Gary E. Jones

A double filter paper technique for plating cultured plant cells

SummaryAn inexpensive method for achieving quantitative clonal growth of single cells and small clumps of cells ofHaplopappus gracilis (Nutt.) Gray on filter paper discs is described. Plating efficiencies of 55–80% are routinely obtained. This method depends upon the presence of a feeder layer of cells only in the initial phases of the plating, allows easy counting of colonies and permits rapid, convenient transfer of groups of colonies to other media without loss of individual colony identity or spatial distribution.

Rapid, Random Evolution of the Genetic Structure of Replicating Tobacco Mosaic Virus Populations

The effects of temperature and type of host on the evolution of variants in replicating populations of tobacco mosaic virus (TMV) originating from an in vitro RNA transcript of a cDNA clone were studied. The phenotypic markers measured were temperature-sensitive (ts) replication and induction of necrotic local lesions (nl) on Nicotiana sylvestris. The proportions of ts variants were about the same under all conditions tested, but changes in temperature or host had strong effects on proportions of nl variants. During replication at elevated temperatures in tobacco or tomato, percentages of nl variants were reduced rapidly to about one tenth their initial values. In contrast, when virus replicated at 25 degrees in tobacco, little change in the average proportion of nl variants was observed, and in tomato, only a slight average reduction occurred. Virus replicating at 25 degrees in Solanum nigrum or Petunia hybrida suffered effects similar to those observed in tobacco or tomato, respectively. However, at 25 degrees in Physalis floridana, there was strong selection against nl variants, which eventually reduced them to essentially undetectable levels. In all hosts tested at 25 degrees, surprisingly large, apparently random changes in the proportion of nl variants occurred in individual plants. These experiments showed that virus populations can evolve rapidly on a time scale of days, and that an element of randomness is an important component in the initiation of change.

The selection of resistance mutants from cultured plant cells

Abstract We have determined the influence of several factors on the recovery of resistance mutants from populations of cultured plant cells. Cells of strains of Haplopappus gracilis resistant to the metabolite-analogues 8-azaguanine, 6-azauracil, or δ-hydroxylysine were mixed with wild-type cells in reconstruction experiments. The colony-forming ability of the resistant cells was measured under various selective conditions. Choice of antimetabolite, size of resistant cell clusters, and overall plating density affect the efficiency of recovery of resistant colonies. The implications of these effects are discussed with respect to the isolation of mutants, the estimation of mutant frequency, and the importance of post-mutagenesis treatment. The azauracil-resistance phenotype should be useful as an indicator of mutagenesis since single resistant cells quantitatively produce visible colonies under selective conditions, and more than 2×106 total cells can be screened per plate.

Haplopappus gracilis cell strains resistant to pyrimidine analogues

SummaryStrains of Haplopappus gracilis (Nutt.) Gray cells resistant to 6-azauracil have been isolated from cultures of diploid cells. These strains are also resistant to 8-azaguanine, as is their parent. The variants are 100- to 125-fold more resistant to 6-azauracil than their parent, and they exhibit different spectra of cross resistance to other pyrimidine analogues. The phenotype of each variant is stable in the absence of selection. The majority of cells in cultures of the variants are diploid; all others examined were tetraploid. Initial rates of uptake of uracil are not reduced in the variants. Fluorouracil, to which two variants are resistant, is taken up by one of them as well as by the parent. Responses of the other two to fluorouracil are not correlated with decreased ability to accumulate this analogue.

Asparaginase II of Saccharomyces cerevisiae: Dynamics of Accumulation and Loss in Rapidly Growing Cells

SUMMARY: Asparaginase II activity in Saccharomyces cerevisiae can be derepressed in stationary phase cells by nitrogen starvation in the presence of an energy source. We have found that high activity of this enzyme is present in early-exponential phase cells even in the presence of abundant nitrogen. In growing cells that contain high asparaginase II activity, further derepression by nitrogen results in the rapid appearance of additional activity. Rapid loss of activity occurs as cultures begin to emerge from exponential growth. Synthesis of protein is required just before loss of activity occurs. Supplementing cultures with l-asparagine or l-glutamine strongly affects the kinetics of loss of activity. Mutation in ASP2 or ASP3, which results in inability to derepress this enzyme in stationary phase cells, also prohibits the development of the enzyme in exponentially growing cells.

nuvA, an Aspergillus nidulans gene involved in DNA repair and recombination, is a homologue of Saccharomyces cerevisiae RAD18 and Neurospora crassa uvs-2

A 40 kb genomic clone and 2.3 kb EcoRI subclone that rescued the DNA repair and recombination defects of the Aspergillus nidulans nuvA11 mutant were isolated and the subclone sequenced. The subclone hybridized to a cosmid in a chromosome-specific library confirming the assignment of nuvA to linkage group IV and indicating its closeness to bimD. Amplification by PCR clarified the relative positions of nuvA and bimD. A region identified within the subclone, encoding a C3HC4 zinc finger motif, was used as a probe to retrieve a cDNA clone. Sequencing of this clone showed that the nuvA gene has an ORF of 1329 bp with two introns of 51 bp and 60 bp. Expression of nuvA appears to be extremely low. The putative NUVA polypeptide has two zinc finger motifs, a molecular mass of 48906 Da and has 39% identity with the Neurospora crassa uvs-2 and 25% identity with the Saccharomyces cerevisiae RAD18 translation products. Although mutations in nuvA, uvs-2 and RAD18 produce similar phenotypes, only the nuvA11 mutation affects meiotic recombination. A role for nuvA in both DNA repair and genetic recombination is proposed.

Asparaginase II of Saccharomyces cerevisiae. Inactivation during the transition to stationary phase

Asparaginase II (L-asparagine amidohydrolase, EC 3.5.1.1) activity of cells from stationary phase cultures of Saccharomyces cerevisiae is very low. When these cells are inoculated into minimal medium, asparaginase II specific activity rises rapidly and reaches a maximum after 9-10 h. During the next 2.5-3 h, a rapid decrease in asparaginase II specific activity occurs. The enzyme does not appear to be secreted into the medium or to be reabsorbed into the cell. Addition of protease inhibitors at the time of maximum activity partially or totally prevents the loss of asparaginase II. L-1-Tosylamide-2-phenylethyl chloromethyl ketone decreases the rate of loss. The sulfhydryl reagents p-hydroxymercuribenzoate and iodoacetamide inhibit the loss of asparaginase II. However, addition of EDTA causes a further increase in activity. This increase is due to de novo protein synthesis. The effect of EDTA can be reversed by the addition of Zn2+. The most likely explanation for the rapid loss of asparaginase II is proteolytic degradation by a Zn2+-dependent, thiol protease or peptidase.

Stability of mutations conferring temperature sensitivity on tobacco mosaic virus.

Among several temperature-sensitive mutants of tobacco mosaic virus, reversion to wild-type phenotype is an event sufficiently rare that essentially pure stocks of mutant virus can easily be maintained. The temperature-sensitive phenotype of many of these mutants is stringently expressed. Our data show that most of our mutants are suitable for use in biochemical experiments.

Saccharomyces cerevisiae to Mutants Resistant -L-Aspartylhydroxamate

Summary: Yeast strains resistant to the L-asparagine analogue -L-aspartylhydroxamate have been shown to have mutations in any of at least three unlinked genes. Mutation in one of these genes, ahrl, is dominantly expressed and affects a function that involves nitrogen metabolism in the cells, including the exclusion of amino acids at the level of transport. The general amino acid permease is rendered sensitive to ammonium ion in strains carrying the dominantly expressed mutations in ahrl, but other functions related to nitrogen metabolism probably are involved as well.

Asparaginase II of Saccharomyces cerevisiae: Characterization of a Mutation that Affects Expression in Rapidly Growing Cells

Summary: A regulatory mutation has been identified that reduces expression of asparaginase II in exponential phase cells of Saccharomyces cerevisiae to 25 % or less of the wild-type activity. Mutants in this gene (ASP4) can be derepressed for asparaginase II by nitrogen starvation and will grow with D-asparagine as the sole nitrogen source. ASP4 assorts independently from ASP2 and ASP3. Mutation in either of the latter two genes results in lack of asparaginase II activity under any growth conditions.