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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)

Transfer of therapeutic genes to human hematopoietic stem cells (HSCs) using complex vectors at clinically relevant efficiencies remains a major challenge. Recently we described a stable retroviral vector that sustains long-term expression of green fluorescent protein (GFP) and a human beta-globin gene in the erythroid progeny of transduced murine HSCs. We now report the efficient transduction of primitive human CD34(+) fetal liver or cord blood cells with this vector and expression of the beta-globin transgene in the erythroid progeny of these human cells for at least 2 months. After growth factor prestimulation and then a 2- to 3-day exposure to the virus, 35% to 55% GFP(+) progeny were seen in assays of transduced colony-forming cells, primitive erythroid precursors that generate large numbers of glycophorin A(+) cells in 3-week suspension cultures, and 6-week long-term culture-initiating cells. In immunodeficient mice injected with unselected infected cells, 5% to 15% of the human cells regenerated in the marrow (including the erythroid cells) were GFP(+) 3 and 6 weeks after transplantation. Importantly, the numbers of GFP(+) human lymphoid and either granulopoietic or erythroid cells in individual mice 6 weeks after transplantation were significantly correlated, indicative of the initial transduction of human multipotent cells with in vivo repopulating activity. Expression of the transduced beta-globin gene in human cells obtained directly from the mice or after their differentiation into erythroid cells in vitro was demonstrated by reverse transcriptase-polymerase chain reaction using specific primers. These experiments represent a significant step toward the realization of a gene therapy approach for human beta-globin gene disorders.
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PMID:Expression of a human beta-globin transgene in erythroid cells derived from retrovirally transduced transplantable human fetal liver and cord blood cells. 1214 6

The thymidine 5'-triphosphate analogue containing a methylene group in place of the 5' oxygen atom can be prepared using modifications of published procedures and can substitute for the natural thymidine triphosphate in chain extension reactions catalyzed by Moloney-MLV reverse transcriptase. Using rabbit beta-globin mRNA as the template together with an appropriate primer, the enzyme readily makes full-length DNA transcripts in which all thymidine 5' oxygen atoms have been replaced with methylene groups. In sequence analyses using the partial depurination procedure, the analogue DNA transcript produces electrophoretic gel patterns identical with those of the corresponding natural DNA transcript. Experiments on second strand synthesis using the four regular triphosphates show that the analogue DNA transcript, like the natural transcript, can serve as a template. The two DNA duplexes (natural/natural and analogue/natural) formed by these reactions produce similar electrophoretic cleavage patterns when treated with either of the endonucleases HaeIII and EcoRI. However, further studies on template properties indicate that, while the enzyme makes a full-length product when using the analogue substrate with a natural DNA strand as template, it appears unable to use the analogue transcript as template with the analogue triphosphate as substrate during second strand synthesis. Preliminary experiments have also been carried out with a DNA polymerase. No products are detected reactions using Taq polymerase with PCR protocols containing the analogue triphosphate as the only source of thymidine.
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PMID:The analogue of thymidine triphosphate containing a methylene group in place of the 5' oxygen can serve as a substrate for reverse transcriptase. 1288 22

The noncritical use of housekeeping genes, RNA mass, or cell number for normalization in quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays has come under scrutiny in recent years, highlighting the need to evaluate references in the immediate context of the relevant samples and experimental design. The purpose of this study was to select appropriate references for normalizing qRT-PCR assays of gene expression in exfoliated cervical cells. We used total nucleic acid extracts from 30 samples, representing the full spectrum of pre-invasive cervical neoplasia. We determined the DNA content by quantitative PCR for the single-copy gene beta-globin and total RNA content using quantitative image analysis of ribosomal bands. In addition, qRT-PCR for 13 candidate housekeeping genes was performed. We used two analysis methods, geNorm and Norm-Finder, to identify the best combination of reference genes and then correlated housekeeping gene expression with DNA content and gel representation of ribosomal RNA. ACTB was the most stable single gene. The addition of PGK1 and RPLP0 increased the robustness in qRT-PCR applications not stratified by disease. These genes also showed the highest correlation with DNA contents in the same samples. If special attention to intraepithelial lesions is appropriate, RPL4 and PGK1 are recommended as the best combination of two genes.
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PMID:DNA and RNA references for qRT-PCR assays in exfoliated cervical cells. 1643 42

Starting from the four DNA bases order in the Boolean lattice, a novel Lie Algebra of the genetic code is proposed. Here, the main partitions of the genetic code table were obtained as equivalent classes of quotient spaces of the genetic code vector space over the Galois field of the four DNA bases. The new algebraic structure shows strong connections among algebraic relationships, codon assignments and physicochemical properties of amino acids. Moreover, a distance defined between codons expresses a physicochemical meaning. It was also noticed that the distance between wild type and mutant codons tends to be small in mutational variants of four genes: human phenylalanine hydroxylase, human beta-globin, HIV-1 protease and HIV-1 reverse transcriptase. These results strongly suggest that deterministic rules in genetic code origin must be involved.
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PMID:A novel Lie algebra of the genetic code over the Galois field of four DNA bases. 1678 Aug 98

A novel algebraic structure of the genetic code is proposed. Here, the principal partitions of the genetic code table were obtained as equivalent classes of quotient spaces of the genetic code vector space over the Galois field of the four DNA bases. The new algebraic structure shows strong connections among algebraic relationships, codon assignment and physicochemical properties of amino acids. Moreover, a distance function defined between the codon binary representations in the vector space was demonstrated to have a linear behavior respect to physical variables such as the mean of amino acids interaction energies in proteins. It was also noticed that the distance between wild type and mutant codons approach to smaller values in mutational variants of four genes, i.e., human phenylalanine hydroxylase, human beta-globin, HIV-1 protease and HIV-1 reverse transcriptase. These results strongly suggest that deterministic rules must be involved in the genetic code origin.
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PMID:A novel algebraic structure of the genetic code over the galois field of four DNA bases. 1682 9


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