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)

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

The genome of the Australian marsupial Macropus robustus contains a highly conserved processed hypoxanthine phosphoribosyltransferase homologue, HPRT-2. Using the techniques of reverse transcriptase-polymerase chain reaction (RT-PCR) and protein isoelectric focusing (IEF) we have shown this processed gene to be fully functional, but liver specific. In contrast, the unprocessed X-linked parent gene HPRT-1 was expressed in all somatic tissues. Expression of the HPRT-2 gene effectively doubles the total HPRT enzyme activity in liver compared to other tissues. Analysis of the 5'-flanking sequence of HPRT-2 revealed regions with homology to the liver-specific regulatory motifs C/EBP, NF-IL6, LF-A1 and LF-B1, although the functional significance of these regions remains unknown. Consistent with X chromosome inactivation in female mammals, transcript levels of the unprocessed X-linked gene HPRT-1 were similar in males and females in all tissues examined. No HPRT-2 activity was detected in testes, indicating that this gene does not compensate for sex chromosome inactivation during spermatogenesis. Moreover, the demonstration of very high HPRT-1 enzyme levels in testes indicated that such a compensatory mechanism may not be required. Phylogenetic analyses attribute considerable antiquity to the processed gene and PCR with conserved primers spanning exons 4-8 of genomic DNA from several different kangaroo species inferring the existence of a conserved processed HPRT-2 homologue in these marsupial species. However, no such conserved PCR product was obtained with DNA from eutherian species, suggesting that integration of HPRT-2 occurred after the separation of the metatherian and eutherian lineages.
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PMID:Identification of a novel tissue-specific processed HPRT gene and comparison with X-linked gene transcription in the Australian marsupial Macropus robustus. 904 50

PEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, was recently identified as the candidate gene for X-linked hypophosphatemia. In the present study, we cloned mouse and human Pex/PEX cDNAs encoding part of the 5' untranslated region, the protein coding region, and the entire 3' untranslated region, determined the tissue distribution of Pex/PEX mRNA, and characterized the Pex mutation in the murine Hyp homologue of the human disease. Using the reverse transcriptase/polymerase chain reaction (RT/PCR) and ribonuclease protection assays, we found that Pex/PEX mRNA is expressed predominantly in human fetal and adult mouse calvaria and long bone. With RNA from Hyp mouse bone, an RT/PCR product was generated with 5' but not 3' Pex primer pairs and a protected Pex mRNA fragment was detected with 5' but not 3' Pex riboprobes by ribonuclease protection assay. Analysis of the RT/PCR product derived from Hyp bone RNA revealed an aberrant Pex transcript with retention of intron sequence downstream from nucleotide 1302 of the Pex cDNA. Pex mRNA was not detected on Northern blots of poly (A)+ RNA from Hyp bone, while a low-abundance Pex transcript of approximately 7 kb was apparent in normal bone. Southern analysis of genomic DNA from Hyp mice revealed the absence of hybridizing bands with cDNA probes from the 3' region of the Pex cDNA. We conclude that Pex/PEX is a low-abundance transcript that is expressed predominantly in bone of mice and humans and that a large deletion in the 3' region of the Pex gene is present in the murine Hyp homologue of X-linked hypophosphatemia.
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PMID:Pex/PEX tissue distribution and evidence for a deletion in the 3' region of the Pex gene in X-linked hypophosphatemic mice. 907 27

Inactivating mutations of the neutral endopeptidase, PEX, have been identified as the cause of X-linked hypophosphatemia (XLH). Though the function of PEX is unknown, current information suggests that impaired renal phosphate conservation in XLH is due to the failure of PEX to either degrade an undefined phosphaturic factor or activate a novel phosphate-conserving hormone. The physiologically relevant target tissue for the XLH mutation has not been identified. An apparent intrinsic defect of osteoblast function in XLH implicates bone as a possible site of PEX expression. In the current investigation, we employed a polymerase chain reaction (PCR) strategy to amplify a PEX cDNA from a human bone cell cDNA library. We found that the human PEX cDNA encodes a 749 amino acid protein belonging to the type II integral membrane zinc-dependent endopeptidase family. The predicted PEX amino acid sequence shares 96.0% identify to the recently cloned mouse Pex cDNA and has 27-38% identity to other members of the metalloendopeptidase family. Using reverse transcriptase (RT)-PCR with PEX-specific primers, we detected PEX transcripts in both human osteosarcoma-derived MG-63 osteoblasts and in differentiated mouse MC3T3-E1 clonal osteoblasts but not in immature MC3T3-E1 preosteoblasts. The association of impaired mineralization of bone in XLH and the apparent developmental stage-specific expression of PEX in osteoblasts suggest that bone is a physiologically relevant site of PEX expression and that PEX may play an active role in osteoblast-mediated mineralization.
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PMID:Cloning and sequencing of human PEX from a bone cDNA library: evidence for its developmental stage-specific regulation in osteoblasts. 919 99

Nucleoside-diphosphate kinase (NDP kinase) from the matrix space of mitochondria in pigeon liver was purified to homogeneity. Degenerate oligonucleotide primers to the N-terminal sequence of the purified protein and the region containing the active site histidine were used in reverse transcriptase-polymerase chain reaction to obtain a major portion of the coding sequence for the mature protein. The sequences of the C and N termini of the mature protein, and eight residues in the signal peptide, were obtained by rapid amplification of cDNA end procedures. The entire coding sequence of a cytosolic form of NDP kinase was also determined. Both isoforms, which share 53% sequence identity, possess the characteristically conserved regions of known NDP kinases. The mature mitochondrial NDP kinase protein migrates in molecular sieving columns with an apparent molecular mass of about 66 kDa. It shows very high thermal stability even though it lacks the proline residue in the killer of prune loop, and the Tyr/Glu C termini that are important in stabilizing other NDP kinases. The affinity of the mitochondrial isoform for adenine and guanine nucleotides is much higher than for pyrimidine nucleotides, but the enzyme is especially susceptible to substrate inhibition by GDP. Semi-quantitative reverse transcriptase-polymerase chain reaction showed that the relative levels of expression of the mitochondrial isoform are liver > kidney >> heart = brain > breast muscle. The cytosolic isoform is strongly and approximately equally expressed in these same five tissues. This work is the first characterization of a NDP kinase isoform that is found in the matrix space of mitochondria.
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PMID:Characterization and cloning of a nucleoside-diphosphate kinase targeted to matrix of mitochondria in pigeon. 930 28

The transition between dependence on maternal transcripts and proteins inherited in the oocyte and embryonic gene expression in the human preimplantation embryo occurs at the four- to eight-cell stage. Recently, studies using reverse transcriptase polymerase chain reaction (RT-PCR) have detected paternal transcripts for the Y-linked genes, ZFY and SRY, and the myotonic dystrophy associated protein kinase gene, DK, as early as the late pronucleate one-cell stage. However, expression at the protein level has not been demonstrated and its function at these early stages is unknown. Using coding sequence polymorphisms to distinguish maternal and paternal transcripts, we have examined the transcription of two ubiquitously expressed genes: X-linked glucose-6-phosphate dehydrogenase (G6PD) and adenosine deaminase (ADA). Both G6PD and ADA are housekeeping genes with TATA-less promoters which, because of their roles in metabolism and ubiquitous expression, may provide a more reliable indication of the timing of activation of the embryonic genome. They also each have biallelic polymorphisms with a high heterozygosity ratio which can be detected by restriction digestion. Couples undergoing in vitro fertilization (IVF) were screened for these polymorphisms. Individual spare oocytes and embryos at different stages of preimplantation development were analyzed by RT-PCR and appropriate restriction digestion in those cases in which the male partner carried a different allele to the female partner. In addition, since only female embryos inherit the paternal allele of X-linked G6PD, cDNA was also analyzed for ZFX/ZFY transcripts to identify the sex of each embryo. One hundred and twenty three individual oocytes and embryos were analyzed by RT-PCR and restriction digestion to detect the paternal transcripts from the polymorphic alleles. Maternal transcripts for G6PD, ADA, and ZFX were detected in all oocytes and embryos and at all stages. Following restriction digestion, paternal G6PD and ZFY transcripts were first detected at the four-cell stage and paternal ADA transcripts in an embryo at the three-cell stage coinciding with the onset of dependency on transcription from the embryonic genome. This approach should be widely applicable to other genes since similar polymorphisms exist in the coding regions of many genes.
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PMID:Paternal transcripts for glucose-6-phosphate dehydrogenase and adenosine deaminase are first detectable in the human preimplantation embryo at the three- to four-cell stage. 936 38

Mutations in PEX, a phosphate-regulating gene with homology to endopeptidase on the X chromosome, were recently identified in patients with X-linked hypophosphatemia (XLH), an inherited disorder of phosphate homeostasis characterized by growth retardation and rachitic and osteomalacic bone disease. To understand the mechanism by which loss of PEX function elicits the mutant phenotype, a study of its mRNA localization and ontogenesis was undertaken. Using the reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) with polyA+ RNA purified from mouse testis, a 337-bp Pex cDNA fragment was generated and cloned in the pCRII plasmid. The cDNA was used to generate sense and anti-sense Pex riboprobes for in situ hybridization (ISH) and Northern analysis. To survey a large number of different tissues, sagittal sections of embryos and newborn mice were examined. ISH showed the presence of Pex mRNA in osteoblasts and odontoblasts. Pex gene expression was detectable on Day 15 of embryonic development, which coincides with the beginning of intercellular matrix deposition in bones. Finally, Northern analysis of total RNA from calvariae and teeth of 3-day-old and adult mice showed that the abundance of the 7-kb Pex transcript is decreased in adult bones and in nongrowing teeth. The present study demonstrates that Pex mRNA is expressed in bones and teeth and suggests that this putative endopeptidase plays an important role in the development of these tissues.
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PMID:Pex mRNA is localized in developing mouse osteoblasts and odontoblasts. 952 91

A genomic clone isolated from an equine genomic library probed with an oligonucleotide (CAG)10 showed high sequence similarity to the human F18 gene and was tentatively named equine F18 gene. Because the human F18 gene is expressed in many tissues, we examined whether this equine clone was also expressed in equine tissues. The cDNA encoding equine F18 was obtained by the reverse transcriptase-polymerase chain reaction (RT-PCR) from equine thymus. The nucleotide sequence of the equine F18 cDNA (1940 bp) was determined and contained both the ATG initiation codon and a poly(A) sequence. The cDNA sequence contained sequence homologous to exon 3 of human F18 gene and a new exon that is not found in the human F18 gene. The equine F18 gene contains a CAG repetitive sequence (CRS) that is translated into a polyglutamine tract. The CRS was analyzed for polymorphism by PCR: four and two different alleles were observed with unrelated east-Asian and thoroughbred horses, respectively. The equine F18 gene was mapped to Xq29.1 by fluorescence in situ hybridization. The human F18 gene is also X-linked. These data strongly supported the conclusion that the clone contains the equine homologue of the human F18 gene.
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PMID:Cloning and characterization of the equine F18 gene, which has a novel exon. 980 Mar 27

The potentially unbalanced expression at preimplantation developmental stages of X-linked genes might be responsible of the faster development of male than female embryos in vitro. Two genes located on the X chromosome, glucose-6-phosphate dehydrogenase (G6PD) and hypoxanthine phosphoribosyl transferase (HPRT), are involved in controlling the amount of oxygen radicals, and hence they might have influence in embryo development. We have quantified mRNA expression of these two genes, using in vitro fertilized-in vitro cultured male and female bovine embryos. In vitro-produced early blastocysts obtained at days 7 and 8 were collected and biopsied for gender determination, and the remaining embryos were kept in LN(2) until RNA purification. After sex determination, embryos were pooled in groups of 3 males or 3 females, and mRNA was purified. Using a semiquantitative sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we detected G6PD and HPRT mRNA expression at the early blastocyst stage in all bovine embryos analyzed. Moreover, mRNA expression of both genes studied was significantly higher in female embryos than in male embryos. The differential expression of G6PD and HPRT at these early stages confirm that sex differences are evident prior to gonadal differentiation and that preimplantation bovine embryos have sexually dimorphic gene expression at least with respect to G6PD and HPRT transcripts. These differences might be responsible of the faster development in culture of in vitro-produced male bovine that has been reported. Mol. Reprod. Dev. 55:146-151, 2000.
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PMID:Differential expression of two genes located on the X chromosome between male and female in vitro-produced bovine embryos at the blastocyst stage. 1061 53

The mechanism by which inactivating mutations of PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) cause X-linked hypophosphatemia remains unknown. However, recent reports suggest errant PHEX activity in osteoblasts may fail to inactivate a phosphaturic factor produced by these cells. To test this possibility, we examined coordinated maturational expression of PHEX and production of phosphate transport inhibitory activity in osteoblasts from normal and hyp-mice. We assessed the inhibitory activity in conditioned medium by examining the effects on opossum kidney cell phosphate transport and osteoblast PHEX expression by reverse transcriptase-polymerase chain reaction during a 17-day maturational period. Inhibitory activity increased as a function of osteoblast maturational stage, with no activity after 3 days and persistent activity by 6 days of culture. More significantly, equal phosphate transport inhibitory activity in conditioned medium from normal and hyp-mouse osteoblasts (control 1.90 +/- 0.12, normal 1.48 +/- 0.10, hyp 1.45 +/- 0.04 nmol/mg of protein/minute) was observed at 6 days. However, by 10 days hyp-mouse osteoblasts exhibited greater inhibitory activity than controls, and by 17 days the difference in phosphate transport inhibition maximized (control 2.08 +/- 0.09, normal 1.88 +/- 0.06, hyp 1.58 +/- 0.06 nmol/mg of protein/minute). Concurrently, we observed absent PHEX expression in normal osteoblasts after 3 days, limited production at 6 days, and significant production by day 10 of culture, while hyp-mouse osteoblasts exhibited limited PHEX activity secondary to an inactivating mutation. The data suggest that the presence of inactivating PHEX mutations results in the enhanced renal phosphate transport inhibitory activity exhibited by hyp-mouse osteoblasts.
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PMID:Coordinated maturational regulation of PHEX and renal phosphate transport inhibitory activity: evidence for the pathophysiological role of PHEX in X-linked hypophosphatemia. 1062 61


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