Two color hybridization analysis using high density oligonucleotide arrays and energy transfer dyes

High density oligonucleotide arrays (DNA chips) have been used in two color mutational analysis of the 3.43 kb exon 11 of the hereditary breast and ovarian cancer gene BRCA1 . Two color analysis allows competitive hybridization between a reference standard and an unknown sample, improving the performance of the assay. Fluorescein and phycoerythrin dyes werepreviously used due to their compatibility with a single line 488 nm excitation source. Here we show that an alternative dye combination, containing the energy transfer dye system phycoerythrin*cy5 along with phycoerythrin, provides more evenly matched signal intensities and decreased spectral overlap between the two fluorophores, while maintaining compatibility with a 488 nm excitation source.     

Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays

We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.     

Cellular gene expression altered by human cytomegalovirus: global monitoring with oligonucleotide arrays

Mechanistic insights to viral replication and pathogenesis generally have come from the analysis of viral gene products, either by studying their biochemical activities and interactions individually or by creating mutant viruses and analyzing their phenotype. Now it is possible to identify and catalog the host cell genes whose mRNA levels change in response to a pathogen. We have used DNA array technology to monitor the level of approximately 6,600 human mRNAs in uninfected as compared with human cytomegalovirus-infected cells. The level of 258 mRNAs changed by a factor of 4 or more before the onset of viral DNA replication. Several of these mRNAs encode gene products that might play key roles in virus-induced pathogenesis, identifying them as intriguing targets for further study.     

Combining the preparation of oligonucleotide arrays and synthesis of high-quality primers

Based on the oligomer-chip technology, oligonucleotide arrays were synthesized directly on polypropylene sheets by a modified phosphoramidite chemistry using beta-eliminating nucleobase-protecting groups in combination with a succinate solid-phase linker. This method decouples the oligonucleotide deprotection from the support cleavage procedure, in contrast to standard phosphoramidite chemistry. In addition to being reliable substrates for hybridization experiments, the arrays also serve as source for the isolation of individual oligonucleotides. Technically, this allowed for a direct control of the quality of the arrayed oligomers. The released compounds were sufficient in amount and purity to work without further purification in PCR and DNA-sequencing reactions, with the results being identical to controls with commercially obtained primer molecules. Consequences for oligomer-chip hybridization procedures, the applicability of such hybrid-function arrays in, for example, diagnostics or comparative biology, and developments toward parallel primer synthesis are discussed.     

Rapid analysis of allelic variants of the sheep PrP gene by oligonucleotide probes

A rapid method to determine the allelic variants of the sheep PrP gene was developed. DNA samples from 128 Suffolk sheep (39 rams and 89 ewes) were screened by using polymerase chain reactions and dot-blot hybridization with 32P-labeled nine allele-specific oligonucleotide probes corresponding to the polymorphic PrP codons 112, 136, 154 and 171. Three allelic variants of the PrP gene, PrP(MARQ), PrP(TARQ) and PrP(MARR), were found in the flocks. Among those variants, nearly half of the ewes had alleles of the 171-Arg variant that is closely associated with resistance to natural scrapie. Assessments of allelic mutations of the PrP gene may help to select the scrapie-resistant progenitors in the flocks.     

Development of an enzyme-labeled oligonucleotide probe for the cholera toxin gene

An alkaline phosphatase-conjugated 30-mer oligonucleotide probe was developed to detect the cholera toxin gene (ctx) in Vibrio cholerae O1. For rapid identification, V. cholerae O1 was grown on selective agar (thiosulfate-citrate-bile salts agar) or in alkaline peptone water and organisms were transferred directly to nylon membranes. Lysis of cells, denaturation of DNA, neutralization, and hybridization were carried out on the membrane. These procedures required only 3 h for completion. The results of the hybridization test with 88 clinical and 20 environmental isolates agreed almost exactly with the results of the immunological tests (anti-cholera toxin antibody-sensitized latex agglutination tests). The specificity of the probe was also tested with strains of enterotoxigenic Escherichia coli, V. cholerae non-O1, and Vibrio mimicus.     

Genomic organization and mutational analysis of KVLQT1, a gene responsible for familial long QT syndrome

BACKGROUND: Turcot's Syndrome is the association of multiple adenomatous polyps of the colon with a primary tumor of the central nervous system. We present the first reported case of Turcot's Syndrome in a patient with malignant ependymomas. Recent advances in the elucidation of the genetic basis for the hereditary forms of colon cancer have provided a clearer understanding of the etiology of Turcot's Syndrome. This new information is relevant to the neurosurgical community and provides updated guidelines in the diagnosis and management of patients with this complex disease process. RESULTS: Turcot's Syndrome is related to two distinct genetic errors. The first involves a germ-line mutation in the adenomatous polyposis coli (APC) gene, which is postulated to act as a tumor suppressor gene. The second is a germ-line defect in one of a group of genes responsible for DNA nucleotide mismatch repair. CONCLUSION: The elucidation of the gene defects responsible for the hereditary forms of colon cancer has provided a clearer understanding of the molecular basis of Turcot's Syndrome. Patients with hereditary forms of colon cancer and neurologic symptoms require immediate and thorough investigation because of their significantly increased risk of developing CNS tumors. Previously healthy patients diagnosed with a CNS tumor with a family history of adenomatous polyposis coli should undergo screening and surveillance colonoscopy as the CNS lesion may precede colonic symptoms. CNS screening guidelines for asymptomatic patients with adenomatous polyposis coli requires further risk analysis studies. All patients diagnosed with Turcot's Syndrome should be tested for the gene defect, including the CNS tumor tissue to provide further data on the genetic relationship between Turcot's Syndrome and the hereditary forms of colon cancer.     

Inactivation of the ATM gene in T-cell prolymphocytic leukemias

T-cell prolymphocytic leukemia (T-PLL) is a rare form of mature leukemia that occurs both in adults as a sporadic disease and in younger patients suffering an hereditary condition, ataxia telangiectasia (AT). The ATM gene, located in the 11q22-23 chromosomal region, is consistently mutated in AT patients. The strong predisposition of AT patients to develop T-PLL and the high frequency of T-cell leukemias/lymphomas observed in atm-deficient mice, together with the known functions of the ATM protein, led us to evaluate the ATM gene as a potential tumor suppressor gene involved in T-PLL. Paired leukemic and nonleukemic cells were obtained from a series of 15 patients suffering sporadic T-PLLs, allowing loss of heterozygosity (LOH) analysis. LOH of the 11q22-23 region was detected in 10 of these 15 cases (67%). The minimal deleted region was defined as an approximately 2.5 Mb interval that contained the ATM gene. No ATM rearrangement or biallelic deletion was detected by Southern blotting in the T-PLL series. However, in five T-PLLs with LOH of the 11q22-23 region, Western blot analysis showed either undetectable (3 cases) or decreased levels (1 case) of ATM protein, whereas ATM was present at high levels in cases without LOH. The protein truncation test (PTT) was then used to search for mutations in the ATM gene. Four mutations (1 nonsense, 2 aberrant splicings, and 1 missense) were detected in patients with LOH and none in patients without LOH of the region. The acquired character of these ATM mutations was demonstrated in three patients. Altogether, allelic ATM inactivations by large deletions or mutations were found in approximately two thirds of T-PLL. ATM is thus a tumor suppressor gene whose inactivation is a key event in the development of T-cell prolymphocytic leukemias.     

Biallelic mutations in the ATM gene in T-prolymphocytic leukemia

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by cerebellar ataxia, oculocutaneous telangiectasia, immune deficiency, genome instability and predisposition to malignancies, particularly T-cell neoplasms. The responsible gene, designated ataxia-telangiectasia mutated (ATM), was recently identified by positional cloning in the chromosomal region 11q22.3-23.1 (ref. 4, 5) ATM is 150 kb in length, consists of 66 exons and encodes a nuclear phosphoprotein of approximately 350 kDa (ref. 4-9). Although ATM is considered to be a tumorigenic factor in several human cancers, it has not yet been found mutated in tumors of non-AT patients. Given the marked predisposition of AT patients to develop neoplasms of the T-cell lineage, we analyzed a series of T-cell leukemias (T-prolymphocytic leukemia, or T-PLL) in non-AT patients in search of genomic changes associated with the development of this disease. Among the recurrent aberrations identified, deletion of the chromosome arm 11q was very frequent. Subsequent molecular cytogenetic analyses allowed us to define a small commonly deleted segment at 11q22.3-23.1 in 15 of 24 T-PLLs studied. Since this critical region contained ATM, we further analyzed the remaining copy of the gene in six cases showing deletions affecting one ATM allele. In all six cases, mutations of the second ATM allele were identified, leading to the absence, premature truncation or alteration of the ATM gene product. Thus, our study demonstrates disruption of both ATM alleles by deletion or point mutation in T-PLL, suggesting that ATM functions as a tumor-suppressor gene in tumors of non-AT individuals.     

Development of a technology for pressing high-density products using gas-draining methods

The factors that hinder the formation of high-density compacts by cold-pressing of powders are considered. The main factors are the gases present in the charge and the technological lubricant introduced to facilitate the work of the press mold. A procedure has been developed for calculating the efficiency of gas drainage from a mold and the pressure in gas pores in the compacts. Recommendations are made for increasing the density of compacts from iron powders by a single cold pressing.Scientific-Industrial FirmBakkonditsioner,Baku. Translated from Poroshkovaya Metallurgiya, No. 5–6, pp. 34–39, May–June, 1994.     

Loss of heterozygosity and mutational analysis of the PTEN/MMAC1 gene in synchronous endometrial and ovarian carcinomas

Mutations of the human putative protein tyrosine phosphatase (PTEN/MMAC1) gene at chromosome 10q23 have been found frequently in type I endometrial carcinomas. Endometrioid adenocarcinoma is the most frequent histology seen in patients with clinically determined synchronous endometrial and ovarian carcinomas. We report a high incidence of PTEN/MMAC1 mutations and 10q23 loss of heterozygosity (LOH) in patients with synchronous endometrial and ovarian carcinomas. Paraffin-embedded precision microdissected tumors were analyzed for 10 matched synchronous endometrial and ovarian cancers and 11 matched control metastatic endometrial cancers. Single-stranded conformation polymorphism analysis was used to screen for mutations in all tumors and corresponding normal lymphocyte DNA. LOH was determined using a panel of four microsatellite markers within the PTEN/MMAC1 locus. PTEN/MMAC1 mutations were found in 43% (9 of 21) of the endometrial cancers studied, similarly represented in the clinically synchronous group (5 of 10 or 50%) and the advanced metastatic group (4 of 11; 36%; P = 0.53). In two of the five cases of clinically synchronous cancers, identical or progressive PTEN mutations were found in both the endometrial and ovarian cancers, suggesting that the ovarian tumor is a metastasis from the endometrial primary. PTEN/MMAC1 mutations in the advanced endometrial cancers were similar in the corresponding metastases. In one case, the mutation was seen in only one of two metastatic lymph nodes. The LOH analysis demonstrated 55% LOH in at least one PTEN/MMAC1 marker. These findings suggest that the putative tumor suppressor gene PTEN/MMAC1 may be a viable molecular marker to differentiate synchronous versus metastatic disease in a subset of clinically synchronous endometrial and ovarian carcinomas.     

Sequence, exon-intron organization, transcription and mutational analysis of prnA, the gene encoding the transcriptional activator of the prn gene cluster in Aspergillus nidulans

Rate coding and temporal coding are two extremes of the neural coding process. The concept of a stationary state corresponds to the information processing approach that views the brain as a decision maker, adopts rate coding as its main strategy and endorses the single- or few neuron approach. If information derived from sensory stimulation is used to continuously update the brain's internal representation of the world, then neural codes may change with time through learning. As a consequence, the same spike sequence may be interpreted differently (or evoke a different behavior) later in the day. This non-stationary viewpoint is embodied in the representational model of brain function that stresses learning and plasticity and employs temporal coding in neural assemblies. We argue that the switching between quasi-stable brain states as a result of learning is more relevant than the neuronal patterns, and the correlations between them, that are found during stationary states. The neural code likely resides in the activity patterns that cause this state-switching.     

Sequence and mutational analysis of the devBCA gene cluster encoding a putative ABC transporter in the cyanobacterium Anabaena variabilis ATCC 29413

The devBCA gene cluster (dev for development), shown to be essential for envelope formation in heterocysts of Anabaena sp. strain PCC 7120, was identified in the gene bank of a second heterocyst-forming strain, Anabaena variabilis ATCC 29413. Sequence and structural organization of the three genes, encoding subunits of a presumptive ABC transporter, were nearly identical in both strains. Mutants of A. variabilis defective in the devA gene were constructed. As devA mutants of Anabaena 7120, A. variabilis mutants were unable to grow on N2 as sole nitrogen source due to incomplete differentiation of heterocysts.     

Identification of two novel LDL receptor gene defects in French-Canadian pediatric population: mutational analysis and biochemical studies

Familial hypercholesterolemia (FH) is at least twofold more prevalent in French Canadians from Québec than in most Western populations. Although our recent data confirmed this high frequency of heterozygous FH in our pediatric population with hypercholesterolemia, none of the five established molecular defects for the French-Canadian population was detected in 29% of the unrelated French-Canadian children characterized by a persistent increase in LDL (low density lipoprotein receptor) cholesterol and a positive parental history of hyperlipidemia (Assouline et al., 1995). To probe for new mutations, six of these molecularly undiagnosed children were investigated as index patients. By using single-strand conformation polymorphism analysis and DNA sequencing, two novel mutations were identified in two of these subjects: (1) 7-base pair (bp) duplication following nucleotide 681 (according to the cDNA sequence) in exon 4 (681ins7), which causes a frameshift, the introduction of a stop at codon 208, and premature chain termination, and (2) A to G change in exon 8 substituting a tyrosine for a cysteine at amino acid 354 (Y354C). A third subject carried the recently reported exon 10 mutation (Y468X), whereas the remaining three patients demonstrated various known polymorphisms with no effect on gene product. Rapid molecular assays were developed to detect the two new mutations as well as the Y468X mutation. Screening of our cohort showed heterozygosity in 1/88, in 2/88, and in 2/88 of patients for the 681ins7, the Y354C, and the Y468X mutations, respectively.     

Immunoglobulin VH gene mutational analysis suggests that primary effusion lymphomas derive from different stages of B cell maturation

Primary effusion lymphoma (PEL) is a recently described distinct subtype of non-Hodgkin's lymphoma associated with infection by the Kaposi's sarcoma-associated herpesvirus, also called human herpesvirus-8. Most cases of PEL are also associated with the Epstein-Barr virus (EBV). In order to better characterize the cellular origin of PEL, we investigated the immunoglobulin (Ig) heavy chain variable region (VH,) genes expressed by tumor cells of the BC-1 and BC-3 cell lines derived from PELs and five original PEL specimens. In the six EBV-positive PELs examined, including the BC-1 cell line, the expressed VH gene sequences showed numerous point mutations relative to the putative germline VH gene sequences. In addition, the VH, segment of one of these cases showed intraclonal sequence heterogeneity, indicating ongoing somatic mutation. In five cases, the distribution and type of mutations indicated that tumor cells had been selected by antigen. Because somatically mutated Ig genes are expressed by B cells that have reached a germinal center/post-germinal center stage of development, these findings suggest that the PEL cell of origin is a germinal center or post-germinal center B cell in most cases. In contrast, the VH gene segment expressed by tumor cells of the BC-3 cell line, which was originated from an EBV-negative PEL obtained from an HIV-negative patient, was unmutated, suggesting a pre-germinal center B cell origin for tumor cells of this particular PEL cell line. Taken together, these findings suggest that development of PELs may not be restricted to one stage of B cell differentiation and may represent transformation of B cells at different stages of ontogeny.     

Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long-distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li-Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.