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)

The haploid nucleus of a human cell contains 3 X 10(9) base pairs. Organized in linear duplex, this DNA would stretch out to a length of some 90 cm. Thus, organization of chromosomes has been a major subject for pioneer cytogenetists. Long lasting controversies on the strandedness of chromosomes, together with newly developed banding techniques, led us to molecular cytogenetics. Next, the discovery of reverse transcriptase, restriction endonucleases, and other recombinant DNA methods have enabled us to isolate and characterize genes from any organism and to determine the DNA sequences and any encoded protein sequences. These new technologies have already helped us to understand many inherited diseases at a molecular level. In sickle cell anemia, thalassemia and in other mendelian disorders we can know their molecular defects by examining the DNA from peripheral leukocytes, without the need for complex biochemical assays or biopsies. Southern blot analysis using restriction endonuclease and a probe is a basic tool for molecular diagnosis. cDNA or DNA fragments are used as probes. Recently, synthesized oligonucleotide probes are available, if the DNA sequence of a gene is determined. In addition, restriction fragment length polymorphisms (RFLPs), play a very important role in the molecular diagnosis. Linkage analysis using RFLPs linked to the gene locus of a certain disease also permits the detection of the patients and carriers within families with genetic diseases of unknown cause. Starting with the genetic map and physical map, genes for cystic fibrosis and Duchenne muscular dystrophy have recently been isolated and cloned.
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PMID:[Recent advances in human molecular genetics]. 197 24

PCR amplification has enabled a variety of studies to be performed on the murine dystrophin transcripts. Figure 7.12 displays a summary of the features of the murine dystrophin mRNA that have been described in this article. The location of the mutation in the original mdx mouse is indicated, as are the different spliced forms of the dystrophin transcript. Also shown are the location of various PCR primer binding sites that were used to deduce the alternative splicing pattern of the gene. It is likely that conventional cloning efforts aimed at identifying the variety of dystrophin spliced forms would have taken years to perform, particularly since several of the isoforms are expressed at levels significantly below the estimated 0.02% of total mRNA that dystrophin represents in skeletal muscle (Hoffman et al., 1987a, b). Amplification of dystrophin mRNA simplifies scanning methods for the identification of DNA sequence variations. Attempts to re-isolate and sequence the 14 kb cDNA to determine the mutation in separate strains of mdx mice are not likely to be time or cost effective. PCR enables these types of questions to be answered in a relatively short period of time, and similar types of analyses can be applied to human DMD tissues. Knowledge of the transcript diversity displayed by the dystrophin gene will enable the role of these separate isoforms to be addressed. Despite considerable effort by a variety of laboratories over the last five years, the precise functional role played by dystrophin remains unclear, and it can only be assumed that the separate isoforms act to modulate the functional role of dystrophin in separate tissues or in response to differing physiological states. PCR amplification of the dystrophin isoforms has enabled the variable regions of the transcript to be subcloned (Bies et al., 1992). These clones have been used to reintroduce the variable regions into full-length mini-gene expression vectors, which are currently being tested for functional activity through the generation of transgenic mdx mice. The transgenic mice can be easily identified through the PCR-ASO assays described in this article, and the reverse transcriptase PCR assays will enable a detailed analysis of the expression pattern of the introduced mini-genes. It is hoped that such analyses will further attempts to determine the feasibility of using gene therapy as a treatment for DMD/BMD.
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PMID:PCR analysis of muscular dystrophy in mdx mice. 811 39

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

Dp71, a C-terminal isoform of dystrophin, has been identified as the major DMD gene product in many nonmuscle tissues. In this report, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to clone and characterize four alternatively spliced Dp71 transcripts from cultured human amniocytes. The cDNAs encoding these Dp71 transcripts were shown to be alternatively spliced for exons 71 and/or 78. RT-PCR analysis also revealed that Dp71 transcripts alternatively spliced for exons 71 and/or 78 were expressed at varying levels in a number of adult human tissues, including muscle, heart, brain, kidney, lung, testis and liver. To investigate size heterogeneity at the translational level, Dp71 cDNAs isolated from amniocytes were expressed in E.coli to generate recombinant Dp71 fusion proteins. These fusion proteins were identified on immunoblots using antibodies specific for the C-terminal sequences of dystrophin that either included (antibody 1461) or excluded exon 78 (antibody 462B). The molecular masses of the Dp71 fusion proteins ranged from 71-75 kDa on SDS-PAGE, consistent with their predicted values. Immunoblot analysis using antibodies 1461 and 462B identified multiple Dp71 isoforms of approximately 70-75 kDa on SDS-PAGE in total protein lysates from amniocytes and various adult human tissues. This variation in molecular mass is consistent with the expression of Dp71 isoforms derived from transcripts alternatively spliced for exons 71 and/or 78. Total protein lysates from normal skeletal muscle, DMD muscle, amniocytes and brain were shown to contain beta-dystroglycan, a component of the dystrophin-associated glycoprotein complex (DGC).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cloning and characterization of alternatively spliced isoforms of Dp71. 854 29

We investigated the profiles of cytokine mRNA expression in muscle in 15 cases of inflammatory myopathy (IM) (5 each of polymyositis, inclusion body myositis, and dermatomyositis) and in 10 controls (5 of Duchenne dystrophy and 5 non-weak subjects). Expressions of the predominantly T cell-derived cytokines (interleukin (IL)-2, IL-4, IL-5, and interferon-gamma (IFN-gamma), of the predominantly macrophage-derived cytokines (IL-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha)), as well as cytokines that can be of either T cell or macrophage origin (granulocyte-macrophage colony stimulating factor (GM-CSF) and transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 2), were monitored by the reverse transcriptase-PCR method. The expression of T cell cytokine mRNAs for IL-2, IL-5, and IFN-gamma was generally weak or inconsistent. IL-4 mRNA expression was consistently moderate to strong in polymyositis but generally weak or absent in the other IMs. The expression of macrophage cytokine mRNAs for IL-1 alpha and IL-1 beta was weak or absent in all cases. Variable TNF-alpha mRNA expression was observed in 12 of 15 IM cases and faint or weak expression in 5 of 10 controls. Very strong GM-CSF expression was detected, but only on boosted PCR, in 12 of 15 cases of IM but in none of the controls. IL-6 was expressed only weakly or inconsistently. In contrast to the variable expression of several of the above mentioned cytokine mRNAs, all IM specimens strongly expressed TGF-beta 1 mRNA and 12 of 15 strongly expressed TGF-beta 2 mRNA. Thus, with the exception of IL-4 expression in polymyositis, a similar pattern of cytokine mRNA expression exists in the different types of IMs. Moreover, this pattern resembles that detected in non-weak and DD controls, although expression is generally weaker in the non-weak controls. The findings suggest that in IM muscle a sustained secretion of cytokines by T cells or of IL-1 by macrophages is not a prerequisite for operation of the immune effector response and that muscle may not be the site of ongoing sensitization.
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PMID:Analysis of cytokine expression in muscle in inflammatory myopathies, Duchenne dystrophy, and non-weak controls. 855 29

Duchenne muscular dystrophy (DMD) is an X-linked lethal disorder caused by a defect in the DMD gene, which encodes the cytoskeletal protein dystrophin. Utrophin is an autosomal homolog of the DMD gene product dystrophin, and augmented expression of endogenous utrophin is expected to provide an alternative therapeutic approach to DMD. We previously reported that an immune response against a beta-galactosidase-expressing adenovirus vector, AxCALacZ, resulted in an accumulation of endogenous utrophin on the extrasynaptic sarcolemma in dystrophin-deficient mdx mice. To determine which cytokine is involved in the regulation of utrophin expression, we directly injected several cytokines separately into neonatal mdx muscles and tested whether the expression of utrophin is increased on the sarcolemma. Importantly, among the cytokines tested, solely interleukin 6 (IL-6) successfully increased expression of utrophin. Moreover, the increase in utrophin mRNA was detected in recombinant IL-6-injected mdx muscles by quantitative real-time reverse transcriptase-polymerase chain reaction. Further, IL-6 expression was elevated in AxCALacZ-infected mdx muscle at an early stage, and anti-IL-6 receptor (IL-6R) antibody treatment blocked enhanced utrophin expression in AxCALacZ-infected mdx muscle. We should point out, however, that overexpression of utrophin due to recombinant IL-6 treatment lasted only 1 week. In addition, expression of utrophin was not evident in normal C57BL/10 neonatal muscles injected with IL-6. Taken together, these results suggest that IL-6 can induce overexpression of utrophin on the extrasynaptic sarcolemma but requires preexisting factors in neonatal mdx muscle to fully regulate utrophin expression.
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PMID:Interleukin 6 induces overexpression of the sarcolemmal utrophin in neonatal mdx skeletal muscle. 1187 29

Dp71 is the major product of the Duchenne muscular dystrophy gene in the brain. In order to study the function of Dp71 in the nervous system we examined the expression of Dp71 isoforms in PC12 rat pheochromocytoma cell line, a well-established system to study neuronal differentiation. We show by reverse transcriptase-polymerase chain reaction and Western blot assays that PC12 cells express two Dp71 isoforms. One isoform lacks exon 71 and the other isoform lacks exons 71 and 78 (Dp71d and Dp71f isoforms respectively). Nerve growth factor-induced neuronal differentiation of PC12 cells results in differential regulation of the expression and subcellular localization of Dp71 isoforms: a) the amount of Dp71f protein increases nine-fold in total extracts while Dp71d increases up to seven-fold in nuclear extracts; b) Dp71f relocates from the cytoplasm to neuritic processes, being prominent at varicosities and the growth cone; c) Dp71d relocates almost entirely to the nucleus and is detected to a lower extent in the cytoplasm and neuritic processes. Dp71f co-localizes with beta-dystroglycan and synaptophysin while Dp71d co-localizes with beta-dystroglycan in the nucleus. Dp71d accumulates at cell-cell contacts where Dp71f is absent. These results suggest that Dp71d and Dp71f associate with different subcellular complexes and therefore may have distinct functions in PC12 cells.
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PMID:Differential expression and subcellular distribution of dystrophin Dp71 isoforms during differentiation process. 1273 41

Dystrophin deficiency leads to the progressive muscle wasting disease Duchenne muscular dystrophy (DMD). Dystrophin-deficient mdx mice are characterized by skeletal muscle weakness and degeneration but they appear outwardly normal in contrast to DMD patients. Mice lacking both dystrophin and the dystrophin homolog utrophin [double knockout (dko)] have muscle degeneration similar to mdx mice, but they display clinical features similar to DMD patients. Dko limb muscles also lack postsynaptic membrane folding and display fiber-type abnormalities including an abundance of phenotypically oxidative muscle fibers. Extraocular muscles, which are spared in mdx mice, show a significant pathology in dko mice. In this study, microarray analysis was used to characterize gene expression differences between mdx and dko tibialis anterior and extraocular skeletal muscles in an effort to understand the phenotypic differences between these two dystrophic mouse models. Analysis of gene expression differences showed that upregulation of slow muscle genes specifically characterizes dko limb muscle and suggests that upregulation of these genes may directly account for the more severe phenotype of dko mice. To investigate whether any upregulation of slow genes is retained in vitro, independent of postsynaptic membrane abnormalities, we derived mdx and dko primary myogenic cultures and analyzed the expression of Myh7 and Myl2. Real-time reverse transcriptase-polymerase chain reaction analysis demonstrates that transcription of these slow genes is also upregulated in dko vs mdx myotubes. This data suggests that at least part of the fiber-type abnormality is due directly to the combined absence of utrophin and dystrophin and is not an indirect effect of the postsynaptic membrane abnormalities.
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PMID:Analysis of gene expression differences between utrophin/dystrophin-deficient vs mdx skeletal muscles reveals a specific upregulation of slow muscle genes in limb muscles. 1652 50

A clinical trial was conducted to test a new protocol of normal muscle precursor cell (MPC) allotransplantation in skeletal muscles of patients with Duchenne muscular dystrophy (DMD). Cultured MPCs obtained from one of the patient's parents were implanted in 0.25 or 1 cm of a Tibialis anterior in 9 patients with DMD. MPC injections were placed 1 to 2 mm from each other, and a similar pattern of saline injections was done in the contralateral muscle. The patients were immunosuppressed with tacrolimus. Muscle biopsies were performed at the injected sites 4 weeks later. In the biopsies of the cell-grafted sites, there were myofibers expressing donor's dystrophin in 8 patients. The percentage of myofibers expressing donor's dystrophin varied from 3.5% to 26%. Evidence of small myofiber neoformation was observed in some patients. Donor-derived dystrophin transcripts were detected by reverse transcriptase-polymerase chain reaction in the cell-grafted sites in all patients. The protocol of immunosuppression was sufficient to obtain these results, although it is not certain whether acute rejection was efficiently controlled in all the cases. In conclusion, intramuscular allotransplantation of normal MPCs can induce the expression of donor-derived dystrophin in skeletal muscles of patients with DMD, although this expression is restricted to the sites of MPC injection.
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PMID:Dystrophin expression in muscles of duchenne muscular dystrophy patients after high-density injections of normal myogenic cells. 1669 Nov 18

Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder caused by mutations in the dystrophin DMD gene located at Xp21.1 region. Up to 65% of the patients present dystrophin gene deletions. Mothers of DMD patients have a two-thirds chance of carrying a dystrophin mutation. The female carrier will transmit the disease gene to half of her sons and half of her daughters. As the recurrence risk for the disease is extremely high, it is very important to detect carrier status among female relatives of the patients to bring an adequate genetic counseling. In this work, results from two methods to identify female carriers are presented. One method is a multicolor fluorescence in situ hybridization (FISH) assay, and the other is reverse transcriptase-polymerase chain reaction (RT-PCR). We showed that FISH is an efficient, sensitive method that brings confident results to detect DMD female carriers as compared to RT-PCR.
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PMID:Identification of duchenne muscular dystrophy female carriers by fluorescence in situ hybridization and RT-PCR. 1847 Oct 87


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