Deoxycoformycin (dCF) is a specific inhibitor of adenosine deaminase (ADA). Rat hepatoma cells deficient in adenosine kinase and growing on adenosine as the sole carbon source are sensitive to the lethal action of dCF. Mutants resistant to dCF arise spontaneously with a frequency of 1.7 x 10(-6). This frequency is increased to 2.6 x 10(-5) by prior mutagenesis with ethyl methane sulfonate. Initially, dCF-resistant cell lines have 3-10 times the level of adenosine deaminase when compared to sensitive parental cells. Subsequent selection of mutants resistant to increased concentrations of dCF results in cells with a 15- to 30-fold increase in ADA levels. Quantitative immunoprecipitation tests indicate that the increase in enzyme activity in one line tested is due to an increase in the number of ADA molecules. These dCF' cell lines may serve as a model system to study the human disease state, hereditary hemolytic anemia, which is associated with increased levels of ADA.

The sequence of the 3' 1184 nucleotides of tobacco vein-banding mosaic virus (TVBMV) genome has been determined. It contains a single open reading frame which encompasses the whole of the coat protein of TVBMV. The sequence of the first 20 amino acids at the N-terminal region of the coat protein has also been determined chemically to be GDDQTVDAGKNVQSNQKQRN. The sequence matches the translation product of the open reading frame starting with amino acid-271; a glycine residue. Thus the coat protein of TVBMV has a calculated M(r) of 30,210. The 3' non-coding region of TVBMV is 185 nucleotides in length. Sequence alignment of the coat proteins or the 3' non-coding regions from TVBMV and other reported potyviruses indicated that TVBMV is a separate species of the potyvirus genus.

Purine nucleoside phosphorylase (PNP) levels are modulated during the growth cycle of rat hepatoma cells and increase two- to three-fold as cells go from early exponential growth phase to stationary growth phase. A mutant of these hepatoma cells has been isolated which is deficient in PNP activity. Quantitative immunoprecipitation tests indicate that the decrease in enzyme activity is due to a decrease in the number of PNP molecules. The low level of PNP enzyme produced by the mutant, however, is indistinguishable from the wild-type enzyme, suggesting that the mutant may be defective in the ability to modulate PNP levels. Fusion of the mutant cells to wild-type parental cells results in hybrids that express the mutant phenotype. Segregants that arise from the hybrids show chromosome loss and reexpression of the wild-type parental phenotype, the mutant parental phenotype, and a 2S wild-type phenotype. These indicate the following about the defect in modulation in the mutant PNP-100: (1) it is trans dominant to the wild-type; (2) its effect is negative; (3) some genomic element is required for its continued effect; and (4) it does not act by obliterating its functioning counterpart in hybrid cells.

Rat hepatoma cells amplified for adenosine deaminase (ADA) gene sequences show the amplified DNA on large, homogeneously staining regions (HSRs). The amplified cells are stable in the absence of selection for 12 mo without loss of ADA activity or gene sequences. However, in hybrids formed between an amplified cell line with a prominent HSR and a nonamplified cell line, rapid loss of ADA activity, as well as gene sequences, occurs. Karyotype analyses of the hybrids indicate that the HSR structures are no longer visible in a large percentage of the hybrid metaphase spreads and appear to have been replaced by DNA structures that resemble double minutes. Our data provide evidence that (1) the extent of the breakdown of the HSR in the hybrids may be affected by the presence of an active adenosine kinase or the level of ATP in the cells and (2) additional unidentified factors are present in the hybrids that affect the integrity of the HSR structure. There is no evidence for a specific transacting factor in nonamplified cells that regulates gene amplification.

Two independently selected series of rat hepatoma cell lines resistant to the drug deoxycoformycin (dCF) were analyzed karyotypically. Several forms of homogeneously staining regions (HSRs) were present on metaphase chromosomes of these cells. In some instances HSRs comprised nearly an entire chromosome, which are among the largest chromosomes in the karyotype. Stable resistance to dCF is acquired in rat cells by overproduction of the enzyme adenosine deaminase (ADA) as a result of amplification of ADA gene sequences. We have localized the amplified ADA gene sequences to HSRs on metaphase chromosomes from both series of dCF-resistant cell lines by in situ hybridization. Based upon the number of ADA gene sequences present and the lengths of the HSRs, we have estimated the size of the amplified unit to range from 450 to 1,000 kb.