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(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.

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.

A sequential hybridization procedure is described which allows the integration sites of viral-specific DNA to be characterized according to their reassociation kinetics. In addition, this approach enables us to estimate the size of the integrated viral DNA. Endogenous virus sequences in normal cells appear to be associated with cell sequences reiterated 1200 times, and each integration unit is approximately equal to one 35S RNA subunit. In AMV-infected cells, the additional AMV-specific DNA sequences reassociate as if they were integrated adjacent to unique cellular DNA or in tandem with the endogenous viral DNA sequences.

Rat lung mitochondrial preparations were incubated in the preasence of pyruvate and malate. The principal metabolic products measured were citrate and CO2. Citrate formation from pyruvate was found to be dependent on the presence of malate. Significant citrate was formed in the presence of isocitrate and the rate of citrate formation was increased by the addition of pyruvate. Small amounts of citrate were formed by lung mitochondrial preparations in the presence of 2-oxoglutarate and succinate only after the addition of pyruvate. The level of acetyl-CoA was significantly greater in the presence of pyruvate than in the presence of pyruvate plus malate. The addition of malate to lung mitoochondrial preparations increased 14CO2 production from [2-14C] pyruvate into malate and citrate. A low level of pyruvate-dependent H14CO3-incorporation into acid-stable products was observed, principally citrate and malate, but this rate did not exceed 5% of the rate of net citrate formation in the presence of malate and pyruvate. The capacity of rate lung mitochondria to form oxaloacetate from pyruvate alone in vitro is very limited, and would appear to cast doubt on a major role of pyruvate carboxylase in citrate formation. It is concluded that the rate of citrate formation from pyruvate is limited by the availability of intramitochondrial oxaloacetate and the rate of citrate efflux across the mitochondrial membrane.