R. meliloti carrying defined deletions that remove fix114 form Fix- nodules which are devoid of intracellular bacteria. Occasionally strains which carry these deletions form pink nodules which appear effective in contrast to the normal white ineffective nodules formed by strains carrying fix114 mutations. Bacteria isolated from these pink nodules retain the original deletion and form effective pink nodules when reinoculated onto alfalfa. It is hypothesized that these isolates carry second site mutations which enable the bacteria to overcome the symbiotic block associated with the fix114 mutation. In this work, five independent isolates were examined and were shown to carry second site mutations that suppress the symbiotic ineffectiveness completely on alfalfa and incompletely on sweet clover. The five independent second site revertants can be divided into two classes based on genetic data and on their sensitivity to detergents and both classes were localized to the chromosome of the wild type Rm1021. One such second site revertant allele, sfx-1, was cloned and localized to a large 18 kb BamHI fragment. / Thesis / Master of Science (MS)

The Salmonella typhimurium/mammalian microsome test has provided a simple and sensitive short‐term assay for the detection of environmental mutagens. Metabolic activation of precarcinogens is usually achieved by incubating the compound to be tested, the bacterial strain and mammalian liver homogenates with NADPH. The results presented here utilize Salmonella typhimurium strain TA100, the precarcinogen 2‐aminofluorene and microsomal enzyme preparations prepared from liver homogenate of carp (Cyprinus carpio) taken from aquatic environments of northeastern Tennessee. Those environments range from virtually unpolluted to extremely polluted. The results show that the precarcinogen 2‐aminofluorene is activated either partially or totally in the presence of liver homogenates of carp taken from polluted aquatic environments (e.g., microsomal enzyme preparations made from rat liver with 2‐aminofluorene produced 808 revertants; whereas the liver preparations made from carp, taken from the Pigeon River, with 2‐aminofluorene produced 2,786 revertants). Revertant colony results correlated well with the degree of pollution within those waters. An increase (data were statistically different at the 0.05 level of significance) of TA100 revertant colonies was observed as aquatic contamination worsened. All data pairs of collecting sites in their order of increasing contamination, as well as those between collecting sites, were statistically different at the 0.05 level of significance.

Generation and characterization of activation tagged potato mutants could aid in functional genomic studies. Morphological and molecular studies were conducted to compare potato cv. Bintje, its two mutants, underperformer (up), and nikku generated using the activation tagging vector pSKI074, and nikku revertant plants. Mutant up exhibited a dwarf phenotype (plant height 42 cm vs. 73 cm in cv. Bintje), abundant axillary shoot growth (3.1 shoots/plant compared to 0.7 shoots/plant in cv. Bintje; in vitro plants), greater tuber yield, altered tuber traits and early senescence compared to wild-type Bintje under in vitro conditions. Under in vivo conditions, the dwarf and early senescence phenotypes of the mutant were consistent, but the tuber yield of up was less (250 g/plant compared to 610 g/plant in wild-type Bintje) and had fewer axillary shoots compared to wild-type (1.9 shoots/plant in up vs. 4.7 shoots/plant in Bintje). Mutant nikku plants exhibited an extremely dwarf phenotype (plant height 2 cm in nikku vs. 6 cm in Bintje), had small hyponastic leaves, were rootless, and infrequently produced small tubers when compared to cv. Bintje. The overall nikku phenotype was suggestive of a constitutive stress response, which was further supported by the higher expression levels of several stress-responsive genes in nikku. The nikku revertant plants exhibited near normal stem elongation, larger leaves and consistent rooting, and it was a case of partial reversion. Southern blot analyses indicated the presence of single T-DNA insertions on chromosome 10 in the up and on chromosome 12 in the nikku mutant. The reversion in the nikku plants was not associated with the loss of enhancer copies from the original nikku mutant. Reverse transcriptase PCR analyses indicated transcriptional activation/repression of several genes in the up and nikku mutants, suggesting pleiotropic effects. In revertant, the expression levels of several genes which were differentially regulated in the nikku mutant were similar to Bintje. The gene immediately flanking the right border of the T-DNA insertion, which encoded a novel BTB/POZ (Broad complex, Tramtrac, Bric a brac; also known as Pox virus and Zinc finger) domain-containing protein, was highly up-regulated in the up mutant. This protein domain plays an important role in several important developmental, transcriptional and regulatory pathways. The mRNA-seq analyses resulted in 1,632 genes that were differentially expressed between mutant up and Bintje and the total number of up-regulated genes (661) were less than the number of genes down-regulated (971 genes) in the up mutant. Further analyses indicated that a variety of biological processes including decreased cell division, cell cycle activity, and abiotic stress responses were modified in the up mutant. In the nikku mutant, two potato genes, encoding an Acyl-CoA N-acyltransferases (NAT) superfamily protein, and a predicted major facilitator superfamily protein (MFS) were identified and overexpression lines Bintje/35S::NAT1 and Bintje/35S::PMT1 were created for recapitulation of the nikku mutant phenotype. Methylated DNA-PCR between the nikku and the revertant indicated a change in methylation status of the 35S enhancers, suggesting that the nikku revertant phenotype may be associated with some epigenetic modification. / Ph. D.