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.     

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.     

Chicken lens delta-crystallin gene expression and methylation in several non-lens tissues.

RNA sequences coding for the most abundant chicken lens proteins, delta-crystallin, were detected at very low levels in day old post hatch chick lung, heart, kidney and liver, and in 6 day embryo headless bodies. The pattern of cytosine methylation within the CCGG sequences of the delta-crystallin genes was also examined and shown to vary in several non-lens tissues, from several stages of development. Embryonic neural retina, which expresses a higher level of delta-crystallin RNA than the above tissues, is no less methylated in the sites studied than the tissues which have no association with the eye, and is actually more heavily methylated than the kidney. Thus no obvious correlation was found between undermethylation and gene expression.     

Cytoplasmic RNA sequences complementary to cloned chick delta-crystallin cDNA show size heterogeneity.

Double-stranded complementary DNA (cDNA) sequences were prepared from day-old chick lens total polysomal RNA and inserted into the unique PstI restriction site of the plasmid pBR322. Colonies containing sequences complementary to abundant lens poly(A)-containing RNA sequences were identified by using lens 32P-labelled cDNA. Some of these clones have been characterized as containing delta-crystallin mRNA coding sequences by genomic DNA blot hybridization and RNA blot hybridizations. Hybridization of labelled DNA from such clones to RNA blots detected four size classes of delta-crystallin RNA sequences, although Southern blots indicated that there are probably only two delta-crystallin genes.     

The pattern of DNA methylation in the delta-crystallin genes in transdifferentiating neural retina cultures

Terminally differentiated lens fibre cells are formed in the vertebrate lens throughout life. Lens fibre cells may also be obtained by an in vitro process termed transdifferentiation, from certain tissues of different developmental origin from lens, such as embryo neural retina. delta-Crystallin is the major protein in the chick embryo lens fibre cells, and also in transdifferentiated lens cells obtained from cultured embryonic neural retina. Lens crystallin proteins and mRNA are present at low levels in the intact embryonic neural retina but are no longer detectable in the early stages of neural retina cell culture. However, levels rise steeply in the later stages and crystallins become the major products in terminally transdifferentiating neural retina cultures. We have used this system to test the hypothesis that the patterns of DNA methylation in particular genes are correlated with gene expression. A number of developmentally regulated genes have been found to be undermethylated in tissues where they are expressed, and methylated in tissues where they are not. However this correspondence does not always hold true. Eight-day-old embryonic neural retina was cultured for the period of time during which crystallin gene expression increases 100-fold. DNA methylation in the delta-crystallin gene region was analysed at several stages of cell culture by using the restriction endonucleases HpaII and MspI which cleave at the sequence CCGG. The former enzyme cannot cleave internally methylated cytosine (CmCGG) while the latter cannot cleave externally methylated cytosine (mCCGG). We detect no change in the methylation of CCGG sites within the delta-crystallin gene regions during transdifferentiation. Since dramatic changes in delta-crystallin gene expression occur during this process we conclude that large scale alterations in the pattern of DNA methylation are not a necessary accompaniment to changes in gene activity.     

Reliability of delta-crystallin as a marker for studies of chick lens induction

Induction of a lens by the optic vesicle of the brain was the first demonstration of how tissue interactions could influence cell fate during development. However, recent work with amphibians has shown that the optic vesicle is not the primary inducer of lens formation. Rather, an earlier interaction between anterior neural plate and presumptive lens ectoderm appears to direct lens formation. One problem with many early experiments was the absence of an unambiguous assay for lens formation. Before being able to test whether the revised model of lens induction applies to chicken embryos, we examined the suitability of using delta-crystallin as a marker of lens formation. Although delta-crystallin is the major protein synthesized in the chick lens, one or both of the two delta-crystallin genes found in chickens is transcribed in many non-lens tissues as well. In studies of lens formation where appearance of the delta-crystallin protein is used as a positive assay, synthesis of delta-crystallin outside of the lens could make experiments difficult to interpret. Therefore, polyacrylamide gel electrophoresis, immunoblotting, and immunofluorescence were used to determine whether the delta-crystallin messenger RNA detected in non-lens tissues is translated into protein, as it is in the lens. On Coomassie-blue-stained gels of several tissues from stage-22 embryos, a prominent protein was observed that co-migrated with delta-crystallin. However, on immunoblots, none of the non-lens tissues tested contained detectable levels of delta-crystallin at this stage. By imunofluorescence, delta-crystallin was observed in Rathke's pouch and in a large area of oral ectoderm near Rathke's pouch, yet none of the cells in these non-lens tissues showed the typical elongated morphology of lens fiber cells. When presumptive lens ectoderm or other regions of ectoderm from stage-10 embryos were cultured and tested for lens differentiation, both cell elongation and delta-crystallin synthesis were observed, or neither were observed. The results suggest that delta-crystallin synthesis and cell elongation together serve as useful criteria for assessing a positive lens response.     

Evidence for the extralenticular expression of members of the β-crystallin gene family in the chick and a comparison with δ-crystallin during differentiation and transdifferentiation

The beta-crystallins are major water soluble proteins of vertebrate lens fibre cells and have previously been regarded as lens-specific proteins: however beta B2-and beta A3/A1-crystallin RNAs are transcribed and beta-crystallin polypeptides are detectable in the developing chick retina. The beta-crystallin RNA is transcribed in a subpopulation of retina cells and the number of transcribing cells and the level of beta-crystallin polypeptides increase during the differentiation of the retina. Several tissues express beta-crystallin polypeptides, but individual tissues are characterised by qualitative and quantitative differences in the beta- and delta-crystallin polypeptides expressed. The expression of beta-crystallins appears to be non-random as defined by tissue distribution, cellular localisation and ontogeny, implying a function for extralenticular beta-crystallins and a complex mechanism for the regulation of their expression.     

Gene conversion and splice-site slippage in the argininosuccinate lyases/delta-crystallins of the duck lens: members of an enzyme superfamily

Argininosuccinate lyase(ASL)/delta-crystallin is a prominent example of an enzyme-crystallin with roles as both a catalyst and a major structural component of the eye lens in birds and reptiles. In chicken it appears that gene duplication and separation of function may have occurred with one gene product acting primarily as a crystallin and one primarily as an enzyme. However, two delta-crystallin-encoding genes are abundantly expressed in the lens of the embryonic duck (Anas platyrhynchos) which has extremely high ASL activity. Here the isolation and sequence analysis of full length cDNA clones for both duck delta-crystallins are described. The two delta-crystallins are highly similar (94% identical in predicted aa sequence), probably as a result of gene conversion. However, the cDNA for duck delta 2-crystallin contains an in-frame insertion of two codons, probably the result of a recent intron boundary slippage. ASL/delta-crystallin belongs to a superfamily of lyases, including fumarases, aspartases and adenylosuccinate lyase which possess some highly conserved blocks of aa sequence. There may be some clues to the tertiary structures of these conserved motifs in otherwise unrelated proteins for which three-dimensional structures are known.     

Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic delta-crystallin.

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens. The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of delta-crystallin, since delta-crystallin peptides could be identified in tryptic peptide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable delta-crystallin peptides, although it is possible that modified delta-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added delta-[35S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of delta-crystallin in lens membrane preparations.