[Molecular-genetic mechanisms of cornea morphogenesis].

In this paper, we analyzed our own results and published data on the expression of regulatory genes encoding transcription factors Pax6/PAX6, Pitx2/PITX2, Fox1/FOXC1, Prox1/PROX1, Oct4/OCT4, Nanog/NANOG, and TGFß2 signaling protein during morphogenesis of the cornea in vertebrates. We considered the results obtained for the cornea of model animals, primarily mice, and human fetal cornea. The main possibility of establishing common mechanisms of eye development in vertebrates in health and disease is comparative studies of eye morphogenesis of humans and animal models.

Cell phenotypes in human amniotic fluid.

Stem cells capable of long-term proliferation and differentiation into different cell types may be a promising source of cells for regenerative medicine. Recently, much attention has been paid to fetal stem cells, among which are cells from amniotic fluid (AF). We have isolated amniotic stem cells from 3 AF samples. Flow cytometry, RT -PCR and immunohistochemistry have shown that these cells express mesenchymal (CD90, CD73, CD105, CD13, CD29, CD44, and CD146), neural (≤3-tubulin, Nestin, and Pax6), epithelial (keratin 19 and p63) markers and also markers of pluripotency (Oct4, Nanog, and Rex-1). Transplantation of the cells to nude mice does not lead to tumor formation. Thus, putative stem/progenitor cells from AF are capable of long-term proliferation in vitro and the profile of gene expression led us to speculate that they have greater differentiation potential than mesenchymal stem cells and may be useful for cell therapy.

O-Crystallin, arginine kinase and ferritin from the octopus lens.

Three proteins have been identified in the eye lens of the octopus, Octopus dofleini. A 22 kDa protein comprising 3-5% of the soluble protein of the lens is 35-43% identical to a family of phosphatidylethanolamine-binding proteins of vertebrates. Other members of this family include the immunodominant antigen of the filarial parasite, Onchocerca volvulus, putative odorant-binding proteins of Drosophila and a protein with unknown function of Caenorhabditis elegans. We have called this protein O-crystallin on the basis of its abundance in the transparent lens. O-Crystallin mRNA was detected only in the lens. Two tryptic peptides of another octopus lens protein, less abundant than O-crystallin, showed 80% identity to arginine kinase of invertebrates, a relative of creatine kinase of vertebrates. Finally, ferritin cDNA was isolated as an abundant cDNA from the octopus lens library. Northern blots showed that ferritin mRNA is not lens-specific.

Characterization of the mouse Prox1 gene.

Prox1, a vertebrate homologue of Drosophila prospero, encodes a divergent homeodomain protein. We have isolated and characterized full length mouse Prox1 cDNA and genomic clones. Mouse Prox1 gene mapped to position 106.3 cM from the centromere of Chromosome 1, which is very close to the retinal degeneration mutation, rd3. Although the coding sequence and exon-intron junctions of the Prox1 genes of wild type and rd3 mutant mice are identical, Northern blot analysis indicated that the ratio of the short (2.3 kb) and long (8 kb) forms of Prox1 mRNA is different in RNA isolated from wild type and rd3 retinas. Immunostaining of the eyes from wild type and rd3 animals also revealed differences in the distribution of Prox1 protein in the retina and lens. These data suggest that the rd3 mutation affects expression of the mouse Prox1 gene.

Structure and chromosomal localization of the human homeobox gene Prox 1.

The genomic organization and nucleotide sequence of the human homeobox gene Prox 1 as well as its chromosomal localization have been determined. This gene spans more than 40 kb, consists of at least 5 exons, and encodes an 83-kDa protein. It shows 89% identity with the chicken sequence at the nucleotide level in the coding region, while the human and chicken proteins are 94% identical. Among the embryonic tissues analyzed (lens, brain, lung, liver, and kidney), the human Prox 1 gene is most actively expressed in the developing lens, similar to the expression pattern of the chicken Prox 1 gene. The Prox 1 gene was mapped to human chromosome 1q32.2-q32.3.

Primary structure and lens-specific expression of genes for an intermediate filament protein and a beta-tubulin in cephalopods.

Intermediate filament (IF) protein and tubulin cDNAs of cephalopod eye lenses were cloned and sequenced. The rod regions of the deduced IF proteins of the squid and octopus were more similar (68% identical) than were head (33% identical) and tail (40% identical) regions. The rod sequences were closer to squid neuronal IF protein (39% identical) than to any other known IF protein. There was only 31% identity between the rod regions, 21-30% identity between the head regions and 23-32% identity between the tail regions of the present IF proteins of cephalopods and other invertebrates. The rod regions of the cephalopod IF proteins contained the 6 heptads characteristic of nuclear lamins, consistent with an evolutionary relationship between IF proteins and lamins. The present octopus alpha-tubulin was 93% and beta-tubulin was 87% identical to the corresponding tubulins of insects and vertebrates. SDS-PAGE and peptide sequencing indicated that the order of abundance of the cephalopod lens cytoskeletal proteins was IF proteins, actin and tubulins. Northern blot hybridization revealed a 4 kb mRNA for the octopus IF protein and 2.9 and 7.3 kb mRNAs for the squid IF protein; the alpha-tubulin mRNA was about 1.8 kb in the octopus and squid, and the beta-tubulin mRNA was about 2.8 kb in the octopus. The alpha-tubulin mRNA was present in all tissues examined; by contrast, the present beta-tubulin and IF protein mRNAs appeared specialized for lens expression.

Abundant mRNAs in the squid light organ encode proteins with a high similarity to mammalian peroxidases.

A library derived from mRNA in the bacterial light organ of the squid, Euprymna scolopes, contained an unexpectedly high proportion of cDNAs that encode proteins with approximately 30% similarity to a family of mammalian peroxidases (PO) including myelo-PO, eosinophil PO, and thyroid PO (donor:hydrogen-peroxide oxidoreductase; EC 1.11.1.7). Two nearly full-length cDNAs were determined to encode putative PO of nearly 93 kDa each that are 97% identical in amino acid sequence to each other. Each contains four potential glycosylation sites, and His416, believed to be within the active site of the human PO, is conserved in the putative PO from the squid light organ. The mRNAs for the putative squid PO were approximately 250 times more abundant in the tissue housing the bacterial symbiont than in the ocular lens or mantle and were undetectable in the light organ lens. By analogy with the bacteriocidal function of PO in mammalian neutrophils, the putative squid PO may be important for modulating or limiting the population of bacteria within the light organ. The possibility that the squid light organ contains a high concentration of PO raises the possibility that the light organ lens is under oxidative stress, providing a possible rationale for the recruitment of its aldehyde dehydrogenase-like crystallin.

Aldehyde dehydrogenase-derived omega-crystallins of squid and octopus. Specialization for lens expression.

omega-Crystallin of the octopus lens is related to aldehyde dehydrogenases (ALDH) of vertebrates (Tomarev, S. I., Zinovieva, R. D., and Piatigorsky, J. (1991) J. Biol. Chem. 266, 24226-24231) and ALDH1/eta-crystallin of elephant shrews (Wistow, G., and Kim, H. (1991) J. Mol. Evol. 32, 262-269). Only very low amounts of omega-crystallin are present in the squid lens. Here, we have cloned omega-crystallin cDNAs of the octopus (Octopus dofleini) and squid (Ommastrephes sloani pacificus) lenses. The deduced amino acid sequences of omega-crystallin from these species are 78% identical to each other, 56-58% identical to cytoplasmic ALDH1 and mitochondrial ALDH2 of vertebrates (which are 66-68% identical to each other), and 40% identical to Escherichia coli and spinach ALDHs. These data are consistent with the idea that the ALDH1/ALDH2 gene duplication in vertebrates occurred after divergence of cephalopods from the line giving rise to vertebrates, but before the separation of squid and octopus. Southern blot hybridization indicated that omega-crystallin is encoded by few genes (possibly just one) in octopus and squid. Northern blot hybridization revealed two bands (2.7 and 9.0 kilobases) of omega-crystallin RNA in the octopus lens and one band (4.2 kilobases) in the squid lens; omega-crystallin RNAs were undetectable in numerous non-lens tissues of octopus and squid, suggesting lens-specific expression of this gene(s). Finally, extracts of the octopus lens had no detectable ALDH activity using different substrates, consistent with omega-crystallin having no enzymatic activity. Taken together, our results suggest that omega-crystallin evolved by duplication of an ancestral gene encoding ALDH and subsequently specialized for refraction in the transparent lens while losing ALDH activity and expression in other tissues.

Squid glutathione S-transferase. Relationships with other glutathione S-transferases and S-crystallins of cephalopods.

Glutathione S-transferase (GST, EC 2.5.1.18) was purified from the digestive gland of the squid Ommastrephes sloani pacificus. It had high enzymatic activity for the 1-chloro-2,4-dinitrobenzene substrate and was composed of a major and a minor polypeptide band, both with molecular masses near 25 kDa on SDS-polyacrylamide gels. GST cDNA clones were derived from the digestive gland mRNA. The deduced GSTs of the longest cDNAs (pGST5 and pGST11) containing the entire coding sequence have a molecular mass near 23 kDa. Sequence comparisons showed that the squid GST is 42-44% identical to both squid and octopus S-crystallins (the major proteins of the lens), 32-34% identical to class pi and 29-32% identical to class alpha GSTs of vertebrates, and 19-23% identical to other GSTs of vertebrates and insects. Northern blot hybridization revealed that GST mRNAs were much more abundant in the digestive gland than in the testis, mantle, or lens. Analysis of a squid GST gene indicated that it has an exon-intron structure similar to that of the vertebrate class pi GST gene. An apparently novel repetitive element was identified in the 5'-flanking sequence of the squid GST gene. Our results suggest that multiple duplications of an ancestral GST gene gave rise to a family of enzymatically inactive crystallins specialized for lens refraction and one (or two) active GST enzyme expressed preferentially, but not exclusively, in the digestive gland in squids. This differs from the innovation of refractive function from a metabolic enzyme by increased expression in the lens with minimal or no gene duplication, as occurred among the enzyme-crystallins of vertebrates.

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.

Crystallins of the octopus lens. Recruitment from detoxification enzymes.

The eye lens crystallins of the octopus Octopus dofleini were identified by sequencing abundant proteins and cDNAs. As in squid, the octopus crystallins have subunit molecular masses of 25-30 kDa, are related to mammalian glutathione S-transferases (GST), and are encoded in at least six genes. The coding regions and deduced amino acid sequences of four octopus lens cDNAs are 75-80% identical, while their non-coding regions are entirely different. Deduced amino acid sequences show 52-57% similarity with squid GST-like crystallins, but only 20-25% similarity with mammalian GST. These data suggest that the octopus and squid lens GST-like crystallin gene families expanded after divergence of these species. Northern blot hybridization indicated that the four octopus GST-like crystallin genes examined are lens-specific. Lens extracts showed about 40 times less GST activity using 1-chloro-2,4-dinitrobenzene as substrate than liver extracts of the octopus, indicating that the major GST-like crystallins are specialized for a lens structural role. A prominent 59-kDa crystallin polypeptide, previously observed in octopus but not squid and called omega-crystallin (Chiou, S.-H. (1988) FEBS Lett. 241, 261-264), has been identified as an aldehyde dehydrogenase. Since cytoplasmic aldehyde dehydrogenase is a major protein in elephant shrew lenses (eta-crystallin; Wistow, G., and Kim, H. (1991) J. Mol. Evol. 32, 262-269) the octopus aldehyde dehydrogenase crystallin provides the first example of a similar enzyme-crystallin in vertebrates and invertebrates. The use of detoxification stress proteins (GST and aldehyde dehydrogenase) as cephalopod crystallins indicates a common strategy for recruitment of enzyme-crystallins during the convergent evolution of vertebrate and invertebrate lenses. For historical reasons we propose that the octopus GST-like crystallins, like those of the squid, are called S-crystallins.

Squid major lens polypeptides are homologous to glutathione S-transferases subunits.

The eye lenses of cephalopods and vertebrates evolved relatively recently and by independent routes. They provide a good experimental model for the study of convergent evolution at the protein level. One proposal is that pre-existing proteins were recruited as structural eye lens proteins during evolution. This has been confirmed for the vertebrate eye lens structural proteins, or crystallins, which have been intensively studied. Despite the limited information about cephalopod eye lenses, it has been suggested that glutathione S-transferases (GSTs) are a possible evolutionary ancestor of the squid major lens proteins. Recently, the N-terminal sequence of the squid major lens protein was shown to be 55% homologous with that of the Ya subunit of the rat GST. Here, we demonstrate that the squid major lens polypeptides are encoded by a gene family of at least three members. We characterize two cDNAs corresponding to these genes and show they probably either are GST subunits themselves, or share an evolutionary ancestor with them.

Frog lens beta A1-crystallin: the nucleotide sequence of the cloned cDNA and computer graphics modelling of the three-dimensional structure.

Four recombinant cDNA clones coding for a 23 kDa beta-crystallin polypeptide of the frog (Rana temporaria) were identified in a collection of cloned cDNA and two of them were sequenced. The cDNA present in these clones codes for a polypeptide 198 amino-acid residues in length, which appears to be the frog beta A1-crystallin because of its high homology with the sequences of beta A1-crystallins from other species. Furthermore, the nucleotide sequence coding for the compact folded region of the protein is highly conserved. Virtually no homology was found in the 3' nontranslated regions of the mRNA. The amino-acid sequence of the Rana beta A1-crystallin was used to build a three-dimensional model based on the coordinates of the homologous bovine gamma II. An analysis of the model shows that the surface residues of the beta A1-crystallin (amphibian, mammalian and bird) are more highly conserved than the buried residues. It is suggested that this is related to the oligomeric nature of the lens beta-crystallins.

A novel type of crystallin in the frog eye lens. 35-kDa polypeptide is not homologous to any of the major classes of lens crystallins.

The nucleotide sequence of a cloned DNA coding for the 35-kDa polypeptide of the eye lens of the frog (Rana temporaria) has been determined. The sequence without connectors and poly(A) tract is 889 nucleotides in length and shows no homology with sequences coding for other classes of crystallins: alpha-, beta-, gamma- or delta-crystallins. The sequence contains one reading frame 675 nucleotides in length, an apparently intact 3'-non-translated region with the polyadenylation signal sequence and a poly(A) tract; the 5'-non-translated region is lost along with part of the coding region; this accounts for about 1/4 of the total mRNA length. The secondary structure prediction according to the Ptitsin - Finkelstein method shows the presence of predominantly beta-strands with only a few alpha-helical regions. We conclude that the 35-kDa polypeptide from the frog eye lens belongs to a new class of eye lens crystallins for which we propose the name epsilon-crystallin.

Multiple genes coding for the frog eye lens gamma-crystallins.

Three new recombinant cDNA clones coding for gamma-crystallins have been identified in the frog (Rana temporaria) clonotheque by hybrid-selected translation/immunoprecipitation experiments, in addition to the gamma-1-crystallin clone that was isolated and sequenced previously [ Tomarev et al., Gene 17 (1982) 131-138; FEBS Letters 146 (1982) 315-318]. mRNA species coding for all these gamma-crystallins are about 650 nucleotides in length, but differ in structure, as follows from restriction and sequence analysis of the cloned cDNAs. The conclusion is that the R. temporaria genome contains a family of at least four similar but not identical gamma-crystallin genes. The complete nucleotide sequence has been determined for the cDNA of one of these clones coding for gamma-2-crystallin. It is 69% homologous with that of R. temporaria gamma-1-crystallin and contains four regions of partial internal homology corresponding to the four structural folding units of the gamma-crystallin molecules. An unusual feature of the gamma-2-crystallin amino acid sequence is the high lysine/arginine ratio equal to 1.1, in contrast to 0.05-0.16 for other known gamma-crystallins.

The absence of the long 3'-non-translated region in mRNA coding for eye lens alpha A2-crystallin of the frog (Rana temporaria).

The nucleotide sequence of a cloned cDNA (clone pRt(1)297; GENE (1982) 17, 131) coding for a 18 kDa polypeptide of the frog eye lens has been determined. The sequence, 791 nucleotide in length has only one long open reading frame (447 nucleotides). The derived amino acid sequence in this frame has greater than 90% homology with the region 25-173 of alpha A2-crystallin amino acid sequence from a related frog species Rana pipiens. The 5'-terminal part of mRNA corresponding to the first 24 amino acids of alpha A2-crystallin has been lost in cloning and substituted by an artefactual sequence. The 3'-terminal part appears to be intact as follows from the presence of the universal poly(A) addition site and poly(A) tract. The 3'-nontranslated region present in frog alpha A2-crystallin mRNA (130 nucleotides) is about 4-times shorter than in mammalian alpha A2-crystallin mRNA. Intact alpha A2-crystallin mRNA with a size of about 700 nucleotides as determined by Northern blot hybridization is about twice smaller than corresponding mammalian mRNAs.

Molecular cloning of double-stranded cDNA from the eye lens of the frog Rana temporaria: construction of the cDNA clonotheque and identification of a clone containing the nucleotide sequences of the lambda-crystallin gene.

Poly(A)+ RNA from the lens of the frog Rana temporaria contains three components (1200 +/- 50, 1000 +/- 50, and 900 +/- 50 bp in size) and a more heterogeneous RNA species with a length of 650-750 nucleotides. This RNA was used as a template for the AMV reverse transcriptase and Escherichia coli DNA-polymerase I and the total cDNA obtained was cloned in the PstI site of the pBR322 plasmid vector. Recombinant plasmids corresponding to abundant poly(A)+ RNA classes contain cDNA inserts from less than or equal to 200 to 1200 nucleotides in length. Part of the library (clonotheque) was divided into classes differing in the presence of absence of the restriction sites for BamHI, EcoRI and HindIII restriction endonucleases. The clones belonging to each of the five classes were characterized by the hybridization-translation test. The translation product of mRNA hybridizing with the clone pRT(1)294 has an M4 of about 22 000 and is specifically precipitated by the antiserum to lambda-crystallins of Rana temporaria. The size of the cDNA present in pRT(1)294, equal to 580 +/- 20 bp, is sufficient for coding the greater part of the lambda-crystallin amino acid sequence. On the basis of these data, we conclude that the clone pRT(1)294 codes for one of the frog lambda-crystallins.