Cell cycle dependence of synthesis of unintegrated viral DNA in mouse cells newly infected with murine leukemia virus.
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E Rassart
Publications and source records attributed to E Rassart.
Fate of unintegrated viral DNA in Fv-1 permissive and resistant mouse cells infected with murine leukemia virus.
We have found that levels of unintegrated linear viral DNA were nearly identical in several Fv-1 resistant cell lines, whereas levels of closed circular viral DNA are markedly reduced in these resistant cells, to the same extent as virus production (P. Jolicoeur and E. Rassart, J. Virol. 33:183-195, 1980). To determine the fate of linear viral DNA made in resistant cells we performed pulse-chase experiments, labeling viral DNA with 5-bromodeoxyuridine and following it with a thymidine chase. 5-Bromodeoxyuridine-labeled viral DNA (HH) recovered by banding on cesium chloride gradients was sedimented on neutral sucrose density gradients or separated by the agarose gel-DNA transfer procedure and detected by hybridization with complementary DNA. Levels of linear viral DNA made in Fv-1(b/b) (JLS-V9 and SIM.R) and Fv-1(n/n) (NIH/3T3 and SIM) cells were found to decrease during the chase period at about the same rate in permissive and nonpermissive conditions, indicating that linear viral DNA is not specifically degraded in Fv-1 resistant cells. Levels of the two species of closed circular viral DNA made in Fv-1 permissive cells increased relative to the levels of linear DNA during the chase period. This confirmed the precursor-product relationship between linear DNA and the two species of circular DNA. In Fv-1 resistant cells, this apparent conversion of linear viral DNA into circular forms was not seen, and no supercoiled viral DNA could be detected. To determine whether the transport of linear viral DNA from the cytoplasm into the nucleus was prevented by the Fv-1 gene product, SIM.R cells were fractionated into cytoplasmic and nuclear fractions, and viral DNA was detected in each fraction by the agarose gel-DNA transfer procedure. Levels of linear viral DNA were nearly identical in both cytoplasmic and nuclear fractions of permissive or resistant cells. Circular viral DNA could be detected in the nuclear fraction of permissive cells, but not in that of resistant cells. A pulse-chase experiment was also performed with SIM.R cells. During the thymidine chase period, linear viral DNA was seen to accumulate in nuclei of both permissive and resistant cells, whereas supercoiled viral DNA accumulated only in nuclei of permissive cells. These results indicate that the Fv-1 gene product does not interfere with the transport of linear viral DNA into the nucleus. Our data also suggest that the Fv-1 restriction does not operate through a degradation process. Therefore, the Fv-1 gene product could either block the circularization of linear viral DNA directly or promote the synthesis of a faulty linear viral DNA whose defect (yet undetected) would prevent its circularization.
Alleles↗
Molecular cloning of B- and N-tropic endogenous BALB/c murine leukemia virus circular DNA intermediates: isolation and characterization of infectious recombinant clones.
The unintegrated closed circular DNA intermediates from B- and N-tropic endogenous BALB/c murine leukemia virus (MuLV) were isolated from acutely infected mouse fibroblasts and cleaved at their unique HindIII sites. The linearized B- and N-tropic MuLV DNAs were then inserted in lambda vector Charon 21A and cloned. Seventeen B-tropic and ten N-tropic recombinants were analyzed. The viral insert of these 27 MuLV recombinants could be grouped into 3 classes. The first class included permuted full-length molecules of 8.8 kilobase pairs (kbp) having two long terminal repeats (LTR) (0.55 kbp) mixed with 8.25-kbp molecules. The second class included 8.25-kbp molecules which harbored only one LTR copy. The third class of inserts was made of molecules with deletions of various lengths (0.1 to 3 kbp). These deletions were localized by restriction enzyme analysis. Significantly more deletions were observed in N-tropic than in B-tropic recombinants, although these two genomes isolated from acutely infected cells seemed to differ only by the absence of a 70-base-pair (bp) sequence in the N-tropic LTR. The biological activity of each of the recombinants of the first and second classes was assayed by transfection on NIH/3T3 cells. Two recombinants, one B-tropic (lambda B-16) and one N-tropic (lambdaN-20) were found to be infectious. Both were able to give rise to replication-competent MuLV which could form large XC plaques and had kept the tropism of its progenitor. Like its progenitor in vivo DNA, lambdaN-20-cloned DNA lacked a 70-bp sequence in its unique LTR. The emerging N-tropic MuLV after transfection with lambdaN-20 was used to acutely infect NIH/3T3 cells. The viral DNA intermediates isolated were now found to have two LTR copies. They had also acquired a 70-bp sequence in each LTR. The acquisition or loss of this 70-bp sequence did not seem to affect the replication, the XC plaque formation, or the tropism of the virus.
Animals↗
Effect of Fv-1 gene product on synthesis of linear and supercoiled viral DNA in cells infected with murine leukemia virus.
Levels of unintegrated viral DNA made in Fv-1b/b (SIM.R, JLS-V9) and Fv-1n/n (NIH/3T3) cell lines after infection with N- or B-tropic murine leukemia virus (MuLV) have been measured. Different forms of viral DNA were sedimented on neutral sucrose or ethidium bromide-cesium chloride density gradients and detected by hybridization with complementary DNA. It was found that the major viral DNA species made in Fv-1 permissive or resistant cells was sedimenting at 20S on neutral sucrose gradient. Levels of this 20S viral DNA species were not significantly different in both systems. However levels of closed circular (form I) viral DNA separated on ethidium bromide-cesium chloride gradients were found to be decreased in Fv-1 resistant cells. Various species of viral DNA were also analyzed by the agarose gel-DNA transfer procedure of Southern. The major viral DNA species was found to migrate as a double-stranded linear DNA of 5.7 x 10(6) daltons. The molecular weight of linear viral DNA molecules extracted from Fv-1 permissive or resistant cells appeared to be the same. Levels of this linear viral DNA were almost identical in both systems except in B-tropic MuLV-infected resistant NIH/3T3 cells in which a moderate decrease has been measured. Two closed circular viral DNA species were observed by this technique. Their levels were markedly decreased in Fv-1 resistant cells. Our results indicate that the Fv-1 restriction does not grossly affect the formation of linear double-stranded viral DNA, but prevents the accumulation of closed circular viral DNA. Therefore the Fv-1 gene product could allow the synthesis of a normal linear viral DNA but interfere with the formation of supercoiled viral DNA. Alternatively, it could promote the synthesis of a faulty linear viral DNA whose defect (yet undetected) would prevent its circularization. In any case, the Fv-1 restriction mechanism appears to occur before the integration event itself.
Alleles↗
Distribution of endogenous murine leukemia virus DNA sequences among mouse chromosomes.
We used mouse-Chinese hamster somatic cell hybrids which lose mouse chromosomes to examine the distribution of murine leukemia virus DNA sequences in the genome of A/HeJ mice. We analyzed total cellular DNA from various hybrid clones for the presence of viral sequences by molecular hybridization and used the Southern blot hybridization procedure to identify viral DNA in cellular restriction endonuclease fragments. Our results show that murine leukemia virus DNA sequences are distributed among many mouse chromosomes in this strain. Chromosome 4 was shown to contain murine leukemia virus DNA sequences.
Animals↗
Restriction endonuclease mapping of unintegrated viral DNA of B- and N-tropic BALB/c murine leukemia virus.
Unintegrated linear and closed circular DNAs of B- and N-tropic endogenous BALB/c murine leukemia virus (MuLV) were extracted from newly infected mouse cells and cleaved with EcoRI, XhoI, PvuI, HindIII, SalI, XbaI, KpnI, SmaI, and PstI restriction endonucleases. The DNA fragments were separated by electrophoresis and analyzed by the Southern blot hybridization procedure. EcoRI did not cleave the two genomes. A physical map of 15 cleavage sites on B- and N-tropic genomes was constructed with the other restriction endonucleases. Identical cleavage sites of B- and N-tropic MuLV DNAs were found with all these enzymes. However, the N-tropic linear genome was found to lack about 75 base pairs at each end of the molecule. PstI, KpnI, and SmaI recognize a cleavage site at both ends of the linear molecules. And sequences derived from the 5' end of the RNA genome were found in the third left end of the linear DNA and at its extreme right-end terminus, suggesting the presence of redundant sequences. Two species of closed circular viral DNA were observed. The larger species has the same size as the linear molecule and appears to be a circularized form of linear DNA. The smaller species contains sequences common to both the linear and the larger circular viral DNA but seems to be deleted from sequences present at either one or both ends of the linear DNA. Therefore, the general structure of the linear and circular DNA species of these B- and N-tropic endogenous BALB/c MuLV appears analogous to the structure found with other retroviruses.
Animals↗
Localization of Escherichia coli RNA polymerase binding sites on bacteriophage S13 and 0X174 DNAs by electron microscopy.
Complexes between Escherichia coli RNA polymerase and bacteriophage S13 and phage phiX174 replicative form III DNAs have been shown to form at specific locations on the phage genomes. The major locations on S13 have been mapped at 8 to 10 and 92 to 96% of the genome length, starting from the unique Pst I cleavage site. The locations correspond to the beginnings of genes D and B, respectively. Four minor locations map at 18 to 22, 28 to 32, 50 to 56, and 70 to 74% of the genome. The 70 to 74% site corresponds to the beginning of the A gene. The major locations on phiX174 are at 8 to 10, 50 to 54, and 92 to 94% of the genome. The 50 to 54% site is at the start of the H gene and has an equivalent minor site on S13, but it is not a promoter site. Three minor sites on phiX174, at 20 to 24, 26 to 32, and 68 to 74% of the genome, correspond to sites on S13. The data confirm the locations of sites identified by restriction fragment binding experiments (E. Rassart and J. H. Spencer, J. Virol. 27:677--687, 1978) and the assignment of putative promoters at the start of genes A, B and D.
Binding Sites↗
Localization of Escherichia coli RNA polymerase binding sites on bacteriophage S13 and phiX174 DNA: alignment with restriction enzyme maps.
Escherichia coli RNA polymerase has been shown to bind to a limited number of Hind and Hae III restriction enzyme fragments. On S13 replicative form DNA there are three major binding sites, and the locations correlate with promoter sites at the beginning of genes a and B and a site overlapping gene D and the beginning of gene E. Two less definite binding sites have been localized, one in gene F and one at the gene G-H junction. In phiX174 replicative form DNA, five sites, each with apparently similar binding properties, have been located, four of which correspond exactly to binding sites in S13. One site, at the beginning of the B gene, could not be assigned to exactly the same location found in S13. This was due in part to differences in the restriction cleavage maps in the area of the DNA and possibly to the higher background of nonspecific binding in the phiX174 experiments. The location of two of the phiX174 sites at the beginning of genes A and D-E corresponds very well with transcription data, but the site at the start of the B gene indicates the promoter site is closer to the initiation sequence of the B protein than was previously suggested on the basis of transcription data.
Binding Sites↗
Purification of Escherichia coli endonuclease specific for apurinic sites in DNA.
The endonuclease specific for apurinic sites in DNA has been isolated from Escherichia coli B41 as a pure monomeric protein of 32,000 daltons. The enzyme hydrolyzes a phosphodiester bond near the apurinic sites in double-stranded DNA; it does not hydrolyze untreated DNA and its action on alkylated DNA is restricted to the apurinic sites always present. This enzyme is not endonuclease II which is most probably a mixture of two enzymes, one a glycosidase (Kirtikar, D. M., and Goldthwait, D. A. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 2022-2026), the other an endonuclease for apurinic sites which is the enzyme isolated in this work.
Amino Acids↗
Gene expression during cold and heat shock in wheat.
Translatable messenger RNAs expression was compared in cold- and heat-stressed winter wheat (Triticum aestivum L. 'Fredrick' and 'Norstar') and spring wheat (T. aestivum L. 'Glenlea'). Polyadenylated RNA isolated from the crown and leaf tissues was translated in a wheat germ cell free system and the acidic and basic in vitro products were resolved by two-dimensional SDS-PAGE and autoradiography. The results showed that low temperature stress rapidly induced two groups of mRNAs. The first group was transient in nature and consists of 18 mRNAs that reached their highest levels of induction after 24 h of low temperature exposure and then decreased to undetectable levels. The second group consists of 53 mRNAs that were also induced or increased rapidly, but maintained their levels of expression during the 4 weeks required to induce freezing tolerance. Among those, at least 34 were expressed at higher levels in the freezing tolerant winter wheat compared with the less tolerant spring wheat. This suggests a possible relation between the expression of these mRNAs and the capacity of each genotype to develop freezing tolerance. In the case of heat shock, 50 mRNAs were induced or increased after 3 h at 40 degrees C. Among these, the expression of only six mRNAs was altered in a similar manner in the three genotypes by both treatments. The remaining mRNAs code for typical heat shock proteins which are different from those induced by low temperature. None of these mRNAs has been associated with the development of freezing tolerance. These results suggest that heat and cold stress are controlled by different genetic systems.
Autoradiography↗