Relationship of avian retrovirus DNA synthesis to integration in vitro.

An in vitro integration system derived from avian leukosis virus-infected cells supports both intra- and intermolecular integration of the viral DNA. In the absence of polyethylene glycol, intramolecular integration of viral DNA molecules into themselves (autointegration) was preferred. In the presence of polyethylene glycol, integration into an exogenously supplied DNA target was greatly promoted. Analysis of integration intermediates revealed that the strand transfer mechanisms of both reactions were identical to those of retroviruses and some transposons: each 3' end of the donor molecule is joined to a 5' end of the cleaved target DNA. The immediate integration precursor appears to be linear viral DNA with the 3' ends shortened by 2 nucleotides. Finally, in the avian system, most cytoplasmic viral DNA appears to be incomplete and further DNA synthesis is required for integration in vitro.     

[3H]SCH 39166, A New D1-Selective Radioligand: In Vitro and In Vivo Binding Analyses

SCH 39166 [(-)-trans-6,7,7a,8,9, 13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5H-benzo-[d]naphtho[2, 1b]azepine] has recently been described as a selective D1 antagonist and has entered clinical trials for the treatment of schizophrenia. The tritiated analogue of this compound, [3H]SCH 39166, has now been synthesized and characterized for its in vitro and in vivo binding profiles. [3H]SCH 39166 binds to D1 receptors in a saturable, high-affinity fashion, with a KD of 0.79 nM. In competition studies, D1-selective antagonists like SCH 23390 displaced the binding of [3H]SCH 39166 with nanomolar affinities, whereas antagonists of other receptors exhibited poor affinity. In vivo, [3H]SCH 39166 bound to receptors in rat striatum in a fashion suggestive of D1 selectivity. Further, when the time course for the binding of [3H]SCH 39166 was compared with the behavioral time course of the unlabeled compound, the two durations of action were virtually indistinguishable. Similar studies were performed for SCH 23390 and its tritiated analogue, but the in vivo binding of this radioligand exhibited a duration of action far greater than the behavioral activity of the unlabeled drug. In concert, these data demonstrate that [3H]SCH 39166 selectively labels D1 receptors in vitro and in vivo, and that this drug is superior for in vivo imaging of the D1 receptor.     

Analysis of mRNA 5'-terminal cap structures and internal N6-methyladenosine by reversed-phase high-performance liquid chromatography

A simple procedure for determining the complete methylation profile of an mRNA molecule in a single chromatographic separation is described. The mRNA is selectively hydrolyzed to its component nucleosides leaving its cap 0 (m⁷GpppN′) or cap 1 (m⁷GpppN′m) structure intact. The hydrolysis products, which can include cap 0, cap 1, 2′-O-methylnucleosides (N″m) of cap 2 (m⁷GpppN′mpN″m) and internal N⁶-methyladenosine, are separated on an octadecyl reverse-phase column using a mobile phase containing acetonitrile and ammonium formate, a weak ion-pairing reagent. methyl-³H-labeled poly(A)-containing mRNA is used to demonstrate the efficacy of the procedure.     

Recombinants between endogenous and exogenous avian tumor viruses: role of the C region and other portions of the genome in the control of replication and transformation.

Endogenous retroviruses of chickens are closely related to exogenous viruses isolated from spontaneous tumors in the same species, yet differ in a number of important characteristics, including the ability to transform cells in culture, ability to cause sarcomas or leukemias, host range, and growth rate in cell culture. To correlate these differences with specific sequence differences between the two viral genomes, the genome RNA of transforming subgroup E recombinants between the Prague strain of Rous sarcoma virus, subgroup B (Pr-RSV-B), and the endogenous Rous-associated virus-0 (RAV-0), Subgroup E, and seven nontransforming subgroup E recombinants between the transformation-defective mutant of Pr-RSV-B and RAV-0 was examined by oligonucleotide fingerprinting. The pattern of inheritance among the recombinant viruses of regions of the genome in which Pr-RSV-B and RAV-0 differ allowed us to draw the following conclusions. (i) Nonselected parts of the genome were, with a few exceptions, inherited by the recombinant virus progeny randomly from either parent, with no obvious linkage between neighboring sequences. (ii) A small region in the Pr-RSV-B genome which maps in the 5' region was found in all transforming but only some of the nontransforming recombinants, suggesting that it plays a role in the control of the expression of transformation. (iii) A region of the Pr-RSV-B genome which maps between env and src was similarly linked to the src gene and may be either part of the structural gene for src or a control sequence regulating the expression of src. (iv) The C region at the extreme 3' end of the virus genome which is closely related in all the exogenous avian retroviruses but distinctly different in the endogenous viruses is the major determinant responsible for the differences in growth rate between RAV-0 and Pr-RSV-B. This latter observation allowed us to redefine the C region as a genetic locus, c, with two alleles cn (in RAV-0) and cx (in exogenous viruses).     

Stable carbon isotope analysis of nucleic acids to trace sources of dissolved substrates used by estuarine bacteria

The natural abundance of stable carbon isotopes measured in bacterial nucleic acids extracted from estuarine bacterial concentrates was used to trace sources of organic matter for bacteria in aquatic environments. The stable carbon isotope ratios of Pseudomonas aeruginosa and nucleic acids extracted from cultures resembled those of the carbon source on which bacteria were grown. The carbon isotope discrimination between the substrate and total cell carbon from bacterial cultures averaged 2.3% +/- 0.6% (n = 13). Furthermore, the isotope discrimination between the substrate and nucleic acids extracted from bacterial cultures was 2.4% +/- 0.4% (n = 10), not significantly different from the discrimination between bacteria and the substrate. Estuarine water samples were prefiltered through 1-micron-pore-size cartridge filters. Bacterium-sized particles in the filtrates were concentrated with tangential-flow filtration and centrifugation, and nucleic acids were then extracted from these concentrates. Hybridization with 16S rRNA probes showed that approximately 90% of the nucleic acids extracted on two sample dates were of eubacterial origin. Bacteria and nucleic acids from incubation experiments using estuarine water samples enriched with dissolved organic matter from Spartina alterniflora and Cyclotella caspia had stable carbon isotope values similar to those of the substrate sources. In a survey that compared diverse estuarine environments, stable carbon isotopes of bacteria grown in incubation experiments ranged from -31.9 to -20.5%. The range in isotope values of nucleic acids extracted from indigenous bacteria from the same waters was similar, -27.9 to -20.2%. Generally, the lack of isotope discrimination between bacteria and nucleic acids that was noted in the laboratory was observed in the field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rate of virus-specific RNA synthesis in synchronized chicken embryo fibroblasts infected with avian leukosis virus.

The rate of avian leukosis virus (ALV)-specific RNA synthesis has been examined in bot- uninfected and ALV-infected synchronized chicken embryo fibroblasts. RNA from cells labeled for 2h with [3H]uridine was hybridized with avian myeloblastosis virus poly(dC)-DNA, and the hybridized RNA was analyzed with poly(I)-spephadex chromatography. Approximately 0.5% of the RNA synthesized in ALV-infected cells was detected as virus specific, and no more than a twofold variation in the rate of synthesis was detected at different times in the cell cycle. In synchronized uninfected chicken embryo fibroblasts, approximately 0.03% of the RNA synthesized was detected as virus specific, and no significant variation in the rate of synthesis was observed during the cell cycle. Treatment of ALV-infected chicken embryo fibroblasts with cytosine arabinoside or colchicine was used to block cells at different stages in the cell cycle. The rates of virus-specific RNA synthesis in cells so treated did not differ significantly from the rates in either stationary or unsynchronized virus-infected chicken embryo fibroblasts. These findings support the conclusion that after the initial division of an ALV-infected chicken embryo fibroblast and the initiation of virus RNA synthesis, the rate of virus-specific RNA synthesis is independent of the cell cycle.     

Rescue of Rous Sarcoma Virus from Rous Sarcoma Virus-Transformed Mammalian Cells

Rat cells transformed by the B77 strain of avian sarcoma virus produce no virus-like particles, yet B77 virus was rescued from these cells by Sendai virus-mediated fusion with chicken cells. This virus rescue was not affected by treatment of the chicken cells with agents that rendered the cells incapable of dividing, although such treatment greatly reduced the ability of the chicken cells to plate as infectious centers after infection with B77 virus. Fusion of R(B77) cells with chicken erythrocytes also led to virus rescue, although with less efficiency than fusion with chicken fibroblasts. Therefore, virus rescue was probably due to a factor or factors contributed by chicken cells which aid in virus production.     

Physical map of the Kirsten sarcoma virus genome as determined by fingerprinting RNase T1-resistant oligonucleotides.

From analysis of the large RNase T1-resistant oligonucleotides of Kirsten sarcoma virus (Ki-SV), a physical map of the virus genome was deduced. Kirsten murine leukemia virus (Ki-MuLV) sequences were detected in T1 oligonucleotides closest to the 3' end of the viral RNA and extended approximately 1,000 nucleotides into the genome. The rat genetic sequences started at this point and extended all the way to the very 5' end of the RNA molecules, where a small stretch of Ki-MuLV sequence was detected. By comparison of the fingerprints of Ki-SV RNA and the RNA of the endogenous rat src genetic sequences, it was found that more than 50% of the T1 oligonucleotides were similar between Ki-SV and the endogenous rat src RNA, suggesting an identical primary nucleotide sequence in over 50% of the viral genomes. The results indicate that Ki-SV arose by recombination between the 5' and 3' ends of Ki-MuLV and a large portion of the homologous sequences of the endogenous rat src RNA.     

Structure of the genome of Moloney murine leukemia virus: a terminally redundant sequence

The genome of the Moloney strain of murine leukemia virus (Mo-MuLV) has been analyzed by digestion with ribonuclease T1 and separation of the digestion products by two-dimensional gel electrophoresis. Thirty large oligonucleotides isolated from such a fingerprint have been characterized. One of these oligonucleotides (number 21) was found to be present in twice the molar yield of the rest. The 30 oligonucleotides were mapped on the genome by determining their yields in various size classes of 3' terminal fragments of Mo-MuLV RNA. The physical map obtained in this way suggested that oligonucletoide 21 was present very near the 3' end of the geome as well as in another location near or at the 5' end. The genome structure suggested by these results was confirmed by analyzing oligonucleotides in Mo-Mulv RNA complementary to strong stop DNA, which is shown to be a copy of the 5' terminal 134 nucleotides of the MoMuLV genome. Some of the oligonucleotides in the RNA protected from RNAase digestion by hybridization to this DNA, including oligonucleotide 21, were present near both the 3' and 5' ends. Comparison of these with the nucleotide sequence of strong stop DNA shows that there is a terminal redundancy of 49-60 nucleotides in the Mo-MuLV genome RNA.