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PubMed1999

Molecular characteristics of poorly differentiated adenocarcinoma and signet-ring-cell carcinoma of colorectum.

In a series of 45 poorly differentiated adenocarcinomas (por) and 7 signet-ring-cell carcinomas (sig) of the colorectum, K-ras gene mutation, p53 immunostaining and microsatellite instability (MSI) were analyzed for a comparison with 46 cases of colorectal carcinomas of the well or moderately differentiated type (well/mod). In addition, the mutations of simple repeated sequences in the transforming-growth-factor-beta type-II receptor (T beta R-II) gene and the BAX gene were analyzed as possible targets for DNA replication errors. Mutation of the K-ras gene in the por, sig and well/mod specimens was detected in, respectively, 22%, 11% and 48%, positive immunostaining for p53 in 41.8%, 28.6% and 60.3%, and MSI in 36%, 30% and 4%. Frameshift mutation of the T beta R-II gene was detected in 27.5% of the por and none of the sig specimens, while corresponding figures for mutation of the BAX gene were 15.7% and 0%. Significant differences between the por and well/mod tumors were found in the occurrence of K-ras mutation at codons 12 and 13, and MSI. Clinicopathologically, the tumor status of por with MSI was found to significantly correlate with the tumor's location in the proximal colon. In cases without MSI and sig, no frameshift mutation of either the T beta R-II or the BAX gene was found. These results suggest that poorly differentiated and signet-ring-cell carcinomas have a genetic background different from that of well or moderately differentiated carcinomas of the colorectum, and that DNA-replication error is at least partly involved in the carcinogenesis of these specific types of colorectal carcinomas.

Adenocarcinoma↗

PubMed1998

Defects in mismatch repair occur after APC mutations in the pathogenesis of sporadic colorectal tumours.

The roles of the intrinsic mutation rate and genomic instability in tumorigenesis are currently controversial. In most colorectal tumours, it is generally supposed that the first mutations occur at the adenomatous polyposis coli (APC) locus; APC mutations are thought to provide cells with a selective advantage but have no known effect on the mutation rate. It has also been suggested that genomic instability is the initiating event in colorectal tumorigenesis and, if this is true, mutations of DNA mismatch repair (MMR) genes (or at similar loci) are the most likely candidates. If defective MMR precedes APC mutations, the APC mutations of colon tumours with defective MMR and hence replication errors (RER+) should differ from those of RER- tumours, in at least three specific ways: (1) a higher frequency of allele loss at APC in RER- tumours; (2) more frameshift than nonsense mutations in RER+ tumours; and (3) APC mutations in simple repeat sequences [(N)n, (N1N2)n, or (N1N2N3)n] in RER+ tumours. We found no evidence that sporadic RER+ and RER- colon cancers (including cell lines) differ in any of these three ways. Although it remains possible that MMR is abnormal in tumours from HNPCC families before APC mutations occur, it is likely that in sporadic colon tumours, APC mutations, rather than genomic instability, are the initiating events in tumorigenesis.

Alleles↗

PubMed1996

Allelotypic and cytogenetic characterization of chemically induced mouse mammary tumors: high frequency of chromosome 4 loss of heterozygosity at advanced stages of progression.

Loss of heterozygosity (LOH) is one of the most common genetic abnormalities in cancer. To define the role of LOH and chromosomal abnormalities at various stages of mouse mammary cancer progression, we analyzed the allelotypes and karyotypes of primary mammary tumors induced in CD2F, mice by two basic protocols, the classical multiple-dose 7,12-dimethylbenz[a]anthracene (DMBA) protocol and a novel protocol of combined medroxyprogesterone acetate (MPA) and DMBA. The advantage of the latter protocol is that its latency for tumor development is much shorter and its tumor incidence is higher than those of DMBA alone. To study more advanced stages of mammary tumor progression, we also analyzed mouse mammary tumors that had acquired autonomous growth and were transplantable into syngeneic hosts. The allelotypic studies were performed by means of microsatellite length polymorphism analysis with a minimum of two simple-sequence repeat markers per chromosome. We observed that MPA-DMBA-induced mammary adenocarcinomas, which in general arose earlier because of the growth promotion exerted by MPA, did not show any significant LOH and were essentially diploid. Tumors induced by DMBA alone, which on average took longer to develop, showed a higher frequency of allelic losses. LOH on chromosome 11 was observed in 30% of the cases. Chromosomes 4 and 8 were affected in 25% and 20% of the tumors, respectively. Interestingly, advanced stages of mammary tumor progression, represented by transplantable mammary tumors, showed a much higher level of genomic instability, specifically a very high frequency (66%) of LOH on chromosome 4. These findings indicate that chromosome 4 harbors a gene whose inactivation may play a role in the acquisition of more aggressive characteristics such as autonomous growth and transplantation ability.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1999

Circular dichroism spectroscopy analysis of conformational transitions of a 54 base pair DNA duplex composed of alternating CGCGCG and TATATA blocks.

CD spectroscopy was used to analyze conformational properties of a self-complementary 54-mer DNA composed of alternating (CG)3 and (TA)3 hexamers. NaCl induced the Z-form in poly(GC), but the 54-mer remained the B-form under the same conditions. The B-Z transition was induced only after the addition of NiCl2. However, the Z-form was adopted by the whole molecule, not by the (CG)3 blocks alone. Two orders of magnitude higher concentrations of NiCl2 were required to induce the Z-form in poly(AT). The Z-form was also induced in poly(GC) by CsF that switched poly(AT) into the X-form, which seems to be a solution counterpart of D-DNA. Under these conditions the CD spectrum of the 54-mer was consistent with the (TA)3 blocks being in the X-form and the (CG)3 blocks in the B-form. At high concentrations of ethanol or trifluoroethanol, poly(AT) was an A-form, while poly(GC) adopted either Z-form, A-form or Z'-form. At the high trifluoroethanol concentrations the 54-mer cooperatively switched into a conformation whose CD spectrum was most consistent with the A-form in the (TA)3 blocks and the Z'-form in the (CG)3 blocks. This suggests that the base pairs are tilted in the Z'-form as in the A-form. The present article illustrates that CD spectroscopy can provide interesting pieces of information about conformational isomerizations and coexistence of multiple conformations in DNA molecules containing blocks of different simple sequence repeats.

Base Pairing↗

PubMed2001

No evidence for BCL10 mutation in endometrial cancers with microsatellite instability.

Previous reports have suggested that the mononucleotide repeats in BCL10 frequently are mutated in both hematologic malignancies and solid tumors. We set out to determine whether these repeats, like simple repeat sequences in other genes, are a target for mutation in endometrial cancers with defective DNA mismatch repair. Primary endometrial cancers (n = 42) and endometrial cancer cell lines (n=5) with microsatellite instability (MSI) were investigated. BCL10 exons 2 and 3 were amplified by PCR and evaluated for mutation using denaturing high-performance liquid chromatography (DHPLC) and single stand conformational variant (SSCV) analysis. Variants were directly sequenced. No BCL10 mutations were detected in exons 2 or 3 by DHPLC or SSCV. A polymorphism in exon 3 (638G-->A) was seen in 4/42 (9.5%) MSI-positive endometrial cancers and 0/5 MSI-positive endometrial cancer cell lines. Thus, mutation in the mononucleotide repeat tracts of BCL10 is not a feature of endometrial cancers with defective DNA mismatch repair.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Somatic Apc mutations are selected upon their capacity to inactivate the beta-catenin downregulating activity.

The APC gene, originally identified as the gene for familial adenomatous polyposis (FAP), is now considered as the true "gatekeeper" of colonic epithelial proliferation. Its main tumor suppressing activity seems to reside in the capacity to properly regulate intracellular beta-catenin signaling. Most somatic APC mutations are detected between codons 1286 and 1513, the mutation cluster region (MCR). This clustering can be explained either by the presence of mutation-prone sequences within the MCR, or by the selective advantage provided by the resulting truncated polypeptides. Here, a Msh2-deficient mouse model (Msh2(delta 7N) ) was generated and bred with Apc(1638N) and Apc(Min) that allowed the comparison of the somatic mutation spectra along the Apc gene in the different allelic combinations. Mutations identified in Msh2(delta 7N/delta 7N) tumors are predominantly dinucleotide deletions at simple sequence repeats leading to truncated Apc polypeptides that partially retain the 20 a.a. beta-catenin downregulating motifs. In contrast, the somatic mutations identified in the wild type Apc allele of Msh2(delta 7N/delta 7N) /Apc(+/1638N) and Msh2(delta 7N/delta 7N) /Apc(+/Min) tumors are clustered more to the 5' end, thereby completely inactivating the beta-catenin downregulating activity of APC. These results indicate that somatic Apc mutations are selected during intestinal tumorigenesis and that inactivation of the beta-catenin downregulating function of APC is likely to represent the main selective factor.

Adenomatous Polyposis Coli↗

PubMed1994

Genomic scan for genes predisposing to schizophrenia.

We initiated a genome-wide search for genes predisposing to schizophrenia by ascertaining 9 families, each containing three to five cases of schizophrenia. The 9 pedigrees were initially genotyped with 329 polymorphic DNA loci distributed throughout the genome. Assuming either autosomal dominant or recessive inheritance, 254 DNA loci yielded lod scores less than -2.0 at theta = 0.0, 101 DNA markers gave lod scores less than -2.0 at theta = 0.05, while 5 DNA loci produced maximum lod scores greater than 1: D4S35, D14S17, D15S1, D22S84, and D22S55. Of the DNA markers yielding lod scores greater than 1, D4S35 and D22S55 also were suggestive of linkage when the Affected-Pedigree-Member method was used. The families were then genotyped with four highly polymorphic simple sequence repeat markers; possible linkage diminished with DNA markers mapping nearby D4S35, while suggestive evidence of linkage remained with loci in the region of D22S55. Although follow-up investigation of these chromosomal regions may be warranted, our linkage results should be viewed as preliminary observations, as 35 unaffected persons are not past the age of risk.

Adolescent↗

PubMed1995

Polymerase chain reaction-based assays for the characterisation of plant genetic resources.

Plant genetic resources are an important component of biodiversity and provide the basic genetic variability that allow new and improved cultivars to be developed. Numerous germplasm collections have been established and it is important to established that such collections are representative and accessible to breeders and biotechnologists. Molecular markers provide the best estimate of genetic diversity since they are independent of the confounding effects of environmental factors. Assays based on the polymerase chain reaction (PCR) are considered to meet both the technical and genetical requirements for the characterisation of plant and animal genetic resources. Two main approaches are described, based on anonymous and defined primers. The use of both randomly amplified polymorphic DNA (RAPD) and microsatellites or simple sequence repeats (SSR) for the characterisation of perennial tree species, and distribution of variability within gene pools is reported. The detection of interspecific gene introgression between coffee species with RAPD markers is described together with the use of microsatellites to genotype potato. The use of PCR-based assays will facilitate the evaluation and utilisation of plant genetic resources.

Base Sequence↗

PubMed1998

Nonradioactive sequence-tagged microsatellite site analyses: a method transferable to the tropics.

Utilization of existing isozyme analysis facilities to detect sequence-tagged microsatellite site (STMS) polymorphism or any simple sequence repeat (SSR) variation is described. Different parameters concerning the difficulties in transferring molecular techniques to less sophisticated laboratory infrastructures (i.e. tropical outstations) are discussed (e.g. reproducibility, efficacy, precision). Nonradioactive STMS analysis is bound to foster collaborative research between "biodiversity" and "biotechnology" centers.

Base Sequence↗

PubMed2003

Loss of heterozygosity analysis of mouse pulmonary adenomas induced by coal tar.

Manufactured gas plant (MGP) residues, commonly known as coal tars, were generated several decades ago as a byproduct of residential and industrial gas production from the distillation of coal. Previous short-term exposure studies have shown MGP residues to be tumorigenic in mouse liver and lung. In order to gain further insight into carcinogenesis by complex mixtures of environmental chemicals containing known carcinogenic polycyclic aromatic hydrocarbons, we investigated mouse pulmonary tumors for loss of heterozygosity (LOH) as a result of multiple exposure to MGP residues. Twenty mouse lung adenomas produced in (C57BL/6 x C3H)F1 hybrid mice and manually microdissected were selected to examine genome-wide allelic losses at 58 microsatellite loci. Regions of chromosomes 1, 4, 5, 8, and 11 were affected in 30-40% of tumors. The elevated rates of allelic imbalance in these chromosomes may indicate the location of unknown tumor suppressor genes significant to neoplastic transformation in mouse lung tissues. Laser capture microdissection-based LOH analysis of pulmonary adenomas showed that contamination of nonneoplastic tissues was not masking the allelic losses in the manually microdissected tumor analysis. The low frequency of chromosome instability in these tumors, measured by means of inter-simple sequence repeat PCR, suggests the presence of discrete regions of LOH instead of extensive structural rearrangements.

Adenoma↗

PubMed2006

Functional polymorphisms in dopamine and serotonin pathway genes.

There is mounting evidence on the functional significance of single nucleotide and simple repeat sequence polymorphisms in both the coding and regulatory regions of genes in the monoamine neurotransmitter pathways. Many of these gene variants have been associated with human behavioral disorders and traits, and thus have important clinical relevance. This review summarizes the literature on the published functional studies from a molecular, cellular, and neurobiological perspective, and notes their possible behavioral consequences. Functional studies have adopted a variety of strategies. Pharmacological studies have focused on the effects of gene variation at the protein level in terms of binding to ligands or drugs. Other key investigations have determined effects on gene expression at the level of transcription in mammalian cell cultures, lymphoblasts, and/or human postmortem brain tissue. This has enabled the comparison of in vitro and in vivo data, and furthermore provides an improved perceptive of their respective advantages. Additionally, molecular biological approaches have identified transcription factors (DNA-binding proteins) that interact with the motifs within the polymorphisms themselves. Various neuroimaging studies have further determined the relationship of genotype with protein availability in the brain, and thus have contributed to our understanding of the in vivo functional significance of gene variants. Finally, there is growing evidence from both human and animal studies on the interaction of functional polymorphisms with the environment in determining a behavioral outcome. Taken together, these findings have contributed to a greater understanding of the plausible molecular mechanisms that underpin the functional significance of polymorphisms in monoamine neurotransmitter pathway genes, and how they may influence behavioral phenotypes.

Animals↗

PubMed2001

Microsatellite instability in squamous cell carcinoma of head and neck from the Indian patient population.

Genomic instability in simple repeated sequences has been observed in several human cancers. We have analyzed 50 squamous cell carcinomas of the head and neck (SCCHN) and 5 pre-malignant severe dysplastic tissues from Indian patient populations for microsatellite instability in 18 different loci spread over eight different chromosomes. Among the tumors analyzed, 45% exhibited instability at two or more loci, and 15% exhibited instability at 40% of the markers tested. Similar analysis of SCCHN tumors from other populations (British, American and French) showed much less frequency of instability. SCCHN tumors in the present study did not show any instability in the mononucleotide repeat sequences. There is also a clear distinction in the nature of the instability in these tumors in comparison with colorectal tumors. These results suggest that the underlying mechanism generating this type of instability is different from those reported for colorectal tumors.

Adult↗

PubMed2007

The impact of epidermal growth factor receptor gene status on gefitinib-treated Japanese patients with non-small-cell lung cancer.

We investigated the relationships between genetic factors and clinical outcome in Japanese non-small-cell lung cancer (NSCLC) patients treated with gefitinib. Ninety-eight NSCLC patients who had been treated with gefitinib, were screened for mutations in epidermal growth factor receptor (EGFR) exons 18-21, KRAS exon2, and polymorphisms including the CA simple sequence repeat in intron1 (CA-SSR1) and single nucleotide polymorphisms in the promoter region (-216G/T and -191C/A), using a PCR-based assay and direct sequencing. The EGFR copy number status was also evaluated using a fluorescence in situ hybridization assay. EGFR and KRAS mutations were found in 38 (38.8%) and 8 (8.2%) of the 98 patients, respectively. A high EGFR copy number status was identified in 31 (41.3%) of the 75 assessable patients. Drug-sensitive EGFR mutations limited to exon19 deletions and L858R were independent predictive factors of a stronger sensitivity to gefitinib (p = 0.0002), the overall survival (OS) (p = 0.0036), and prolonged progression-free survival (PFS) (p < 0.0001). The EGFR copy number status was not related to a sensitivity to gefitinib and prolonged OS and PFS. Regarding polymorphisms, patients with a short CA-SSR1 showed a prolonged OS as compared with those with a long length in patients with a drug-sensitive EGFR mutation, although this difference was not significant (p = 0.13). Thus, drug-sensitive EGFR mutations predict a favorable clinical outcome and a high EGFR copy number may not be related to clinical benefits in gefitinib-treated Japanese patients with NSCLC. Our findings also suggest that the CA-SSR1 length may influence the clinical outcome in patients with a drug-sensitive EGFR mutation.

Aged↗

PubMed1995

Utilization of ribonucleotides and RNA primers by Tetrahymena telomerase.

Telomerase is a ribonucleoprotein (RNP) DNA polymerase involved in telomere synthesis. A short sequence within the telomerase RNA component provides a template for de novo addition of the G-rich strand of a telomeric simple sequence repeat onto chromosome termini. In vitro, telomerase can elongate single-stranded DNA primers processively: one primer can be extended by multiple rounds of template copying before product dissociation. Telomerase will incorporate dNTPs or ddNTPs and will elongate any G-rich, single-stranded primer DNA. In this report, we show that Tetrahymena telomerase was able to incorporate a ribonucleotide, rGTP, into product polynucleotide. Synthesis of the product [d(TT)r(GGGG)]n was processive, suggesting that the chimeric product remained associated with the enzyme both at the active site and at a second, previously characterized, template-independent product binding site. As predicted by this finding, RNA-containing oligonucleotides served as primers for elongation. More than 3 nt of RNA at a primer 3' end decreased the quantity of product synthesis but increased the affinity of the primer for telomerase. Thus, RNA-containing primers were effective as competitive inhibitors of DNA primer elongation by telomerase. These results support the possible evolutionary origin of telomerase as an RNA-dependent RNA polymerase.

Animals↗

PubMed1993

Utility of a C-jun microsatellite marker in determining gene dosage for fatty (fa).

The Zucker fatty (fa) mutation provides a genetic model for obesity and non-insulin dependent diabetes mellitus. The molecular pathogenesis of the metabolic phenotype of these animals is not known. Detailed molecular maps of the region surrounding the fa locus on rat chromosome 5 can be used for positional cloning experiments as well as to permit genotyping of animals from appropriate crosses before the confounding metabolic effects of obesity have occurred. We describe the development of a polymerase chain reaction (PCR) assay for a polymorphic simple sequence repeat (SSR) in the promoter region of the protooncogene c-Jun. This assay was used to position c-Jun 4.5cM proximal to the fa locus in 111 F2 progeny of a 13MBN fa/+ F1 intercross. Concurrent use of the c-Jun SSR with a previously described assay for a microsatellite in the glucose transporter, Glut1, permits rapid and accurate assessment of genotypes at the fa locus in animals of any age using minimal amounts of DNA. A strategy is described which minimizes the error rate in assigning genotype at the fatty locus for backcross and intercross progeny.

Animals↗

PubMed1996

Linkage studies in alcohol-responsive myoclonic dystonia.

A large German family with "myoclonic dystonia with lightning jerks responsive to alcohol" was identified. Eleven affected pedigree members and six obligate gene carriers from five generations were identified. A description of one branch of this pedigree was published in 1964. Our examination 30 years after the initial report confirms the clinical syndrome of a nonprogressive movement disorder characterized by myoclonic jerks affecting the proximal muscles and the muscles of the trunk, accompanied by mild dystonic features in some affected family members. Segregation analysis favors autosomal dominant inheritance with high, but incomplete, penetrance in males and much lower penetrance in females. Linkage analysis was performed using simple sequence repeat polymorphisms (CA repeats) closely associated with or spanning the chromosomal regions containing 15 candidate genes: the gene for early-onset generalized torsion dystonia, DYT1 (chromosome 9q34); the genes for subunits alpha 2, beta 1, and gamma 1 (chromosome 4p12-4q13); for alpha 1, alpha 6, beta 2, and gamma 2 (chromosome 5q31.1-5q31.3); for alpha 4, alpha 5, beta 3, and gamma 3 (chromosome 15q11-15q13); for rho 1 and rho 2 (chromosome 6q14-6q21) of the gamma-aminobutyric acid A receptor; and for the alpha subunit of the glycine receptor (chromosome 5q31). By a combination of pairwise and multipoint linkage analysis, it could be excluded that any of these candidate gene-bearing chromosomal regions contain the disease gene in this family. We also excluded major portions of three chromosomal regions syntenic with mouse chromosome 3, which carries the murine beta subunit of the glycine receptor.

Adolescent↗

PubMed1996

The region responsible for stroke on chromosome 4 in the stroke-prone spontaneously hypertensive rat.

To detect genetic loci responsible for stroke susceptibility, we produced 107 male F2 progenies crossed between stroke-prone spontaneously hypertensive rats (SHRSP/Izm) and normotensive Wistar Kyoto rats (WKY/Izm) and followed them up until they developed cerebral stroke. One hundred and twenty-five simple sequence repeat (SSR) markers were analyzed in these F2 rats. Nine of 107 F2 rats suffered from macroscopically overt stroke. In these 9 rats, the segregation ratio of 3 genotypes at 6 SSR marker loci on chromosomes 2, 4, 9, and 10 was highly distorted from the expected value (the observed sp/sp:sp/wky:wky/ wky ratio was either 6:3:0 or 6:2:1, while the expected was 1:2:1, p < 0.01 by chi 2 test). Further, the brain weight was significantly heavier (p < 0.001) in the F2 rats suffering from stroke, suggesting that the brain weight was a parameter for stroke. The brain weight of F2 rats cosegregated with D4Mit19, D4Mgh7, and D4Mgh8 (p = 0.0015, 0.0014, and 0.0040 by ANOVA, respectively) on chromosome 4 supporting genetic effects of this genetic loci on the pathogenesis of cerebral stroke. Blood pressure did not cosegregate with these markers on chromosome 4. These results suggest that a region on chromosome 4, independently of hypertension, determines genetic susceptibility to cerebral stroke.

Animals↗

PubMed1998

Isolation of full-length cDNA of mouse PAX4 gene and identification of its human homologue.

Recent genetic studies have suggested that PAX4, a member of the paired box (PAX) gene family, is involved in the mechanism regulating the fate of pancreatic islet endocrine progenitor cells. Murine PAX4 was originally identified by genomic screening and, to date, only a partial sequence of PAX4 has been reported. In this study, we cloned the full-length cDNA of mouse PAX4 by RACE (rapid amplification of cDNA ends) using RNA from MIN6 cells, a mouse insulinoma cell line. The full length of cDNA was 1.38 kb, consistent with the estimated size of the transcript by Northern blot. The deduced mouse PAX4 protein was 349 amino acids and had the predicted molecular weight of 38 kDa. Two DNA binding motifs, a 128-amino acid paired domain and a 61-amino acid paired-type homeodomain exhibit the highest amino acid homology with PAX6 (71.2%, 65.0%, respectively), another member of the PAX gene family. However, the sequence of the C-terminal segment of PAX4 diverged and showed no significant homology with any other known PAX genes. As to the genomic DNA, the coding region of the mouse PAX4 gene spanned approximately 5.5 kb and was composed of 10 exons. In the public DNA database, a human cosmid (g1572c264), which was localized on human chromosome 7q31.3, was found to contain a gene homologous to PAX4. The nucleotide and protein sequence homologies between mouse PAX4 and its human homologue were 83.1% and 80.0%, respectively. Interestingly, the ARP5 (ADP-ribosylation factor 5) gene was also found in the same cosmid g1572c264, suggesting the ARP5 gene to be adjacent to the human PAX4 homologue. The human cosmid g1572c264 contains at least four SSRPs (simple sequence repeat polymorphism), which could be used for genetic linkage studies of the locus. The results of this study, i.e. isolation of the full-length cDNA sequence of PAX4 and identification of the homologous human gene, will facilitate further functional and genetic studies of the PAX4 gene.

Alternative Splicing↗