PubMed Health⌕ Search

Biomedical subjects

S M Lipkin

Publications and source records attributed to S M Lipkin.

11 recordsLinked to original sources
PubMed2001

A nonsense mutation in MLH1 causes exon skipping in three unrelated HNPCC families.

Germline mutations in the DNA mismatch repair genes MSH2 and MLH1 are responsible for the majority of hereditary nonpolyposis colorectal cancer (HNPCC) families. A common mutation mechanism is to disrupt MLH1 and MSH2 mRNA splicing. The disruption creates aberrant mRNAs lacking specific coding exons (exon skipping). Here, we report a novel skipping of MLH1 exon 12 caused by an AAG to TAG nonsense mutation at codon 461 in three HNPCC families of North American origins. The nonsense codon was found in a conserved haplotype in the three unrelated families and seems to represent a founder mutation. The skipping created an aberrant MLH1 mRNA transcript lacking exon 12. The effect of the codon 461 nonsense mutation on exon 12 skipping is evident even though it was placed in a minigene construct containing entirely different coding sequences. Notably, the effect of the nonsense mutation on exon skipping is incomplete. Accordingly, a second aberrant MLH1 transcript encompassing the nonsense codon is also produced. Whereas the latter transcript is unstable, presumably because of nonsense-mediated mRNA decay, neither of the aberrant transcripts seems to affect the stability of wild-type MLH1 mRNA. This study demonstrates that the germ-line nonsense mutation at codon 461 of MLH1 disrupts normal MLH1 mRNA processing, and that exon skipping underlies pathogenesis in these HNPCC families.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Germline and somatic mutation analyses in the DNA mismatch repair gene MLH3: Evidence for somatic mutation in colorectal cancers.

DNA mismatch repair is of considerable scientific and medical importance because of its essential role in maintaining genomic integrity, and its association with hereditary non-polyposis colon cancer (HNPCC). Germline mutations in five mismatch repair genes (MLH1, MSH2, PMS1, PMS2, and MSH6) have been associated with HNPCC susceptibility. Our laboratory recently identified MLH3, a novel DNA mismatch repair gene. We screened the MLH3 coding sequence in 60 probands with increased genetic risk factors for colorectal cancer susceptibility and no mutations in the other candidate genes. No definite MLH3 germline mutations were found. We subsequently screened 36 colon tumors, and discovered an appreciable frequency of somatic MLH3 coding mutations in MSI-H tumors (25%). In four of six tumors, evidence of biallelic inactivation was noted. Furthermore, MLH3 nonsense mutations were identified in two of 12 microsatellite stable (MSS) tumors with 14q24 loss of heterozygosity. While our analyses do not exclude the existence of germline MLH3 mutations in patients with increased genetic risk factors for colorectal cancer susceptibility, they suggest such mutations are uncommon in this patient population. The finding of an appreciable frequency of somatic MLH3 mutations is consistent with a possible role for this gene in the progression of colorectal cancer tumorigenesis. Hum Mutat 17:389-396, 2001. Published 2001 Wiley-Liss, Inc.

Adaptor Proteins, Signal Transducing↗
PubMed2000

MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability.

DNA mismatch repair is important because of its role in maintaining genomic integrity and its association with hereditary non-polyposis colon cancer (HNPCC). To identify new human mismatch repair proteins, we probed nuclear extracts with the conserved carboxy-terminal MLH1 interaction domain. Here we describe the cloning and complete genomic sequence of MLH3, which encodes a new DNA mismatch repair protein that interacts with MLH1. MLH3 is more similar to mismatch repair proteins from yeast, plants, worms and bacteria than to any known mammalian protein, suggesting that its conserved sequence may confer unique functions in mice and humans. Cells in culture stably expressing a dominant-negative MLH3 protein exhibit microsatellite instability. Mlh3 is highly expressed in gastrointestinal epithelium and physically maps to the mouse complex trait locus colon cancer susceptibility I (Ccs1). Although we were unable to identify a mutation in the protein-coding region of Mlh3 in the susceptible mouse strain, colon tumours from congenic Ccs1 mice exhibit microsatellite instability. Functional redundancy among Mlh3, Pms1 and Pms2 may explain why neither Pms1 nor Pms2 mutant mice develop colon cancer, and why PMS1 and PMS2 mutations are only rarely found in HNPCC families.

Amino Acid Sequence↗
PubMed1996

Constitutive retinoid receptors expressed from adenovirus vectors that specifically activate chromosomal target genes required for differentiation of promyelocytic leukemia and teratocarcinoma cells.

Sufficient knowledge of transcription factor structure and function has accumulated to allow attempts at the rational design of novel transcription factors for the study of gene regulation and potential application in gene therapy. In the present studies, we have systematically evaluated the function of chimeric retinoid receptors generated by fusion with the transactivation domain of VP16 and expression in adenovirus vectors. By varying the location of fusion of the VP16 transactivation domain with the retinoic acid receptor (RAR) or retinoid X receptor (RXR), marked differences in the specificity of gene activation were obtained. Although several chimeric proteins activated both RAR and RXR target genes, fusion of the NT16 transactivation domain to the N terminus of RAR permitted specific activation of reporter genes containing retinoic acid response elements. In contrast, fusion of the VP16 transactivation domain to the C terminus of RXR permitted specific activation of reporter genes containing RXR response elements. When tested for their ability to activate chromosomal targets, the chimera consisting of VP16 linked to the N terminus of PAR was much more active in promoting the differentiation of HL-60 cells and NTera-2 cells than the chimera consisting of VP16 linked to the C terminus of RXR. These observations support the existence of two distinct retinoid signalling pathways predicted on the basis of biochemical and pharmacologic studies and provide direct evidence that the programs of differentiation elicited by retinoic acid in these cells are mediated by a specific subset of binding sites for RAR-RXR heterodimers. VP16-RAR and VP16-RXR fusion proteins should be of further use in dissecting the relative contributions of RARs and RXRs to specific programs of gene expression. Constitutive retinoid receptors may also be considered for use as novel tumor suppressor genes for genetically based treatment of retinoid-responsive cancers.

Adenoviridae↗
PubMed1993

Identification of a novel zinc finger protein binding a conserved element critical for Pit-1-dependent growth hormone gene expression.

The growth hormone (GH) and prolactin genes require the pituitary-specific POU domain transcription factor Pit-1 for their activation. However, additional factors are necessary for the effective expression of these genes. Analysis of evolutionarily conserved sequences in the proximal GH promoter suggests the critical importance of one highly conserved element located between the two Pit-1 response elements. Mutation of this site decreases expression of a transgene in mice > 100-fold. We have identified a major activity binding to this site as a novel member of the Cys/His zinc finger superfamily, referred to as Zn-15. The Zn-15 DNA-binding domain comprises three zinc fingers separated by unusually long linker sequences that would be expected to interrupt specific DNA site recognition. Zn-15 synergizes with Pit-1 to activate the GH promoter in heterologous cell lines in which this promoter is only minimally responsive to Pit-1 alone. Our data suggest that functional interactions between the tissue-specific POU domain factor Pit-1 and this novel zinc finger factor binding to an evolutionarily conserved region in the GH promoter may constitute an important component of the combinatorial code that underlies the effective expression of the GH gene.

Amino Acid Sequence↗
PubMed1992

A negative retinoic acid response element in the rat oxytocin promoter restricts transcriptional stimulation by heterologous transactivation domains.

Retinoic acid receptors are ligand-dependent transcription factors that stimulate gene transcription from promoters containing retinoic acid or thyroid hormone response elements. We describe a high-affinity binding site from the rat oxytocin promoter that mediates negative transcriptional regulation by the retinoic acid receptor. To examine whether strong, constitutive transactivation domains would be capable of stimulating gene transcription when bound to this DNA binding site that normally mediates transcriptional repression, we fused the transactivation domain of the herpes simplex viral protein VP16 to the amino terminus of the retinoic acid receptor and tested the activity of the chimeric protein on the negative retinoic acid response element. This chimeric retinoic acid receptor acted as a strong, constitutive transactivator when bound to promoters containing palindromic thyroid hormone/retinoic acid response elements but surprisingly it still repressed gene transcription when bound to promoters containing the oxytocin-negative retinoic acid response element. These results suggest that a negative DNA binding site itself can inhibit the function of even potent constitutive transactivation domains, and provide evidence that tethering of a constitutive transactivation domain to DNA is insufficient to activate gene transcription.

Animals↗
PubMed1991

The orientation and spacing of core DNA-binding motifs dictate selective transcriptional responses to three nuclear receptors.

Characterization of several thyroid hormone (T3), retinoic acid, and estrogen response elements has led to the identification of conserved DNA half-sites (core binding motifs). We present evidence that differences in both the relative orientation and spacing of these motifs within hormone response elements determine the distinct transcriptional responses of three members of the nuclear receptor superfamily. When separated by 3 bp, direct repeat, palindromic, and inverted palindromic arrangements of these motifs impart selective transcriptional responses to retinoic acid, estrogen, and T3 receptors, respectively. Varying the spacing between core motifs alters the specificity. Without spacing, a direct repeat of the core motif paradoxically configures the T3 receptor to confer transactivation in the absence of T3 and repression in its presence. Such an element occurs naturally in the mouse beta-thyrotropin promoter, physiologically under negative regulation by T3. The orientation and spacing of core binding motifs may thus function in concert as a code that accounts for the selective patterns of transcriptional responses of hormonally regulated promoters.

Animals↗
PubMed1989

Positive and negative regulation of gene transcription by a retinoic acid-thyroid hormone receptor heterodimer.

We present evidence that the human thyroid hormone receptor forms a heterodimer with the human retinoic acid receptor. This interaction results in a cooperative increase in binding of the alpha retinoic acid receptor to a subset of thyroid hormone response elements. Mutations within the DNA binding domain or near the C-terminus abolish either receptor's ability to interact cooperatively on these elements. The thyroid hormone-retinoic acid receptor heterodimer exhibits novel transcriptional properties in that coexpression of both receptors at low levels in Green monkey kidney (CV1) cells results in a positive transcriptional effect on promoters containing a palindromic thyroid hormone response element, but has a surprisingly negative effect on a thyroid hormone response element derived from the alpha myosin heavy chain gene. These results suggest that by forming heterodimers, more elab-orate control of transcription can be achieved by creating receptor combinations with differing activities.

Amino Acid Sequence↗