(1/401) Transformation by v-Myb.
The v-myb oncogene of the avian myeloblastosis virus (AMV) is unique among known oncogenes in that it causes only acute leukemia in animals and transforms only hematopoietic cells in culture. AMV was discovered in the 1930s as a virus that caused a disease in chickens that is similar to acute myelogenous leukemia in humans (Hall et al., 1941). This avian retrovirus played an important role in the history of cancer research for two reasons. First, AMV was used to demonstrate that all oncogenic viruses did not contain a single cancer-causing principle. In particular, although both Rous sarcoma virus (RSV) and AMV could replicate in cultures of either embryonic fibroblasts or hematopoietic cells, RSV could transform only fibroblasts whereas AMV could transform only hematopoietic cells (Baluda, 1963; Durban and Boettiger, 1981a). Second, chickens infected with AMV develop remarkably high white counts and therefore their peripheral blood contains remarkably large quantities of viral particles (Beard, 1963). For this reason AMV was often used as a prototypic retrovirus in order to study viral assembly and later to produce large amounts of reverse transcriptase for both research and commercial purposes. Following the discovery of the v-src oncogene of RSV and the demonstration that it arose from the normal c-src proto-oncogene, a number of acute leukemia viruses were analysed by similar techniques and found to also contain viral oncogenes of cellular origin (Roussel et al., 1979). In the case of AMV, it was shown that almost the entire retroviral env gene had been replaced by a sequence of cellular origin (initially called mab or amv, but later renamed v-myb) (Duesberg et al., 1980; Souza et al., 1980). Remarkably, sequences contained in this myb oncogene were shared between AMV and the avian E26 leukemia virus, but were not contained in any other acutely transforming retroviruses. In addition, the E26 virus contained a second sequence of cellular origin (ets) that was unique. The E26 leukemia virus was first described in the 1960s and causes an acute erythroblastosis in chickens, more reminiscent of the disease caused by avian erythroblastosis virus (AEV) than by AMV (Ivanov et al., 1962). (+info)
(2/401) Retrovirus DNA termini bound by integrase communicate in trans for full-site integration in vitro.
Integration of linear retrovirus DNA involves the concerted insertion of the viral termini (full-site integration) into the host chromosome. We investigated the interactions that occur between long terminal repeat (LTR) termini bound by avian retrovirus integrase (IN) for full-site integration in vitro. Wild-type (wt) or mutant LTR donors that possess gain-of-function ("G") or loss-of-function ("L") for full-site integration activity were used. G LTR termini are characterized as having significantly higher strand transfer activity than the wt and the L LTR termini. L LTR mutations are classified as partially or extremely defective for strand transfer activity. The L mutations were further classified by their ability to either permit or block the assembly of G or wt LTR termini into nucleoprotein complexes capable of full-site strand transfer. We demonstrated that avian myeloblastosis virus IN bound to G LTR termini increased the incorporation of partially defective L LTR termini into nucleoprotein complexes that were capable of full-site integration. The observed full-site integration activity of these assembled nucleoprotein complexes appeared to be influenced by each individual IN-LTR complex in trans. In contrast, extremely defective L LTR termini exhibited the ability to effectively block the assembly of wt LTR termini into nucleoprotein complexes capable of full-site strand transfer. Data from nonspecific DNA competition experiments suggested that IN had an apparent higher affinity for G LTR donor termini than for partially defective L LTR donor termini as measured by full-site integration activity. However, assembled nucleoprotein complexes containing either two G or two L LTR donors were stable, having a similar half-life of approximately 2 h on ice. The results suggest that LTR termini bound by IN exhibit an allosteric effect to modulate full-site integration in vitro. Similar regulatory controls also appear to exist in vivo between the wt U3 and wt U5 LTR termini in retroviruses as well as purified retrovirus preintegration complexes that promoted full-site integration in vitro. (+info)
(3/401) Retinoid X receptor suppresses transformation by the v-myb oncogene.
The v-myb oncogene of avian myeloblastosis virus causes acute monoblastic leukemia in vivo and transforms myelomonocytic cells in culture. Retinoids are potent regulators of proliferation and differentiation in various cell types, and they can initiate differentiation in certain types of leukemic cells. However, the BM2 v-myb-transformed chicken monoblastic cell line is resistant to retinoic acid treatment. We found that overexpression of the retinoid X receptor confers sensitivity of BM2 cells to retinoic acid, resulting in induction of growth arrest and terminal differentiation. In contrast, the frequency of apoptosis was not affected by the retinoid X receptor in this cell type. We also demonstrated that suppression of transformation by v-Myb results from the negative effect of retinoid X receptor on v-Myb transactivation function, similar to that previously described for the retinoic acid receptor. The retinoid X receptor-induced inhibition of transactivation by v-Myb seems to be enhanced by a cell type-specific factor(s), which is not required by retinoic acid receptor. (+info)
(4/401) Metal-induced infidelity during DNA synthesis.
The effect of several divalent cations on the accuracy of DNA replication in vitro has been examined. Only Be2+ altered the accuracy of DNA synthesis using purified DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 188.8.131.52) from avian myeloblastosis virus. The Be2+-induced base substitutions occurred with all templates and with all nucleotides tested. Analysis of the product by equilibrium density centrifugation and processive hydrolysis with snake venom phosphodiesterase suggested that the noncomplementary nucleotides were present in phosphodiester linkage. Nearest neighbor studies indicated that many of the Be2+-induced errors were present as single base substitutions. The enhancement of error frequency could be duplicated by the pretreatment of the enzyme, but not the template, with Be2+. Glycerol gradient centrifugation dissociated the Be2+-DNA polymerase complex and restored the initial error frequency of the polymerase. Thus, the weak binding of a metal cation to a DNA polymerase could alter the accuracy with which that polymerase copied DNA. Beryllium is a known carcinogen. The potential use of this system as a screening technique to detect chemical mutagens and carcinogens is considered. (+info)
(5/401) Chromatin as a template for RNA synthesis in vitro.
RNA transcribed in vitro from myeloblast chromatin by exogenously added RNA polymerase B predominantly consists of short chains that remain in hybrid structure with the template; the remainder of the product is free RNA of heterogeneous size. Addition of polyanions during synthesis caused an increase in the size and amount of free RNA with a concomitant decrease in the proportion of small RNA. The large molecular weight RNA is derived from the short RNA chains, which are synthesized de novo during the reaction in vitro. The effect of polyanions on the size and nature of the product may be related to structural changes induced in the template rather than to an inhibition of nuclease activity. (+info)
(6/401) A new chemical procedure for 32P-labeling of ribonucleic acids at their 5'-ends after isolation.
A new technique, which utilizes the chemical reaction between [32P]diimidazolidate of orthophosphate and the cetyltrimethylammonium salt of high-molecular-weight RNA in nonaqueous dimethyl formamide, has been developed for the 32P-labeling of RNAs after isolation. The radioactive label of high specific activity is introduced onto a phosphorylated 5'-end of the RNA and renders it suitable for 5'-terminal group analysis. When the labeling reaction was applied to the 70S RNA of avian myeloblastosis virus, a labeled 35S RNA was isolated on sucrose-dimethyl sulfoxide gradients without apparent degradation. (+info)
(7/401) Avian retrovirus DNA internal attachment site requirements for full-site integration in vitro.
Concerted integration of retrovirus DNA termini into the host chromosome in vivo requires specific interactions between the cis-acting attachment (att) sites at the viral termini and the viral integrase (IN) in trans. In this study, reconstruction experiments with purified avian myeloblastosis virus (AMV) IN and retrovirus-like donor substrates containing wild-type and mutant termini were performed to map the internal att DNA sequence requirements for concerted integration, here termed full-site integration. The avian retrovirus mutations were modeled after internal att site mutations studied at the in vivo level with human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). Systematic overlapping 4-bp deletions starting at nucleotide positions 7, 8, and 9 in the U3 terminus had a decreasing detrimental gradient effect on full-site integration, while more internal 4-bp deletions had little or no effect. This decreasing detrimental gradient effect was measured by the ability of mutant U3 ends to interact with wild-type U3 ends for full-site integration in trans. Modification of the highly conserved C at position 7 on the catalytic strand to either A or T resulted in the same severe decrease in full-site integration as the 4-bp deletion starting at this position. These studies suggest that nucleotide position 7 is crucial for interactions near the active site of IN for integration activity and for communication in trans between ends bound by IN for full-site integration. The ability of AMV IN to interact with internal att sequences to mediate full-site integration in vitro is similar to the internal att site requirements observed with MLV and HIV-1 in vivo and with their preintegration complexes in vitro. (+info)
(8/401) Modeling the late steps in HIV-1 retroviral integrase-catalyzed DNA integration.
Model oligodeoxyribonucleotide substrates representing viral DNA integration intermediates with a gap and a two-nucleotide 5' overhang were used to examine late steps in human immunodeficiency virus, type 1 (HIV-1) retroviral integrase (IN)-catalyzed DNA integration in vitro. HIV-1 or avian myeloblastosis virus reverse transcriptase (RT) were capable of quantitatively filling in the gap to create a nicked substrate but did not remove the 5' overhang. HIV-1 IN also failed to remove the 5' overhang with the gapped substrate. However, with a nicked substrate formed by RT, HIV-1 IN removed the overhang and covalently closed the nick in a disintegration-like reaction. The efficiency of this closure reaction was very low. Such closure was not stimulated by the addition of HMG-(I/Y), suggesting that this protein only acts during the early processing and joining reactions. Addition of Flap endonuclease-1, a nuclease known to remove 5' overhangs, abolished the closure reaction catalyzed by IN. A series of base pair inversions, introduced into the HIV-1 U5 long terminal repeat sequence adjacent to and/or including the conserved CA dinucleotide, produced no or only a small decrease in the HIV-1 IN-dependent strand closure reaction. These same mutations caused a significant decrease in the efficiency of concerted DNA integration by a modified donor DNA in vitro, suggesting that recognition of the ends of the long terminal repeat sequence is required only in the early steps of DNA integration. Finally, a combination of HIV-1 RT, Flap endonuclease-1, and DNA ligase is capable of quantitatively forming covalently closed DNA with these model substrates. These results support the hypothesis that cellular enzyme(s) may catalyze the late steps of retroviral DNA integration. (+info)