Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have determined the frequency of EWS fusion transcripts in a series of primary Ewing's sarcomas and peripheral primitive neuroectodermal tumors and cells lines. Type 1 and 2 EWS-Fli1 fusions were demonstrated in 8 cell lines and 14 patient samples. Five patients with cytogenetically characterized rearrangements of chromosome 22 that did not involve chromosome 11 were included in these studies. A novel EWS-Fli1 in-frame isoform fusing EWS to exon 8 of Fli1 was isolated from a tumor with a variant t(12;22;22)(q14;p1;q12) translocation. Three in-frame isoforms of a novel hybrid transcript derived from the fusion of EWS with the ETS domain of the human erg gene were identified in patient samples and a cell line with cytogenetically unidentified or cryptic translocations involving chromosomes 21 and 22. Interphase analysis by fluorescent in situ suppression hybridization using two overlapping erg yeast artificial chromosome clones demonstrated disruption of the erg gene on chromosome 21 in a patient sample with monosomy 22. Our results provide new information about the involvement of EWS in small round cell tumors involving exchange of its putative RNA-binding domain with DNA-binding domains derived from different members of the ETS family of transcription factors. These studies emphasize the utility of reverse transcriptase PCR analysis and fluorescent in situ hybridization as additional diagnostic tools for differential diagnosis among small round cell tumors.
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PMID:EWS-erg and EWS-Fli1 fusion transcripts in Ewing's sarcoma and primitive neuroectodermal tumors with variant translocations. 804 Mar 1

The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of > 6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-point sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS.
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PMID:Differential splicing of human androgen receptor pre-mRNA in X-linked Reifenstein syndrome, because of a deletion involving a putative branch site. 812 58

A cDNA for integrin beta 3 isolated from a human erythroleukaemia (HEL) cell library contained a 340 bp insert at position 1281. This mRNA, termed beta 3c, results from the use of a cryptic AG donor splice site in intron 8 of the beta 3 gene, and is different from a previously described alternative beta 3 mRNA. The predicted open reading frame of beta 3C stops at a TAG stop codon 69 bp downstream from position 1281. It starts with the signal peptide and the 404 N-terminal extracellular residues of beta 3, encompassing the ligand binding sites, followed by 23 C-terminal intron-derived residues, corresponding to a truncated form of beta 3 lacking the cysteine-rich, transmembrane and cytoplasmic domains. Expression of beta 3C mRNA was demonstrated in human platelets, megakaryocytes, endothelial cells and HEL cells by reverse transcriptase/PCR. The beta 3C transcript was also demonstrated in the mouse, suggesting its conservation through evolution. Finally, a 60 kDa polypeptide corresponding to the beta 3C alternative transcript was demonstrated in platelets by Western blotting using a polyclonal antibody raised against a synthetic peptide designed from the beta 3C intronic sequence. Taken together, these results suggest a biological role for beta 3C, the first alternative transcript showing an altered extracellular domain of a beta integrin.
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PMID:A new alternative transcript encodes a 60 kDa truncated form of integrin beta 3. 819 53

Duchenne and Becker muscular dystrophies (DMD and BMD) are allelic X-linked disorders arising from mutations in the (2.4 Mb) dystrophin gene at Xp21. We have applied the reverse transcriptase-polymerase chain reaction (RT-PCR) to identify a larger than normal dystrophin mRNA from a male with Duchenne muscular dystrophy and his younger affected brother. The increased size of the dystrophin mRNA was due to a splice-site mutation at the exon 26:intron 26 junction where a T to G substitution prevented normal RNA processing. A cryptic splice-site, downstream of the mutation, was activated during processing, resulting in the inclusion of 117 bases of intron 26. This insertion introduced an in-frame stop codon into the mature dystrophin mRNA. An allele-specific test was developed to identify the mutation and was applied to this family. Interestingly, the mother of the two affected boys did not carry the mutation, as determined by allele-specific amplification and direct DNA sequence analysis, indicating gonadal mosaicism. Her eldest daughter, designated as a carrier based upon conventional testing and haplotype analysis, also did not carry the family mutation. Initial haplotyping of the family appeared to be straightforward with gonadal mosaicism becoming evident only after allele-specific analysis. The application of linked markers to identify the disease allele for conventional genetic counselling would have been erroneous in this family and highlights the diagnostic power of precise identification of the disease-causing mutation.
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PMID:Identification of a point mutation and germinal mosaicism in a Duchenne muscular dystrophy family. 819 94

Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence.
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PMID:Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells. 833 36

Despite its rarity by routine karyotypic analysis, cryptic t(12;21)(p12-13;q22) translocation leading to TEL/AML1 fusion has been recognized as the most frequent genetic rearrangement in childhood acute lymphoblastic leukemia (ALL) in two recent studies, one from France and the other from the United States. To estimate the frequency of this abnormality in the Chinese population, we studied 41 children with ALL and 17 with acute myeloid leukemia (AML) in two medical centers in Taiwan, using the reverse transcriptase polymerase chain reaction (RT-PCR) assay. Results of this analysis demonstrated a 17% frequency of this translocation in the ALL population overall and 19% in patients with B-lineage ALL, similar to previous findings in Caucasian children. None of the patients with AML had TEL/AML1 fusion transcripts. In addition to its association with the B-lineage immunophenotype, TEL/AML1 was also correlated with a low presenting leukocyte count and favorable age (1-10 years). These findings, combined with earlier reports, indicate that TEL/AML1 fusion is the most frequent genetic abnormality in childhood ALL, regardless of race. Molecular diagnosis of t(12;21)-positive ALL may identify a subgroup of patients who do not require intensive treatment for cure.
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PMID:High incidence of TEL/AML1 fusion resulting from a cryptic t(12;21) in childhood B-lineage acute lymphoblastic leukemia in Taiwan. 866 57

A cryptic splice donor site in the first intracytoplasmic (IC) exon of the murine Fc gamma RIIB gene generates a previously unknown Fc gamma RIIB isoform. Three membrane Fc gamma RIIB polypeptides of 37, 32, and 30 kDa were immunoprecipitated by Fc gamma RIIB-specific Abs, and three Fc gamma RIIB cDNAs of 1071, 987, and 930 bp were amplified by reverse transcriptase-PCR with Fc gamma RIIB-specific oligonucleotides from the mastocytoma cells P815. The 1071-bp cDNA contains all sequences of IC exons and encodes the 37-kDa Fc gamma RIIB1 isoform. The 930-bp cDNA lacks sequences of the first IC exon and encodes the 30-kDa Fc gamma RIIB2 isoform. The 987-bp cDNA has an 84-nucleotide deletion of the first IC exon 3' sequences. When stably transfected in the lymphoma B cells IIA1.6, or in the mast cells RBL-2H3, this cDNA encoded 32-kDa Fc gamma RIIB whose biologic properties were undistinguishable from those of Fc gamma RIIB1: they inhibited B cell activation when coaggregated to B cell receptors and capped when aggregated at 37 degrees C, but failed to mediate endocytosis or phagocytosis. Sequences responsible for capping and inhibition of internalization, previously assigned to sequences encoded by the first IC exon, can thus be mapped in the 19 N-terminal residues. These residues are highly conserved in human Fc gamma RIIB1. The 87-bp first IC exon of the human gene ends by a single splice site in the downstream intron and encodes a 19-amino acid insertion. The 32-kDa Fc gamma RIIB is the murine homologue of human Fc gamma RIIB1. We propose to name it Fc gamma RIIB1'. Fc gamma RIIB1' was expressed in myeloid and lymphoid cell lines, in normal spleen cells, and in resting or LPS-activated B cells.
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PMID:Identification, molecular cloning, biologic properties, and tissue distribution of a novel isoform of murine low-affinity IgG receptor homologous to human Fc gamma RIIB1. 868 14

In human foamy virus (HFV) the reverse transcriptase is expressed independently of the Gag protein as a 127-kDa Pol precursor molecule. Evaluating the mechanism of Pol expression we identified a spliced mRNA which uses the main 5' splice donor and a splice acceptor site located in the gag gene. The significance of this spliced transcript for HFV Pol expression was studied by constructing a virus with a mutated splice acceptor site. This virus was unable to express detectable Pol proteins after transient transfection. Replication of the mutant was studied by a sensitive assay based on HFV transactivator-stimulated expression of an integrated lacZ gene under control of the HFV long terminal repeat. Whereas in the first 2 weeks after transfection the mutant replicated 3 to 5 order of magnitude less well than wild-type virus, extracellular titers obtained thereafter were similar to those of wild-type virus. This increase in replication competence was accompanied by a reversion of the mutated splice acceptor site. The results underlined the importance of the spliced pol transcript for HFV replication and pointed to a second mechanism of Pol expression. Indicator gene assays suggest that this other mechanism is likely to be a transactivator-dependent cryptic promoter in the gag gene which gives rise to Pol-encoding transcripts.
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PMID:Expression of human foamy virus reverse transcriptase involves a spliced pol mRNA. 886 27

Previously we created two strains of factor VIII-deficient mice by insertion of a neo gene into (1) the 3' end of exon 16 and (2) exon 17 of the factor VIII gene. Affected mice of both strains have no plasma factor VIII activity, yet are healthy with no spontaneous bleeding. Factor VIII-deficient females bred with affected males survive pregnancy and delivery. We used reverse transcriptase-polymerase chain reaction of liver RNA to characterize factor VIII mRNA processing. Factor VIII mRNA of the exon 16 knockout strain contains neo sequences plus 17 bp of intron 16 due to use of a cryptic donor site in intron 16. All factor VIII mRNA of the exon 17 knockout strain lacks exon 17 and neo sequences. In skipping exon 17, the intron 16 donor site or a cryptic donor site 46 bp 3' to the intron 16 donor site are used. Thus, factor VIII deficiency in exon 16 knockout mice is due to truncated protein, while in exon 17 knockout mice it is due to either truncated or partially deleted protein. After immunizing exon 16 knockout mice with human recombinant factor VIII, two monoclonal antibodies were obtained that recognize < 100 pg of mouse factor VIII light chain. Assay of cryoprecipitate from the plasma of affected mice failed to show factor VIII light chain.
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PMID:Further characterization of factor VIII-deficient mice created by gene targeting: RNA and protein studies. 889 9

The recurrent (12;21)(p13;q22) translocation fuses the two genes TEL and AML1 that have previously been cloned from translocation breakpoints in myeloid leukemias. Using mainly reverse transcriptase-polymerase chain reaction (RT-PCR), the TEL-AML1 chimeric transcript has been observed in 22-27% of pediatric patients with acute lymphoblastic leukemia (ALL), in particular in the early B-lineage ALL subtype, making it the most common genetic lesion in these patients. The vast majority of acute myeloid leukemias, other ALL subtypes and even adults with early B-lineage ALL were TEL-AML1-negative. We determined whether the TEL-AML1 fusion gene can also be observed in continuous human leukemia cell lines with an early B-lineage phenotype. Twenty-nine such cell lines established from children (n = 13) or adults (n = 13) with early B-lineage ALL and five cell lines derived from chronic myeloid leukemia in blast crisis or B cell non-Hodgkin's lymphoma were investigated for the occurrence of the TEL-AML1 rearrangement by RT-PCR. While all 13 adult early B-lineage ALL cell lines and the five cell lines from other leukemias or lymphomas were negative, 1/13 pediatric cell lines (cell line REH) was found to be positive for TEL-AML1; though neither reciprocal AML1-TEL, nor normal TEL, mRNA was detectable by RT-PCR in this cell line. These findings agreed with the results of conventional cytogenetic and FISH analysis of REH which was found to carry the der(21) partner only of t(12;21)(p13;q22), probably resulting from a complex translocation, t(4;12;21;16)(q32;p13;q22;q24.3). Hybridization with flanking cosmid clones (179A6 and 148B6), covering exons 1 and 8 respectively of TEL, confirmed a rearrangement accompanying the t(12;21), and showed cryptic deletion of the residual allele resulting from an apparently reciprocal t(5;12)(q31;p13). These findings in REH provide a further example of, and possible cytogenetic mechanism for, the paradigm of TEL-AML1 fusion accompanied by deletion of the residual TEL allele. The low rate of early B-lineage ALL cell lines carrying this translocation contrasts clearly with the relative high frequency of TEL-AML1-positive cases in primary material. It is possible that expression of the fusion product hampers the in vitro growth and establishment in culture of such leukemic cells. Nevertheless, the cell line REH represents a powerful tool for the further molecular characterization of this unique breakpoint and can serve as a positive control in routine PCR reactions.
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PMID:Occurrence of TEL-AML1 fusion resulting from (12;21) translocation in human early B-lineage leukemia cell lines. 906 87


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