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Y Bernstein

Publications and source records attributed to Y Bernstein.

At least 19 recordsLinked to original sources
PubMed2001

The RUNX3 gene--sequence, structure and regulated expression.

The RUNX3 gene belongs to the runt domain family of transcription factors that act as master regulators of gene expression in major developmental pathways. In mammals the family includes three genes, RUNX1, RUNX2 and RUNX3. Here, we describe a comparative analysis of the human chromosome 1p36.1 encoded RUNX3 and mouse chromosome 4 encoded Runx3 genomic regions. The analysis revealed high similarities between the two genes in the overall size and organization and showed that RUNX3/Runx3 is the smallest in the family, but nevertheless exhibits all the structural elements characterizing the RUNX family. It also revealed that RUNX3/Runx3 bears a high content of the ancient mammalian repeat MIR. Together, these data delineate RUNX3/Runx3 as the evolutionary founder of the mammalian RUNX family. Detailed sequence analysis placed the two genes at a GC-rich H3 isochore with a sharp transition of GC content between the gene sequence and the downstream intergenic region. Two large conserved CpG islands were found within both genes, one around exon 2 and the other at the beginning of exon 6. RUNX1, RUNX2 and RUNX3 gene products bind to the same DNA motif, hence their temporal and spatial expression during development should be tightly regulated. Structure/function analysis showed that two promoter regions, designated P1 and P2, regulate RUNX3 expression in a cell type-specific manner. Transfection experiments demonstrated that both promoters were highly active in the GM1500 B-cell line, which endogenously expresses RUNX3, but were inactive in the K562 myeloid cell line, which does not express RUNX3.

Alternative Splicing↗
PubMed1998

Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors.

The mammalian AML/CBFalpha runt domain (RD) transcription factors regulate hematopoiesis and osteoblast differentiation. Like their Drosophila counterparts, most mammalian RD proteins terminate in a common pentapeptide, VWRPY, which serves to recruit the corepressor Groucho (Gro). Using a yeast two-hybrid assay, in vitro association and pull-down experiments, we demonstrate that Gro and its mammalian homolog TLE1 specifically interact with AML1 and AML2. In addition to the VWRPY motif, other C-terminal sequences are required for these interactions with Gro/TLE1. TLE1 inhibits AML1-dependent transactivation of the T cell receptor (TCR) enhancers alpha and beta, which contain functional AML binding sites, in transfected Jurkat T cells. LEF-1 is an additional transcription factor that mediates transactivation of TCR enhancers. LEF-1 and its Drosophila homolog Pangolin (Pan) are involved in the Wnt/Wg signaling pathway through interactions with the coactivator beta-catenin and its highly conserved fly homolog Armadillo (Arm). We show that TLE/Gro interacts with LEF-1 and Pan, and inhibits LEF-1:beta-catenin-dependent transcription. These data indicate that, in addition to their activity as transcriptional activators, AML1 and LEF-1 can act, through recruitment of the corepressor TLE1, as transcriptional repressors in TCR regulation and Wnt/Wg signaling.

Amino Acid Sequence↗
PubMed1996

Expression of the human acute myeloid leukemia gene AML1 is regulated by two promoter regions.

The human chromosome 21 AML1 gene is expressed predominantly in the hematopoietic system. In several leukemia-associated translocations AML1 is fused to other genes and transcription of the fused regions is mediated by upstream sequences that normally regulate the expression of AML1. The 5' genomic region of AML1 was cloned and sequenced. The two 5' untranslated regions (UTRs) previously identified in AML1 cDNAs were located in this region and the distance between them was established. The distal 5' UTR maps over 7 kb upstream of the proximal one. Using primer extension with mRNA, transcription start sites were identified at two distinct sites above these 5' uTRs. Sequence analysis revealed the absence of a TATA motif and the presence of Sp1, PU.1, Oct, CRE, Myb, Ets, and Ets-like binding sites in both upstream regions. Several initiator elements (Inr) that overlap the transcription start sites were also identified. These proximal and distal upstream regions and their deletion mutants were cloned in front of a luciferase reporter gene and used in transfection assays. We demonstrate that both upstream regions function as promoters in hematopoietic (Jurkat) and nonhematopoietic (HEK) cell lines. The activity of both promoters was orientation dependent and was enhanced, in a cell-type specific manner, by a heterologous enhancer sequence. These results indicate that additional control elements, either negative or positive, regulate the tissue-specific expression of AML1.

Base Sequence↗
PubMed1996

A large variety of alternatively spliced and differentially expressed mRNAs are encoded by the human acute myeloid leukemia gene AML1.

The human chromosome 21 acute myeloid leukemia gene AML1 is frequently rearranged in the leukemia-associated translocations t(8;21) and t(3;21), generating fused proteins containing the amino-terminal part of AML1. In normal blood cells, five size classes (2-8 kb) of AML1 mRNAs have been previously observed. We isolated seven cDNAs corresponding to various AML1 mRNAs. Sequencing revealed that their size differences were mainly due to alternatively spliced 5' and 3' untranslated regions, some of which were vast, exceeding 1.5 kb (5') and 4.3 kb (3'). These untranslated regions contain sequences known to control mRNA translation and stability and seem to modulate AML1 mRNA stability. Further heterogeneity was found in the coding region due to the presence of alternatively spliced stop codon-containing exons. The latter led to production of polypeptides that were smaller than the full-length AML1 protein; they lacked the trans-activation domains but maintained DNA binding and heterodimerization ability. The size of these truncated products was similar to the AML1 segment in the fused t(8;21) and t(3;21) proteins. In thymus, only one mRNA species of 6 kb was detected. Using in situ hybridization, we showed that its expression was confined to the cortical region of the organ. The 6-kb mRNA was also prominent in cultured peripheral blood T cells, and its expression was markedly reduced upon mitogenic activation by phorbol myristate acetate (TPA) plus concanavalin A (ConA). These results and the presence of multiple coding regions flanked by long complex untranslated regions, suggest that AML1 expression is regulated at different levels by several control mechanisms generating the large variety of mRNAs and protein products.

Alternative Splicing↗
PubMed1995

Effect of mouthguard bleaching on enamel surface.

PURPOSE: To evaluate in vitro the effect of 10% carbamide peroxide in a mouthguard on the shear bond strength of resin composite to bleached enamel surfaces and morphological changes in these surfaces. MATERIALS AND METHODS: Thirty anterior extracted were divided into a control and a test group. Plastic trays were made for each tooth and the test group treated with Opalescence bleaching gel and incubated at 37 degrees C humid environment for 8 hours. The bleaching agent was washed and the trays replaced with water-soaked cotton wool for another 16 hours in the incubator. This procedure was repeated every day for 21 days. The control group was just treated with the water-soaked cotton wool. Selected teeth from both groups were evaluated with the SEM and 72 hours after the bleaching treatment, Scotchbond 2/Silux Plus rings were bonded to the enamel surfaces in both groups and sher bond strength testing performed. RESULTS: Bleaching created some enamel porosity and significant reduction in the resin composite shear bond strength.

Carbamide Peroxide↗
PubMed1995

Common promoter features in human and mouse liver type phosphofructokinase gene.

The liver-type phosphofructokinase is a key glycolytic enzyme encoded by genes residing on human and mouse chromosomes 21 and 10 respectively. Genomic DNA regions upstream of the initiator ATG spanning 2.6Kb and 3.4Kb of human and mouse liver-type phosphofructokinase gene were sequenced and analyzed. The proximal 0.4Kb region of both genes featured a CpG island containing 60%-73% GC residues. The first 120 nucleotides preceding the ATG are highly conserved displaying 73% of sequence similarity between human and mouse genes. While this region lacks TATA and CAAT boxes it contains four Sp1 binding sites and was capable of promoting a non regulated expression of the reporter gene chloramphenicol acetyl transferase, in transfection assays. Additional conserved elements were found further upstream at the 5'-region of both the human and mouse genes. They consisted of two Alu repeats and several sequence motifs known to serve as transcription factors binding sites.

Animals↗
PubMed1994

AML1, AML2, and AML3, the human members of the runt domain gene-family: cDNA structure, expression, and chromosomal localization.

cDNAs corresponding to three human runt domain containing genes, AML1, AML2, and AML3, were isolated and characterized. In addition to homology in the highly conserved runt domain, extensive sequence similarities were also observed in other parts of the proteins. All three carried an identical, putative ATP binding site -GRSGRGKS-, and their C-terminal halves were particularly rich in proline and serine residues. While AML1 cDNAs were cloned by others, AML2 represents a new member, not previously described, of the runt domain gene family, and AML3 was identified as the human homologue of mouse PEB-P2 alpha A. The chromosomal location of AML1 to chromosome 21q22 was confirmed, while AML2 and AML3 were mapped to chromosome regions 1p36 and 6p21, respectively. Analysis of AML1 and AML2 expression in hematopoietic cell lines revealed a distinct pattern of expression.

Amino Acid Sequence↗
PubMed1994

Isolation, chromosomal localization, and sequence analysis of human chromosome 21 zinc finger domains.

The zinc finger element is a conserved motif among a group of proteins involved in binding to nucleic acid. This motif has been detected in many regulatory factors and is highly represented in the human genome. To investigate the presence of zinc-finger-encoding genes on human chromosome 21, chromosome-specific libraries were screened with an oligodeoxynucleotide probe representing the conserved H-C link region between adjacent fingers. Three distinct genomic clones, designated ZF21-1, ZF21-2, and ZF21-3, were isolated and mapped to the long arm of chromosome 21 as well as to the heterochromatic short arm of several other chromosomes. DNA sequence analysis has shown that these genomic clones contain multiple zinc finger elements of the Kruppel type with only partial similarity to other known zinc finger genes. However, in each clone, few fingers were degenerated; they contain inframe stop codons and frameshifts that would preclude their translation. It seems therefore, that these chromosome 21 zinc finger sequences are not parts of functional genes. Nevertheless, the possibility that these domains are transcribed, and thus might have a regulatory role, is considered.

Animals↗
PubMed1992

Gene dosage and Down's syndrome: metabolic and enzymatic changes in PC12 cells overexpressing transfected human liver-type phosphofructokinase.

Down's syndrome (DS) is a human genetic disease caused by triplication of the distal third of chromosome 21 and overexpression of an unknown number of genes residing in it. The gene for the liver-type subunit of phosphofructokinase (PFKL), a key glycolytic enzyme, maps to this region and the product is overproduced in DS erythrocytes and fibroblasts. These facts, together with abnormalities which occur in DS glycolysis, make PFKL overexpression a candidate for causing some aspects of the DS phenotype. A cellular model for examining the consequences of PFKL overexpression in DS was constructed by transfecting rat PC12 cells with the human PFKL cDNA. Phosphofructokinase (PFK) isolated from PFKL-overexpressing clones was more inhibited by ATP and citrate and less activated by fructose-6-phosphate than control PFK; similar results were obtained when PFK preparations from DS and control fibroblasts were compared. In vivo NMR measurements determined that cells overexpressing PFKL performed glycolysis 40% faster than controls. These results show that overexpression of PFKL is the cause for altered biochemical regulatory characteristics of PFK in DS fibroblasts and can result in enhancement of glycolysis rates. It is also shown that increased gene dosage can exert its influence not merely by enhancing the amounts of gene products but also by altering their biochemical nature.

Adenosine Triphosphate↗
PubMed1990

The structure of the human liver-type phosphofructokinase gene.

We have isolated the gene for the human liver-type phosphofructokinase, from upstream to the 5' mRNA terminus to beyond the polyadenylation site. The gene is at least 28 kb long and is divided into 22 exons; it contains conventional splice-junction sequences and one polyadenylation signal. Exons and introns are quite rich in G and C residues; some 60% of all nucleotides are either G or C. Five possible sites of polymorphism have been found. The gene structure reveals no signs of internal similarities despite protein sequence evidence which suggests that the PFK molecule is divided into two similar halves. The structure and organization of the human liver-type PFK gene are shown to be extremely similar to those of the rabbit muscle-type PFK.

Animals↗
PubMed1989

The primary structure of human liver type phosphofructokinase and its comparison with other types of PFK.

The complete mRNA sequence of the human liver-type phosphofructokinase (hPFKL) was determined. The sequence included 55 nucleotides of 5' and 515 of 3' noncoding regions, as well as 2,337 nucleotides encoding the 779 amino acids of the hPFKL. Extensive similarity (approximately 90%) in the coding region was observed between the hPFKL and the mouse PFKL, whereas the degree of similarity between different types of PFK, i.e., hPFKL and human muscle-type PFK (hPFKM), was merely 68%. Nevertheless, striking similarity between these different types of PFK was noticed when the amino acid residues creating the various active sites of the enzyme were compared. Human PFK L- and M-specific probes were constructed and used to quantitate the mRNA levels in fetal and adult brains and fetal liver. It was found that while relative amount of PFKL mRNA in adult brain was one-fourth of that detected in fetal brain the level of PFKM mRNA in adult brain was slightly higher than in fetal tissue, suggesting that PFK expression might be controlled at the transcriptional level.

Amino Acid Sequence↗
PubMed1986

Overproduction of human Cu/Zn-superoxide dismutase in transfected cells: extenuation of paraquat-mediated cytotoxicity and enhancement of lipid peroxidation.

The 'housekeeping' enzyme Cu/Zn-superoxide dismutase (SOD-1) is encoded by a gene residing on human chromosome 21, at the region 21q22 known to be involved in Down's syndrome. The SOD-1 gene and the SOD-1 cDNA were introduced into mouse L-cells and human HeLa cells, respectively as part of recombinant plasmids containing the neoR selectable marker. Human and mouse transformants were obtained that expressed elevated levels (up to 6-fold) of authentic, enzymatically active human SOD-1. This enabled us to examine the consequences of hSOD-1 gene dosage, apart from gene dosage effects contributed by other genes residing on chromosome 21. Human and mouse cell clones that overproduce the hSOD-1 had altered properties; they were more resistant to paraquat than the parental cells and showed an increase in lipid peroxidation. The data are consistent with the possibility that gene dosage of hSOD-1 contributes to some of the clinical symptoms associated with Down's syndrome.

Animals↗
PubMed1986

Human Cu/Zn superoxide dismutase gene family: molecular structure and characterization of four Cu/Zn superoxide dismutase-related pseudogenes.

Four distinct human Cu/Zn superoxide dismutase (SOD1; EC 1.15.1.1)-related sequences were isolated from genomic DNA libraries. Genomic blots, heteroduplex analyses, and DNA sequencing showed that they are processed pseudogenes not residing on chromosome 21. Three of them originated from the 0.7-kilobase SOD1 mRNA, while the fourth was derived from the 0.9-kilobase mRNA species. Comparison between the coding sequences of the functional gene and two of the processed genes suggested that they integrated into the genome about 25 million years ago.

Animals↗
PubMed1985

Architecture and anatomy of the chromosomal locus in human chromosome 21 encoding the Cu/Zn superoxide dismutase.

The SOD-1 gene on chromosome 21 and approximately 100 kb of chromosomal DNA from the 21q22 region have been isolated and characterized. The gene which is present as a single copy per haploid genome spans 11 kb of chromosomal DNA. Heteroduplex analysis and DNA sequencing reveals five rather small exons and four introns that interrupt the coding region. The donor sequence at the first intron contains an unusual variant dinucleotide 5'-G-C, rather than the highly conserved 5'-GT. The unusual splice junction is functional in vivo since it was detected in both alleles of the SOD-1 gene, which were defined by differences in the length of restriction endonuclease fragments (RFLPs) that hybridize to the cDNA probe. Genomic blots of human DNA isolated from cells trisomic for chromosome 21 (Down's syndrome patients) show the normal pattern of bands. At the 5' end of gene there are the 'TATA' and 'CAT' promoter sequences as well as four copies of the -GGCGGG- hexanucleotide. Two of these -GC- elements are contained within a 13 nucleotide inverted repeat that could form a stem-loop structure with stability of -33 kcal. The 3'-non coding region of the gene contains five short open reading-frames starting with ATG and terminating with stop codons.

Bacteriophage lambda↗