PubMed Health⌕ Search

Biomedical subjects

J Piatigorsky

Publications and source records attributed to J Piatigorsky.

At least 19 recordsLinked to original sources
PubMed1995

The chicken beta A4- and beta B1-crystallin-encoding genes are tightly linked.

Analysis of the 5' flanking region of the chicken beta B1-crystallin-encoding gene (beta B1-cry) revealed regions of sequence homology with the bovine beta A4-crystallin-encoding gene (beta A4-cry). Subsequently, the chicken beta A4-cry cDNA sequence was determined, and it was demonstrated that beta A4- and beta B1-cry are linked head to head in the chicken chromosome with 2147 nucleotides (nt) of intergenic spacer. Chicken beta A4-cry contains six exons, with the first exon being noncoding. Chicken beta A4-cry is the smallest beta-cry ever described, due to the small size of its introns which range in length from 68 to 96 nt. While three polymorphisms were noted between some cDNA clones and the genomic sequence, Southern blot analysis demonstrated that beta A4-cry exists as a single copy in the chicken genome. Northern blot analysis indicated that beta A4-cry is a lens-specific transcript which is expressed at higher levels in the embryo than in the adult. The beta A4-cry mRNA is present at 400-fold lower levels than the beta B1-cry mRNA in the 14-day embryonic chicken lens, and at 2000-fold lower levels than the beta B1-cry mRNA in the adult lens. These results are consistent with the idea that the beta-cry family was once clustered in the chromosome as the gamma-cry family is today, and raises the possibility that the relatively low expression of beta A4-cry is mechanistically linked to the high expression of beta B1-cry in the chicken lens.

Animals↗
PubMed1995

Conversion from oligomers to tetramers enhances autophosphorylation by lens alpha A-crystallin. Specificity between alpha A- and alpha B-crystallin subunits.

Previously we showed that alpha-crystallins are autophosphorylated (Kantorow, M., and Piatigorsky, J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3112-3116). Here we report that addition of 1% deoxycholate converted alpha A-crystallin aggregates into 80-kDa tetramers which were 10-fold more active for autophosphorylation. Circular dichroism (CD) spectra of alpha-crystallin revealed little or no change in secondary and tertiary structures in 1% deoxycholate, alpha A2D, a truncated form of bovine alpha A that exists as a tetramer, was as active for autophosphorylation in the absence of deoxycholate as intact alpha A was in the presence of deoxycholate. At least one serine between amino acids 131 and 145 of bovine alpha A was autophosphorylated in peptide mapping experiments. Chicken alpha A-crystallin, which lacks the Ser-122 cAMP-dependent kinase site of bovine alpha A, was also autophosphorylated in the presence of deoxycholate. In contrast to alpha A-crystallin, autophosphorylation by alpha B-crystallin was not activated by deoxycholate despite its conversion to a tetrameric form, and alpha B was also more efficiently phosphorylated by cAMP-dependent kinase than alpha A. These data suggest metabolic differences between the alpha-crystallin subunits that may be related to specific expression of alpha A in the lens and ubiquitous expression of alpha B in numerous normal and diseased tissues.

Animals↗
PubMed1995

Chromosomal localization of the genes for five zinc finger proteins expressed in mouse lens.

Based on sequence similarity to a consensus zinc finger domain, we have identified cDNAs encoding five proteins containing zinc finger nucleic acid binding motifs from a newborn mouse lens library. Utilizing these cDNAs as hybridization probes, we have mapped two of the corresponding genes to mouse Chr (chromosome) 11, two to mouse Chr 7, and one to mouse Chr 4 using two multilocus crosses. Because the zinc finger proteins encoded by these genes may be involved in regulating other genes that are expressed in lens, they can be considered candidates for the large number of yet unmapped cataract loci.

Animals↗
PubMed1995

Pax-6 and lens-specific transcription of the chicken delta 1-crystallin gene.

The abundance of delta-crystallin in the chicken eye lens provides an advantageous marker for tissue-specific gene expression during cellular differentiation. The lens-specific expression of the delta 1-crystallin gene is governed by an enhancer in the third intron, which binds a positive (delta EF2) and negative (delta EF1) factor in its core region. Here we show by DNase I footprinting, electrophoretic mobility-shift assays, and cotransfection experiments with the delta 1-promoter/enhancer fused to the chloramphenicol acetyltransferase reporter gene that the delta 1-crystallin enhancer has two adjacent functional Pax-6 binding sites. We also demonstrate by DNase I footprinting that the delta EF1 site can bind the transcription factor USF, raising the possibility that USF may cooperate with Pax-6 in activation of the chicken delta 1- and alpha A-crystallin genes. These data, coupled with our recent demonstration that Pax-6 activates the alpha A-crystallin gene, suggest that Pax-6 may have been used extensively throughout evolution to recruit and express crystallin genes in the lens.

Animals↗
PubMed1995

Three-dimensional structure, catalytic properties, and evolution of a sigma class glutathione transferase from squid, a progenitor of the lens S-crystallins of cephalopods.

The glutathione transferase from squid digestive gland is unique in its very high catalytic activity toward 1-chloro-2,4-dinitrobenzene and in its ancestral relationship to the genes encoding the S-crystallins of the lens of cephalopod eye. The three-dimensional structure of this glutathione transferase in complex with the product 1-(S-glutathionyl)-2,4-dinitrobenzene (GSDNB) has been solved by multiple isomorphous replacement techniques at a resolution of 2.4 A. Like the cytosolic enzymes from vertebrates, the squid protein is a dimer. The structure is similar in overall topology to the vertebrate enzymes but has a dimer interface that is unique when compared to all of the vertebrate and invertebrate structures thus far reported. The active site of the enzyme is very open, a fact that appears to correlate with the high turnover number (800 s-1 at pH 6.5) toward 1-chloro-2,4-dinitrobenzene. Both kcat and kcat/KmCDNB exhibit pH dependencies consistent with a pKa for the thiol of enzyme-bound GSH of 6.3. The enzyme is not very efficient at catalyzing the addition of GSH to enones and epoxides. This particular characteristic appears to be due to the lack of an electrophilic residue at position 106, which is often found in other GSH transferases. The F106Y mutant enzyme is much improved in catalyzing these reactions. Comparisons of the primary structure, gene structure, and three-dimensional structure with class alpha, mu, and pi enzymes support placing the squid protein in a separate enzyme class, sigma. The unique dimer interface suggests that the class sigma enzyme diverged from the ancestral precursor prior to the divergence of the precursor gene for the alpha, mu, and pi classes.

Amino Acid Sequence↗
PubMed1995

cDNA encoding a chicken protein (CRP1) with homology to hnRNP type A/B.

The sequence of a cDNA encoding a putative chicken RNA-binding protein is reported. The C-terminal portion of the predicted protein is similar to a family of nucleic acid binding proteins that includes murine CArG box-binding factor CBF-A, human hnRNP A/B, hepatitis B enhancer-binding protein E2BP, and AU-rich RNA-binding protein AUF1. These proteins all have two consecutive RNA recognition motifs. However, the N-terminal 72 amino acids of this deduced chicken protein show no relation to the N-terminal sequences of the other proteins. We call this protein chicken ribonucleoprotein, CRP1.

Amino Acid Sequence↗
PubMed1995

Differential use of the regulatory elements of the alpha B-crystallin enhancer in cultured murine lung (MLg), lens (alpha TN4-1) and muscle (C2C12) cells.

The mouse alpha B-crystallin-encoding gene (alpha B-cry) is highly expressed in the lens and expressed to lesser extents in other tissues. Here, we investigated alpha B-cry expression in mouse-lung-derived MLg cells. Two sizes of MLg alpha B-cry transcripts comigrated with alpha B-cry transcripts contained in total and poly(A)+RNA from mouse lung, with preference for the larger species in the MLg cells. Expression of both alpha B-cry promoter/cat reporter gene constructs and alpha B-cry enhancer (nt -427/-259)/herpes simplex virus (HSV) thymidine kinase promoter (ptk)/human growth hormone reporter gene (hGH) constructs was studied in transfected MLg cells and the results compared with those obtained from alpha TN4-1 lens and C2C12 muscle cells. The alpha B-cry enhancer increased activity of the endogenous and tk promoters approx. 2-fold in the MLg cells, in contrast to its 3-7-fold effect in alpha TN4-1 cells and 17-20-fold effect in C2C12 myotubes. Site-specific mutagenesis of the previously identified enhancer control elements, alpha B-E-1 (nt -407 to -397), alpha BE-2 (-360 to -327) and MRF (-300 to -288), decreased enhancer strength in transfected MLg cells. DNase I footprinting showed that MLg nuclear proteins occupy only alpha BE-1 and alpha BE-2. Previous data have shown that lens cells use alpha BE-1, alpha BE-2 and alpha BE-3, while muscle cells use, in addition, the muscle regulatory factor-binding site (MRF). Thus, the present experiments correlate tissue-specific enhancer strength and the number of control elements utilized.

Animals↗
PubMed1995

Transient expression of glutamate decarboxylase and gamma-amino butyric acid in embryonic lens fibers of the rat.

We have determined the localization and developmental expression of glutamate decarboxylase (GAD67) in the rat lens. Immunofluorescence experiments showed that GAD67 was transiently expressed in the nuclear fiber cells of the lens between embryonic days (E) 15 and 20, with maximal immunostaining occurring on E17 and E18. gamma-amino butyric acid (GABA) co-localized with GAD67 in the embryonic nuclear fiber cells. Reverse transcription-polymerase chain reaction (RT-PCR) tests showed that at least three alternatively spliced forms of GAD67 mRNA, including mRNAs with and without the I80 and the I86 insert, were transiently co-expressed with GAD67 in the embryonic lens. The major GAD67 protein in the lens was 67 kDa. We conclude that enzymatically active GAD67 is transiently expressed in the lens nuclear fiber cells of the embryonic rat. The transient expression is regulated by transcriptional and/or posttranscriptional processes. We speculate on the basis of possible common gene regulatory elements for glutamate and ornithine decarboxylases and the involvement of these enzymes with polyamine synthesis, that the transient expression of GAD67 may be connected to nuclear and/or DNA breakdown during lens fiber cell differentiation.

Animals↗
PubMed1993

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.

Aldehyde Dehydrogenase↗
PubMed1992

Structure and lens expression of the gene encoding chicken beta A3/A1-crystallin.

The beta A1- and beta A3-crystallins are major polypeptides in the lenses of vertebrates. We present evidence that a single beta A3/A1 gene encodes these two proteins in the chicken. The beta A3/A1 gene has been sequenced and its functional promoter identified in transfection experiments. The chicken beta A3/A1 gene has the same structure as the human orthologue: six exons with standard splice sites and two alternative start codons from which the protein products are apparently translated. Northern analysis revealed an abundant 0.9-kb transcript in the lenses of 1-2-day-old chickens and no detectable transcripts in the rest of the eye, brain, heart, kidney, liver or skeletal muscle. The 5'-flanking sequence of the chicken beta A3/A1 gene is very similar to that of the human and mouse genes, suggesting conservation of important putative regulatory sequences in addition to the TATA box. A thymidine-rich element (bp -218 to -163) and a potential AP-1-binding site (bp -264 to -258) are present within the chicken 5'-flanking region. A DNA fragment from -382 to +22 of the chicken beta A3/A1 gene is sufficient to promote expression of the bacterial cat gene in transfected chicken primary lens epithelial cells, but not in transfected dermal fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Taxon-specific recruitment of enzymes as major soluble proteins in the corneal epithelium of three mammals, chicken, and squid.

Studies of others have shown that class 3 aldehyde dehydrogenase is a major component of the epithelial cells of the mammalian cornea. Here we demonstrate by peptide sequencing that other major proteins of the corneal epithelium are also identical or related to enzymes in the human, mouse, kangaroo, chicken, and squid. Aldehyde dehydrogenase class 3 was found to be the major protein of human, mouse, and kangaroo corneal epithelial cells. Peptidyl prolyl cis-trans isomerase (cyclophilin) or a homologue thereof is strikingly abundant in the corneal epithelial cells of chicken, but not mammals, and appears to be absent from the cornea of squid. By contrast, enolase or its homologue is relatively abundant in both the mammalian and chicken corneal epithelial cells. In some instances, abundant enzymes are common to cornea and lens in the same species--for example, arginino-succinate lyase/delta 1-crystallin in the chicken and glutathione S-transferase-like protein in the squid; in other cases, the abundant proteins in the cornea have not been found as lens crystallins in any species--for example, aldehyde dehydrogenase class 3 and cyclophilin. These data suggest that enzymes and certain enzyme-crystallins have been recruited as major corneal proteins in a taxon-specific manner and may serve structural rather than, or as well as, enzymatic roles in corneal epithelial cells.

Amino Acid Sequence↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

Molecular analyses of carbonic anhydrase-II expression and regulation in the developing chicken lens.

The expression of carbonic anhydrase-II (CA-II) in the developing chicken lens was examined and compared with that in the retina of the chicken embryo. CA-II expression was measured by immunohistochemistry and radioimmunoassay during development, and CA-II mRNA was quantified by Northern blot and densitometric scanning and localized by in situ hybridization. A functional promoter of the chicken CA-II gene was identified by transfection of primary embryonic chicken lens epithelial cells and analyzed in deletion mutants. The results establish that CA-II makes up about 0.1% of the total soluble protein of the embryonic chicken lens, an amount insufficient to make it a candidate for an enzyme crystallin in this species. Lens fiber differentiation coincided with a loss of CA-II mRNA and protein; by contrast, CA-II persisted in the epithelial cells of the embryonic and mature lens. This and previous studies showed that CA-II amounts to as much as 3% of the protein of the embryonic chicken retina and follows a different developmental time course of expression; like the lens, CA-II decreases until day 10 in the embryonic retina, but, unlike the lens, it increases thereafter and plateaus at hatching. Progressive deletions of the 5' flanking regions (from position -1314 to +32) of the CA-II gene fused to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene resulted in a gradual loss of promoter activity, consistent with an additive effect of putative cis-regulatory elements found in many crystallin genes. These experiments provide the foundation for a molecular analysis of the developmental and differential regulation of the CA-II gene in lens and retina.

Amino Acid Sequence↗
PubMed1992

Conservation of mouse alpha A-crystallin promoter activity in chicken lens epithelial cells.

Previous transfection experiments have shown that 162 base pairs (bp) of the 5' flanking sequence of the chicken alpha A-crystallin gene are required for promoter activity in primary chicken lens epithelial cells (PLE), while only 111 bp of the 5' flanking sequence are needed for activity of the mouse alpha A-crystallin promoter in transfected chicken PLE cells or in a SV40 T-antigen-transformed transfected mouse lens epithelial cell line (alpha TN4-1). The effect of site-directed mutations covering positions -111 to -34 of the mouse alpha A-crystallin promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) gene was compared in transfected chicken PLE cells and mouse alpha TN4-1 cells; selected mutations were also examined in a nontransformed rabbit lens epithelial cell line (N/N1003A). In general, the same mutations reduced promoter activity in the transfected lens cells from all three species, although differences were noted. The mutations severely affected regions -111/-106 and -69/-40 regions in all the transfected cells examined; by contrast, mutations at positions -105/-99 and -87/-70 had a somewhat greater effect in the chicken PLE than the mouse alpha TN4-1 cells, while mutations of the -93/-88 sequence reduced expression in the alpha TN4-1 but not the PLE cells. A partial cDNA with sequence similarity to alpha A-CRYPB1 of the mouse has been isolated from a chicken lens library; mouse alpha A-CRYBP1 is a putative transcription factor which binds to the -66/-55 sequence of the mouse alpha A-crystallin promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

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.

Aldehyde Dehydrogenase↗