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A B Chepelinsky

Publications and source records attributed to A B Chepelinsky.

At least 37 records · Page 2Linked to original sources
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

The alpha A-crystallin gene: conserved features of the 5'-flanking regions in human, mouse, and chicken.

Approximately 2 kb of 5'-flanking sequences of the lens-specific alpha A-crystallin genes from human and mouse are presented and compared with similar regions of the chicken gene. A repetitive element was found approximately 1 kb upstream from the coding sequences of the alpha A-crystallin gene in all three species (Alu in human, B2 in mouse, and CR1 in chicken), suggesting that they may have an important functional or structural role. Despite the ability of alpha A-crystallin promoters to function across species, dot matrix analyses show only limited similarity among the 600 bp 5' to the structural genes of these three species. The human 5'-flanking sequence is more similar to that of the mouse and chicken than the mouse and chicken are to each other. Numerous short sequences (8-13 bp) are common to all three genes but are distributed differently in each species. The locations and conservation of these sequence motifs suggest functional roles, possibly as cis-regulatory elements of transcription. One motif is similar to the alpha A-CRYBP1 binding site implicated earlier in the transcriptional regulation of the mouse alpha A-crystallin gene, and other motifs correspond to sites previously mapped by methylation interference studies in the mouse alpha A-crystallin promoter. The modular arrangement of conserved sequence motifs is consistent with evolutionary changes occurring at the level of gene regulation.

Animals↗
PubMed1991

Genomic cloning, complete nucleotide sequence, and structure of the human gene encoding the major intrinsic protein (MIP) of the lens.

Major intrinsic protein (MIP, also called MP26) is the predominant fiber cell membrane protein of the ocular lens. MIP has been suggested to play a role in cell-cell communication in the lens. Its expression is tissue-specific and developmentally regulated. We have isolated and characterized the human gene encoding MIP and report here its genomic structure and entire nucleotide sequence. The gene is 3.6 kb, contains four exons separated by introns ranging in size from 0.4 to 1.6 kb, and is present in single copy per haploid human genome. Primer extension of human lens RNA indicates that transcription of the gene initiates from a single site 26 nt downstream from the TATA box. Three complete Alu repetitive elements are found in tandem in the 5'-flanking region of the gene, and a single complete Alu sequence is present in the third intron. The interspecies comparisons of the MIP gene coding sequence and homologies to other members of a putative transmembrane channel protein superfamily are also discussed.

Amino Acid Sequence↗
PubMed1991

Tandem sequence repeats in transmembrane channel proteins.

The flow of ions and small molecules out of and between cells is mediated by various classes of transmembrane proteins. One group of putative channel proteins, including the abundant lens protein MIP, is widely distributed from prokaryotes to vertebrates. This article suggests that these proteins contain a structural twofold repeat and may have arisen by gene duplication. Such a model has implications for the tertiary structures of these important proteins.

Amino Acid Sequence↗
PubMed1991

Lens protein gene expression: alpha-crystallins and MIP.

The crystallin genes encode the major soluble proteins of the lens. Some of the crystallin genes are expressed exclusively in the lens while others are also expressed in different tissues. The two alpha-crystallin genes, alpha A and alpha B, differ in their tissue specificity. Transcription of the alpha A-crystallin gene occurs only in the lens, while the alpha B-crystallin gene is also expressed in other tissues, including heart, skeletal muscle, kidney, lung and brain. MIP (also called MP26), the major intrinsic protein of the lens fiber membranes, is also expressed exclusively in the lens. Correct expression of both alpha-crystallin and MIP are required for normal lens function. Here we review our studies on the molecular basis of expression of the alpha-crystallin and MIP genes in the lens. The 5' flanking sequences containing the initiation site of transcription of the alpha A-crystallin, alpha B-crystallin and MIP genes were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene, and the expression of this reporter gene was studied in transient assays and transgenic mice. DNA sequences flanking the 5' end of the alpha A-crystallin gene contain regulatory elements responsible for the lens-specific expression and developmental regulation of the CAT gene in transgenic mice. Interestingly, although some of the murine alpha A-crystallin regulatory sequences are conserved in the human and chicken genes, different functional regulatory elements appear to control the expression of the murine and chicken alpha A-crystallin genes. The 5' flanking sequence of the alpha B-crystallin gene preferentially directs expression of the CAT gene to the lens and to skeletal muscle. Different regulatory elements of the alpha B-crystallin gene appear to be responsible for its transcription in various tissues. The 5' flanking sequence of the MIP gene also contains regulatory elements that direct expression of the CAT gene to lens cells; these sequences are not functional in transfected non-lens cells and are different from the cis regulatory elements controlling alpha-crystallin gene expression. The multiplicity of cis-regulatory elements controlling the transcription of these three genes indicates the complexity of the mechanisms that regulate gene expression in the lens.

Animals↗
PubMed1990

Regulation of the murine alpha A-crystallin promoter in transgenic mice.

To identify sequences necessary for lens-specific gene expression, lines of transgenic mice were generated which contain murine alpha A-crystallin promoter sequences [-111 to +46 (alpha 111), -88 to +46 (alpha 88), and -34 to +46 (alpha 34)] fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and CAT expression was analyzed. Mice carrying the alpha 111-CAT or the alpha 88-CAT fusion transgene expressed CAT exclusively in lens, except for one line containing alpha 111-CAT, which expressed low levels of CAT in several nonlenticular tissues. Transcription from these promoters in lens initiated at the same site as the endogenous alpha A-crystallin promoter. In one line of mice alpha 88-CAT transgene became active in the lens during embryonic development at approximately the same time that the alpha A-crystallin gene normally begins to be expressed. In contrast, the alpha 34-CAT fusion transgene, containing the TATA box but no sequences further upstream, was inactive in transgenic mice. Our data suggest that 134 bp of sequence (-88 to +46) in the murine alpha A-crystallin gene is sufficient to provide lens specificity, although we cannot rule out the possibility that other sequences also contribute to promoter function.

Animals↗
PubMed1988

Binding of nuclear proteins to promoter elements of the mouse alpha A-crystallin gene.

We have investigated the binding of nuclear proteins from the embryonic chicken lens to synthetic oligonucleotides derived from sequence -111/-55 of the murine alpha A-crystallin gene. These sequences were shown previously to consist of a distal (-111/-88) and a proximal (-88/-60) region which are required for expression of this gene (Chepelinsky, A. B., Sommer, B., and Piatigorsky, J. (1987) Mol. Cell. Biol. 7, 1807-1814). Here we use gel retardation and methylation interference experiments to provide evidence for selective binding of different nuclear proteins to oligonucleotides containing sequences -111/-84, -83/-55, and -111/-55. Similar (although not necessarily identical) proteins were found in nuclear extracts of chicken erythrocytes and HeLa cells. Despite this fact, the alpha A-crystallin promoter (-111/+46) did not function in transfected HeLa cells; moreover, deletion experiments showed that only the TATA box is required for activity of this promoter in a HeLa whole cell extract, the distal (-111/84) and proximal (-83/-55) elements having no positive effect on transcription in the HeLa cell extract. These experiments support the idea that the same or related nuclear proteins found in many tissues are necessary but not sufficient for expression of the murine alpha A-crystallin gene.

Animals↗
PubMed1987

Oncogenesis of the lens in transgenic mice.

Neoplastic tumors of the ocular lens of vertebrates do not naturally occur. Transgenic mice carrying a hybrid gene comprising the murine alpha A-crystallin promoter (-366 to +46) fused to the coding sequence of the SV40 T antigens developed lens tumors, which obliterated the eye cavity and even invaded neighboring tissue, thus establishing that the lens is not refractive to oncogenesis. Large-T antigen was detected early in lens development; it elicited morphological changes and specifically interfered with differentiation of lens fiber cells. Both alpha- and beta-crystallins persisted in many of the lens tumor cells, while gamma-crystallin was selectively reduced. Accessibility, characteristic morphology, and defined protein markers make this transparent epithelial eye tissue a potentially useful system for testing tumorigenicity of oncogenes and for studying malignant transformation from its inception until death of the animal.

Animals↗
PubMed1987

Interaction between two different regulatory elements activates the murine alpha A-crystallin gene promoter in explanted lens epithelia.

Previous experiments have indicated that 5' flanking DNA sequences (nucleotides-366 to +46) are capable of regulating the lens-specific transcription of the murine alpha A-crystallin gene. Here we have analyzed these 5' regulatory sequences by transfecting explanted embryonic chicken lens epithelia with different alpha A-crystallin-CAT (chloramphenicol acetyltransferase) hybrid genes (alpha A-crystallin promoter sequences fused to the bacterial CAT gene in the pSVO-CAT expression vector). The results indicated the presence of a proximal (-88 to +46) and a distal (-111 to -88) domain which must interact for promoter function. Deletion experiments showed that the sequence between -88 and -60 was essential for function of the proximal domain in the explanted epithelia. A synthetic oligonucleotide containing the sequence between -111 and -84 activated the proximal domain when placed in either orientation 57 base pairs upstream from position -88 of the alpha A-crystallin-CAT hybrid gene.

Acetyltransferases↗
PubMed1987

Crystallin genes: lens specificity of the murine alpha A-crystallin gene.

The abundant soluble proteins of the eye lens, the crystallins, are encoded by several gene families which are developmentally regulated in the embryonic lens. We have studied the expression of the murine alpha A-crystallin gene. Transfection experiments using the pSVO-CAT vector and explanted lens epithelia from embryonic chickens demonstrated proximal (-88 to -60) and distal (-111 to -85) regulatory sequences which interact when the alpha A-crystallin promoter is activated in the lens cells. Transgenic mouse experiments showed that the sequence between positions -366 to +46 of the alpha A-crystallin promoter can drive foreign genes selectively in the lens. A fusion gene consisting of this alpha A-crystallin promoter sequence and the T-antigen gene of SV40 produced a lens tumor in transgenic mice. Thus, crystallin promoters provide a useful model for tissue-specific gene expression and permit targeting the expression of foreign genes to a highly differentiated tissue during development.

Animals↗
PubMed1986

Retention of lens specificity in long-term cultures of diploid rabbit lens epithelial cells.

Rabbit lens epithelial cells from newborn animals exhibited limited growth when cultured under standard conditions. Cell lines were generated when explants from individual lenses were cultured in medium supplemented with conditioned medium or untreated rabbit serum. All lines exhibited a stable epithelial morphology. One line, N/N1003A, was examined extensively with respect to its growth, ploidy, and maintenance of lens-specific functions. Cells at population-doubling level (pdl) 120 exhibited a normal chromosomal banding pattern, were diploid, were non-tumorigenic in vivo, did not grow in suspension culture, and did not exhibit sustained growth in medium supplemented with low concentrations of serum. The shape of the growth curves and the final density for cells at pdl 24 and 181 exposed to various concentrations of serum were identical. The cells showed no diminution in growth as a function of in vitro age. The cells retained lens-specific functions. Proteins were isolated from cells at pdl 40 and 170, and were separated on polyacrylamide gels. Western immunoblot analysis using antiserum to alpha-crystallin, a tissue-specific protein found in lens epithelial cells in vivo, indicated the presence of alpha-A- and alpha-B-crystallin polypeptides. The cells also contained the transcription factors required for activating the murine alpha-A-crystallin gene promoter, which is known to function with precise tissue specificity. When an expression vector including the bacterial chloramphenicol acetyltransferase (CAT) gene controlled by the alpha-A-crystallin gene promoter was introduced into the lens epithelial cells, the CAT gene was expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Structural and functional evidence for differential promoter activity of the two linked delta-crystallin genes in the chicken.

There are two delta-crystallin genes (delta 1 and delta 2) in the chicken which are oriented with the same transcriptional polarity (5' delta 1-delta 2-3') and are separated by approximately 4.2 kb of DNA. Existing evidence indicates that delta 1 is very active in the embryonic lens; in contrast, delta 2 appears very inactive, if expressed at all. We have sequenced the 5' regions of delta 1 and delta 2 and tested their ability to promote chloramphenicol acetyltransferase (CAT) activity using the pSVO-CAT expression vector in transfected embryonic lens epithelia. The sequence data establish that the previously reported delta-crystallin cDNAs were derived from mRNAs encoded in delta 1 and not in delta 2. The transfection experiments indicated that the delta 1 promoter is appreciably stronger than the delta 2 promoter. Interestingly, a consensus CCAAT sequence (position - 71) and two consensus viral core-enhancer-like sequences (positions - 308 and +350) are confined to the more active, delta 1 gene. delta 1 and delta 2 have similar TATA boxes (TAAAA) at positions - 28 and - 27, respectively. Exon 1 in delta 1 (35 bp) and in delta 2 (34 bp) are extremely homologous, containing just two mismatches; exon 2 in delta 1 (64 bp) and in delta 2 (51 bp) are only 56% homologous and contain the putative translation initiating codon and three encoded amino acids. Unexpectedly, intron 1 of both genes has numerous pentameric repeats present in the murine immunoglobulin heavy-chain switch regions (GAGCT, GGGGT and GGGCT). Other direct repeats were found in delta 1 and delta 2, and short homologies were noted between the chicken delta-crystallin and murine alpha A-crystallin genes.

Animals↗
PubMed1985

Lens-specific expression and developmental regulation of the bacterial chloramphenicol acetyltransferase gene driven by the murine alpha A-crystallin promoter in transgenic mice.

Two lines of transgenic mice with one to two copies of a DNA fragment containing nucleotides -364 to +45 of the murine alpha A-crystallin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene expressed the CAT gene only in their eye lenses. Both CAT activity and alpha A-crystallin were first detected in eyes at approximately 12.5 days of embryonic development, suggesting that the alpha A-CAT fusion gene and the endogenous alpha A-crystallin gene are co-regulated during lens development in the transgenic mice. These experiments show that the murine alpha A-crystallin gene contains a short, cis-acting, tissue-specific regulatory sequence at its 5' end that can target the expression of the bacterial CAT gene, and probably foreign eukaryotic genes, specifically to the ocular lens.

Acetyltransferases↗
PubMed1985

Lens-specific expression of the chloramphenicol acetyltransferase gene promoted by 5' flanking sequences of the murine alpha A-crystallin gene in explanted chicken lens epithelia.

We have developed a system using explanted embryonic chicken lens epithelia to express foreign recombinant genes containing crystallin DNA regulatory sequences introduced by calcium phosphate transfection. Optimal results were obtained with lens epithelia from 14-day embryos transfected 1 day after explantation and assayed 3 days later. When DNA sequences (-364 to +45) of the murine alpha A-crystallin gene were inserted in the pSVO-CAT expression vector of Gorman et al. [Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051] in the same orientation as in the crystallin gene, they promoted chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) activity in the transfected epithelia. Sequences 87 to 364 base pairs upstream from the murine gene cap site were required for CAT gene expression. These crystallin gene regulatory sequences did not promote CAT expression in primary cultures of embryonic chicken fibroblasts or other nonlens cells. By contrast, the long terminal repeat of Rous sarcoma virus and the early promoter of simian virus 40 promoted CAT activity in lens and nonlens cells. Our experiments thus demonstrate that the explanted embryonic chicken lens epithelium is an advantageous recipient for identifying lens-cell-specific regulatory sequences of crystallin genes and implicate a DNA region upstream of the "TATA box" for regulation of the murine alpha A-crystallin gene. These experiments also suggest that explanted epithelia from other tissues may be useful for studying the expression of foreign genes.

Acetyltransferases↗
PubMed1985

Lens-specific promoter activity of a mouse gamma-crystallin gene.

Crystallins are the major water-soluble proteins in vertebrate eye lenses. These lens-specific proteins are encoded by several gene families, and their expression is differentially regulated during lens cell differentiation. Here we show that a cloned mouse gamma-crystallin promoter is active in lens explants derived from 14-day-old chicken embryos but inactive in a variety of cells of non-lens origin. We also show that sequences required for proper utilization of this promoter are contained between nucleotide positions -392 and +47 relative to the transcription initiation site; deletion of sequences from positions -392 to -171 completely abolishes promoter activity. Since chickens do not have gamma-crystallin genes, the expression of a mouse gamma-crystallin promoter in chicken lens cells suggests that different classes of crystallin genes may be regulated by common lens tissue-specific mechanism(s) independent of species.

Animals↗
PubMed1983

Studying promoters and terminators by gene fusion.

Prokaryotic gene control signals can be isolated, compared, and characterized by precise fusion in vitro to the Escherichia coli galactokinase gene (galK), which provides both a simple assay and genetic selection. This recombinant galK fusion vector system was applied to the study of promoters and terminators recognized by the Escherichia coli RNA polymerase. Three promoters created by mutation from DNA sequences having no promoter function were characterized. Mutations that inactivate promoter function were selected, structurally defined, and functionally analyzed. Similarly, transcription termination was examined, and mutations affecting terminator function were isolated and characterized.

Base Sequence↗