UMLS:C0023418 - FACTA Search

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Query: UMLS:C0023418 (leukemia)
93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Construction and using of retrovirus vectors derived from the Moloney murine leukemia virus (MoMuLV) are described. These vectors, designated minimal vectors, contain the left and right long terminal repeats (LTRs), a binding site for proline tRNA, a polypurine tract (PPT), and a dominant marker for selective introduction of vectors into a packaging cell line, but lack the internal sequences of the virus genome. The experiments showed that the minimal vectors can be replicated and that their titer was approximately 1500-fold lower than that of wild-type vectors. The minimal vectors were shown to contain all the cis-acting sequences necessary for correct reverse transcription. One infectious virion. like wild-type viruses, produced only one provirus. Unlike the avian reticuloendotheliosis virus (REV), psi+ and psi(-)-genomes of MoMuLV did not compete for virion proteins in the psi2 packaging cell line. When an insert was introduced into a central part of the lTR U5 region, the titer of the minimal vector remained the same, while the titer of the wild-type vector decreased approximately 40-fold.
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PMID:[Effect of internal sequences in the Moloney murine leukemia virus genome on replication]. 866 18

We have determined the solution structure of the synthetic chimeric duplex r(ccca)d(AATGA).d(TCATTTGGG) by two-dimensional NMR, distance geometry, restrained molecular dynamics, and full relaxation matrix simulation of the two-dimensional nuclear Overhauser effect spectra at various mixing times. The chimeric strand of this duplex consists of the last four residues of the tRNA(Pro) primer for (-) strand DNA synthesis of Moloney murine leukemia virus and the first five residues of the (-) strand DNA produced by extending this primer; the complementary DNA strand corresponds to the (+) strand product from this template. The hybrid section of this chimeric duplex assumes a structure similar to that found for pure hybrid duplexes of mixed sequence, while the DNA section assumes a conformation closer to B-form DNA. There is significant distortion of the duplex at the hybrid-DNA junction which is manifested in marked changes in the helical parameters buckle, roll, and tip, changes in glycosidic torsion angles, and changes in the backbone torsion angles delta, epsilon, and zeta. The sugar conformations also undergo large changes, from heteromerous puckers in the hybrid section to a more B-form in the DNA section. Furthermore, the intrastrand phosphate separation in the chimeric strand is more typical of A-form duplexes in the RNA section but more like B-form duplexes in the DNA section. In the DNA section the minor groove width changes gradually from B-form at the periphery and approaches hybrid-like dimensions closer to the junction. The structural discontinuities act synergistically to produce a bend of 18 +/- 3 degrees at the junction. The global structure of this sequence is similar to that previously found in the chemically analogous Okazaki fragment r(gcg)d(TATACCC).d(GGGTATACGC) in solution. Such structure homology suggests a possible link between structure and function with respect to the recognition and cleavage of the junction RNA residues in both retroviral chimeras and Okazaki fragments during reverse transcription and normal DNA replication.
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PMID:Structure of chimeric duplex junctions: solution conformation of the retroviral Okazaki-like fragment r(ccca)d(AATGA).d(TCATTTGGG) from Moloney murine leukemia virus. 867 64

Infecting mice with a mutant Moloney murine leukemia virus which contains the bacterial suppressor tRNA supF in its LTR allows rapid cloning of proviral integration sites from genomic tumour DNA. In a previous study Emu pim-1/Emu L-myc bitransgenic mice had been inoculated neonatally with MoMuLV supF virus. The retroviral infection led to acceleration of lymphomagenesis indicating the proviral activation of further oncogenes cooperating with myc and pim-1 in tumour development. Using a functional supF screen for analysis of genomic mouse tumour DNA libraries which had been constructed in the phage vector EMBL3A, a common proviral integration site on mouse chromosome 5 was cloned and found to be identical to the proviral integration site evi-5 which has recently been identified in an AKXD T-cell lymphoma and which is located 18 kb upstream of the gfi-1 gene. Tumours bearing evi-5 integrations showed an enhanced gfi-1 expression level suggesting that gfi-1 is the target gene for insertions at the evi-5 locus. Together with three other previously described Moloney integration clusters all responsible for enhanced gfi-1 expression the number of tumours from infected double transgenic Emu L-myc/Emu pim-1 transgenic mice with retrovirally activated gfi-1 added up to 53% underscoring the role of GFI-1 as an effective collaborator for MYC and PIM-1 in the process of lymphomagenesis.
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PMID:MoMuLV proviral integrations identified by Sup-F selection in tumors from infected myc/pim bitransgenic mice correlate with activation of the gfi-1 gene. 869 92

Nucleocapsid protein NCp10 of murine leukemia virus (MuLV) is encoded by the 3' domain of gag and contains a zinc finger of the form Cys-X2-Cys-X4-His-X4-Cys flanked by basic amino acids. In the course of virus assembly, NCp10 is necessary for core formation, and the zinc finger flanked by the basic residues is required for the packaging of the genomic RNA dimer. In vitro, NCp10 exhibits strong nucleic acid binding and annealing activities that appear to be critical for virus infectivity since NCp10 promotes dimerization of the viral RNA containing the E/DLS packaging-dimerization signal and annealing of replication primer tRNA(Pro) to the initiation site of reverse transcription (PBS). Recent in vitro studies have suggested that NCp10 may also play a role in proviral DNA synthesis. To investigate the function of NCp10 in proviral DNA synthesis in vivo, we developed a simple and convenient genetic packaging system consisting of two DNA constructs expressing the packaging components gag-pol and env of Friend MuLV and a Moloney MuLV-based lacZ vector with either the MuLV E+ or a rat VL30 E packaging signal. This system allowed us to examine the consequences of a set of mutations in NCp10 on a single round of recombinant virus replication. Most mutations in the N- or C-terminal domain of NCp10 do not significantly alter infectivity, while those in the zinc finger drastically impair infectivity. Analysis of the viral RNA content in virions showed that all mutations in the zinc finger decrease but do not abolish packaging of the recombinant genome. Interestingly enough, mutation of Y-28 to S (mutation Y28S) in the zinc finger results in RNA packaging at a level similar to that observed upon deletion of three prolines and three arginines in the C-terminal domain of NCp10 (mutant delta PR3). However, mutant Y28S is noninfectious while mutant delta PR3 is only threefold less infectious than the wild-type virus, which prompted us to examine the role of NCp10 protein in proviral DNA synthesis in vivo using these nucleocapsid mutants. PCR amplification was used to analyze viral DNA synthesized in newly infected cells, and results indicate that the Y28S zinc finger mutation impairs reverse transcription, thus suggesting that the nucleocapsid protein zinc finger plays a key role in proviral DNA synthesis in vivo.
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PMID:The zinc finger of nucleocapsid protein of Friend murine leukemia virus is critical for proviral DNA synthesis in vivo. 870 95

Initiation of minus (-) strand DNA synthesis was examined on templates containing R, U5, and primer-binding site regions of the human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV) genomic RNA. DNA synthesis was initiated from (i) an oligoribonucleotide complementary to the primer-binding sites, (ii) synthetic tRNA(3Lys), and (iii) natural tRNA(3Lys), by the reverse transcriptases of HIV-1, FIV, EIAV, simian immunodeficiency virus, HIV type 2 (HIV-2), Moloney murine leukemia virus, and avian myeloblastosis virus. All enzymes used an oligonucleotide on wild-type HIV-1 RNA, whereas only a limited number initiated (-) strand DNA synthesis from either tRNA(3Lys). In contrast, all enzymes supported efficient tRNA(3Lys)-primed (-) strand DNA synthesis on the genomes of FIV and EIAV. This may be in part attributable to the observation that the U5-inverted repeat stem-loop of the EIAV and FIV genomes lacks an A-rich loop shown with HIV-1 to interact with the U-rich tRNA anticodon loop. Deletion of this loop in HIV-1 RNA, or disrupting a critical loop-loop complex by tRNA(3Lys) extended by 9 nt, restored synthesis of HIV-1 (-) strand DNA from primer tRNA(3Lys) by all enzymes. Thus, divergent evolution of lentiviruses may have resulted in different mechanisms to use the same host tRNA for initiation of reverse transcription.
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PMID:Initiation of (-) strand DNA synthesis from tRNA(3Lys) on lentiviral RNAs: implications of specific HIV-1 RNA-tRNA(3Lys) interactions inhibiting primer utilization by retroviral reverse transcriptases. 881 51

The reverse transcriptase-associated RNase H activity of Moloney murine leukemia virus specifically cleaves within the polypurine tract region of the viral genome to generate the primer for plus-strand DNA synthesis and removes the tRNA primer after minus-strand initiation by preferentially cleaving the RNA one nucleotide before the RNA-DNA junction. Moreover, the enzyme is unable to cleave the extended tRNA substrate at the RNA-DNA junction even at high enzyme concentrations. The RNase H domain of the reverse transcriptase was expressed as a glutathione S-transferase fusion protein and purified from Escherichia coli extracts. Following removal of the glutathione S-transferase portion of the protein, the specificity of the isolated RNase H domain was determined in the plus-strand primer reaction and in the tRNA primer removal reaction. Although the isolated domain lacked specificity in both cases, it was still unable to cleave the tRNA substrate precisely at the RNA-DNA junction. Specificity in both cases could be restored by adding back a truncated form of Moloney murine leukemia virus reverse transcriptase lacking the RNase H domain. These results implicate the polymerase domain as a specificity determinant for the RNase H activity of reverse transcriptase. The isolated RNase H domain had higher activity in the presence of Mn2+ than in the presence of Mg2+, but neither the RNase H domain alone nor the RNase H domain coupled to the polymerase domain in wild-type protein exhibited the normal cleavage specificities in the presence of the nonphysiological divalent cation.
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PMID:RNase H domain of Moloney murine leukemia virus reverse transcriptase retains activity but requires the polymerase domain for specificity. 897 Sep 88

Reverse transcription of retroviral genomes is primed by a tRNA annealed to an 18-nucleotide primer binding site. Here, we present a primer complementation system to study molecular interaction of the replication machinery with the primer and primer binding site in vivo. Introduction of eight base substitutions into the primer binding site of a murine leukemia virus-based vector allowed efficient RNA encapsidation but resulted in severely reduced vector replication capacity. Replication was restored upon complementation with a synthetic gene designed to encode a complementary tRNA-like primer, but not with a noncomplementary tRNA-like molecule. The engineered primer was shown to be involved in both the initiation of first-strand synthesis and second-strand transfer. These results provide an in vivo demonstration that the retroviral replication machinery may recognize sequence complementarity rather than actual primer binding site and 3' primer sequences. Use of mutated primer binding site vectors replicating via engineered primers may add additional control features to retroviral gene transfer technology.
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PMID:Complementation of a primer binding site-impaired murine leukemia virus-derived retroviral vector by a genetically engineered tRNA-like primer. 899 41

RNA-DNA hybrid model substrates which mimic an intermediate of Moloney murine leukemia virus (M-MuLV) reverse transcription at the stage where the tRNAPro is removed were constructed. This substrate was used to assay the ability of M-MuLV reverse transcriptase (RT) to cleave the RNA portion of the substrate. The cleavage specificities of the cognate M-MuLV RT and the heterologous enzyme from the human immunodeficiency virus type-1 (HIV-1) were compared. M-MuLV and HIV-1 RT recognize and cleave the RNA at distinct positions. The site of the initial RNase H cleavage in vitro was determined using 3' end nearest neighbor analysis of the initial cleavage product. M-MuLV RT/RNase H removed the model tRNAPro between the terminal ribo-A and ribo-C, resulting in a terminal ribo-A attached to the viral DNA, whereas HIV-1 RT/RNase H was shown to cleave at the RNA-DNA junction. Analysis of the DNA over time indicated that the ribo-A is subsequently removed by M-MuLV RT. In vivo analysis from double-LTR circle junctions illustrated that 16 of the 23 clones isolated possessed the predicted junction if complete removal of the tRNA primer were to occur. The predicted junction for complete removal of the tRNA primer was CATT-AATG. One aberrant circle junction was isolated which could result from the use of an alternative primer. In contrast with HIV, no M-MuLV circle junctions were isolated which indicated processing of a single-LTR terminus by integrase. Analysis from in vivo and in vitro studies indicate that the M-MuLV tRNAPro primer is completely removed after plus-strand strong-stop synthesis.
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PMID:RNase H cleavage of tRNAPro mediated by M-MuLV and HIV-1 reverse transcriptases. 912 56

We cloned a tumorigenic phenotype-associated cDNA encoding a tRNA synthetase-like protein from an acute-phase human myeloid leukemia cell line. The cDNA was isolated by reiterative subtraction of cDNAs synthesized from tumor-generating parental leukemia cells versus those from a nontumorigenic variant of the same cells. The selected cDNA encodes a protein that is a close homolog of one subunit of prokaryote and yeast phenylalanyl-tRNA synthetase (PheRS). The expressed protein reacts specificially with polyclonal antibodies raised against mammalian phenylalanyl-tRNA synthetase. Expression of the gene (designated CML33) was directly confirmed by Northern blot hybridization to be substantially enhanced in the tumorigenic cells compared with the nontumorigenic variant. In addition, expression of CML33 in myeloid leukemia cells was sensitive to the stage of the cell cycle and to induction of differentiation. Although the relationship between these observations and the tumorigenic state of the human myeloid leukemia cell line used in these studies is unknown, to our knowledge, this is the first demonstration in mammalian cells of tumor-selective and cell cycle stage- and differentiation-dependent expression of a member of the tRNA synthetase gene family.
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PMID:Expression of a gene encoding a tRNA synthetase-like protein is enhanced in tumorigenic human myeloid leukemia cells and is cell cycle stage- and differentiation-dependent. 917 88

During reverse transcription of retroviral RNA, synthesis of (-) strand DNA is primed by a cellular tRNA that anneals to an 18-nt primer binding site within the 5' long terminal repeat. For (+) strand synthesis using a (-) strand DNA template linked to the tRNA primer, only the first 18 nt of tRNA are replicated to regenerate the primer binding site, creating the (+) strand strong stop DNA intermediate and providing a 3' terminus capable of strand transfer and further elongation. On model HIV templates that approximate the (-) strand linked to natural modified or synthetic unmodified tRNA3Lys, we find that a (+) strand strong stop intermediate of the proper length is generated only on templates containing the natural, modified tRNA3Lys, suggesting that a posttranscriptional modification provides the termination signal. In the presence of a recipient template, synthesis after strand transfer occurs only from intermediates generated from templates containing modified tRNA3Lys. Reverse transcriptase from Moloney murine leukemia virus and avian myoblastosis virus shows the same requirement for a modified tRNA3Lys template. Because all retroviral tRNA primers contain the same 1-methyl-A58 modification, our results suggest that 1-methyl-A58 is generally required for termination of replication 18 nt into the tRNA sequence, generating the (+) strand intermediate, strand transfer, and subsequent synthesis of the entire (+) strand. The possibility that the host methyl transferase responsible for methylating A58 may provide a target for HIV chemotherapy is discussed.
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PMID:Posttranscriptional modification of retroviral primers is required for late stages of DNA replication. 920 70

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