Addition of deoxynucleotides to an RNA primer by a DNA polymerase in human platelets.
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Biomedical subjects
Publications and source records attributed to D Gillespie.
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Newly evolved, tandemly arrayed, highly repeated DNAs from three primates were compared using Markov-chain and random-simulation approaches. Markov-chain calculations suggested that the repeated DNA sequences derived from the amplification of a progenitor sequence some 55 million years ago. Divergence of the products of this ancestral amplification could have created a library of related DNA sequences from which newly evolved repeated DNA was drawn - by recent amplifications of library members. The ancestral DNA amplification may have provided the genetic flexibility for creating the primate order; more recent amplifications in old-world primates probably led to the present subfamilies. The random-simulation approach verified the idea that repeated DNA evolves nonrandomly. A variable region was identified within the sequence.
A highly repeated DNA was isolated from the West African baboon (Papio papio) as a 343-base-pair fragment after digestion of total baboon DNA with the restriction endonuclease BamHI. The DNA sequence of this fragment was obtained by chemical cleavage methods and is compared with the DNA sequence of related highly repeated primate DNAs from African green monkey (Cercopithecus aethiops) and man. The 343-base-pair baboon repeat consists of two related but nonidentical wings of 172 and 171 base pairs, respectively. The baboon 172-base-pair wing shares more homology with the African green monkey 172-base-pair repeat than with the baboon 171-base-pair wing. Comparison with the previously published monkey and human DNA sequences indicates that: (i) All the DNA sequences apparently arose from a common ancestral sequence. (ii) Evolution of the primate DNA sequences can be explained by a model involving unequal crossovers at specific points within the repeated DNA, possibly mediated by the sequence 5'-AAGG-3' 3'-TTCC-5' or its invert 5'-GGAA-3' 3'-CCTT-5'. (iii) There are alternating domains of conserved and divergent DNA sequences within each greater than 170-base-pair wing sequence. Taken together, the DNA sequences of these primates suggest a model whereby highly repeated DNAs are established and evolve as a consequence of unequal nonrandom exchanges of DNA duplexes. These exchanges may be mediated by short repeated nucleotide sequences and involve exchanges within and between the greater than 170-base-pair wings.
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Two procedures were developed for removing DNA from agarose after electrophoretic separation of DNA fragments according to size. Both involve dissolving the DNA-containing agarose in NaI. The preparative technique uses binding of DNA to glass in the presence of NaI. The method is rapid and convenient, and DNA of all molecular weight ranges can be recovered in high yield and without degradation. The DNA is free of agarose and remains susceptible to digestion by restriction enzymes. The analytical technique uses selective precipitation of DNA with acetone and has been adapted to molecular hybridization scans of sequences in agarose gels. The sequence-monitoring system is quantitative, directly measuring the proportion of the probe complementary to a given DNA fragment and vice versa. It is especially suitable for analyzing restriction enzyme digests of DNA in mapping experiments.
Repeated DNA sequences in primates having identical or nearly identical members and exhibiting unusual phylogenetic specificity were analyzed. They appeared in repeated DNA sequences in each group of primates, probably within the last 10 to 15 million years, and are conserved to the same extent as unique DNA sequences. The finding allows a new approach to the construction of evolutionary trees.
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RNA purified from two related RNA tumor viruses, one isolated from a baboon, Papio anubis, and the second from cultured blood leukocytes of a patient with acute myelogenous leukemia, was labeled with 125I and hybridized to DNA from different primates. RNA from both viruses showed maximum sequence homology with genes in baboons and little homology with genes of humans. The results confirm earlier suggestions that both viruses originated by transcription of baboon virogenes, and that one was transmitted to humans in nature. Hybridization of the viral RNA to cell DNA followed complicated kinetic patterns, indicating the presence of both repeated and infrequent virogene elements. This conclusion was verified in experiments using varied DNA:RNA ratios. It is proposed that virogenes, though composed of genes repeated 10 times or more, consist of some sequences more preferentially conserved than others. The non-uniformity of virogene sequence conservation limits the use of viral probes in studies concerning certain aspects of virogene evolution.
Hybridisation of RNA from a baboon endogenous type C RNA virus to DNA from tissues of leukaemic patients indicates that a virus of this type is horizontally transmitted among humans. DNA from several patients with leukaemia hybridised 70% of the hybridisable RNA from baboon endogenous type C RNA virus (BaEV) and yielded hybrids of high tm, whereas DNA from normal human tissues hybridised only 23% of the BaEV RNA, and the tm of these hybrids was lower.
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The results of molecular hybridization experiments with high-molecular-weight RNA isolated from RNA tumor viruses and DNA from normal cells suggest that RNA tumor virus genomes originate from cell genes. Some RNA tumor viruses (here called class 1) appear to have been generated in recent times in that their RNA is closely related in nucleotide sequence to certain cell genes (class 1 genes). A second class of RNA tumor viruses (here called class 2) is more distantly related to genomic information of normal cells. Structural properties of the RNA of RNA tumor viruses lead us to propose that the tumor virus RNA is originated when RNA transcripts of class 1 genes are processed by a mechanism we call "paraprocessing." We postulate that RNA paraprocessing is normally used only at particular times during differentiation and is characterized by the cytoplasmic appearance of high-molecular-weight RNA chains containing terminal polyadenylic acid (200 residues). Paraprocessing of class 1 gene transcripts in committed or differentiated cells is considered to be aberrant in transcription that can lead to the generation of an RNA tumor virus genome. If the paraprocessed class 1 gene transcript codes for a reverse transcriptase, replication of the RNA becomes possible. Transfer of the replicating RNA to a new cell can result in genetic change such that the virus genome mutates, differing from the original progenitor genes. We propose that this genetic change causes class 1 viruses to become class 2. These ideas are applied to evidence concerning the biology of infection of RNA tumor viruses and concerning the involvement of RNA tumor viruses in human cancer. Genetic change can also occur during the origination of an RNA tumor virus genome by repeated reverse transcription and recombination (45) or by genetic alteration of particularly changeable cell genes ("hot spots") (43).
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The reverse transcriptase and endogenous DNA product synthesized by virus-like particles in the cytoplasm of human leukemic cells have been studied for their genetic relatedness to homologous components obtained from several animal RNA tumor viruses. The human reverse transcriptase activity was inhibited by antibodies prepared against reverse transcriptase from some animal RNA tumor viruses. The DNA molecules synthesized endogenously by the human cytoplasmic particle in the presence of actinomycin D, using the reverse transcriptase enzyme and RNA template residing in the particle, hybridized to 70S RNA purified from certain animal RNA tumor viruses. Both the human reverse transcriptase and DNA product are closely related to homologues from primate type-C viruses, more distantly related to those from murine type-C viruses, and essentially unrelated to similar structures from feline or avian type-C viruses. They are not related to type-B RNA tumor viruses. The results demonstrate that the components from the human leukemic cells are viral (type-C) and primate in nature.
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[(3)H]DNA copies of avian, feline, murine, and primate RNA tumor virus genomes were synthesized in vitro by an RNA-dependent DNA polymerase reaction. These DNAs were hybridized to 60-70S RNA that had been purified from the viruses. The amount of the [(3)H]DNA hybridized yielded a measure of the genetic relatedness among the DNA preparations synthesized by the viruses. When many combinations of DNA and RNA were analyzed, the pattern of hybridization showed in some cases that the DNA copies of the viral RNA were related to each other in the same way that the natural hosts of the viruses are phylogenetically related. This pattern was observed only among the RNA leukemia viruses. The sarcoma component in sarcoma-leukemia viruses from rats and primates appeared to be unusually closely related. The mouse mammary carcinoma virus and two unclassified viruses (MPMV and Visna) appeared to be genetically distinct.A similar analysis of DNA synthesized by an RNA-dependent DNA polymerase associated with a viral-like particle obtained from the cytoplasm of human leukemic white blood cells demonstrated that this DNA occupied a space in the affinity pattern of leukemia viruses which is expected of a nucleic acid from a primate-type-C RNA tumor virus. This observation strengthens earlier evidence that components of RNA tumor viruses are associated with human leukemia.
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Visna virus 70S RNA contains long stretches of polyadenylic acid [poly(A)]. The homogeneity in length of poly(4) regions is observed in 70S RNA from visna virus and all RNA tumor viruses tested, and not with other types of RNA. By this criterion visna virus resembles RNA tumor viruses.