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Biomedical subjects

A Chenchik

Publications and source records attributed to A Chenchik.

16 recordsLinked to original sources
PubMed2004

Disease profiling arrays: reverse format cDNA arrays complimentary to microarrays.

The identification of differentially expressed genes enables us to understand the molecular mechanisms associated with disease, conditions of stress, drug treatments and developmental processes. Microarrays provide a powerful tool for studying these complex phenomena. Verification of differentially expressed genes and correlation with biological function, which is usually done by northern blot analysis, RNase protection assay or RT-PCR, is the bottleneck in all these protocols. We developed a new type of cDNA array for high-throughput expression profiling of multiple tissues and blood samples (i) for confirmation analysis of statistically significant number of clinical samples (ii) from limited amount of starting material, and (iii) with detailed clinical data from each individual. In contrast to traditional cDNA arrays, these arrays are spotted with a complex cDNA representing the entire mRNA message expressed in a given tissue or blood sample. cDNAs for these arrays were generated using SMART technology and accurately represent the original mRNA population, producing specific and quantitative signals during hybridization. cDNAs on Disease Profiling Arrays were derived from total RNAs of diseased and normal tissues or different blood fractions of patients. These cDNAs were spotted onto nylon membranes along with positive and negative controls. The arrays were then hybridized with gene-specific probes. Hybridization results revealed disease-related as well as patient-specific gene expression patterns between different disease types for these genes. These studies demonstrate that Disease Profiling Arrays are suitable for high-throughput studies comparing the relative abundance of a target gene, for simultaneously detecting differentially expressed genes in a wide variety of tissues and blood samples, and can be used down-stream from cDNA microarrays for confirmation analysis.

Autoimmune Diseases↗
PubMed2001

Gene expression profiling in postmortem Rett Syndrome brain: differential gene expression and patient classification.

The identification of mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MECP2) gene in Rett Syndrome (RTT) suggests that an inappropriate release of transcriptional silencing may give rise to RTT neuropathology. Despite this progress, the molecular basis of RTT neuropathogenesis remains unclear. Using multiple cDNA microarray technologies, subtractive hybridization, and conventional biochemistry, we generated comprehensive gene expression profiles of postmortem brain tissue from RTT patients and matched controls. Many glial transcripts involved in known neuropathological mechanisms were found to have increased expression in RTT brain, while decreases were observed in the expression of multiple neuron-specific mRNAs. Dramatic and consistent decreases in transcripts encoding presynaptic markers indicated a specific deficit in presynaptic development. Employing multiple clustering algorithms, it was possible to accurately segregate RTT from control brain tissue samples based solely on gene expression profile. Although previously achieved in cancers, our results constitute the first report of human disease classification using gene expression profiling in a complex tissue source such as brain.

Adolescent↗
PubMed2001

Use of SMART-generated cDNA for gene expression studies in multiple human tumors.

We demonstrate here that SMART PCR-amplified cDNAs arrayed on a nylon membrane are suitable for high-throughput tissue expression profiling when starting biological materials are limited. We show that SMART cDNA accurately reflects gene expression patterns found in total RNA by comparing the expression level of several target genes in SMART PCR-amplified cDNAs and their corresponding total RNAs. We also arrayed cDNAs from 68 matched tumor and normal samples on a nylon membrane to determine whether SMART PCR-amplified cDNA could be used for detecting differentially expressed genes in these tissues. These arrays containing normalized tumor and normal cDNAs were hybridized with probes for glutathione peroxidase and gelsolin. The hybridization results revealed cancer-related and patient-specific gene expression differences between tumor and normal tissues for these genes. These studies show that SMART PCR-amplified cDNAs maintain the complexity of the original mRNA population and are thus suitable for high-throughput studies to compare the relative abundance of target genes and to detect differentially expressed genes in a wide variety of tissues simultaneously.

DNA, Complementary↗
PubMed2001

Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction.

Here, we describe a fast, simple method for constructing full-length cDNA libraries using SMART technology. This novel procedure uses the template-switching activity of Moloney murine leukemia virus (MMLV) reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, three cycles of PCR are performed using a modified oligo(dT) primer and an anchor primer to enrich the cDNA population for full-length sequences. Starting with 1 microgram human skeletal muscle poly(A)+ RNA, a cDNA library was constructed that contained 3 x 10(6) independent clones with an average insert size of 2 kb. Sequence analysis of 172 randomly selected clones showed that 77% of cDNA clones corresponding to known genes contained intact open reading frames. The average length of complete open reading frames was 2.4 kb. Furthermore, 86% of the full-length clones retained longer 5' UTR sequences than the longest 5' end deposited in the GenBank database. cDNA libraries generated using this method will be useful for accelerating the collection of mRNA 5' end sequence information, which is currently very limited in GenBank.

Cloning, Molecular↗
PubMed2000

Specific pattern of p53 phosphorylation during nitric oxide-induced cell cycle arrest.

Nitric oxide (NO) is an efficient inhibitor of cell proliferation. Here we show that part of the antiproliferative activity of NO in fibroblasts is mediated through p53 signaling pathway. Cells from p53-/- knockout mice are compromised in their ability to stop dividing in the presence of NO. NO strongly induces expression of genes which are transcriptional targets of p53, and p53 is necessary for some, but not all, of the transcription activation effects of NO. Furthermore, NO strongly increases the cellular level of p53 protein. Since phosphorylation of particular residues of the p53 molecule has been correlated with its functional activity, we determined the phosphorylation pattern of p53 molecule after exposure to NO and compared it with the phosphorylation patterns that develop upon treatment with gamma-irradiation, UV light, and adriamycin. We found that NO induces a specific signature pattern of p53 phosphorylation, distinct from the patterns evoked by other inducers. This study suggests that NO activates specific signaling pathways that may partially overlap, but that do not coincide, with signaling pathways activated by other known inducers of p53 activity.

3T3 Cells↗
PubMed1999

cDNA expression array reveals heterogeneous gene expression profiles in three glioblastoma cell lines.

Tumor cell lines are an indispensable tool for cancer research. However, among cell lines of the same pathological group, heterogeneity has been detected in gene expression, gene mutation, and cellular response to various treatments. In this study, we systematically investigated the extent of heterogeneity of gene expression in three glioblastoma cell lines using cDNA array technology in which the expression of 588 cellular genes is studied simultaneously. Comparison of the expression profiles revealed substantial qualitative and quantitative heterogeneity. Among the 588 genes, 197 genes were expressed in all three lines and 56 genes were not expressed in any of the three lines; total of 222 genes were expressed in only two of the three cell lines, and 113 genes were expressed in only one of the three cell lines. These results provide molecular evidence that cell lines of the same pathological origin can be highly heterogeneous.

DNA, Complementary↗
PubMed1999

Amplification of cDNA ends based on template-switching effect and step-out PCR.

A new method for amplifying cDNA ends is described which requires only first-strand cDNA synthesis and a single PCR to generate a correct product with very low or no background. The method can be successfully applied to total RNA as well as poly A+ RNA. The same first-strand cDNA can be used to amplify flanking sequences of any cDNA species present in the sample.

DNA, Complementary↗
PubMed1999

RecA-mediated affinity capture: a method for full-length cDNA cloning.

We describe an improved method for rapid cloning of full-length cDNA from cDNA libraries. This approach is based on the ability of Escherichia coli RecA protein to form a stable nucleoprotein complex with a linear single-stranded DNA probe and homologous sequences in circular double-stranded DNA. Hybridization of RecA-coated biotinylated DNA probes to homologous plasmid DNA creates triple-stranded complexes, which are then captured on streptavidin-coated magnetic beads. Following magnetic separation of the hybrid molecules, the enriched plasmid population is recovered by alkaline treatment, precipitated, resuspended and used to transform bacteria. Typically, many clones can then be recovered by colony hybridization screening of a single plate of the enriched library. We have used this technology to clone full-length and alternatively spliced forms of the human bcl-xL cDNA from a human liver cDNA library.

Alternative Splicing↗
PubMed1997

Construction of cDNA libraries from small amounts of total RNA using the suppression PCR effect.

Here we describe a method for preparing high-quality cDNA libraries from total RNA. By this method, double-stranded (ds) cDNA ligated with a specially designed ds adaptor is amplified by PCR using a modified T-primer and another primer corresponding to the outer part of the adaptor. The suppression PCR effect strongly inhibits the amplification of poly(A) RNA, thereby reducing background. This method leads to amplification of high-quality cDNA, facilitating the construction of representative cDNA libraries from as little as 10-100 ng of total RNA.

DNA, Complementary↗
PubMed1996

Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate.

The major drawback of subtractive cDNA libraries is that the original disproportion in concentrations of different types of transcripts is preserved. This usually makes the isolation of specific rare transcripts extremely difficult. To overcome this difficulty, we propose a strategy that introduces the equalization of concentrations (normalization) of specific transcripts during the subtractive process. This makes possible obtaining both rare and highly abundant transcripts in the resulting subtracted library. This technique has been applied for isolation of transcripts activated upon induction of Jurkat cells by phytohemaglutinin and phorbol 12-myristate 13-acetate. Six novel up-regulated sequences belonging to a low-abundance class of transcripts have been obtained.

Blotting, Northern↗
PubMed1996

Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.

Base Sequence↗
PubMed1996

Full-length cDNA cloning and determination of mRNA 5' and 3' ends by amplification of adaptor-ligated cDNA.

An efficient cDNA amplification procedure is described for determining of the 5' and 3' ends of mRNAs and cloning full-length cDNAs. In this approach, a double-stranded (ds) adaptor is ligated to both ends of a library of ds cDNA by T4 DNA ligase. This adaptor-ligated ds cDNA is then used to selectively amplify 5'- or 3'-cDNA fragments by PCR with a combination of gene-specific and adaptor-specific primers. This is a unified method for 5' and 3' rapid amplification of cDNA ends (RACE) from the same adaptor-ligated ds cDNA template. A specially designed adaptor combines features of "vectorette PCR" and "suppression PCR" technologies that significantly reduce background during amplification. The application of "long and accurate PCR" (LA PCR) technology makes possible the amplification of large RACE products and full-length cDNAs with high fidelity to the original mRNA. We investigated efficacy and limitations of this PCR-based approach for cDNA cloning by amplification of 5'- and 3'-RACE fragments and full-length cDNAs of three members of the abundant human actin gene family (1.3-1.9 kb), the medium abundance transferrin receptor mRNA (5.0 kb) and the low-medium abundance insulin-like growth factor II receptor mRNA (9.1 kb).

Actins↗
PubMed1994

TaqStart Antibody: "hot start" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase.

The specificity and DNA yield of PCRs are often improved by the "hot start" technique and analogous methods. The intent of the approach is to eliminate or prevent the generation of nonspecific PCR templates that may be synthesized at ambient temperature prior to thermal cycling. Monoclonal antibodies (MAbs) raised in mice to the purified DNA polymerase of Thermus aquaticus (Taq) were selected for their ability to reversibly block polymerase activity. The MAbs, incubated with Taq DNA polymerase and added to PCR tubes at ambient temperature, yield specific DNA fragments upon amplification when using high numbers of temperature cycles and a very low copy number of target DNA in a complex DNA background. This approach, using the TaqStart Antibody, permits the preparation of reaction mixtures at ambient temperatures without the subsequent opening of reaction tubes, use of grease or waxes, or of degradative enzymes and deoxyribonucleotide analogs.

Antibodies, Monoclonal↗
PubMedDate not supplied

Characterization of cellular pathways involved in glioblastoma response to the chemotherapeutic agent 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) by gene expression profiling.

The effectiveness of chemotherapy for human cancers is limited by pharmacokinetic parameters such as variation in metabolism and is determined by the cellular response. In this work, we aimed to gain a more holistic understanding of the molecular basis of glioma response to the DNA-alkylating agent 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) by using a systematic approach: we investigated the expression of 588 genes with various cellular functions in a BCNU-resistant glioblastoma cell line and a BCNU-sensitive subline before and after treatment with BCNU. Our gene expression profiling revealed major differences in gene expression between these two cell lines, especially after treatment with BCNU. One striking example was that BCNU decreased the expression of six DNA-repair genes in sensitive but not in resistant cells. In sensitive cells, BCNU treatment resulted in the induction of two MAP kinase genes; this finding suggests that the specific response to BCNU in sensitive cells may involve the Jun kinase signal transduction pathway. After BCNU treatment, marked induction of tumor necrosis factor was detected only in sensitive cells, suggesting that tumor necrosis factor is a mediator of BCNU-induced cell death. Bcl-2 family members were not altered by BCNU in sensitive cells, suggesting that BCNU-induced cell death may be independent of the bcl-2 pathway. Results of the present study demonstrate that gene expression profiling may facilitate identification of cellular pathways associated with specific responses to chemotherapeutic agents and contribute to an understanding of the molecular basis of drug action.

Antineoplastic Agents, Alkylating↗