Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

alpha A-crystallin is an abundant soluble protein of the vertebrate eye lens. In addition to the TATA box, four positive cis-regulatory elements of the chicken alpha A-crystallin gene have been identified by linker scanning mutagenesis, DNase I footprinting, and gel mobility shift experiments. The regulatory elements described here have been named DE2A (at positions -144 to -134), DE2B (at positions -128 to -118), and DE1A (at positions -114 to -103). DE2A and DE2B form a dyad of symmetry between positions -141 and -118 (5'-AGACTGTCAT....AGGTCAGTCT-3'), consistent with the close similarity in the mobility of complexes formed with lens nuclear proteins by these two elements. Mutations in DE2A, DE2B, and DE1A leading to loss of promoter activity using the bacterial chloramphenicol acetyltransferase reporter gene transfected into primary embryonic chicken lens epithelial cells resulted in a corresponding loss in the ability to compete for complex formation with lens nuclear proteins in gel mobility shift assays. Mutation of the alpha A-CRYBP1-like site (-67/-57), necessary for function of the mouse alpha A-crystallin promoter, did not affect the activity of the chicken promoter. The DNase I footprinting and gel mobility shift experiments confirmed the previously noted binding of nuclear proteins to a dyad of symmetry at positions -153 to -140. In contrast to DE2A, DE2B, and DE1A, mutagenesis and gel mobility shift experiments failed to correlate function and protein binding for the -153/-140 dyad. DE2A, DE2B, and DE1A agree well with the regulatory elements alpha CE1 (-162/-134), alpha CE3 (-135/-121), and alpha CE2 (-119/-99) (Matsuo, I., and Yasuda, K. (1992) Nucleic Acids Res. 20, 3701-3712) for this gene. The present results suggest, however, that the lens enhancer activity of alpha CE1 is due to the sequence -141/-134, which forms the upper half of the DE2A/DE2B dyad of symmetry, rather than the -153/-140 dyad as previously suspected.
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PMID:Functional elements DE2A, DE2B, and DE1A and the TATA box are required for activity of the chicken alpha A-crystallin gene in transfected lens epithelial cells. 845 50

Beta A3/A1-crystallin is one of the major refractive proteins of the chicken eye lens. Previously we showed that a fragment from -382 to +22 bp of the beta A3/A1-crystallin gene functions as a promoter in transfected lens cells. Here we show by use of the bacterial chloramphenicol acetyltransferase reporter gene that the -143/+22 fragment is sufficient for lens-specific promoter activity in transgenic mice. DNase I footprinting shows that lens nuclear proteins protect several regions within the minimal promoter fragment.
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PMID:Lens-specific expression of a chicken beta A3/A1-crystallin promoter fragment in transgenic mice. 863

We have demonstrated previously that a transgene comprising the -164/+44 fragment of the murine alphaB-crystallin gene fused to the bacterial chloramphenicol acetyltransferase (cat) gene is lens-specific in transgenic mice. The -147 to -118 sequence was identified as a lens-specific regulatory region and is called here LSR1 for lens-specific region 1. In the present experiments, a -115/+44-cat transgene was also lens-specific in transgenic mice, although the average activity was 30 times lower than that derived from the -164/+44-cat transgene. The -115/+44 alphaB-crystallin fragment contains a highly conserved region (-78 to -46) termed here LSR2. A -68/+44-cat transgene, in which LSR2 is truncated, was inactive in transgenic mice. DNase I footprinting indicated that LSR1 and LSR2 bind partially purified nuclear proteins from either alphaTN4-1 lens cells or the mouse lens as well as the purified paired domain of Pax-6. Site-specific mutation of LSR1 eliminated both Pax-6 binding and promoter activity of the -164/+44-cat transgene in transgenic mice. Finally antibody/electrophoretic mobility shift assays and cotransfection experiments indicated that Pax-6 can activate the alphaB-crystallin promoter via LSR1 and LSR2. Our data strengthen the idea that Pax-6 has had a major role in recruiting genes for high expression in the lens.
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PMID:Pax-6 and alphaB-crystallin/small heat shock protein gene regulation in the murine lens. Interaction with the lens-specific regions, LSR1 and LSR2. 879 91

There are two tandemly linked delta-crystallin genes [5' delta 1 -delta 2 3'] in the chicken, with the delta 1-crystallin gene being expressed much more highly (50-100-fold) in the embryonic lens than the delta 2-crystallin gene. Previous transfection experiments have shown that a lens-preferred enhancer exists in the third intron of each chicken delta-crystallin gene. In the present investigation we have used transgenic mice to establish that both the chicken delta 1- and delta 2-crystallin enhancers are preferentially active in the mouse lens in combination with their homologous promoter and the chloramphenicol acetyltransferase (CAT) reporter gene. The promoter/ CAT constructs lacking the enhancers were inactive in the transgenic mice. In one case, a truncated delta 2-crystallin promoter (-308/+24) in combination with the enhancer was also active in the Purkinje cells of the cerebellum of the transgenic mice, which could prove useful in future experiments. Finally, retinoic acid receptors (RAR beta) activated the delta 1-crystallin, but not the delta 2-crystallin enhancer in teh recombinant plasmids in cotransfected embryonic chicken lens epithelial cells treated with retinoic acid. This activation did not occur when using the care enhancer (fragment B4) lacking surrounding flanking sequences (fragment B3 and B5) of the enhancer. Together these experiments show that the chicken delta-crystallin enhancers show lens-preference in transgenic mice despite the absence of delta-crystallin in this species and add retinoic acid nuclear receptors to the growing list of transcription factors (including Pax-6, Sox-2, and delta EF3) that directly or indirectly contribute to the high expression of the delta 1-crystallin gene in the lens.
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PMID:Lens-preferred activity of chicken delta 1- and delta 2-crystallin enhancers in transgenic mice and evidence for retinoic acid-responsive regulation of the delta 1-crystallin gene. 921 65

It has been demonstrated previously that Pax-6, a paired domain (PD)/homeodomain (HD) transcription factor critical for eye development, contributes to the activation of the alphaB-, alphaA-, delta1-, and zeta-crystallin genes in the lens. Here we have examined the possibility that the inverse relationship between the expression of Pax-6 and beta-crystallin genes within the developing chicken lens reflects a negative regulatory role of Pax-6. Cotransfection of a plasmid containing the betaB1-crystallin promoter fused to the chloramphenicol acetyltransferase reporter gene and a plasmid containing the full-length mouse Pax-6 coding sequences into primary embryonic chicken lens epithelial cells or fibroblasts repressed the activity of this promoter by as much as 90%. Pax-6 constructs lacking the C-terminal activation domain repressed betaB1-crystallin promoter activity as effectively as the full-length protein, but the PD alone or Pax-6 (5a), a splice variant with an altered PD affecting its DNA binding specificity, did not. DNase footprinting analysis revealed that truncated Pax-6 (PD+HD) binds to three regions (-183 to -152, -120 to -48, and -30 to +1) of the betaB1-crystallin promoter. Earlier experiments showed that the betaB1-crystallin promoter sequence from -120 to -48 contains a cis element (PL2 at -90 to -76) that stimulates the activity of a heterologous promoter in lens cells but not in fibroblasts. In the present study, we show by electrophoretic mobility shift assay and cotransfection that Pax-6 binds to PL2 and represses its ability to activate promoter activity; moreover, mutation of PL2 eliminated binding by Pax-6. Taken together, our data indicate that Pax-6 (via its PD and HD) represses the betaB1-crystallin promoter by direct interaction with the PL2 element. We thus suggest that the relatively high concentration of Pax-6 contributes to the absence of betaB1-crystallin gene expression in lens epithelial cells and that diminishing amounts of Pax-6 in lens fiber cells during development allow activation of this gene.
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PMID:Dual roles for Pax-6: a transcriptional repressor of lens fiber cell-specific beta-crystallin genes. 971 Jun 41

alpha-Crystallin consists of two subunits, alphaA and alphaB, and each can form an oligomer by itself or with the other. The aggregation arises from interdomain interactions. However, it is not known whether such interactions also exist among alpha-, beta-, and gamma-crystallins. This heterogeneous crystallin interaction is far weaker than the homogeneous crystallin interaction and is difficult to detect by conventional spectroscopic measurements. We used a mammalian two-hybrid system in this study. The major crystallin components, alphaA-, alphaB-, betaB2-, and gammaC-crystallin genes, were subcloned into the DNA binding domain and transcription activation domain vectors of the two-hybrid system, and they were cotransfected along with a chloramphenicol acetyltransferase (CAT) reporter vector into HeLa cells. Chloramphenicol acetyltransferase activity indicated that there were interactions between alphaA- (or alphaB-) and betaB2- or gammaC-crystallins but with an intensity of one-third that of alphaA-alphaB interactions. Hsp27, a member of the family of the small heat-shock proteins, showed a similar interaction property with alphaB-crystallin. Using the N- and C-terminal domain-truncated mutants, we demonstrated that both domains were important in the alphaA-crystallin self-interaction, but that only the C-terminal domain was important in the alphaB-crystallin self-interaction. These results show that the two-hybrid system can detect interactions among various crystallins and may be used in mapping interaction domains.
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PMID:Detection of protein-protein interactions among lens crystallins in a mammalian two-hybrid system assay. 1170 Mar 27


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