Trichloroethylene (TCE) is a toxic pollutant that has become a widespread problem by seeping into groundwater across the developed world. Clean-up of sites contaminated with TCE is extremely difficult due to the absence of an efficient and cost-effective method for clean-up. Bioremediation efforts include a variety of potential microbial candidates with various metabolic capabilities as clean up options of contaminated sites. Cupriavidus necator, a soil bacterium was found to possess the ability to degrade TCE via a phenol-dependent pathway. Previous research by Ayoubi and Harker (1998) created a strain (MM02) capable of constitutive TCE degradation but the underlying genetic alteration causing constitutive production of the phenol hydroxylase pathway (PHL) and TCE breakdown was poorly characterized. We attempted to gain further understanding of the alterations that occurred in the PHL pathway to cause TCE to break down and replicate constitutive TCE degradation in a new strain with reduced foreign elements that may be introduced into the environment. Strain MM02 possessing this constitutive degradation activity and strain MM01were sequenced and compared to discover the source of this variation. A 210 base-pair deletion in the beginning of the PHL operon was identified and is likely the cause of this altered activity. The new strain of C. necator (MM14) was created using traditional bacterial mating methods and included a cleanly introduced kanamycin resistance gene and its associated promoter which could drive constitutive expression of the PHL pathway. The TCE degradation abilities of strains MM01, MM02, and MM14 were evaluated through the TCE degradation assay and gas chromatography. We had difficulty accurately measuring the concentration of TCE due to its volatile nature and dramatically altered the method ultimately reducing variation and capturing TCE concentrations in assays. When accurate readings were obtained, none of the strains measured exhibited quantifiable TCE degradation activity when compared to controls. Our results showed .08% of the degradation by strain MM02 measured previously (P. J. Ayoubi, 1997). Based on our findings, we were unable to replicate the TCE degradation caused my MM02 and our genetically modified strain also failed to breakdown TCE.

There are four members involved in the breeding complexes of poeciliid fishes found in the freshwaters of northeastern Mexico: males and females of a bisexual species, and diploid and triploid unisexuals. Both unisexuals reproduce by gynogenesis, i.e., an asexual type of reproduction where the sperm triggers egg development but the male genome is excluded to produce clonal offspring. The three types of females are closely related, which suggests that they are potential competitors since all three require the service of the same males. The potential for competition is compounded by a highly skewed sex ratio in favor of females. On the average the unisexuals comprise about 30% of the Poecilia females. This high frequency coupled with a close genetic relatedness to their bisexual hosts, raises the question of how the unisexuals are maintained in nature. Other investigators who work with bisexual/unisexual complexes in the related genus, Poeciliopsis, have postulated that male dominance hierarchies are responsible for restricting the access of subordinate males to their conspecific females. Consequently, these subordinate males mate with unisexual females. The current report tests whether or not this hypothesis applies to bisexual/unisexual complexes of Poecilia. We have found that linear dominance hierarchies appear to function in the defense of home ranges and do not restrict access of males to females. Dominant males exhibit less mating activity than subordinate males towards females. Previous reports showed that males are reproductively competent throughout the year, whereas females show striking asynchrony in their reproductive readiness. Such asynchrony limits the proportion of receptive females at any one time. Consequently, there are more males ready to mate than there are females receptive to their mating attempts. This may lead to mating frenzies. We postulate that these indiscriminate matings maintain the fertility of both unisexuals. When the relative reproductive outputs of adult females are compared, both unisexuals appear as fit as their bisexual congeners.

Genetic changes likely occur in wild fish populations as a consequence of interactions with cultured fish, but to what extent do those changes threaten the maintenance of natural genetic diversity and population viability? Following a review and categorization of numerous processes suspected of being agents of post-release genetic change in recipient wild populations (Chapter 1), I focus on risks relating to the magnitude and duration of releases -- but with a twist. That is, I assume that the mean fitness of released, cultured individuals does not differ from that of the recipient natural population. Throughout, attention is devoted to potential post-release changes in inbreeding (NeI) and variance (NeV) effective population sizes -- indicators of expected rates of population-level change in inbreeding and drift variance, respectively. The reductive effect that large-scale releases exert on NeI in recipient populations can be significant. The effect is shown to be a threshold process (Chapter 2) and thus suggestive of an approach for determining risk-adverse stocking (or release) rates. This approach is utilized in Chapter 3, which describes genetic recommendations for an incipient marine stocking program. Several discordant contemporary NeI models are examined mathematically and by computer simulation (Chapter 4). I show that certain published results pertaining to the effect of multiple paternity on NeI are erroneous; a general model is described which accounts for inbreeding and relatedness in and among parents. That model is utilized in an empirical study of gene correlation in a hatchery cohort (Chapter 5). Propagation-related causes of reductions in NeI are also investigated in this cohort. Finally, extending mutational meltdown theory to accommodate fluctuating population sizes and recessive selective effects, I show that when large reductions in NeV occur (such as those that accompany admixtures of cultured and wild fish), the expected time to population inviability is significantly reduced (Chapter 6). Although a more comprehensive theoretical approach is needed, a precautionary inference may be drawn -- aquaculture-induced reductions in Ne, even though they may be transient, can lead to adverse genetic impacts. Avoidance of Ne-reductions cannot be accomplished, in a practical sense, without considering the stocking or release rates of cultured fish.

In a territorial population of pronghorn from Antelope Island, UT, interaction between male dominance and territory defense was examined. High-ranking males were more likely to defend territories. Closely ranked animals engaged in more dominance interactions than distantly ranked individuals, and middle-ranked animals were involved in disproportionately more interactions than either high- or low-ranking animals. Large males possessed large horns and prongs as well as small cheek patches. Results from a factor analysis suggested that large males defended territories with a high density of sage. However, in this study, we did not observe pronghorn feed on sage during the territorial season. Though male pronghorn practiced resource defense polygyny, large, dominant males did not defend territories with a high density of green vegetation or green forbs. Large males appeared to defend territories with low visibility. In 1996, intruders entered areas that contained females throughout the territorial season. During the next year, highly visible, small territories received the most intrusions. Together, these observations suggest defense of tactical locations. Defending a tactical location may help females avoid harassment and males hide the presence of females. Different populations of pronghorn practice different mating systems. To understand this variation, we examined the behavior patterns/rates of individual territorial and bachelor males. The highest rates of activity and behavior patterns occurred in March/April and in September. Territorial males cheek rubbed at a higher rate than bachelors. Territorial males were more active and SPUD (sniff, paw, urinate, and defecate) marked at a higher rate than bachelor males in 1996. After the formation of a bachelor herd in 1997, bachelor males showed higher rates of male-male interactions than territorial males. Territorial males maintained the same activity and behavioral rates in the presence and absence of females. Dispersion pattern of scent marks was more clumped in the presence of females. These findings suggest cheek rubs function more as a space-claiming behavior while SPUD marking is more strongly associated with male-male interactions. Comparison to male behavior in nonterritorial populations indicates that the behavioral mechanisms are present in all populations to accommodate shifts in social systems.

The methylotrophic yeast, Pichia pastoris, is the most successful and favored microbial eukaryotic expression system for the production of recombinant proteins for biopharmaceutical or industrial purposes. P. pastoris has the ability to produce foreign proteins at high levels extracellularly, and since it secretes few endogenous proteins, this ability eliminates the need for expensive purification costs. It also combines the ease of genetic manipulation with rapid growth to high cell densities and provides complex posttranslational modifications. The most commonly utilized secretion signal leader in P. pastoris is the MATα prepro signal leader, originally found in S. cerevisiae. However, because some proteins cannot be secreted efficiently by P. pastoris, strategies to enhance secretion efficiency have involved the modification of the MATα prepro secretion signal leader. The study focuses on using site-directed mutagenesis of specific sets of amino acids of MATα prepro secretion leader to evaluate the correlation between secondary structure and secretion level. MATα pro-HRP mutants were created, in order to analyze the export of heterologous proteins in P. pastoris. In addition, structural analysis through circular dichroism was performed on mutant MATα pro-peptides to evaluate differences in secondary structure as a result of the mutagenesis. Mutants, pSC6 (Δ57-65) and pSC7 (Δ66-70) did not generate the same HRP secretion level as Δ57-70. In addition, a new proposed model of MATα pro-peptide signal leader was created. This new model suggests that the N and C terminus of MATα pro-peptide need to be presented correctly for proper interaction with secretion machinery and for efficient protein secretion. With these analyses, optimization of secretion systems can be achieved to impact the fields of science, industry, healthcare, and economics worldwide.

The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation with rapid growth to high cell densities and provides complex posttranslational modifications. The most successful and commonly used secretion signal leader in Pichia pastoris has been the MAT α prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently even with the MAT α prepro secretion signal. Some strategies to enhance secretion efficiency involved modifying the secretion signal leader. Based on the knob-socket model and Jpred3 ( a secondary structure predictor), eleven deletions of MAT α prepro secretion signal and one MAT α pre double pro-peptide mutant was engineered and assayed with either horseradish peroxidase (HRP), or Candida antarctica lipase B reporter protein to evaluate the correlation between secondary structure and secretion level. In addition, structural analysis through circular dichroism was completed for the wild type pro-peptide and a mutant pro-peptide to evaluate differences in secondary structure. Results suggest pro-peptide amino acids 75-78 play an important role in determining secretion level and a higher secretion level tends to associate with secondary structures that are less defined. With these analyses, optimization of secretion systems can be achieved to impact the fields of science, industry, healthcare, and economics worldwide.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18826号 / 理博第4084号 / 新制||理||1587(附属図書館) / 31777 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 曽田 貞滋, 准教授 渡辺 勝敏, 教授 高橋 淑子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20048号 / 理博第4233号 / 新制||理||1609(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 中川 尚史, 教授 沼田 英治, 教授 中務 真人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20218号 / 理博第4303号 / 新制||理||1618(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 古市 剛史, 教授 Fred Bruce BERCOVITCH, 教授 平井 啓久 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20636号 / 農博第2243号 / 新制||農||1053(附属図書館) / 学位論文||H29||N5080(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 北山 兼弘, 教授 田中 千尋, 教授 松浦 健二 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM