size between delta 1 and delta 2, and the sequence homology ranges from 70% (exon 2) to 100% (exons 7, 12, and 15), with the remaining exons having 89-98% identity between the delta 1 and delta 2 genes. Consequently, the encoded delta 2 polypeptide is 91% identical to the delta 1 polypeptide. Considerable similarity also exists between homologous introns of delta 1 and delta 2, with most of the differences accounted for by insertions and/or deletions. The presence of a TATA box, consensus splice junctions (almost identical to the delta 1 gene), lariat branch sequences, and a polyadenylation signal strengthen the possibility that delta 2 is a functional gene.     

Two delta-crystallin polypeptides are derived from a cloned delta 1-crystallin cDNA

Previous studies have shown that there are 2 similar delta-crystallin genes (delta 1 and delta 2) and at least 2 delta-crystallin polypeptides in the chicken eye lens. We show here that both delta-crystallin polypeptides can be synthesized from mRNA transcribed in vitro from a cloned delta 1-crystallin cDNA. Both polypeptides co-migrate in SDS-urea-polyacrylamide electrophoresis with their authentic counterparts isolated from 15-day-old embryonic chicken lenses, and both react with sheep anti-chicken delta-crystallin serum. Screening nearly 900 delta-crystallin cDNA clones from a 15-day-old embryonic lens library with an oligonucleotide probe specific for exon 2 of the delta 2-crystallin gene failed to detect any delta 2 cDNA clones, indicating that the delta 2 gene produces little or no mRNA in the lens at this stage of development. Our results suggest that both of the observed delta-crystallin polypeptides are derived from mRNA transcribed from the delta 1 gene, with heterogeneity arising at the translational or co-translational level.     

Conservation of δ-crystallin gene structure between ducks and chickens

A cloned chicken delta-crystallin cDNA was used to identify two putative delta-crystallin genes in the duck by Southern blot hybridization. A DNA fragment containing most of one of these genes was isolated from a library made in bacteriophage lambda Charon 28A containing genomic DNA from 14-day-old embryonic ducks. Electron microscopy, partial gene sequencing, primer extension analysis using duck mRNA, and comparison with the well-characterized chicken delta-crystallin genes suggest that our cloned duck delta-crystallin gene, like the chicken delta-crystallin genes, is 8-10 kb long and contains 17 exons. Hybridization and sequencing data show great similarity between the homologous 5' untranslated and coding exons of the duck and chicken delta-crystallin genes. Overall, the homologous introns also appear to have approximately 30% sequence similarity, and have been subject to deletion/insertion events. Our partial characterization of duck delta-crystallin gene sequences suggests that this avian and reptilian crystallin family has been conserved during evolution, as have the other crystallin gene families that are expressed in the eye lens.     

Tissue-specific regulation of a chicken delta-crystallin gene in mouse cells: involvement of the 5' end region.

A cloned delta-crystallin gene of the chicken is preferentially expressed in lens cells after introduction into various mouse tissues. The level of expression in the lens epithelium is 20 times higher than in fibroblasts. Taking advantage of this system, we attempted to define regulatory regions of the delta-crystallin gene using a variety of deletion and substitution mutants. The results indicate that tissue-specific regulation of the delta-crystallin gene is mediated by the 5' end region of the gene; sequences upstream from -93 are not required for expression and sequences downstream from +58 are not involved in tissue specificity. The high expression in lens cells requires 5' flanking sequences of 80-bp long from the cap site, whereas the low expression in fibroblasts requires an additional 12 bp upstream sequence. Expression of both types is lost in a mutant with only 51 bp of the 5' flanking sequence. Thus, fine deletion analysis demonstrated that expression in lens cells and expression in fibroblasts are distinct not only in level but in regulation.     

Complete amino acid sequence of human cartilage link protein (CRTL1) deduced from cDNA clones and chromosomal assignment of the gene

Little is known about the primary amino acid structure of human cartilage link protein (CRTL1). We screened a human genomic library with a cDNA encoding the 3' untranslated region and the adjoining B1 domain of chicken link protein. One clone was isolated and characterized. A 3.5-kb EcoRI-KpnI fragment from this genomic clone that contains the human B1 exon was used to map the gene to chromosome 5q13----q14.1. The same fragment was used to screen a cDNA library prepared from mRNA of Caco-2, a human colon tumor cell line. Two overlapping clones were isolated and shown to encode all of CRTL1. The deduced amino acid sequence is 354 residues long. The amino acid sequence shows a striking degree of identity to the porcine (96%), rat (96%), and chicken (85%) link protein sequences. Furthermore, there is greater than 86% homology between the 3' untranslated region of the genes encoding human and porcine link proteins. These results indicate that there has been strong evolutionary pressure against changes in the coding and 3' untranslated regions of the gene encoding cartilage link protein.     

Expression of duck lens delta-crystallin cDNAs in yeast and bacterial hosts. Delta 2-crystallin is an active argininosuccinate lyase.

The major soluble protein in the lenses of most birds and reptiles is delta-crystallin. In chickens and ducks the delta-crystallin gene has duplicated, and in the duck both genes contribute to the protein in the lens, while in the chicken lens there is a great preponderance of the delta 1 gene product. Purified delta-crystallin has previously been shown to possess the enzymatic activity of argininosuccinate lyase. In order to determine the enzymatic properties of the two duck delta-crystallins their corresponding cDNA molecules were placed in yeast and bacterial expression plasmids. In Saccharomyces cerevisiae, the activity of each crystallin was assessed by transformation of the expression plasmids into a strain deficient for argininosuccinate lyase activity. The ability of the resulting yeast to grow on arginine deficient medium was used as a measure of enzymatic activity. Yeast expressing the duck delta 2-crystallin protein grew rapidly, while those expressing delta 1-crystallin failed to grow. Enzyme activity measurements confirmed the presence of activity in the delta 2-crystallin-expressing yeast, and no detectable activity could be demonstrated in the delta 1-crystallin-expressing yeast. Northern blotting of RNA from the transformed yeast revealed equal levels of mRNA species from the two constructs. For further analysis, the delta 2-crystallin cDNA was placed in the bacterial expression plasmid, pET-3d. The delta 2-crystallin protein produced in Escherichia coli was purified to homogeneity and analyzed to determine the kinetic properties. A Km of 0.35 mM was determined for argininosuccinate and a Vm of 3.5 mumols/min/mg was determined. These data demonstrate that, following duplication of the primordial argininosuccinate lyase gene, one of the genes maintained its role as an enzyme (delta 2-crystallin) while also serving as a crystallin and the other has evolved to specialize as a structural protein in the lens (delta 1-crystallin), presumably losing most or all of its catalytic capacity.     

Structural and functional similarities of delta-crystallin messenger ribonucleic acids from duck and chicken lenses

delta-Crystallin of the embryonic duck lens was compared with that of the embryonic chicken lens with respect to polypeptide composition, synthesis, and messenger ribonucleic acid (mRNA) sequences. Labeling experiments with [35S]methionine revealed that the duck delta-crystallin is composed of minor amounts of polypeptides with molecular weights near 50000 (50K) and 49000 (49K) and much greater amounts of polypeptides with molecular weights near 48000 (48K) and 47000 (47K), as judged by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. All four sizes of polypeptides were synthesized in similar relative proportions as found in vivo in a rabbit reticulocytes lysate supplemented with delta-crystallin mRNA isolated from the embryonic duck lens. Synthesis of the 48K and 47K delta-crystallin polypeptides was differentially reduced in duck lenses cultured in the presence of ouabain. This is similar to the differential reduction of synthesis of the lower molecular weight delta-crystallin peptides in embryonic chicken lenses demonstrated previously. R loops formed between duck or chicken delta-crystallin mRNA and a cloned chicken delta-crystallin cDNA and heteroduplexes formed between duck or chicken delta-crystallin mRNA and cloned chicken genomic DNAs containing delta-crystallin sequences showed that, except for the putative 5' leader sequence, the duck and chicken delta-crystallin mRNAs have extremely similar nucleotide sequences. These data indicate considerable conservation of delta-crystallin throughout the approximately 100 million years of divergence between ducks and chickens. The findings also suggest a possible relationship between the structure of delta-crystallin mRNA and the differential reduction in synthesis of the lower molecular weight delta-crystallin polypeptides in ouabain-treated lenses of ducks and chickens.     

Differentiation-dependent expression of the chicken delta-crystallin gene introduced into mouse teratocarcinoma stem cells.

PCC3 mouse teratocarcinoma (TCC) stem cells were cotransfected with either the plasmid p delta C-1A or p delta C-1B carrying the chicken delta-crystallin gene, and with the plasmid pSV2gpt containing the selectable bacterial xanthine-guanine phosphoribosyltransferase (XGPRT) gene, using the calcium phosphate technique. Nine transformed PCC3 stem cell lines, each of which was clonally derived from respective colonies surviving after the selection process, were isolated. Southern blot analysis revealed that all of them stably maintained delta-crystallin sequences associated with high mol. wt. cellular DNA after propagation in non-selective medium in vitro, and after the production of solid tumors in the syngenic host mice. Six cell lines contain the intact delta-crystallin gene sequence and eight contain the gpt sequence. The number of delta-crystallin DNA copies was highly variable among transformed lines, 1-500 delta-crystallin genes per diploid mouse genome. No expression of the exogenous genes was detected in the transformed cells as long as they were in the undifferentiated state. However, the synthesis of delta-crystallin in certain types of cells was detected immunohistologically in three lines after the differentiation. The positive cell types were unique to each line, skeletal muscle in Y delta-9, certain columnar epithelia in Y delta-2, and unidentified spindle-shaped cells in Y delta-3. Authentic delta-crystallin polypeptides with a mol. wt. of 48 000 are synthesized upon differentiation of line Y delta-3 in solid tumors in syngenic mice.