Delta-crystallin mRNA Research Articles

Insulin-like Growth Factor I and Insulin Regulate δ-Crystallin Gene Expression in Developing Lens

Abstract Normal development of the chicken embryo requires insulin and insulin receptors. Insulin and also insulin-like growth factor I (IGF-I) can stimulate embryonic growth when applied in vivo at the beginning of organogenesis (Girbau, M., Gomez, J. A., Lesniak, M. A., and De Pablo, F. (1987) Endocrinology 121, 1477-1482). In the present work we chose the developing eye lens, an avascular organ composed of a single cell type, to characterize further the specific effects of insulin and IGF-I upon cell differentiation and gene expression. Epithelial cells (before terminal differentiation) and fiber cells (terminally differentiated) were cultured in the presence of the hormones. delta-Crystallin mRNA steady-state levels as well as nuclear delta-crystallin gene transcription were measured. Either insulin or IGF-I (0.1-10 ng/ml) increased (2-4-fold) delta-crystallin mRNA in epithelial and fiber lens cells from day 6 embryos. The effect of insulin was largely blocked by the Fab fragment of anti-insulin receptor antibody (B-10). By contrast, as it had been shown for metabolic actions in other systems, bivalent B-10 IgG itself mimicked insulin action, i.e. it induced an increase on delta-crystallin mRNA levels. Thus, insulin appears to act through its own receptor in regulating the levels of delta-crystallin mRNA. There was a differential transcriptional component in insulin and IGF-I effects on delta-crystallin gene expression. IGF-I induction of transcription, as measured by nuclear run-on assay, is greater than insulin induction (approximately 2.5-fold versus 1.4-fold) and faster. The delta-crystallin gene will provide the opportunity to analyze the action of insulin and IGF-I on the expression of a structural protein marker of cell differentiation during early embryonic development.

Crystallin gene expression and lentoid body formation in quail embryo neuroretina cultures transformed by the oncogenic retrovirus Mill Hill 2 or Rous sarcoma virus.

The lens-specific proteins alpha and delta crystallins and lentoid bodies, structures that follow a differentiation pathway similar to that of the lens, regularly appear after 4 to 5 weeks in quail embryo neuroretina monolayer cultures. We have investigated the effects of the avian oncogenic retroviruses Mill Hill 2 and Rous sarcoma virus on this process. Quail embryo neuroretina cells transformed by Mill Hill 2 virus were established into permanent cultures that synthesized alpha and delta crystallins and contained stem cells for the production of lentoid bodies. In contrast, transformation with the Rous sarcoma virus mutant tsNY-68 blocked the appearance of mRNA crystallins, but cytoplasmic alpha and delta crystallin mRNA and alpha crystallin appeared 44 h after a shift to the nonpermissive temperature. However, delta crystallins and lentoid bodies were only present after 7 days. The crystallins of transformed quail neuroretina cultures were immunologically indistinguishable from those of quail lenses and of normal quail embryo neuroretina cultures.

Crystallin gene expression and lentoid body formation in quail embryo neuroretina cultures transformed by the oncogenic retrovirus Mill Hill 2 or Rous sarcoma virus.

The lens-specific proteins alpha and delta crystallins and lentoid bodies, structures that follow a differentiation pathway similar to that of the lens, regularly appear after 4 to 5 weeks in quail embryo neuroretina monolayer cultures. We have investigated the effects of the avian oncogenic retroviruses Mill Hill 2 and Rous sarcoma virus on this process. Quail embryo neuroretina cells transformed by Mill Hill 2 virus were established into permanent cultures that synthesized alpha and delta crystallins and contained stem cells for the production of lentoid bodies. In contrast, transformation with the Rous sarcoma virus mutant tsNY-68 blocked the appearance of mRNA crystallins, but cytoplasmic alpha and delta crystallin mRNA and alpha crystallin appeared 44 h after a shift to the nonpermissive temperature. However, delta crystallins and lentoid bodies were only present after 7 days. The crystallins of transformed quail neuroretina cultures were immunologically indistinguishable from those of quail lenses and of normal quail embryo neuroretina cultures.

Alterations in crystallin gene expression during subculture of chick lens cells

We report here on the changes in crystallin gene expression during serial subculture of lens epithelial cells derived from day-old post-hatch chicks. Total cellular RNA from mass cultures were analysed by in vitro cell-free translation and by RNA blot (Northern) hybridization using a cloned delta-crystallin cDNA. Our results indicate that following subculture, lens epithelial cells which still retain the capacity for lens fibre differentiation (lentoid body formation) show a selective loss of delta-crystallin synthesis, and that this is related to the loss of delta-crystallin mRNA. The data suggest that older epithelial-cell populations give rise to lentoid bodies which in terms of crystallin gene expression closely resemble the later-formed cortical fibres of the adult chick lens. Tertiary cultures had an accelerated growth rate, formed no lentoids, contained no translatable alpha- or delta-crystallin mRNAs but still contained translatable beta-crystallin mRNAs.

The expression of chick alpha A2-crystallin RNA during lens development and transdifferentiation.

During development of the vertebrate lens, the lens epithelium undergoes a final stage of differentiation into lens fibre cells, during which the major lens proteins, the crystallins, are synthesised. Lentoids, comprising clusters of lens fibre cells can also be produced by transdifferentiation from certain non-lens tissues, including neural retina and pigmented retinal epithelium. We have isolated an alpha A2-crystallin genomic probe and used it to study the transcription and processing of alpha A2-crystallin mRNA both during lens development and transdifferentiation. We relate these results to earlier measurements in this laboratory of delta-crystallin transcription and alpha- and delta-crystallin protein synthesis, to compare the expression of these two sets of genes. Tissue specific differences in gene expression were found. delta-crystallin mRNA is transcribed before alpha A2-crystallin RNA in the lens, but after it in transdifferentiating neural retina and pigmented epithelia.

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.

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.

Organization of delta-crystallin genes in the chicken.

Double-stranded DNA was synthesized from delta-crystallin mRNA prepared from lens fibers of 15-day-old chick embryos and cloned at the Pst I site of the plasmid pBR322. Using the cloned cDNA and single-stranded cDNA as hybridization probes, a number of genomic DNA fragments containing delta-crystallin gene sequences have been cloned from the partial and complete EcoRI digests of chick brain DNA. One of the clones from the partial digests contains a DNA fragment that consists of four EcoRI fragments of 7.6 kb, 4.0 kb, 2.6 kb, and 0.8 kb. The gene sequences reside in the (5')7.6 kb - 0.8 kb - 4.0 kb (3') fragments. Electron microscopy has provided evidence that the cloned DNA fragment includes the entire gene sequences complementary to delta-crystallin mRNA except for the 3' terminal poly(A) tail, and that the delta-crystallin gene is interrupted by at least 13 intervening sequences. Another clone contains a genomic fragment that consists of two EcoRI fragments of 3.0 kb and 11 kb. The DNA fragment in the latter clone represents a different delta-crystallin gene, as judged by restriction endonuclease mapping and by electron microscopy.

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.

Chicken lens crystallin DNA sequences show at least two delta-crystallin genes.

Restriction analysis of chicken DNA using probes derived from a cloned cDNA and analysis of cloned genomic DNA fragments containing delta-crystallin gene sequences have indicated the presence of at least two non-allelic genes for delta-crystallin, the first and principal crystallin synthesised in the embryonic chicken lens. Electron microscopic analyses of three cloned genomic fragments revealed that the delta-crystallin mRNA gene sequences are interrupted at least 14 times in one of the delta-crystallin genes.

Molecular cloning and partial characterization of delta-crystallin cDNA sequences in a bacterial plasmid.

Double-stranded cDNA synthesized from delta-crystallin mRNA isolated from lens fiber cells of 15-day-old embryonic chicken was cloned in Escherichia coli chi 1776 in the Pst I site of the plasmid pBR322 by using the oligo(dC) . oligo(dG) joining procedure. Twelve Amps Tetr transformants contained sequences complementary to purified delta-crystallin [32P]cDNA. One of the recombinant clones (p delta Cr-2) had an insert of 1241 +/- 240 base pairs, as judged by R-looping analysis with purified delta-crystallin mRNA. The inserted cDNA represents at least 69% of the delta-crystallin coding sequences. p delta Cr-2 was further characterized by restriction analysis, protection of delta-crystallin [3H]cDNA from digestion by S1 nuclease, and hybrid-mediated arrest of delta-crystallin mRNA translation in vitro. p delta Cr-2 provides an invaluable probe for additional analysis of the primary structure, gene organization, and regulated synthesis of delta-crystallin, the principal protein synthesized during lens differentiation in the chicken embryo.

Metabolism of m 7GMP-containing 5′terminal mRNA CAP sequences in extracts of embryonic chick lens cells

A pyrophosphatase activity in 15-day embryonic chick lens cells, previously shown to release m 7GMP from 5′terminal mRNA cap, m 7G(5′)pppG(m), has now been examined for its action on a variety of 5′terminal capped sequences of different lengths. The m 7GpppN-pyrophosphatase activity releases m 7GMP from m 7GMP-containing caps m 7GpppN (N = A, Am), and capped sequences (N = GmpCp, AmpApC and AmpApCpApG) but not from m 7GpppN-terminated delta-crystallin mRNA obtained from 15-day lenses. m 7GMP-release from methylated purine caps is inhibited by cleavage product m 7GMP, but is not significantly inhibited by ADP, GDP, PPi, EDTA, poly(A) or by analogous cap molecules FAD and NAD+. m 7GMP, released from caps by the m 7GpppN-pyrophosphatase, can be converted to ribose-5-phosphate and other compounds by the cryoplasmic preparation.

Quantitation of delta-crystallin messenger RNA during lens induction in chick embryos.

Embryonic chick cells of the presumptive lens ectoderm are induced to differentiate into a lens by the optic vesicle, an outgrowth from the developing brain. This lens induction involves formation of the lens placode by cell elongation between about 44 and 50 hr of development, followed by invagination of the placode between about 50 and 55 hr of development. The amount of delta-crystallin messenger RNA (mRNA) in heads of embryos between 48 and 72 hr of development was determined by molecular hybridization with [3H]DNA complementary to purified delta-crystallin mRNA. An average of approximately 0.5 pg of delta-crystallin mRNA was found per embryonic head at 48 hr of development. This leads to approximately 265 molecules of delta-crystallin mRNA per lens cell, if one assumes that the mRNA is confined to the lens rudiment, as is indicated by immunofluorescence studies of delta-crystallin performed by other investigators. Pulse-labeling experiments with [35S]methionine indicated that the delta-crystallin mRNA is being translated already at this time. delta-Crystallin mRNA accumulated at an average rate of 1 molecule per cell per minute between 48 and 72 hr of development. Thus, by extrapolation, the initiation of delta-crystallin mRNA accumulation coincides with the initiation of lens placode formation at approximately 43-44 hr of development, which is some 8-9 hr after the initiation of lens induction by the optic vesicle. These data suggest that delta-crystallin synthesis is regulated by the accumulation of delta-crystallin mRNA during lens induction in chick embryos.

Reiteration frequency of delta-crystallin DNA in lens and non-lens tissues of chick embryos. delta-Crystallin gene is not amplified during lens cell differentiation.

The differentiation of embryonic chick lens fibers is characterized by an increased rate of synthesis of the lens protein, delta-crystallin, and accumulation of delta-crystallin mRNA. In the present study, the number of delta-crystallin genes in lens and non-lens tissues of embryonic chicks has been determined to test whether the accumulation of delta-crystallin mRNA during lens fiber differentiation is associated with an amplification of delta-crystallin genes. DNA from embryonic chick lens fibers, embryonic chick lens epithelia, or whole chick embryos with the eyes removed, was annealed with [3H]DNA complementary to delta-crystallin mRNA. Analysis of the annealing reactions indicated that the sequences for delta-crystallin are in the unique fraction of the chick genome, and are not amplified in the lens during embryonic development.

Isolation and in vitro translation of delta-crystallin mRNA from embryonic chick lens fibers.

Of the protein synthesized and accumulated during differentiation of embryonic chick lens fibers, 70-80% is the tissue specific protein delta-crystallin. We have isolated and partially characterized the total cytoplasmic mRNA from purified lens fibers of 15-day-old embryos as an initial step toward understanding the regulated expression of delta-crystallin during development. Each lens fiber mass contained an average of 10 mug of cytoplasmic RNA; approximately 0.1 mug per fiber mass was recovered in the mRNA fraction by oligo(dT)-cellulose chromatography. The mRNA electrophoresed primarily as a single peak on a polyacrylamide-agarose gel with an apparent molecular weight of about 9 x 10(5) estimated by comparison with 28S and 18S rRNA markers. Of the protein synthesized in response to the mRNA in cell-free systems derived from Krebs II ascites tumor cells or rabbit reticulocytes, 70-80% comigrated with delta-crystallin on sodium dodecyl sulfatepolyacrylamide-agarose gels. Comparison of the tryptic peptides of delta-crystallin with those of the in vitro products from both heterologous systems established that the lens fiber mRNA contained delta-crystallin mRNA, and that no other functional mRNAs were present in detectable quantities. Thus, the specialization of protein synthesis in embryonic chick lens fibers apparently results from an accumulation of delta-crystallin mRNA in the cytoplasm.