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.     

The 23-kDa polypeptide from Common Frog lens: isolation,characteristics and cellular localization

The major water-soluble polypeptide with molecular weight of approximately 23 kDa (the 23-kDa polypeptide) was identified in the lens of common frog Rana temporaria L. According to the gel filtration data, the peptide is a part of an oligomeric protein with molecular weight of more than 300 kDa (alpha-crystallin fraction). A highly pure fraction of the 23-kDa polypeptide was isolated by two-step ion-exchange chromatography and SDS electrophoresis and the specific antibodies were obtained. Immunohistochemistry showed the presence of the 23-kDa polypeptide in the cytoplasm of lens epithelial cells (including its central region) and in the zones neighbouring the plasma membranes in cortical fibers. The 23-kDa polypeptide was not found in the lens central zone (nucleus). It was also present in the retina (in the cells of inner nuclear layers), but not in the other tissues and organs of adult frog. Immunochemical analysis showed that the 23-kDa polypeptide was different from all known crystallins of frogs and other animals (bull, mouse, rat, and chicken). The nature of the 23-kDa polypeptide and the relation of its expression with lens cell differentiation are discussed.     

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.     

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.     

Taxon-specific zeta -crystallin in Japanese tree frog (Hyla japonica) lens

The present study demonstrated that the 38-kDa protein, instead of rho-crystallin (36 kDa), is expressed taxon specifically in the lens of Japanese tree frog (Hyla japonica). The 38-kDa protein was distinguished from rho-crystallin expressed in the lenses of bullfrog (Rana catesbeiana) and European common frog (Rana temporaria) immunochemically. Although the N terminus of the 38-kDa protein was blocked, the analyses of partial amino acid sequences showed that the protein was zeta-crystallin. Analysis of cDNA sequence encoding zeta-crystallin of the tree frog lens demonstrated that the deduced protein consisted of 329 amino acids including initial methionine and having 62.2 and 62.9% identity with zeta-crystallin of camel and guinea pig lenses, respectively. The molecular mass of the deduced structure was calculated to be 35,564 Da. zeta-Crystallin of the tree frog lens exhibited the intrinsic enzymatic activity of quinone reductase (EC, NADPH:quinone oxidoreductase). The crystallin specifically catalyzed the reduction of 9,10-phenanthrenequinone (Km, 42 microm) using NADPH (Km, 60 microm) as a cofactor. The enzymatic activity was inhibited by dicumarol, anti-coagulant drug, with IC50 of 4 microm. On gel filtration chromatography, the crystallin was recovered as 150-kDa molecular mass complex, indicating that the crystallin was homotetramer consisting of 38-kDa subunits. The crystallin gene was expressed specifically in the lens. These results show that taxon-specific crystallins such as zeta- and rho-crystallins may be available for the biochemical discrimination of Hyla- and Rana groups among frogs.     

Comparative study of the crystallin supramolecular structure in the carp, frog, and rat lenses by small-angle roentgen ray scattering

The supramolecular structure of crystallins in intact ocular lenses of carp, frog and rat as well as in the interior (nuclear) and outer (cortical) parts of these lenses was studied by the small-angle X-ray scattering method. The results show that the supramolecular structure of crystallins substantially varies both in lenses of different vertebrate species and in various parts of the same lens. In carp lens and in the cortical part of rat lens, crystallins have an ordered supramolecular structure, as indicated by a small-angle X-ray diffraction maximum in the region of Bragg distances 15-20 nm, whereas in frog lens and in the nuclear part of rat lens, the supramolecular structure of these proteins is disordered. The power-law X-ray scattering by rat lens nucleus may be evidence of fractal structures in the lens. A comparison of these results with literary data indicates that there is no obvious correlation between the type of supramolecular structure of crystallins and their polypeptide composition in lenses of different vertebrate species. The results suggest that the supramolecular ordering (short-range order) of crystallins is not a necessary condition for lens transparency.     

Deletion mutation in an eye lens beta-crystallin. An animal model for inherited cataracts.

The most prevalent proteins in the lens of the eye are called crystallins, and it is thought that aberrant crystallins may cause opacification of lens tissue. The Philly mouse, a strain with an inherited cataract, has an abnormal beta B2-crystallin, the principal beta-crystallin in the mouse. The cDNA that codes for the beta B2-crystallin protein has been cloned and sequenced from both the normal and the cataractous Philly mouse. The normal mouse beta B2 cDNA is 756 nucleotides in length with 618 nucleotides of open reading frame. An in-frame deletion of 12 nucleotides has occurred in the Philly mouse cDNA, which results in the loss of 4 amino acids. The sequence of the mutant beta B2 was analyzed against the reported structure of the normal bovine beta B2-crystallin determined by x-ray crystallography. The region, in which the deletion of the amino acids occurs near the COOH terminus, is essential for the formation of the tertiary structure of the beta B2-crystallin. The loss of these residues could explain the alterations that are seen with the Philly beta B2 protein and may account for the instability of the Philly beta B2 protein. This abnormal beta B2-crystallin may be the cause of the cataract in this animal.     

The structure and expression of a distantly related member of the beta-gamma crystallin super gene family from Xenopus.

In an attempt to understand amphibian crystallin gene regulation, we have isolated and partially characterized several genomic clones which hybridized to the gamma 1 cDNA probe from Rana temporaria. A complete sequence of one of these clones showed slight homology to the mammalian beta and gamma crystallins. The deduced amino acid sequence of the coding region and its alignment as a folding unit indicated that all the topologically equivalent residues involved in maintaining the protein folding pattern are highly conserved. Northern blot analysis of total RNAs derived from several adult tissues, including eye lens, suggested that an RNA of approximately 700 nucleotides long is present in lens, heart, spleen and embryos of later stages of development but not in retina, oocytes and embryos of early developmental stages.     

Lens crystallin changes associated with amphibian metamorphosis: involvement of a beta-crystallin polypeptide

Lens crystallins isolated from the tadpole and frog lenses were compared with regard to the developmental changes of crystallin compositions. The major changes during the process of metamorphosis were (1) the total contents of alpha- and gamma-crystallins decrease from more than 70% to less than 60% and (2) one of the major beta-crystallin polypeptides increases from less than 1% to about 6% and (3) an amphibian-specific rho-crystallin also increases from about 6% to more than 10% of total soluble proteins of the lens. We have characterized the metamorphosis-dependent beta-crystallin polypeptide by peptide mapping and sequence determination of the protease-digested fragments. This polypeptide showed very high sequence homology to that of the major beta Bp-crystallin chain reported for the mammalian lenses. The changes of the relative abundance of various crystallins and the gradually-elevated levels of the expression of this beta Bp-like crystallin in the developing lens during metamorphosis may also have some bearing on the maintenance of lens stability in the adult frog lenses.     

Comparative x-ray diffraction study of the crystalline lens in a number of vertebrates including man

X-ray diffraction method has been applied for comparative investigation of native structure of eye lens proteins (crystallins). X-ray diffraction patterns of the whole lenses and/or their nuclear parts were obtained for man and vertebrate animals. Crystalline lenses of the fishes Acerina cernua and Pelmatochromis kribensis, frog Rana temporaria, bull and man contain crystallins with a very similar secondary and tertiary structure, whereas lenses of chicks and the tortoise Testudo horsfieldi contain mainly crystallins with other structure. The results obtained reveal evolutionary conservatism of crystallin structure in fishes, amphibians and mammals. It was also concluded that there is no correlation between crystallin structure of the lens, elasticity of the latter and accommodation mechanism.     

An unusually long non-coding region in rat lens alpha-crystallin messenger RNA.

Most of the mRNA sequence coding for the alpha A2 chain of the ocular lens protein alpha-crystallin from rat, has been determined by sequencing cloned DNA copies of this mRNA. The 892-base pair cDNA sequence encompasses all but 52 N-terminal amino acids of the alpha A2 chain. It lacks the sequence characteristic for the 22 extra amino acids inserted in the alpha A2 -like chain, named alpha AIns. A stretch of 583 nuceotides, representing more than 50% of the entire mRNA sequence, is located 3' wards of the alpha A2 coding sequence. It contains the characteristic AAUAAA signal involved in poly(A) -addition and represents an unexpectedly long non-coding region. Examination of the total cytoplasmic poly(A) RNA of rat lens by filter-hybridization and subsequent translation of the electrophoretically separated mRNA fractions shows that the alpha A2 chain is encoded by mRNA species which are distinct from the alpha AIns encoding mRNA. No evidence is obtained for an extensive size heterogeneity in the 3' untranslated regions of these two different rat lens mRNAs.     

The complete nucleotide sequence of RNa1 of barley mild mosaic virus (asl1) and attempts at bacterial expression of the P3 protein

The complete nucleotide sequence of RNA1 of an Aschersleben isolate of barley mild mosaic virus (BaMMV) was determined. It consists of 7263 nucleotides (nt) excluding the 3' poly (A) tail. The 5' and 3' nontranslated regions (NTR) are 148 and 338 nt in length, respectively, and flank a single large open reading frame coding for a precursor polypeptide with a calculated molecular mass of 256 kDa. Sequence comparison revealed a 96% amino acid (aa) identity to RNA1 translation products of Japanese and French BaMMV isolates. Conserved nucleotide motifs in the 3' sense and 5' complementary sense NTR of the two genomic RNAs were identified that may represent the polymerase recognition sites. A range of constructs containing various parts of the coding region of the P3 nonstructural protein was prepared for expression in Escherichia coli . A short stretch of 35 aa in the C-proximal region of P3 appeared to be highly toxic to the bacterium.     

Rabbit C-reactive protein. Biosynthesis and characterization of cDNA clones

To study the biosynthesis of rabbit C-reactive protein (CRP), a cDNA library was constructed from CRP mRNA-enriched polysomal poly(A) RNA. Four recombinant plasmids, designated pCX9, pCX23, pCX28, and pCX39, from 39 positive clones were sequenced and found to represent overlapping clones. DNA sequencing of CRP cDNA and primer extension of the 5'-end of CRP mRNA have demonstrated that the complete length of rabbit CRP mRNA consists of 2331 nucleotides and a terminal poly(A) segment. Analysis of the resulting sequence indicated that rabbit CRP mRNA contained a 5'-noncoding region of 107 nucleotides, a leader sequence encoding 20 amino acids, a coding region covering 205 amino acids, and a 3'-noncoding region of 1549 nucleotides. The 3'-noncoding region contained a consensus AAUAAA sequence that is 105 nucleotides upstream from the 3'-terminal poly(A) segment. Using an in vitro translation system, we have confirmed that CRP is synthesized as a precursor polypeptide (Mr approximately equal to 26,000) which undergoes processing to form the mature polypeptide (Mr approximately equal to 23,500). The CRP precursor failed to display a calcium-dependent affinity for phosphorylcholine ligand as demonstrated by mature CRP, suggesting that the phosphorylcholine-binding site of CRP only formed after processing. Northern blot analysis suggested that following induction with turpentine, liver was the only site where CRP mRNA synthesis could be demonstrated and that the change in mRNA concentration correlated with the course of CRP production. Southern blot analysis of liver genomic DNA indicated a single gene copy for CRP.     

Isolation and characterization of a cDNA encoding the KS1/4 epithelial carcinoma marker

The mAb KS1/4 recognizes a novel cell surface glycoprotein on a variety of epithelial carcinomas which may be a useful target Ag for antibody-directed diagnostic and therapeutic approaches. Here we report the isolation and characterization of a full length cDNA clone coding for the KS1/4 Ag, as well as, physical and biochemical studies on the antigen derived from an adenocarcinoma of the lung cell line. Affinity purification of the KS1/4 Ag reveals three glycosylated species by NaDodSO4 PAGE with molecular weights of 42, 40, and 35 kDa. The 42- and 40-kDa species are similar at the protein level, differing by their degree of glycosylation, and the 35-kDa protein results from a dibasic proteolytic cleavage of the larger m.w. species. Although both the 42- and 40-kDa forms are found on the cell surface, the 40-kDa protein appears to be the predominant species. A cDNA clone containing the complete KS1/4 coding sequence and the 5'- and 3'-non-translated regions was isolated from a library constructed from the human adenocarcinoma of the lung derived cell line, UCLA-P3. The cDNA clone contains an open reading frame of 314 amino acids which includes a putative signal sequence of 23 amino acids. Northern blot analysis shows a single RNA species of 1.5-kb. Sequence analysis of the 5' and 3' noncoding regions of the KS1/4 cDNA revealed homologies to known proto-oncogenes and inflammatory mediators.     

Evolution of eye lens crystallins: the stress connection

Crystallins, the structural proteins of the eye lens, ensure the transparency and integrity of the lens throughout life. Recent sequence comparisons have shown that evolution has recruited crystallins among already existing heat-shock proteins and stress-inducible enzymes.     

Purification and characterization of rho-crystallin from Japanese common bullfrog lens

In a previous paper, we reported that the partial amino acid sequence (225 residues) from the COOH terminus of rho-crystallin from European common frog lens shows 77% similarity to that of prostaglandin (PG) F synthetase, an aldo-keto reductase, from bovine lung (Watanabe, K., Fujii, Y., Nakayama, K., Ohkubo, H., Kuramitsu, S., Kagamiyama, H., Nakanishi, S., and Hayaishi, O. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 11-15). Here rho-crystallin was purified to apparent homogeneity from the eye lens of the Japanese common bullfrog (Rana catesbeiana) by four sequential chromatographies using Sephadex G-100, Red Sepharose, and dual Mono S. Two types of rho-crystallin, RHO-I and RHO-II, named according to their elution order from a Mono S column, are essentially identical in terms of immunochemical properties, amino acid composition, and partial amino acid sequence. But the NH2-terminal Thr of RHO-I is blocked with an acyl group, while that of RHO-II is free. Both crystallins as well as PGF synthetase are monomeric proteins with a molecular weight of about 35,000 and they have the ability to bind NADPH with a stoichiometry of 0.75 mol of cofactor/mol of protein. Although rho-crystallin does not cross-react with antibody against PGF synthetase, the NH2-terminal amino acid sequence (107 residues) of rho-crystallin shows 77% similarity to that of the enzyme. However, PGD2, PGE2, 9,10-phenanthrenequinone, p-nitrobenzaldehyde, DL-glyceraldehyde, D-glucuronic acid, D-glucose, D-xylose, menadione, p-nitroacetophenone, dihydroxyacetone, succinic semialdehyde, phenylglyoxal, and testosterone were not substrates for these crystallins. PGH2 9,11-endoperoxide reductase activities of RHO-I and RHO-II were 1.3 and 1.0 milliunits/mg of protein, respectively, which are only about 2% of that of bovine lung PGF synthetase. These results indicate that the rho-crystallins RHO-I and RHO-II belong to a group of aldo-keto reductases based on primary structure, molecular properties, and NADPH-binding ability, but show only low PGH2 9,11-endoperoxide reductase activity.     

A novel crystallin from octopus lens

Lens crystallins were isolated from cephalopods, octopus and squid. Two protein fractions were obtained from the octopus in contrast to only one crystallin from the squid. The native molecular mass for these purified fractions and their polypeptide compositions were determined by gel filtration, sedimentation analysis, and SDS-gel electrophoresis. Octopod and decapod lenses share one common major squid-type crystallin of 29 kDa, with one additional novel crystallin present only in the octopus lens. This newly-characterized crystallin (termed omega-crystallin) exists as a tetrameric protein of 230 kDa, consisting of 4 identical subunits of approx. 59 kDa. It is distinct from the previously known crystallins both in amino acid composition and subunit structure. N-terminal sequence analysis indicated that the omega-crystallin is N-terminally blocked, whereas the major octopus crystallin is identical to the reported squid crystallin with regard to the first 25 residues of protein sequence. Sequence similarity between this major cephalopod crystallin and glutathione S-transferase were found, which suggested some enzymatic role of crystallins inside the cephalopod lens.     

A complete complementary DNA for the oncodevelopmental calcium-binding protein, oncomodulin

RNA from a rat liver tumor (Morris hepatoma 5123tc) was used to construct cDNAs together comprising the complete coding sequence of rat oncomodulin mRNA. Information obtained from these cDNAs as well as from primer extension analysis gave a deduced length for the complete oncomodulin mRNA of approximately 680 nucleotides (excluding the poly(A) tail) including a 5'-untranslated region of 97 +/- 2 nucleotides, a 324-nucleotide-coding sequence and a 259-nucleotide 3'-noncoding region. Comparison of the oncomodulin cDNA sequence with those coding for other members of the calcium-binding protein family shows little homology with the exception of a recently reported parvalbumin cDNA where the oncomodulin and parvalbumin nucleotide sequences are 59% identical in the protein-coding region. RNA blot analysis of poly(A+) RNA from normal adult rat liver gave no evidence of oncomodulin expression in this tissue. A single RNA species was detected, however, in RNA extracts from the hepatoma and from rat and human placentas. A probe prepared from one of the rat oncomodulin cDNAs hybridized with a single DNA species in restriction digests of hepatoma and normal DNA from rat and sequences in DNA of humans and other mammals. A 38-nucleotide sequence spanning the 5'-untranslated region and the first seven codons of the oncomodulin cDNA, was far less homologous than was the same region of a parvalbumin cDNA, to a chicken calmodulin cDNA sequence coding for the first calcium-binding domain. The oncomodulin gene appears to have diverged more from that of calmodulin than has the parvalbumin gene.     

Evolution of crystallins for a role in the vertebrate eye lens

The camera eye lens of vertebrates is a classic example of the re‐engineering of existing protein components to fashion a new device. The bulk of the lens is formed from proteins belonging to two superfamilies, the α ‐crystallins and the β γ ‐crystallins. Tracing their ancestry may throw light on the origin of the optics of the lens. The α ‐crystallins belong to the ubiquitous small heat shock proteins family that plays a protective role in cellular homeostasis. They form enormous polydisperse oligomers that challenge modern biophysical methods to uncover the molecular basis of their assembly structure and chaperone‐like protein binding function. It is argued that a molecular phenotype of a dynamic assembly suits a chaperone function as well as a structural role in the eye lens where the constraint of preventing protein condensation is paramount. The main cellular partners of α ‐crystallins, the β ‐ and γ ‐crystallins, have largely been lost from the animal kingdom but the superfamily is hugely expanded in the vertebrate eye lens. Their structures show how a simple Greek key motif can evolve rapidly to form a complex array of monomers and oligomers. Apart from remaining transparent, a major role of the partnership of α ‐crystallins with β ‐ and γ ‐crystallins in the lens is to form a refractive index gradient. Here, we show some of the structural and genetic features of these two protein superfamilies that enable the rapid creation of different assembly states, to match the rapidly changing optical needs among the various vertebrates.