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Biomedical subjects

A Lasham

Publications and source records attributed to A Lasham.

8 recordsLinked to original sources
PubMed2000

Regulation of the human fas promoter by YB-1, Puralpha and AP-1 transcription factors.

Fas (CD95/Apo-1) gene expression is dysregulated in a number of diseased states. Towards understanding the regulation of fas gene expression, we previously identified activator and repressor elements within the human fas promoter. Using a combination of expression screening and reporter gene assays, we have identified transcription factors which bind to these elements and thereby regulate transcription of the fas promoter. These are three single-stranded DNA binding proteins, YB-1, Puralpha and Purbeta and two components of the AP-1 complex, c-Fos and c-Jun. c-Jun is a potent transcriptional activator of fas and stimulated expression levels up to 184-fold in reporter gene assays. Co-expression with c-Fos abrogated c-Jun-mediated activation. YB-1 and Puralpha are transcriptional repressors of fas and decreased basal transcription by 60-fold in reporter gene assays. Purbeta was predominantly an antagonist of YB-1/Puralpha-mediated repression. Overexpression of YB-1 and Puralpha in Jurkat cells was shown to reduce the level of cell surface Fas staining, providing further evidence that these proteins regulate the fas promoter. It has been suggested that YB-1 plays a role in cell proliferation as an activator of growth-associated gene expression. We have shown that YB-1 is a repressor of a cell death-associated gene fas. These results suggest that YB-1 may play an important role in controlling cell survival by co-ordinately regulating the expression of cell growth-associated and death-associated genes.

Base Sequence↗
PubMed1998

Silencer and enhancer regions in the human CD95 (Fas/APO-1) gene with sequence similarity to the granulocyte-macrophage colony-stimulating factor promoter: binding of single strand-specific silencer factors and AP-1 and NF-AT-like enhancer factors.

The CD95 (Fas/APO-1) apoptosis receptor is expressed in a variety of tissues and transiently upregulated in lymphocytes during activation-induced cell death. A silencer (S1; -1035 to -1008) and an adjacent enhancer (E1; -1007 to -964) region have been mapped in the CD95 gene. The S1 region shows similarity to binding sites for the transcriptional repressor NF-GMb, which prefers binding to single-stranded DNA. The E1 contains an everted repeat of two CATTA/T elements spaced by 2 bp (ER2). Such motifs are directly repeated in the CLE0 region of the human granulocyte-macrophage colony-stimulating factor (huGM-CSF) promoter. A motif (TGATGTCA) which matches a CREB site and is similar to an AP-1 site is embedded within ER2. Sequence-specific binding of nuclear factors to single-stranded S1 probes involved, to some extent, a central heptamer motif (ATCCAAA) also present in E1. Competition binding studies suggested that AP-1 or AP-1 components, as well as factors related, but not identical, to NF-AT bound to E1 probes. S1-binding-proteins/complexes of 47, 77, and 100 kDa were detected by Southwestern analysis and ultraviolet crosslinking. Complexes of 70 and 80 kDa were formed with a double-stranded E1 probe in UV-crosslinking, whereas Southwestern analysis with this probe revealed single binding species of 59 and 113 kDa. ER2 autonomously enhanced transcription from the heterologous HSV tk promoter in a cell type-specific manner only in the absence of the S1 region. This analysis has identified a small region in the CD95 gene containing adjacent opposing regulatory elements which are likely to be involved in the cell type- and activation state-specific gene expression under physiologic conditions.

Animals↗
PubMed1993

Direct sequencing of lambda DNA from crude lysates using an improved linear amplification technique.

We describe an improved method for directly sequencing lambda (lambda) DNA that has been isolated from either crude cleared lysates or plate lysates. This protocol does not require that the DNA be obtained from bacteriophage particles that have been purified by caesium chloride centrifugation. Nanogram quantities of lambda DNA are unidirectionally amplified using a radioactively-labelled oligonucleotide primer, and Thermus aquaticus (Taq) DNA polymerase, in the presence of T4 gene 32 protein (gp 32). The amplification/sequencing reactions are then incubated with terminal deoxynucleotidyl transferase (TdT) and all four deoxynucleotide triphosphates to elongate any prematurely-arrested products. This procedure, which is a modification of a previously-published method, results in a significant improvement in the quality and amount of DNA sequence information that can be obtained from lambda templates. Although it was developed to sequence DNA directly from lambda EMBL3 recombinants, it can also be used with cosmid DNA, M13 and plasmid DNA, and polymerase chain reaction (PCR) amplification products, yielding excellent ladders in each case. In addition, our method resolves the nucleotide sequences of double-stranded plasmid templates that are difficult to determine by conventional dideoxynucleotide sequencing protocols because of 'stalling', in which bands appear at the same position in all four lanes.

Bacteriophage lambda↗
PubMed1991

Conserved organization of gamma-aminobutyric acidA receptor genes: cloning and analysis of the chicken beta 4-subunit gene.

A series of genomic clones containing DNA that encodes the chicken gamma-aminobutyric acidA (GABAA) receptor beta 4 subunit have been isolated. These have been restriction mapped and partially sequenced to determine the structural organization and the size of the beta 4-subunit gene. This gene, which comprises nine exons, spans more than 65 kb. The organization of the chicken GABAA receptor beta 4-subunit gene has been compared to that of the murine GABAA receptor delta-subunit gene and to those of the genes that encode other members of the ligand-gated ion-channel superfamily, namely muscle and neuronal nicotinic acetylcholine receptors (AChRs). Although the positions of the intron/exon boundaries of GABAA receptor subunit genes are seen to be highly conserved, there are significant differences between the genes that encode GABAA receptor and AChR subunits. These results are discussed in relation to the proposal that this superfamily of ligand-gated ion-channel receptor genes arose by duplication of an ancestral receptor gene.

Amino Acid Sequence↗
PubMed1991

gamma-Aminobutyric acidA receptor heterogeneity is increased by alternative splicing of a novel beta-subunit gene transcript.

DNA sequences encoding two variants of a novel gamma-aminobutyric acidA (GABAA) receptor beta subunit were isolated from an embryonic chicken whole-brain cDNA library and a chicken genomic library. The coding regions of these variants only differ from each other by the absence or presence of 12 bp in the region that encodes the presumed intracellular loop between transmembrane domains M3 and M4; the encoded subunits have been named beta 4 and beta 4', respectively. The predicted mature polypeptides are 72-77% identical to the previously characterized mammalian and chicken beta 1, beta 2, and beta 3 subunits. Analysis of the beta 4-subunit gene reveals that the different transcripts encoding the two variants arise by the use of one of two 5'-donor splice sites that are separated by 12 bp. This is the first demonstration of alternative splicing of a GABAA receptor subunit gene transcript and represents a further mechanism for the generation of GABAA receptor heterogeneity.

Animals↗