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Biomedical subjects

M A Baluda

Publications and source records attributed to M A Baluda.

At least 73 records · Page 4Linked to original sources
PubMed1975

Acquisition of viral DNA sequences in target organs of chickens infected with avian myeloblastosis virus.

The distribution of oncornavirus DNA sequences in various tissues of normal chickens and of chickens with leukemia or kidney tumors induced by avian myeloblastosis virus (AMV) was analyzed by DNA-RNA hybridization using 35S AMV RNA as a probe. All the tissues from normal chickens which were tested contained the same average cellular concentration of endogenous oncornavirus DNA. In contrast, different tissues from lekemic chickens and from chickens bearing kidney tumors contained different concentrations of AMV homologous DNA: in some tissues there was no increase whereas other tissues acquired additional AMV-specific DNA sequences. The increase was the greatest in tissues which can become neoplastic after infection, such as myeloblasts, erythrocytes, and kidney cells. It was directly demonstrated that DNA from AMV-induced kidney tumor contains AMV sequences which are absent in DNA from normal cells. A similar finding had been previously obtained with leukemic cells (15). 3H-labeled 35S RNA from purified AMV was exhaustively hybridized with an excess of normal chicken DNA to remove all the viral RNA sequences which are complementary to DNA from uninfected cells. The 3H-labeled RNA which failed to hybridize was isolated by hydroxylapatite column chromatography which separates DNA-RNA hybrids from single-stranded RNA. The residual RNA hybridized to chicken kidney tumor DNA but did not rehybridize with normal chicken DNA.

Animals↗
PubMed1975

Homology between avian oncornavirus RNAs and DNA from several avian species.

3H-labeled 35S RNA from avian myeloblastosis virus (AMV), Rous associated virus (RAV)-0, RAV-60, RAV-61, RAV-2, or B-77(w) was hybridized with an excess of cellular DNA from different avian species, i.e., normal or leukemic chickens, normal pheasants, turkeys, Japanese quails, or ducks. Approximately two to three copies of endogenous viral DNA were estimated to be present per diploid of normal chicken cell genome. In leukemic chicken myeloblasts induced by AMV, the number of viral sequences appeared to have doubled. The hybrids formed between viral RNA and DNA from leukemic chicken cells melted with a Tm 1 to 6 C higher than that of hybrids formed between viral RNA and normal chicken cell DNA. All of the viral RNAs tested, except RAV-61, hybridized the most with DNA from AMV-infected chicken cells, followed by DNA from normal chicken cells, and then pheasant DNA. RAV-61 RNA hybridized maximally (39%) with pheasant DNA, followed by DNA from leukemic (34%), and then normal (29%) chicken cells. All viral RNAs tested hybridized little with Japanese quail DNA (2 to 5%), turkey DNA (2 to 4%), or duck DNA (1%). DNA from normal chicken cells contained only 60 to 70% of the RAV-60 genetic information, and normal pheasant cells lacked some RAV-61 DNA sequences. RAV-60 and RAV-61 genomes were more homologous to the RAV-0 genome than to the genome of RAV-2, AMV, or B-77(s). RAV-60 and RAV-61 appear to be recombinants between endogenous and exogenous viruses.

Alpharetrovirus↗
PubMed1975

Ribonucleotide sequence homology among avian oncornaviruses.

RNA sequence relatedness among avian RNA tumor virus genomes was analyzed by inhibition of DNA-RNA hybrid formation between 3H-labeled 35S viral RNA and an excess of leukemic or normal chicken cell DNA with increasing concentrations of unlabeled 35S viral RNA. The avian viruses tested were Rous associated virus (RAV)-3, avian myeloblastosis virus (AMV), RAV-60, RAV-61, and B-77 sarcoma virus. Hybridization of 3H-labeled 35S AMV RNA with DNA from normal chicken cells was inhibited by unlabeled 35S RAV-0 RNA as effeciently (100%) as by unlabeled AMV RNA. Hybridization between 3H-labeled 35S AMV RNA and DNA from leukemic chicken myeloblasts induced by AMV was suppressed 100 and 68% by unlabeled 35S RNA from AMV and RAV-0, respectively. Hybridization between 3H-labeled RAV-0 and leukemic chicken myeloblast DNA was inhibited 100 and 67% by unlabeled 35S RNA from RAV-0 and AMV, respectively. It appears therefore that the AMV and RAV-0 genomes are 67 to 70% homologous and that AMV hybridizes to RAV-0 like sequences in normal chicken DNA. Hybridization between AMV RNA and leukemic chicken DNA was inhibited 40% by RNA from RAV-60 or RAV-61 and 50% by B-77 RNA. Hybridization between RAV-0 RNA and leukemic chicken DNA was inhibited 80% by RAV-60 or RAV-61 and 70% by B-77 RNA. Hybridization between 3H-labeled 35S RNA from RAV-60 or RAV-61 and leukemic chicken myeloblast DNA was reduced equally by RNA from RAV-60, RAV-61, AMV or RAV-0; this suggests that RNA from RAV-60 and RAV-61 hybridizes with virus-specific sequences in leukemic DNA which are shared by AMV, RAV-0, RAV-60, and RAV-61 RNA'S. Hybridization between 3H-labeled 35S RNA from RAV-61 and normal pheasant DNA was inhibited 100% by homologous viral RNA, 22 TO 26% BY RNA from AMV or RAV-0, and 30 to 33% by RNA from RAV-60 or B-77. Nearly complete inhibition of hybricization between RAV-0 RNA and leukemic chicken DNA by a mixture of AMV and B-77 35S RNAs indicates that the RNA sequences shared by B-77 virus and RAV-0. It appears that different avian RNA tumor virus genomes have from 50 to 80% homology in nucleotide sequences and that the degree of hybridization between normal chicken cell DNA and a given viral RNA can be predicted from the homology that exists between the viral RNA tested and RAV-0 RNA.

Animals↗
PubMed1975

Quantitative and qualitative differences in DNA complementary to avian myeloblastosis virus between normal and leukemic chicken cells.

Hybridization of avian myeloblastosis virus (AMV) RNA with DNA immobilized on filters or in liquid with a vast DNA excess was used to measure the viral specific DNA sequences in chicken cells. Newly synthesized viral DNA (v-DNA) appears within an hour after infection of chicken embryo fibroblasts (CEF) with avian oncornaviruses. A fraction of newly synthesized v-DNA becomes integrated into the cellular genome and the remainder gradually disappears. A covalent linkage between v-DNA and cellular DNA was demonstrated to exist in CEF and in leukemic myeloblasts by alkaline sucrose velocity sedimentation. Hybridization of AMV RNA in DNA excess has revealed that there are 2 clases of viral specific sequences within normal as well as in leukemic cells. The 2 types of sequences differ in their rate of hybridization. The amount of both types of DNA sequences is about 2 times higher in leukemic cells than in normal cells. Both the fast- and slowly reacting sequences in leukemic cells exhibit a higher Tm (2 degrees C) than the respective DNA sequences in normal cells. Furthermore, when nucleotide sequences in AMV RNA complementary to normal DNA are removed first by exhaustive hybridization with normal DNA, the residual RNA only hybridizes with leukemic DNA but not with normal DNA. These results suggest that leukemic cells contain viral specific DNA sequences which are absent in normal cells. Endogenous v-DNA has been shown to be integrated in cellular DNA region(s) with a reiteration frequency of approximately 1,200 copies per cell and each integration unit appears to have a size approximately equivalent to the 35S RNA subunit of the viral genome. Viral sequences acquired after infection appear to be integrated in the unique region of cell DNA, or in tandem with the endogenous viral sequences.

Animals↗
PubMed1974

Differences between the integration of avian myeloblastosis virus DNA in leukemic cells and of endogenous viral DNA in normal chicken cells.

The nature of integrated viral DNA in normal and leukemic chicken cells has been studied by sequential nucleic acid hybridization procedures that localize the viral specific DNA in cellular DNA regions differing in reiteration frequency. First, DNA.DNA reassociation was employed to fractionate cellular DNA sequences according to their reiteration frequencies. Next, the DNA in each fraction was denatured, immobilized on nitrocellulose filters, and then hybridized with viral [(3)H]RNA. In normal cells, endogenous viral DNA appears to be associated with cell sequences reiterated 1200 times, and each integration unit appears to have a maximal size approximately equivalent to the 35S RNA subunit of the virion. In infected cells, additional viral sequences are found which reassociate as if they integrated adjacent to unique cellular DNA, or in tandem with endogenous viral DNA.

Animals↗
PubMed1974

Acquisition of new DNA sequences after infection of chicken cells with avian myeloblastosis virus.

DNA-RNA hybridization studies between 70S RNA from avian myeloblastosis virus (AMV) and an excess of DNA from (i) AMV-induced leukemic chicken myeloblasts or (ii) a mixture of normal and of congenitally infected K-137 chicken embryos producing avian leukosis viruses revealed the presence of fast- and slow-hybridizing virus-specific DNA sequences. However, the leukemic cells contained twice the level of AMV-specific DNA sequences observed in normal chicken embryonic cells. The fast-reacting sequences were two to three times more numerous in leukemic DNA than in DNA from the mixed embryos. The slow-reacting sequences had a reiteration frequency of approximately 9 and 6, in the two respective systems. Both the fast- and the slow-reacting DNA sequences in leukemic cells exhibited a higher T(m) (2 C) than the respective DNA sequences in normal cells. In normal and leukemic cells the slow hybrid sequences appeared to have a T(m) which was 2 C higher than that of the fast hybrid sequences. Individual non-virus-producing chicken embryos, either group-specific antigen positive or negative, contained 40 to 100 copies of the fast sequences and 2 to 6 copies of the slowly hybridizing sequences per cell genome. Normal rat cells did not contain DNA that hybridized with AMV RNA, whereas non-virus-producing rat cells transformed by B-77 avian sarcoma virus contained only the slowly reacting sequences. The results demonstrate that leukemic cells transformed by AMV contain new AMV-specific DNA sequences which were not present before infection.

Journal Article↗
PubMed1974

Synthesis of avian oncornavirus DNA in infected chicken cells.

The intracellular synthesis and integration of viral DNA (vDNA) into the host cell genome was studied in cultured chicken embryo fibroblasts infected with avian sarcoma or leukemia viruses. The newly synthesized vDNA was detected by hybridization with 70S viral RNA. Extraction of infected cell DNA by the selective procedure of Hirt resulted in the enrichment of newly synthesized vDNA in the low molecular weight supernatant fraction while leaving the bulk of cellular DNA containing integrated vDNA in the high molecular weight pellet fraction. This approach led to detection of intracellular vDNA synthesis within 1 h after infection and to vDNA integration into cellular DNA within 24 h. There was a several-fold increase in the vDNA content of infected cells during the initial phase of virus infection. But only a part of this newly synthesized vDNA appeared to become covalently linked with high molecular weight cellular DNA. Most of the remaining unintegrated vDNA gradually disappeared. The sedimentation profiles of minimally sheared cellular DNA in alkaline sucrose velocity gradients suggest that vDNA is synthesized as free linear molecules of approximately 3 x 10(6) daltons which subsequently are covalently linked to host cell DNA.

Alpharetrovirus↗
PubMed1974

Presence in leukemic cells of avian myeloblastosis virus-specific DNA sequences absent in normal chicken cells.

(3)H-labeled 35S RNA from purified avian myeloblastosis virus (AMV) was exhaustively hybridized with an excess of normal chicken DNA to remove all viral RNA sequences which are complementary to DNA from uninfected cells. The [(3)H]RNA which failed to hybridize was isolated by hydroxylapatite column chromatography which separates DNA-RNA hybrids from single-stranded [(3)H]RNA. The residual RNA hybridized to leukemic chicken DNA but did not rehybridize with normal chicken DNA. This demonstrates conclusively that DNA from AMV-induced leukemic cells contain viral-specific sequences which are absent in DNA from normal cells.

Animals↗
PubMed1974

Reliability of the RNA-DNA filter hybridization for the detection of oncornavirus-specific DNA sequences.

Denatured DNA from leukemic myeloblasts or uninfected chicken embryos, immobilized on nitrocellulose filters, was hybridized to a vast excess of [(3)H]70S RNA from purified avian myeloblastosis virus. The viral RNA was eluted from the RNA-DNA hybrids, purified, and then rehybridized in solution to an excess of either leukemic or normal chicken embryonic DNA. This study revealed that all the slow and the fast hybridizing viral RNA sequences detectable by liquid hybridization in DNA excess had hybridized to the filter bound DNA. Both techniques also gave similar values for the number of 28S ribosomal RNA genes contained in a chicken cell genome: 210 by the liquid hybridization procedure and 218 by the filter hybridization technique. Therefore, filter hybridization can accurately detect DNA sequences present in relatively few numbers in the genome of higher organisms.

Animals↗
PubMed1973

Separation of DNA sequences complementary to the RNA of avian myeloblastosis virus from chicken DNA by alkaline cesium chloride density sedimentation.

Density gradient sedimentation in alkaline cesium chloride of DNA from normal chicken embryos or leukemic myeloblasts fragmented to a size of 13S revealed that the DNA sequences complementary to 70S avian myeloblastosis virus RNA sedimented in the high guanine plus cytosine region ahead of the main peak of cellular DNA. When the DNA was fragmented into pieces of 6.6S there was a broader distribution of the DNA sequences complementary to the viral RNA. This technique could be employed as a step towards the isolation of DNA copies of the entire viral RNA genome from the mass of host cellular DNA.

Animals↗
PubMed1973

Integrated state of oncornavirus DNA in normal chicken cells and in cells transformed by avian myeloblastosis virus.

The covalent linkage of oncornavirus-specific DNA to chicken DNA was investigated in normal chicken embryo fibroblasts (CEF) and in virus-producing leukemic cells transformed by avian myeloblastosis virus (AMV). The virus-specific sequences present in cellular DNA fractionated by different methods were detected by DNA-RNA hybridization by using 70S AMV RNA as a probe. In CEF and in leukemic cells, the viral DNA appeared to be present only in the nucleus. After cesium chloride-ethidium bromide density equilibrium sedimentation, the viral DNA was present as linear, double-stranded molecules not separable from linear chicken DNA. After extraction by the Hirt procedure, the viral DNA precipitated with the high-molecular-weight DNA. After alkaline sucrose velocity sedimentation, the viral DNA cosedimented with the high-molecular-weight cellular DNA. The results indicate that in both types of cells studied, the oncornavirus-specific DNA sequences were linked by alkali stable bonds to nuclear cellular DNA of high molecular weight and did not appear to be present in free form of any size.

Animals↗
PubMed1972

Widespread presence, in chickens, of DNA complementary to the RNA genome of avian leukosis viruses.

DNA-RNA hybridization experiments have demonstrated the widespread presence in chickens of DNA complementary to the RNA of avian myeloblastosis virus. All apparently normal chicken embryos, or adult chickens that were tested, contained viral DNA in amounts ranging from 1.7 to 4.6 viral genome equivalents per cell. Embryos that were negative or positive for the group-specific antigen of avian leukosis viruses contained the same amount of viral DNA. Embryos from a strain of chickens free of leukosis viruses of groups A and B that develop 40% fewer spontaneous leukotic tumors than the original strain contained an average of 2.1 viral DNA equivalents per cell, whereas the original strain contained 3.2 viral DNA equivalents. By comparison, leukemic cells and cells infected with avian myeloblastosis virus or Rous sarcoma virus contained between 4 and 13 viral DNA equivalents. This study provides direct biochemical evidence for the presence in normal chicken cells of avian leukosis virus DNA that is inheritable and that represents either complete or incomplete viral genomes.

Animals↗
PubMed1972

Distribution of deoxyribonucleic acid complementary to the ribonucleic acid of avian myeloblastosis virus in tissues of normal and tumor-bearing chickens.

(3)H-labeled 70S ribonucleic acid (RNA) from purified avian myeloblastosis virus (AMV) was used as a probe in deoxyribonucleic acid (DNA)-RNA hybridization experiments to detect the presence of DNA complementary to the AMV genome in various tissues from noninfected normal chickens and from chickens infected with AMV. There was a remarkable constancy in the average cellular concentration of virus-specific DNA found in every tissue from the same uninfected chicken, and even in different chickens from the same strain. In contrast, different tissues from chickens bearing AMV-induced kidney tumors (embryonal nephromas) revealed an unequal distribution in the average virus-specific DNA content per cell. The increase was limited to tumor cells and to tissues that contain target cells for AMV, i.e., red blood cells, kidney cells, and possibly leukocytes. The red blood cells from AMV-infected chickens suffering from acute myeloblastic leukemia, although producing no virus, contained as many viral genome equivalents per cell as did leukemic myeloblasts known to produce large quantities of AMV. An increased viral DNA content was observed in the target cells of chickens that did not show any sign of tumor formation 6 months after infection with AMV. This study demonstrates that vertically transmitted viral DNA is uniformly and stably distributed among all tissues of the offspring, but that horizontal infection after hatching results in an increase in viral DNA content only in some dividing, target tissues that may or may not give rise to neoplasias.

Animals↗
PubMed1970

DNA complementary to viral RNA in leukemic cells induced by avian myeloblastosis virus.

Nucleic acid hybridization studies were made between 71S-AMV-RNA and DNA from leukemic myeloblasts and from normal chicken cells. There was homology between the viral RNA and chicken cell DNA and to a greater extent between viral RNA and leukemic cell DNA. Leukemic cell DNA hybridized approximately twice as much viral RNA as did normal chicken DNA. Thermal melting studies showed that the viral RNA bound to normal and leukemic cell DNA consists of long polynucleotides (T(m) = 87 degrees and 92 degrees C, respectively, in 2x saline citrate). This suggests that the leukemic cells contain a DNA template of the viral RNA.

Animals↗
PubMed1969

Characterization of influenza virus ribonucleic acid duplex produced by annealing in vitro.

A ribonuclease-resistant ribonucleic acid (RNA) with a sedimentation coefficient of 12S was obtained by self-annealing influenza virus-specific RNA isolated from infected cells. It had the properties of double-stranded RNA. (i) Sedimentation behavior in sucrose gradient was independent of salt concentration. (ii) Thermal transition profile was sharp; the melting temperature is 83 C in 0.1 SSC (0.15 m NaCl plus 0.015 m sodium citrate) and 98 C in SSC. (iii) Buoyant density in cesium sulfate was 1.58 g/cm(3) compared to 1.64 g/cm(3) for single-stranded RNA. (iv) It gave rise to single-stranded RNA after denaturation. (v) The 12S RNA duplex contained both plus and minus strands of influenza virus. Labeled plus strands could be displaced by extraneous cold plus strands and extraneous (32)P-labeled plus strands could be incorporated into duplex after denaturation and reannealing.

Animals↗