In order to identify potential cis-acting elements responsible for the correct expression of the mosquito trypsin genes we have resorted to an evolutionary approach. This approach is based on the identification of DNA footprints that are conserved in homologous genes isolated from different species. Several trypsin clones have been isolated from Aedes species specific genomic DNA libraries, and by sequencing been shown to contain an ORF coding for the late trypsin gene. Analysis of the 5' FLR's of the species specific late trypsin genes, reveals the presence of a conserved TATA-box and transcriptional initiator. A phylogenetic footprinting analysis detected some evolutionarily conserved sequence elements in the 5' regulatory regions of the late trypsin gene. A cis-element that bears sequence similarity with the target of the transcription factor Tinman has been identified. Analysis of the trypsin coding region shows that the late trypsin from the most distant species retains approximately 83% amino acid identity with late trypsin from Ae aegypti. Furthermore, unique features of the late trypsin specificity pocket from Ae aegypti are retained in all species examined making this a unique evolutionary molecular tag for this serine protease. The dicotomy of a positive/negative charge observed in the specificity pocket of trypsin members of the chymotrypsin family of serine proteases is retained; however, the conserved aspartate is replaced by a glutamate. The position of this glutamate is displaced towards the N-terminus by a serine, which is characteristic of chymotrypsin-like enzymes from the same family. Moreover, there is an insertion of a proline after the serine amino acid at the C-terminal end of the pocket. Alignment of this trypsin to other members of the same protease family strongly suggests that it is related to a unique group of proteases called serine collagenases. The most important enzymatic characteristic of enzymes belonging to this group is there lack of substrate specificity. Outside of the typical metalloproteases, these are the only proteases known to cleave collagen. What these observations mean in terms of the evolution of enzyme specificity aid structural fold remain to be elucidated.

Mitochondria are the organelles where respiration occurs. The yeast mitochondrial genome encodes only 8 proteins, therefore the organelle depends on the nuclear genome for many proteins required in different steps of mitochondrial gene expression. Regulation of mRNA stability, processing and translation are important steps in gene expression within the mitochondrion. Cbp1, a protein encoded by the nuclear gene CBP1, is required specifically for stabilization of precursor and mature cytochrome b (COB) RNA, which is encoded in the mitochondrial genome. Previous work identified a cis-element, CCG, in the 5' untranslated region (UTR) of COB, that is critical for the Cbp1-dependent stability of COB mRNA. Mutation of any single nucleotide resulting in an A̲CG, CA̲G or CCU̲ triplet causes destabilization of COB mRNA and concomitant loss of respiratory capability. In the present study, suppressors were selected in the CCU strain in an effort to define important sites in Cbp1 for protection of COB mRNA. The mitochondrial mutant strain CCU is conditional; it grows slowly at 25°C but does not grow at 18°C or 33°C on the non-fermentable carbon source glycerol. Twelve dominant suppressors in CBP1 were obtained. They define two main groups, based on the pattern of growth on glycerol at different temperatures. The CBP1-encoded suppressors make strains containing the mitochondrial CCU̲ mutation respiratory competent at 33°C by allowing accumulation of mature COB mRNA. Suppressors that map to the carboxyl half of Cbp1, such as S289G, S330R, Q358K, Q358R, L489W, K532M, D533Y and I638M, rescue the temperature-sensitive (ts) phenotype caused by the CCU̲ mutation. The suppressors that map to the amino half of Cbp1, such as K205R, E241G, I249T, N281D and I293L, rescue the ts phenotype to a lesser extent than the suppressors that map to the carboxyl half of Cbp1. These results suggest that the two halves of Cbp1 have different functions in the processing and stability of COB transcripts. The hypothesis that Cbp1 has a role in translation of cytochrome b (COB) mRNA was not testable previously, since disruption of CBP1 results in instability and degradation of COB mRNA. In a Δpet127 strain, COB precursor mRNA is not processed to the mature 5' site and thus accumulates to levels equivalent to that of the wild-type mature mRNA (Wiesenberger and Fox, 1997). Null alleles of pet127 were selected as suppressors of A̲CG and CCU̲ mutations in COB. COB precursor mRNA levels in these strains were similar to the Δpet127 strain with a wild-type mitochondrial genome (Chen et al., 1999). In the present study, the effect of deleting CBP1 in a Δpet127 strain was measured. Strain Δcbp1 Δpet127 accumulated no mature COB mRNA but high levels of COB precursor mRNA. The levels of precursor mRNA in the Δcbp1 Δpet127 strain were 3-fold higher than in wild-type strain and approaching 60% of wild-type mature levels (wild-type COB precursor is 18% of mature COB mRNA). Absolutely no apocytochrome b protein was detected in the Δcbp1 Δpet127 strain. This result suggests that Cbp1 is required for translation of the COB message. Future experiments to determine the role of Cbp1 in the translation of COB mRNA are described.

This dissertation investigated the evolution and ecology of male sterility in the gynodioecious Bouteloua chondrosioides (Poaceae) by studying the distribution, inheritance, phenotypes of male sterility, mitochondrial DNA polymorphisms, and the distribution and effect polyploidy has on sex type expression in B. chondrosioides. B. chondrosioides has two male sterile types, one of which is described for the first time in this dissertation. Field studies determined that the proportion of male sterility was highly variable among populations and non-randomly distributed within populations. Investigations of the progeny of individuals of known sex type rejected models of simple nuclear recessive and dominant inheritance of male sterility. Examination of characters that may affect reproduction demonstrated that there were few significant reproductive differences explaining the maintenance of the two male sterile forms. In order to investigate if male sterility is due to cytoplasmic factors, mitochondrial DNA restriction fragment length polymorphisms were examined to determine if there were correlations between unique restriction fragment patterns and male sterile forms. These studies demonstrated that some, but not all, male sterile individuals do have unique mitochondrial restriction fragments. In addition to these investigations, the distribution of polyploidy was characterized and investigations performed to determine if there are correlations between sex type and ploidy level and if polyploidy has evolved once or multiple times in B. chondrosioides. Flow cytometry resulted in data that demonstrated no correlation between male sterility and ploidy level, and that while most populations are either only diploid or tetraploid, some populations had both diploid and polyploid individuals. The examination of the relationships of cpDNA sequences from individuals of known ploidy level demonstrated that polyploidy appears to have originated and established more than once in the history of B. chondrosioides. The results from these three studies exhibit patterns that are in accord with the hypothesis that male sterility in B. chondrosioides is due to cytoplasmic male sterility.

Investigated in laboratory column experiments were the effects of 0.01 M and 0.001 M CaPO₄ concentrations and pH on the attachment-detachment of bacteriophages PRD-1 and MS-2. Bacteriophages PRD-1 and MS-2 exhibited attachment to the soil at concentrations of 0.01M CaPO₄ and 0.5M NaCl. Release of attached phage at 0.001 M CaPO₄ and without NaCl was observed. The pH was also found to affect the attachment-detachment of PRD-1 and MS-2. However, they attached at pH 5.5 and detached at pH 8.0 at a limited extent and over an extented long period of time. The effect of salt concentration on deattachment was greater than the effect of pH. Similar results were obtained when glass beads were used as the adsorbent. These results suggest that changes in pH and ionic strength (as might occur after a rainfall) can result in the rapid release of previously adsorbed virus. (Abstract shortened by UMI.)

Guayule (Parthenium argentatum Gray), a semidesert shrub was evaluated as a potential rubber crop. Since polyploid guayule reproduces by apomixis, progeny of individual plants should duplicate the characteristics of their parent. This study was conducted to estimate variations within and among progeny families from single-plant selections. Ten progeny per family were individually evaluated for plant height, width, fresh and dry weight of clipped branches, rubber and resin content, and yield. Leaves of selected plants were analyzed by gradient polyacrylamide gel electrophoresis for esterase and peroxidase to estimate genetic relatedness between parents and progenies. Negative correlation was between parents and progenies for most characters, but none were statistically significant from zero. This suggests a low heritability for the characters measured. Esterase and peroxidase detected variation in banding between parents and progenies and among progenies. This study suggested that out-crossing and meiotic reduction have occurred among apomictic guayule lines.

Muscle atrophy is a serious side effect seen with extended time in space. Proteolytic degradation of specific muscle proteins leads to smaller, weaker muscles that are structurally more susceptible to damage. Calpains are proteases that specifically degrade target proteins of the myofibril, and have been implicated in many types of muscle atrophy. Calpain activity is regulated by a combination of activation by calcium and inhibition by calpastatin, its endogenous inhibitor. This dissertation describes the generation of a skeletal muscle-specific, doxycycline (Dox) controlled, calpastatin over-expression system in transgenic mice to regulate calpain activity. A dual construct system, the transactivator line utilizes an optimized tet-on system and a modified muscle creatine kinase promoter to create muscle specific expression of a tet transactivator. The second transgenic line, consisting of a bi-directional promoter centered on a tet responder element controlling both a luciferase reporter gene and a tagged calpastatin, is transcriptionally silent until activated by a dox induced transactivator molecule. Compound hemizygous mice showed high level, Dox dependent, muscle-specific overexpression of luciferase and transgenic calpastatin, demonstrating the effectiveness and flexibility of the tet-on system to provide a tightly regulated overexpression system in adult skeletal muscle. Consistent overexpression of calpastatin was hard to maintain, however, and not all of the proposed experiments could be achieved. Additional studies compared the degradation of hindlimb suspended mouse muscle proteins (weightlessness model) to those of ground control muscle proteins briefly incubated in Ca²⁺ (to initiate calpain degradation). Four proteins known as targets for calpain degradation were selected for analysis. Degradation responses of myofibrillar proteins titin, nebulin, and troponin T in hindlimb suspension clearly mimicked that seen with the calcium incubations. The cytoskeletal calpain target protein, desmin, however did not respond the same to both treatments showing moderate degradation with calcium and no degradation with hindlimb suspension. These data suggest that myofibrillar calpain target proteins, but not necessarily cytoskeletal proteins, are rapidly targeted for degradation in hindlimb suspension in a manner similar to that induced by calcium, implicating calpain as a mediator of this degradation.

Since the mid-1970's, there has been active research on the development of Vernonia galamensis (Cass.) Less. as a potential new oilseed crop. Vernolic acid (cis-12:13-epoxy-cis-9-octadecenoic acid) comprises 70--75% of vernonia oil and is chemically reactive, affording it a variety of industrial applications. A concern in the domestication of an oilseed crop is to establish a breeding program to improve oil quality traits. The objectives of this research were to (1) develop a rapid procedure for seed analyses; (2) determine the development of vernonia oil; and (3) estimate the narrow-sense heritability (h 2) of oil quality traits. Successful separation of free fatty acids (FFA) and acylglycerides from small vernonia seed samples was achieved using aminopropyl solid phase extraction columns. Acylglycerides were eluted with a mixture of chloroform and isopropanol, while FFA were eluted with a mixture of acetone and trifluoroacetic acid. Four breeding lines from a collection of Vernonia galamensis held at the US Water Conservation Laboratory in Phoenix, AZ were used for the oil development study and grown in field trails in Phoenix and Tucson, Arizona. Seeds were collected on nine different days after flowering over the course of seed maturation. Seed samples were analyzed for FFA, acylglyceride constituent fatty acids, total acylglycerides, and total oil. In each breeding line, FFA content changed significantly throughout the course of the measurement period, and synthesis of acylglycerides constituent fatty acids followed a previously described pathway proceeding from C16:0 to C18:0 to C18:1 to C18:2 to C18:1 epoxy. Vernolic acid increased late in the measurement period, while total acylglycerides and total oil increased steadily over the period. Mature vernonia seed exhibited substantial variation in the amount of FFA, acylglyceride constituent fatty acids, total acylglycerides, and total oil. Sixty-nine half-sib families were created to study the heritability of FFA, vernolic acid, acylglycerides, and total oil production. Mature capitula were collected and analyzed individually for oil constituents. Narrow sense heritability estimates for these four oil quality traits were: FFA = 33%, vernolic acid = 65%, acylglycerides = 47%, and total oil = 50%. The results indicate potential for progress in selection for these traits.

Sharks, the most primitive vertebrates that have been found to possess bona fide immunoglobulins, are known as "living fossils". This dissertation describes my studies on the characterization of carcharhine shark Ig heavy chain genes. Shark V sc H genes have been cloned from both cDNA and genomic DNA, and the sequence data reveals that these carcharhine shark Ig V scHS(Vmu) contain essentially all V scH canonical structures and are associated with a four domain IgM-like constant region. Shark Vmus are shown to be highly diversified and can be divided into six families based on the sequences of this work. Another class of V scHS (Vo) associated with six C-region domains has been found in the carcharhine shark. This molecule represents a new isotype of heavy chain (IgW). The higher homology of the Vo with its own constant regions may indicate that Vo is more primitive. Phylogenetic analysis reveals that the V scHS of the horned shark and the skate can also be divided into two major classes (Vo and Vmu). The divergence of these two major V scH classes must have occurred prior to the divergence of the sharks and the skates, which is about 350 million years ago. The V scHS of all other vertebrates are found located between these two major shark/skate classes on phylogenetic trees. Here, I suggest that Vo is the primordial V sc H-region based on my phylogenetic analysis. The cDNA encoding a whole IgM heavy chain was cloned and sequenced. This heavy chain has four C-region domains which are homologues to the IgM C-region domains of representative vertebrate classes. Phylogenetic analysis shows that the divergence of IgM C-region domains from the primordial C-domain occurred rapidly prior to the divergence of the sharks and other vertebrates. Afterwards, C-region domains have evolved independently of each other with a constant rate. Here it is shown that IgM C-region domains can be used in the studies of vertebrate phylogeny. Although it is not clear at present how the hypothesized precursor Ig gene diverged into separate V- and C-domain genes, it is clear from this work that this divergence occurred rapidly during the hypothetical "big bang".

An essential component of development is the accumulation of specific metabolites in a temporal and tissue-specific manner. The growth regulator abscisic acid (ABA), which accumulates at a specific time during seed development, is required for seed maturation and prevents the premature developmental switch from dormancy to germination ABA accumulates differently in two tissues of the seed; levels in the embryo are several-fold higher than in the endosperm and the temporal accumulation of ABA is also different between these tissues. To begin to understand how ABA accumulation is regulated during seed development, the regulation of ABA biosynthesis was investigated. The approach taken was to examine the expression of the biosynthetic enzyme, phytoene desaturase (PDS), which catalyzes a regulated step in ABA synthesis in several other organisms (Bramley, 1985, Sandmann et al., 1989, Hugueney et al., 1992 and Giuliano et al., 1993). Unlike ABA accumulation, PDS transcript and protein levels were higher in the endosperm than in the embryo. The spatial difference in PDS levels did correlate with levels of the pathway intermediate, beta-carotene, suggesting that PDS may control the synthesis of ABA precursors while subsequent enzymes may regulate ABA accumulation. The temporal expression of Pds was also unrelated to ABA accumulation. In the endosperm, transcript levels were initially high and declined during desiccation while protein levels remained high throughout development. In the embryo, transcript levels were low and constant while protein levels declined. There are several maize mutants (viviparous mutants) disrupted in ABA biosynthesis, resulting in decreased levels of ABA and premature germination. Analysis of the Pds allele and transcript in the viviparous-5 mutant showed that the gene contains multiple insertions and deletions, giving rise to a larger transcript. In addition, the 55 kDa PDS protein was not detected in the vp5 mutant by immunoblot analysis, indicating that the vp5 phenotype results from a mutation at the PDS locus. To determine whether the wild type protein encoded by the ABA mutant, vp2, or the pathway intermediate, lycopene, regulate PDS, transcript and protein levels were compared in wild type and mutant (vp2 and vp7, respectively) seeds. The levels of PDS were not significantly different in vp2 or vp7 wild type and mutant seeds, suggesting that neither the VP2 protein nor lycopene regulate PDS at the steady-state transcript or protein level.

Bordetella pertussis is the causative agent of the childhood disease whooping cough or pertussis. The regulation of virulence factor expression by two proteins (Baf and Btr) in this organism was studied. Pertussis toxin (ptx) transcription is activated by the two component BvgAS system. BvgAS-dependent ptx-lacZ expression in Escherichia coli can be achieved by DNA gyrase inhibitors, growth in minimal medium, or addition of the B. pertussis baf gene in trans. The mechanism by which Baf affects pertussis toxin expression in E. coli and B. pertussis is the subject of this investigation. In E. coli, wild type Baf increases ptx-lacZ expression, has no effect on bvgA-lacZ expression, yet increases BvgA protein levels 2-fold. In B. pertussis, ptx-lacZ expression is increased 10-fold when baf is overexpressed. However, BvgA protein levels are unaffected in B. pertussis as are pertussis toxin, adenylate cyclase toxin, and filamentous hemagglutinin levels. The effects of Baf on pertussis toxin expression in E. coli and B. pertussis is not due to a slowing of growth since overexpression of Baf does not affect growth rate. Finally, baf is shown to be an essential gene for viability, suggesting that Baf regulates the expression of other genes in addition to pertussis toxin in B. pertussis. Available data suggests Baf enhances BvgA function through an unknown mechanism. Possible models for Baf's role in pertussis toxin expression are discussed. Btr is an oxygen responsive transcriptional regulator homologous to the FNR protein of E. coli. Btr was shown to be important in growth and survival of B. pertussis in a mouse model. Btr-regulated genes were identified using a titration assay and include B. pertussis sodB and btr, E. coli aspA and a newly identified B. pertussis gene, brg1. brg1 is unaffected by Btr aerobically but is activated 3-fold by Btr under anaerobic growth conditions. brg1 encodes a protein similar to the LysR family of transcriptional regulators. The nucleotide sequence adjacent to brg1 was determined and found to encode proteins with similarity to various metabolic enzymes.