Search PubMed⌕ Search

Biomedical subjects

S A Bossone

Publications and source records attributed to S A Bossone.

7 recordsLinked to original sources
PubMed1997

A far upstream cis-element is required for Wilms' tumor-1 (WT1) gene expression in renal cell culture.

To identify novel cis-regulatory elements responsible for the tissue-restricted expression pattern of the Wilms' tumor-1 (WT1) gene, we mapped a total of 11 DNase I-hypersensitive sites in the 5'-flanking region and first intron of the human gene, six of which were specific for WT1 expressing cell lines. A 1.4-kilobase (kb) fragment from the mouse wt1 5'-flanking region contained cross-hybridizing sequence with significant homology to a region of DNase I hypersensitivity in the human WT1 gene which bound to nuclear matrix in human fetal kidney 293 cells. None of the DNase I-hypersensitive sites/matrix attachment regions, either alone or in combination, were sufficient for tissue-specific WT1 expression in transient and stably transfected cell lines. However, stable transfection of an approximately 620-kb yeast artificial chromosome (YAC) that carried the entire mouse wt1 locus into 293 cells resulted in wt1 (trans)gene expression at a level of approximately 30% of the endogenous human gene. Deletion of the 1.4-kb cross-hybridizing mouse fragment, located approximately 15 kb upstream of the transcription start site, caused complete loss of wt1 gene expression in the YAC-transfected 293 cells. In summary, we have identified a far upstream element that contains a region of DNase I hypersensitivity and that binds to nuclear matrix. This element includes phylogenetically conserved sequence and is required, although not sufficient, for mouse wt1 gene expression in human fetal kidney cells in culture.

Animals↗
PubMed1997

Sp1 is a critical regulator of the Wilms' tumor-1 gene.

We performed deletion analysis of WT1-reporter constructs containing up to 24 kilobases of 5'-flanking and first intron WT1 sequence in stably transfected cultured cells as an unbiased approach to identify cis elements critical for WT1 transcription. Although not a tissue-specific element, a proximate 9-base pair CTC repeat accounted for approximately 80% of WT1 transcription in this assay. Enhancer activity of the element and mutated versions correlated completely with their ability to form a DNA-protein complex in gel shifts. Antibody supershift, oligonucleotide competition, and Southwestern studies indicated that the CTC-binding factor is the transcriptional activator Sp1. Sp1 binds the CTC repeat with an affinity, KD = 0.37 nM, at least as high as the consensus GC box. Similar CTC repeats are found in promoters of other growth-related genes. Because Sp1 is important for WT1 expression, we examined Sp1 immunohistochemistry in fetal and adult kidney. In a pattern that precedes that of WT1 message, Sp1 immunostaining was highest in uninduced mesenchyme, early tubules, developing podocytes, and mature glomeruli, but was minimal in mature proximal tubules. This work suggests abundant Sp1 may be a prerequisite for WT1 expression, and that Sp1 may have a wider role in nephrogenesis.

Animals↗
PubMed1994

MAZ-dependent termination between closely spaced human complement genes.

The zinc finger protein MAZ, originally identified as a factor that binds to the c-myc P2 promoter, is associated with transcriptional termination. As shown in these studies, a termination sequence between the closely spaced human complement genes C2 and Factor B contains a protein binding site which interacts with three different proteins in vitro. Binding of one of these factors, MAZ, correlates with activity of the C2 termination sequence in vivo. Cloned MAZ was used to obtain a consensus binding site, G5AG5. This allowed identification of new sites, between the closely spaced human genes g11 and C4 and within an intron of the mouse IgM-D gene, where termination is known to occur and regulate the expression of IgD. The g11 and IgM MAZ sites lie within sequences that have activity in a termination assay and, furthermore, mutation of C2 or g11 MAZ sites severely reduces termination activity. MAZ bends DNA, and inherently bent DNA is highly active as a terminator, suggesting that MAZ-induced bending is important for C2 and g11 termination. We propose that MAZ sites exist in promoters which require protection against transcriptional interference, such as those of closely spaced genes, to cause efficient termination. The MAZ consensus sequence will facilitate the identification of further sites.

Base Sequence↗
PubMed1992

MAZ, a zinc finger protein, binds to c-MYC and C2 gene sequences regulating transcriptional initiation and termination.

ME1a1, a 16-base-pair nuclear factor binding site residing between the c-MYC P1 and P2 transcription initiation sites, is required for P2 activity. A cDNA encoding a 477-amino acid zinc finger protein designated MAZ (MYC-associated zinc finger protein) was cloned from a HeLa lambda gt11 library by screening with a concatamerized ME1a1 binding site probe. In addition to six potential zinc fingers of the Cys2His2 type, MAZ contains an amino-terminal proline-rich domain and several polyalanine tracts. Its mRNA was present in all human tissues tested except for kidney, as a doublet of approximately 2.5 and 2.7 kilobases, along with differentially expressed minor species. MAZ bound specifically to the wild-type ME1a1 sequence but not to a ME1a1 mutant that also failed to yield P2 activity. When expressed as a fusion protein in a pMAL-c vector, MAZ binds with specificity to a GA box sequence (GGGAGGG) found in the c-MYC P2 promoter, to the P2 attenuator region within the gene's first exon, and to a related sequence involved in the transcriptional termination of the C2 gene. MAZ may encode a transcription factor with dual roles in transcription initiation and termination.

Amino Acid Sequence↗
PubMed1987

Nerve-related 3S acetylcholinesterase in murine thymus.

One form of acetylcholinesterase (AChE), 3S, has been identified in the thymus of normal mice as the predominant species. Histochemical studies show that the AChE is localized to nerves or to nerve-related tissues. The form isolated is composed of a salt and a detergent-sensitive fraction. Since the sedimentation values and the kinetics of the two fractions are identical, it is proposed that only one gene encodes for this globular 3S species of AChE.

Acetylcholinesterase↗