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Biomedical subjects

V P Stanton

Publications and source records attributed to V P Stanton.

At least 19 recordsLinked to original sources
PubMed1999

Single-nucleotide polymorphisms can cause different structural folds of mRNA.

Single-nucleotide polymorphisms (SNPs) are the most common type of genetic variation in man. Genes containing one or more SNPs can give rise to two or more allelic forms of mRNAs. These mRNA variants may possess different biological functions as a result of differences in primary or higher order structures that interact with other cellular components. Here we report the observation of marked differences in mRNA secondary structure associated with SNPs in the coding regions of two human mRNAs: alanyl tRNA synthetase and replication protein A, 70-kDa subunit (RPA70). Enzymatic probing of SNP-containing allelic fragments of the mRNAs revealed pronounced allelic differences in cleavage pattern at sites 14 or 18 nt away from the SNP, suggesting that a single-nucleotide variation can give rise to different mRNA folds. By using phosphorothioate oligodeoxyribonucleotides complementary to the region of different allelic structures in the RPA70 mRNA, but not extending to the SNP itself, we find that the SNP exerts an allele-specific effect on the accessibility of its flanking site in the endogenous human RPA70 mRNA. This further supports the allele-specific structural features identified by enzymatic probing. These results demonstrate the contribution of common genetic variation to structural diversity of mRNA and suggest a broader role than previously thought for the effects of SNPs on mRNA structure and, ultimately, biological function.

Alanine-tRNA Ligase↗
PubMed1999

Selective killing of cancer cells based on loss of heterozygosity and normal variation in the human genome: a new paradigm for anticancer drug therapy.

Most drugs for cancer therapy are targeted to relative differences in the biological characteristics of cancer cells and normal cells. The therapeutic index of such drugs is theoretically limited by the magnitude of such differences, and most anticancer drugs have considerable toxicity to normal cells. Here we describe a new approach for developing anticancer drugs. This approach, termed variagenic targeting, exploits the absolute difference in the genotype of normal cells and cancer cells arising from normal gene sequence variation in essential genes and loss of heterozygosity (LOH) occurring during oncogenesis. The technology involves identifying genes that are: 1) essential for cell survival; 2) are expressed as multiple alleles in the normal population because of the presence of one or more nucleotide polymorphisms; and 3) are frequently subject to LOH in several common cancers. An allele-specific drug inhibiting the essential gene remaining in cancer cells would be lethal to the malignant cell and would have minimal toxicity to the normal heterozygous cell that retains the drug-insensitive allele. With antisense oligonucleotides designed to target two alternative alleles of replication protein A, 70-kDa subunit (RPA70) we demonstrate in vitro selective killing of cancer cells that contain only the sensitive allele of the target gene without killing cells expressing the alternative RPA70 allele. Additionally, we identify several other candidate genes for variagenic targeting. This technology represents a new approach for the discovery of agents with high therapeutics indices for treating cancer and other proliferative disorders.

Antineoplastic Agents↗
PubMed1996

High-resolution physical mapping by combined Alu-hybridization/PCR screening: construction of a yeast artificial chromosome map covering 31 centimorgans in 3p21-p14.

We describe an integrated approach to large-scale physical mapping using an Alu-PCR hybridization screening strategy in conjunction with direct PCR-based screening to construct a continuous yeast artificial chromosome map covering >20 mb in human chromosome 3, bands p14-p21, composed of 205 loci, connected by 480 yeast artificial chromosomes, with average interlocus distance of approximately equal to 100 kb. We observe an inverse distribution of Alu-PCR and (CA)n markers. These results suggest that the two screening methods may be complementary and demonstrate the utility of Alu-PCR hybridization screening in the closure of high-resolution human physical maps.

Animals↗
PubMed1996

The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9.

The t(7;11)(p15;p15) translocation is a recurrent chromosomal abnormality associated primarily with acute myeloid leukaemia (FAB M2 and M4). We present here the molecular definition of this translocation. On chromosome 7 positional cloning revealed the consistent rearrangement of the HOXA9 gene, which encodes a class I homeodomain protein potentially involved in myeloid differentiation. On chromosome 11 the translocation targets the human homologue of NUP98, a member of the GLFG nucleoporin family. Chimaeric messages spliced over the breakpoint fuse the GLFG repeat domains of NUP98 in-frame to the HOXA9 homeobox. The predicted NUP98-HOXA9 fusion protein may promote leukaemogenesis through inhibition of HOXA9-mediated terminal differentiation and/or aberrant nucleocytoplasmic transport.

Amino Acid Sequence↗
PubMed1996

The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein.

The recurrent translocation t(8;16)(p11;p13) is a cytogenetic hallmark for the M4/M5 subtype of acute myeloid leukaemia. Here we identify the breakpoint-associated genes. Positional cloning on chromosome 16 implicates the CREB-binding protein (CBP), a transcriptional adaptor/coactivator protein. At the chromosome 8 breakpoint we identify a novel gene, MOZ, which encodes a 2,004-amino-acid protein characterized by two C4HC3 zinc fingers and a single C2HC zinc finger in conjunction with a putative acetyltransferase signature. In-frame MOZ-CBP fusion transcripts combine the MOZ finger motifs and putative acetyltransferase domain with a largely intact CBP. We suggest that MOZ may represent a chromatin-associated acetyltransferase, and raise the possibility that a dominant MOZ-CBP fusion protein could mediate leukaemogenesis via aberrant chromatin acetylation.

Acetyltransferases↗
PubMed1995

Ordered restriction endonuclease maps of yeast artificial chromosomes created by optical mapping on surfaces.

We have developed a surface mounting technology for the rapid construction of ordered restriction maps from individual DNA molecules. Optical restriction maps constructed from yeast artificial chromosome DNA molecules mounted on specially derivatized glass surfaces are accurate and reproducible, and the technology is amenable to automation. The mounting procedures described here should also be useful for fluorescence in situ hybridization studies. We believe these improvements to optical mapping will further stimulate the development of nonelectrophoretic approaches to genome analysis.

Automation↗
PubMed1995

Large-scale cloning of human chromosome 2-specific yeast artificial chromosomes (YACs) using an interspersed repetitive sequences (IRS)-PCR approach.

We report here an efficient approach to the establishment of extended YAC contigs on human chromosome 2 by using an interspersed repetitive sequences (IRS)-PCR-based screening strategy for YAC DNA pools. Genomic DNA was extracted from 1152 YAC pools comprised of 55,296 YACs mostly derived from the CEPH Mark I library. Alu-element-mediated PCR was performed for each pool, and amplification products were spotted on hybridization membranes (IRS filters). IRS probes for the screening of the IRS filters were obtained by Alu-element-mediated PCR. Of 708 distinct probes obtained from chromosome 2-specific somatic cell hybrids, 85% were successfully used for library screening. Similarly, 80% of 80 YAC walking probes were successfully used for library screening. Each probe detected an average of 6.6 YACs, which is in good agreement with the 7- to 7.5-fold genome coverage provided by the library. In a preliminary analysis, we have identified 188 YAC groups that are the basis for building contigs for chromosome 2. The coverage of the telomeric half of chromosome 2q was considered to be good since 31 of 34 microsatellites and 22 of 23 expressed sequence tags that were chosen from chromosome region 2q13-q37 were contained in a chromosome 2 YAC sublibrary generated by our experiments. We have identified a minimum of 1610 distinct chromosome 2-specific YACs, which will be a valuable asset for the physical mapping of the second largest human chromosome.

Animals↗
PubMed1994

YAC contigs for 4q35 in the region of the facioscapulohumeral muscular dystrophy (FSHD) gene.

We report here the construction of a genetic linkage map and an overlapping set of clones containing DNA markers linked to the causative locus for facioscapulohumeral muscular dystrophy (FSHD) on 4q35. Multipoint linkage analysis placed eight loci in the following order with odds greater than 1000:1: cen-D4S171-FXI-D4S426-D4S187-D4S130-D4S 163-D4S139-D4F35S1-qter. The most likely position of D4S809 was distal to D4F35S1. Thirty-four yeast artificial chromosomes (YACs) were isolated by PCR-based assays for STSs derived from DNA markers with known genetic and physical order. Walking from the insert ends of 2 YACs identified 7 additional YACs, bridging the gaps between three of the markers. Two new YACs were found by hybridization of a cosmid inter-Alu PCR product to dot blots of inter-Alu PCR products of YAC DNA pools. All YAC clones were positioned using the genetic and physical order of the STSs and inter-Alu PCR fingerprint data. Eleven of the YACs and two cosmids were mapped by fluorescence in situ hybridization to confirm the location of the clones and to detect chimerism. The 43 YACs were assembled into two contigs. The larger contig spans approximately 2.4 Mb and contains markers closest to the FSHD gene.

Base Sequence↗
PubMed1994

Rapid and efficient construction of yeast artificial chromosome contigs in the mouse genome with interspersed repetitive sequence PCR (IRS-PCR): generation of a 5-cM, > 5 megabase contig on mouse chromosome 1.

We have developed a new technique for the generation of YAC contigs in the mouse genome that is based on the ability to detect overlapping clones by hybridization of shared IRS-PCR products. As a demonstration of the technique, a 5-cM, > 5 megabase contig was developed on the distal half of mouse Chromosome (Chr) 1, spanning the region from Lamb2 to At3. The contig covers roughly 5% of the genetic distance of the chromosome and is comprised of more than 80 clones; 71 probes were assigned physical order to the chromosome, of which 59 were new markers generated in this study. Eight of the new probes were shown to be polymorphic between C3H/HeJ-gld and M. spretus. Three probes were mapped on a [(C3H/HeJ-gld x M. spretus) x C3H/HeJ-gld] interspecific backcross to integrate the physical map with a high-resolution genetic map of the region.

Animals↗
PubMed1993

Rapid identification of overlapping YACs in the MEN2 region of human chromosome 10 by hybridization with Alu element-mediated PCR products.

An overlapping set of 21 yeast artificial chromosomes (YACs) spanning the RET proto-oncogene [Takahashi et al., Oncogene 3 (1988) 571-578] and D10S102 markers on human chromosome 10 was isolated in a series of hybridization-based chromosomal walks in a YAC library. Genetic linkage analyses implicate this chromosomal region as the location of the gene (MEN2A) responsible for multiple endocrine neoplasia type 2A. Four YACs carrying a RET sequence-tagged site (STS) and two YACs carrying a D10S102 STS were used to initiate chromosome walks. These were based on hybridization of Alu element-mediated polymerase chain reaction (Alu-PCR) products from YACs to dot blots of Alu-PCR products from complex pools of YAC clones. The hybridization anchor content of YACs identified in the walks was confirmed by probing blots of Alu-PCR products from individual YACs and by comparing Alu-PCR fingerprints of each YAC. Ten hybridization-based Alu-PCR anchors and three STS anchors were ordered within eleven intervals created by the 21 overlapping YACs. The order of anchors requiring the fewest gaps in the YACs is consistent with the walking results and establishes the STS anchor order as D10S102-D10S94-RET. The overlapping set of YACs represents about 1.55 Mb of the human genome according to restriction mapping of four representative YACs in the contig. These results demonstrate the power of Alu-PCR hybridization for chromosomal walking and provide a rich source of overlapping YACs which can be used to identify candidate MEN2A genes.

Base Sequence↗
PubMed1993

Clinical and genetic studies of renal cell carcinomas in a family with a constitutional chromosome 3;8 translocation. Genetics of familial renal carcinoma.

OBJECTIVE: To describe the clinical course and genetic studies of renal carcinoma in members of a family with the constitutional chromosome translocation, t(3;8) (p14;q24). DESIGN: A follow-up study that updates our 1979 report of renal carcinoma in 10 of these relatives. SETTING: A cancer center and university hospital. PATIENTS: Members of the family, including five carriers of the 3;8 translocation who were in remission of renal cancer. MEASUREMENTS: Clinical follow-up of the family and genetic analyses of the renal cancer specimens of three patients. RESULTS: Renal carcinoma recurred in all five patients in the family at 1 to 16 years of follow-up. Three patients have died of renal cancer, and two are in a second remission. The renal cancers from three family members consistently reveal loss of the entire derivative chromosome 8, which bears the chromosome 3p segment spanning band p14 to the telomere. In contrast, no genetic change was detected in the derivative chromosome 3 or in normal chromosomes 3 and 8. CONCLUSIONS: This family illustrates the importance of clinical follow-up of patients with a hereditary cancer that can develop at multiple foci and recur over time. The inherited 3;8 translocation and loss of the translocated distal chromosome 3p in tumor specimens of family members may help localize the gene or genes involved in the pathogenesis of both familial and sporadic renal carcinoma.

Adult↗
PubMed1993

Isolation and FISH mapping of 80 cosmid clones on the short arm of human chromosome 3.

We have isolated and mapped by fluorescence in situ hybridization 80 new cosmids on the short arm of chromosome 3. These markers were isolated from a radiation-reduced hybrid, DM1, made from a cell line that was monochromosomal for human chromosome 3. Selected cosmids were used in double-label cohybridization experiments in which polymerase chain reaction products, generated by an Alu oligonucleotide primer from genomic DNA, were used for chromosome banding. Fifty-six of the cosmid probes map between 3p14.3 and 3p22 while 24 other probes cluster around bands 3p23-3p25. Three probes that appeared to map close to the chromosome 3 region bearing a t(3,8)p14.2; q24.1 translocation associated with renal cell carcinoma were analyzed by interphase mapping techniques and hybridized to metaphase spreads from the translocation cell line. These 80 probes will be useful in the elucidation of genetic alterations associated with diseases such as small cell lung carcinoma, renal cell carcinoma, and von Hippel-Lindau disease.

Animals↗
PubMed1992

Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member.

Using positional cloning strategies, we have identified a CTG triplet repeat that undergoes expansion in myotonic dystrophy patients. This sequence is highly variable in the normal population. PCR analysis of the interval containing this repeat indicates that unaffected individuals have been 5 and 27 copies. Myotonic dystrophy patients who are minimally affected have at least 50 repeats, while more severely affected patients have expansion of the repeat containing segment up to several kilobase pairs. The CTG repeat is transcribed and is located in the 3' untranslated region of an mRNA that is expressed in tissues affected by myotonic dystrophy. This mRNA encodes a polypeptide that is a member of the protein kinase family.

Amino Acid Sequence↗
PubMed1992

Sperm typing allows accurate measurement of the recombination fraction between D3S2 and D3S3 on the short arm of human chromosome 3.

The interval between the D3S2 and D3S3 loci on human chromosome 3p is a frequent site of deletions in a number of different cancers and contains the most common fragile site in man. Both loci have been physically mapped to 3p but because heterozygosity for D3S3 is so infrequent, recombination between them could not be determined accurately by using family studies. Sperm typing can measure recombination between DNA polymorphisms even in a single individual and thus can make use of polymorphisms with a low PIC. The recombination fraction between D3S2 and D3S3 was estimated to be 0.28 based on analyzing 189 and 77 sperm from two doubly heterozygous donors, respectively. These results demonstrate one of the ways in which sperm typing can complement pedigree analysis in constructing genetic maps.

Base Sequence↗
PubMed1992

The peripheral myelin gene PMP-22/GAS-3 is duplicated in Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with a DNA duplication at chromosome 17p11.2. In view of the point mutation in the gene for peripheral myelin protein pmp-22/gas-3 in Trembler mice, a murine model for CMT1A, we have analysed whether this gene is altered in CMT1A. Here we show that the human homologue of the murine pmp-22 gene is located within the CMT1A DNA duplication, which is a direct repeat and does not interrupt the coding region of PMP-22. Expression of PMP-22 in CMT1A fibroblasts is similar to expression in control fibroblasts. Increased gene dosage or altered PMP-22 expression in the peripheral nervous system are therefore possible mechanisms by which PMP-22 is involved in CMT1A.

Base Sequence↗
PubMed1992

Phosphorylation occurs in the amino terminus of the Raf-1 protein.

The ability of the Raf-1 protein to morphologically transform murine fibroblasts can be activated by amino-terminal deletions or substitutions. We have compared the phosphorylation states of full-length and representative transforming and non-transforming amino-terminal deletion mutants of the Raf-1 protein using phosphoamino acid analysis and tryptic phosphopeptide mapping. Several [32P]orthophosphate-labeled tryptic phosphopeptides that were present in the full-length Raf-1 protein were absent from the highly transforming 22W Raf-1 mutant (lacking 305 amino-terminal residues). Peptide-specific antisera localized Raf-1 phosphorylation sites to several amino-terminal cyanogen bromide and tryptic peptides that are deleted from the 22W protein. A major phosphorylated tryptic peptide of the Raf-1 protein was immunoprecipitated by antiserum directed against amino acid residues 257-275, a highly conserved region of the raf family. This tryptic peptide is entirely deleted from the highly transforming 22W protein. Subtractive Edman degradation and electrophoretic analysis of the immunoprecipitated tryptic peptide indicated that phosphorylation of the Raf-1 protein occurs at serine 259. Multiple phosphorylated tryptic peptide forms were immunoprecipitated by antiserum directed against Raf-1 residues 283-309. The majority of this tryptic peptide is also deleted from the highly transforming Raf-1 mutant 22W.

3T3 Cells↗