Biomedical subjects

W W Wasserman

Publications and source records attributed to W W Wasserman.

6 recordsLinked to original sources

PubMed1998

Organization of the ABCR gene: analysis of promoter and splice junction sequences.

Mutations in the human ABCR gene have been associated with the autosomal recessive Stargardt disease (STGD), retinitis pigmentosa (RP19), and cone-rod dystrophy (CRD) and have also been found in a fraction of age-related macular degeneration (AMD) patients. The ABCR gene is a member of the ATP-binding cassette (ABC) transporter superfamily and encodes a rod photoreceptor-specific membrane protein. The cytogenetic location of the ABCR gene was refined to 1p22.3-1p22.2. The intron/exon structure was determined for the ABCR gene from overlapping genomic clones. ABCR spans over 100kb and comprises 50 exons. Intron/exon splice site sequences are presented for all exons and analyzed for information content (Ri). Nine splice site sequence variants found in STGD and AMD patients are evaluated as potential mutations. The localization of splice sites reveals a high degree of conservation between other members of the ABC1 subfamily, e.g. the mouse Abc1 gene. Analysis of the 870-bp 5' upstream of the transcription start sequence reveals multiple putative photoreceptor-specific regulatory elements including a novel retina-specific transcription factor binding site. These results will be useful in further mutational screening of the ABCR gene in various retinopathies and for determining the substrate and/or function of this photoreceptor-specific ABC transporter.

ATP-Binding Cassette Transporters↗

PubMed1998

Identification of regulatory regions which confer muscle-specific gene expression.

For many newly sequenced genes, sequence analysis of the putative protein yields no clue on function. It would be beneficial to be able to identify in the genome the regulatory regions that confer temporal and spatial expression patterns for the uncharacterized genes. Additionally, it would be advantageous to identify regulatory regions within genes of known expression pattern without performing the costly and time consuming laboratory studies now required. To achieve these goals, the wealth of case studies performed over the past 15 years will have to be collected into predictive models of expression. Extensive studies of genes expressed in skeletal muscle have identified specific transcription factors which bind to regulatory elements to control gene expression. However, potential binding sites for these factors occur with sufficient frequency that it is rare for a gene to be found without one. Analysis of experimentally determined muscle regulatory sequences indicates that muscle expression requires multiple elements in close proximity. A model is generated with predictive capability for identifying these muscle-specific regulatory modules. Phylogenetic footprinting, the identification of sequences conserved between distantly related species, complements the statistical predictions. Through the use of logistic regression analysis, the model promises to be easily modified to take advantage of the elucidation of additional factors, cooperation rules, and spacing constraints.

Binding Sites↗

PubMed1998

Molecular cloning of a novel mouse gene with predominant muscle and neural expression.

Because numerous diseases affect the muscle and nervous systems, it is important to identify and characterize genes that may play functional roles in these tissues. Sequence analysis of a 106-kb region of human Chromosome (Chr) 19q13.2 revealed a novel gene with homology to the Neuroendocrine-specific protein (NSP), and it has, therefore, been designated NSP-like 1 (Nspl1). We isolated the mouse homolog of this gene and performed extensive expression analysis of both the mouse and human genes. The mouse Nspl1 gene is alternatively spliced to produce two major transcripts: a 2.1-kb mRNA that is expressed at highest levels in the brain, and a 1.2-kb transcript that is primarily expressed in muscle. The larger message contains 10 exons, whereas the smaller transcript contains 7 exons. The last 6 exons, which are present in both transcripts, share significant amino acid sequence identity with the endoplasmic reticulum-bound portion of NSP. Mouse and human Nspl1/NSPL1 genes have expression patterns that are similar to that of the dystrophin gene. In addition, the putative regulatory domains of Nspl1 appear similar in composition and distribution to the defined dystrophin regulatory sequences.

Amino Acid Sequence↗

PubMed1997

CBP/cycA, a CCAAT-binding protein necessary for adhesion-dependent cyclin A transcription, consists of NF-Y and a novel Mr 115,000 subunit.

Cells of most tissues, with the exception of hematopoietic cells, require adhesion to an appropriate surface to grow. Cyclin A is needed for cell cycle progression at the G1-S transition, and appearance of cyclin A mRNA and protein in late G1 has been shown to be dependent on adhesion-initiated signals in normal rat kidney fibroblasts. Previously, we have reported that the adhesion-dependent activation of cyclin A transcription in late G1 is mediated by CBP/cycA (CCAAT-binding protein for cyclin A gene), a novel CCAAT-binding protein. Specific binding of CBP/cycA, a Mr 30,000/40,000/115,000 heterotrimeric protein complex, to the CCAAT element of the cyclin A promoter was detectable in growing but not in G0-arrested or nonadherent normal rat kidney cells. Here, we demonstrate that the Mr 30,000/40,000 subunits of CBP/cycA are identical with NF-YA and NF-YB, the two subunits of NF-Y. In addition, we show that, aside from CBP/cycA, NF-Y itself also binds to the CCAAT element of the cyclin A promoter. But, whereas the binding of CBP/cycA is adhesion and cell cycle dependent and correlates with the expression of cyclin A in late G1 phase, NF-Y itself seems to bind in a cell cycle-independent manner.

Animals↗

PubMed1997

Comprehensive analysis of proteins which interact with the antioxidant responsive element: correlation of ARE-BP-1 with the chemoprotective induction response.

Transcriptional activation of the mouse glutathione S-transferase Ya gene by chemoprotective molecules is mediated through the interaction of trans-acting factors with an antioxidant responsive element (ARE) in the promoter region of this gene. In a step toward identifying those factors which bind productively to the GST Ya ARE, all of the discernible, specific ARE-binding proteins (ARE-BP) in nuclear extracts from HepG2 cells were systematically characterized. By gel-mobility-shift analysis, seven specific ARE-BPs, termed ARE-BP-1 through 7 in order of increasing mobility, were observed that did not vary in concentration or migration between induced and uninduced cell extracts. The molecular weights of the individual ARE-BP subunits were determined by a two-dimensional electrophoresis protocol. Ferguson gel analysis of native protein size indicated that several of the ARE-BP-DNA complexes are composed of multiple protein subunits. Wild-type AREs and GST Ya ARE fragments and mutant sequences were evaluated for their ability to mediate induction in a reporter gene system in HepG2 cells. This same panel of sites was tested in an in vitro binding assay for the ability to compete for the ARE-BPs. A binding profile for each ARE-BP was compiled. Correlation between the ARE-BP binding profiles and induction results indicated that: (i) the ARE-BP-1 and ARE-BP-2 complexes formed only with AREs that supported induction, and (ii) the ARE-BP-4 complex formed with all inducible AREs, but it also bound to ARE mutants that failed to support induction. Based on the studies, an early composite regulatory element model for ARE-mediated expression is presented. ARE-BP-1 is proposed to be the mediator of the ARE's unique induction response to chemoprotective agents.

Antioxidants↗

PubMed1997

Functional antioxidant responsive elements.

Exposure of human and rodent cells to a wide variety of chemoprotective compounds confers resistance against a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes like glutathione S-transferases (GST). Antioxidant responsive elements (AREs) mediate the transcriptional induction of a battery of genes which comprise much of this chemoprotective response system. Past studies identified a necessary ARE "core" sequence of RTGACnnnGC, but this sequence alone is insufficient to mediate induction. In this study, the additional sequences necessary to define a sufficient, functional ARE are identified through systematic mutational analysis of the murine GST Ya ARE. Introduction of the newly identified necessary nucleotides into the regions flanking a nonresponsive, ARE-like, GST-Mu promoter sequence produced an inducible element. A screen of the GenBank database with the newly identified ARE consensus identified 16 genes which contained the functional ARE consensus sequence in their promoters. Included within this group was an ARE sequence from the murine ferritin-L promoter that mediated induction when tested. In an electrophoretic mobility-shift assay, the ferritin-L ARE was bound by ARE-binding protein 1, a protein previously identified as the likely mediator of the chemoprotective response. A three-level ARE classification system is presented to account for the distinct induction strengths observed in our mutagenesis studies. A model of the ARE as a composite regulatory site, where multiple transcription factors interact, is presented to account for the complex characteristics of ARE-mediated chemoprotective gene expression.

Animals↗