Terminal exon definition occurs cotranscriptionally and promotes termination of RNA polymerase II.
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Analysis of nascent transcription from the human epsilon- and beta-globin genes shows that transcriptional termination occurs within 1.5 kb of the poly(A) site and is dependent on the presence of functional poly(A) signals. Even so, transcripts that have not been cleaved at the poly(A) site are detected up to the termination region, suggesting that there is a kinetic lag between transcription over the poly(A) signal and its effect on transcriptional termination. Surprisingly, mutation of the splice acceptor (SA) of the beta-globin IVS2 also abolishes transcriptional termination. Our results emphasize the interconnection of transcription and RNA processing by showing that the enhancement of 3' end processing by the terminal splice acceptor occurs cotranscriptionally. (+info)
Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells.
(42/18626)
A PCR strategy is described for global amplification of DNA from a single eukaryotic cell that enables the comprehensive analysis of the whole genome. By comparative genomic hybridization, not only gross DNA copy number variations, such as monosomic X and trisomic 21 in single male cells and cells from Down's syndrome patients, respectively, but multiple deletions and amplifications characteristic for human tumor cells are reliably retrieved. As a model of heterogeneous cell populations exposed to selective pressure, we have studied single micrometastatic cells isolated from bone marrow of cancer patients. The observed congruent pattern of comparative genomic hybridization data, loss of heterozygosity, and mutations as detected by sequencing attests to the technique's fidelity and demonstrates its usefulness for assessing clonal evolution of genetic variants in complex populations. (+info)
Engagement of natural cytotoxicity programs regulates AP-1 expression in the NKL human NK cell line.
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NK cell cytotoxicity is a fast and efficient mechanism of target cell lysis. Using transcription analysis, such as multiplex messenger assays, we show here that natural cytotoxicity exerted by the human NKL cell line correlates with mRNA accumulation of very early activator protein (AP)-1 transcription factor genes such as JunB, FosB and c-Fos. In addition, DNA-binding activities of Jun-Fos heterodimers were observed by electrophoretic mobility shift assays during the course of natural cytotoxicity. Interaction between immunoglobulin-like transcript-2/leukocyte Ig-like receptor 1 on NKL cells and HLA-B27 on target cells leads to an impairment of NKL natural cytotoxicity, which correlates with an absence of JunB, FosB, and c-Fos transcription, as well as an absence of their DNA-binding activity. Our studies thus indicate that, despite the rapidity of NK cell-mediated lysis, AP-1 transcription factor is activated during the early stage of NK cell cytolytic programs and that engagement of NK cell inhibitory receptors for MHC class I molecules impairs the very early activation of AP-1. (+info)
Comparative genomic hybridization detects many recurrent imbalances in central nervous system primitive neuroectodermal tumours in children.
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A series of 23 children with primitive neuroectodermal tumours (PNET) were analysed with comparative genomic hybridization (CGH). Multiple chromosomal imbalances have been detected in 20 patients. The most frequently involved chromosome was chromosome 17, with a gain of 17q (11 cases) and loss of 17p (eight cases). Further recurrent copy number changes were detected. Extra copies of chromosome 7 were present in nine patients and gains of 1q were detected in six patients. A moderate genomic amplification was detected in one patient, involving two sites on 3p and the whole 12p. Losses were more frequent, and especially involved the chromosomes 11 (nine cases), 10q (eight cases), 8 (six cases), X (six patients) and 3 (five cases), and part of chromosome 9 (five cases). These recurrent chromosomal changes may highlight locations of novel genes with an important role in the development and/or progression of PNET. (+info)
Mapping of novel regions of DNA gain and loss by comparative genomic hybridization in esophageal carcinoma in the Black and Colored populations of South Africa.
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Esophageal cancer (EC) is the leading cause of cancer death in the Black male population in South Africa. Although several oncogenes and tumor suppressor genes have previously been found altered in this cancer, many novel genes remain to be identified. To identify the chromosomal location of these unknown genes, we have analyzed DNA of 29 South African EC patients by comparative genomic hybridization. Frequent loss occurred at chromosome 1p (52%), 4p (52%), 18q (48%), 19p (52%), 19q (55%), and 22q (41%). The most common gains were detected at 1q (41%), 2q (52%), 3q (72%), 5p (31%), 7p (48%), 7q (45%), 8q (55%), and Xq (69%). High level amplification was detected at 2q24-33, 6p21.1-q14, 7p12-q21, 7q11.2-31, 8q22-24, 8q13-qter, 13q21-34, and at 13q32-34. The present comparative genomic hybridization study opens the way for additional targeted studies on these particular chromosomal regions to identify the specific genes involved in the higher susceptibility to specific subtypes of esophageal carcinoma in different geographical regions. The loss of 8p (28%) and Xp (17%) in tumors of male individuals may provide clues to the basis of the sex-biased frequency of occurrence of EC favoring men. (+info)
Construction and sequence analysis of subtraction complementary DNA libraries from human preimplantation embryos.
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PURPOSE: Because stage-specific genetic expression in human preimplantation development is not sufficiently studied, we have undertaken the construction of a subtraction complementary DNA (cDNA) library enriched for transcripts specific for human blastocysts. METHODS: For this purpose individual pools of cDNAs synthesized from four hatched blastocysts and three cleaving 8- to 10-cell embryos were exposed to suppression subtractive hybridization to minimize the presence of transcripts of housekeeping genes and other genes of maternal origin known to be expressed earlier in preimplantation development. Random clones of this library were sequenced and analyzed using the BLAST algorithm. RESULTS: The resulting subtraction library had a complexity of 3 x 10(5) and an average size of inserts of about 0.8 kb. Sequencing of random library clones revealed the following human genes: CD9 antigen, fatty acid binding protein, ferritin heavy chain, amyloid precursor, MAP kinase messenger RNAs, DNA clone 127H14, messenger RNA for diacylglycerol kinase, a sequence homologous to C1 inhibitor, messenger RNA for the KIAA0145 gene, and others. CONCLUSIONS: The presence of these genes in human preimplantation development suggests expression specific to the blastocyst stage. (+info)
Prospective comparison of the Gen-probe PACE 2 assay and the Abbott ligase chain reaction for the direct detection of Chlamydia trachomatis in a low prevalence population.
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In a prospective study, the Gen-Probe PACE 2 (GP) assay was compared with Abbott Laboratories' ligase chain reaction (LCR) assay for the detection of Chlamydia trachomatis. A total of 493 female patients consented to collection of two cervical samples; a first-void urine (FVU) sample was collected also from 446 of the participants. Cervical samples were tested by both GP and LCR; 16 samples (3.1%) tested positive by both methods and no discrepant results were observed. All but one of the FVU samples collected from patients with a positive cervical sample was positive for C. trachomatis by LCR. The stability of FVU samples over time in the LCR test was also evaluated and proved to be significantly longer than the 4 days stated by the manufacturer. While LCR proved to be highly sensitive in detecting chlamydial infection in FVU samples, no difference was noted between LCR and GP in the detection of cervical C. trachomatis infection in this study population. (+info)
The mechanism of intrinsic transcription termination.
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In bacteria, an intrinsic transcription termination signal appears in RNA as a hairpin followed by approximately eight uridines (U stretch) at the 3' terminus. This signal leads to rapid dissociation of the ternary elongation complex (TEC) into RNA, DNA, and an RNA polymerase. We demonstrate that the hairpin inactivates and then destabilizes TEC by weakening interactions in the RNA-DNA hybrid-binding site and the RNA-binding site that hold TEC together. Formation of the hairpin is restricted to the moment when TEC reaches the point of termination and depends upon melting of four to five hybrid base pairs that follow the hairpin's stem. The U stretch-induced pausing at the point of termination is crucial, providing additional time for hairpin formation. These results explain the mechanism of termination and aid in understanding of how cellular factors modulate this process. (+info)