It has been one hundred years since mitochondria were first observed and recorded by Altman in 1890, when they were named bioblasts. Ten years later, "mitochondrion", which means threadlike granule, started to be used. It is still an unanswered question where this organelle originated. More and more evidence and enthusiasm favor the hypothesis that mitochondria have evolved from engulfed prokaryotic symbionts (Martin and Muller, 1998). An opposing idea proposed that mitochondria just represent another kind of intracellular membrane system, like Golgi (Cavalier-Smith, 1987). Whichever is true, it is known today that mitochondria are well-defined and ubiquitous cellular structures compartmentalized by double membranes. They not only provide some of their own genetic information, but also are the site of cellular lipid synthesis and oxidative phosphorylation. Since mitochondria are such complex functional units, the study of mitochondrial biogenesis (a process to produce a respiratory competent organelle) is a combined issue of genetics, biochemistry and chemistry. It aims to answer questions regarding mitochondrial morphology, continuity, protein and phospholipid syntheses, protein transport, etc. This study is concentrated on a since regulatory step of a single mitochondrial gene in yeast, i.e. the stabilization of the cytochrome b (COB) mRNA, which requires the nuclear-encoded Cbp1 protein. The results of my study support that the nuclear-encoded Cbp1 protein stabilizes COB messages in two different ways: First, it processes the 5'-untranslated region (UTR); second, it is required after formation of the mature 5'-end of COB mRNA. Evidence is provided that Cbp1 physically interacts with a CCG element in the COB 5'-UTR, and the maintenance of this interaction is critical for COB mRNA accumulation. Suppressor analysis of COB 5'-UTR mutations identified factors in general mitochondrial mRNA turnover pathways. Thus, in addition to studying the mechanism of Cbp1-dependent COB mRNA stabilization, the further analysis of genes identified by mutation in this work may reveal previously uncharacterized components in the general pathways of yeast mitochondrial mRNA decay.

The Glucocorticoid Receptor (GR) activates apoptosis in immature thymocytes through a mechanism that requires transactivation and repression of gene transcription. Several targets of GR regulation have been identified, including Glutathione-S-Transferase M1 (GSTM1) and Nur77 expression, and NFB function, however no specific mechanism for activating GR-regulated apoptosis has been determined. Apoptosis is also induced in immature mouse thymocytes by ceramide analogues, staurosporine, and Fas stimulation, through diverse signal transduction pathways, ultimately resulting in caspase protease activation. Glucocorticoids (GC), ceramide analogues, and staurosporine pathways are inhibited by the Bcl-2-class of apoptosis regulator proteins. In some cell lines, Fas-dependent apoptosis is also blocked by Bcl-2. To identify the involvement of specific genes and components of the apoptosis machinery in this GR-dependent system, a somatic cell genetic approach was taken. A panel of Dexamethasone (Dex)-resistant cell lines (Apt-) isolated in a previous study were characterized with respect to their sensitivity to GR-independent apoptotic signals. While W7.2 cells were sensitive to staurosporine, ceramide, and Fas stimulation, Apt3.8, Apt4.8 and Apt5.8, were found to be resistant to some or all of these treatments. Apt4.8 and Apt5.8, but not Apt3.8, were found to be sensitive to staurosporine-induced apoptosis, whereas, all three Apt- mutants were found to be resistant to ceramide and Fas-mediated apoptosis. Measurement of steady state and Dex-regulated transcripts for Bcl-2-related genes, and Fas-interacting genes, and the caspase gene family, indicated that the observed differential resistance of the Apt- cell lines was not due to altered steady-state levels of any of these gene transcripts. GR-dependent regulation of GSTM1 and Nur77 transcription, and NFkB function was also tested in the Dex-resistant cell lines. GSTM1 expression was not induced in the Apt- cell lines by Dex. TNF a -induced activation of NFkB function was not observed in Apt3.8, and GR-dependent repression of Nur77 expression is defective in Apt4.8. From this genetic analysis of thymocyte apoptosis I conclude that GR-dependent apoptosis occurs via a mitochondria-dependent pathway which overlaps with many other apoptotic pathways including Fas in the WEHI7.2 cell system.

The genes cappuccino (capu) and spire (spir) are required for establishment of the anterior/posterior (A/P) and dorsal/ventral (D/V) axes during oogenesis in Drosophila melanogaster. In the oocytes of capu and spir mutant females, axis-defining determinants are either mislocalized or not localized at all. Mounting evidence suggests that this localization defect is due to misregulation of the microtubule and actin cytoskeletons, and by extension, suggests that the wild type function of capu and spir is to regulate the cytoskeleton during oogenesis. In support of this hypothesis, previous data from two-hybrid experiments have suggested that SPIR binds to actin through its two WH2 domains. Here I show the interaction between SPIR and ACTIN is direct using in vitro binding assays. Both genetic and yeast two-hybrid evidence suggested that CAPU and SPIR also interact with Rho family GTPases, which include RHOA, RHOL, CDC42 and RAC1. GST pull-down experiments, performed to test the directness of these interactions, revealed that CAPU and SPIR both bind strongly to RHOA and weakly to CDC42. SPIR also binds strongly to RHOL and weakly to RAC1. I also present here the first evidence for capu function outside of oogenesis and for three splice variants of capu. Searches of the Drosophila genome database reveal that two splice variants of capu are expressed in the adult head, and in situ hybridization results reveal that capu message is expressed in the developing larval brain. Additionally, phenotypes in the adult wing are described. One representative of each of two classes of EST from the database that appeared to define two novel classes of capu splice forms are sequenced and compared to the existing capu splice form. Taken together, the data described here help demonstrate that capu and spir play a role in early axis determination in Drosophila, and in regulation of the cytoskeleton. It remains to be determined if capu and spir act to regulate the actin cytoskeleton which in turn regulates the microtubule cytoskeleton, or if these genes directly regulate the microtubule cytoskeleton.

The diversity of many groups of organisms is related to the evolution of features that contribute to rapid radiations. This project reconstructed the phylogeny of carabid beetles in the subfamily Harpalinae, a speciose group of terrestrial predators. The phylogenetic inference focused on the sister group relationships, the monophyly of the subfamily and the tribal relationships within harpalines. Molecular sequence data, primarily from 28S ribosomal DNA and the wingless gene, were collected from more than 200 carabid beetles. Parsimony, minimum evolution distance, maximum likelihood, and Bayesian phylogenetic analysis methods were used to reconstruct the phylogeny of harpalines. Brachinine bombardier beetles and austral psydrines were found to be closely related to the harpaline clade. Within harpalines, zuphiites formed a clear clade as did pseudomorphines + graphipterines + orthogoniines. However the lebiomorph assemblage and the tribe Lebiini were not monophyletic. With the use of harpaline phylogenetic hypotheses, the evolution of the arboreal lifestyle was elucidated within the subfamily, including the rate and number of origins and losses of arboreality. Correlated evolution of several morphological characters and habitat was explored. Significant correlation of adhesive subtarsal setae and bilobed fourth tarsomeres on carabid legs were found with arboreality and may be arboreal adaptations, while long legs and long elytra are probably not associated with arboreality. The relationship of other morphological characters with arboreality is not clear. Harpalines may have been part of a rapid radiation of species diversity, where many lineages invaded new ecological niches and evolved novel morphological features to become adapted to their environment.

A complete understanding of the speciation process requires elucidation of the underlying genetic details. Considerable empirical and theoretical research has revealed two important patterns that characterize the genetic basis of reproductive isolation. First, reproductive isolation is usually caused by incompatible substitutions at different, interacting loci. Second, the locations of genes contributing to reproductive isolation are biased toward the X chromosome. These observations suggest that reproductive barriers between young species are frequently due to disrupted interactions involving genes on the X chromosome. This idea motivates a detailed study of introgression for X-linked loci across a European hybrid zone between two species of house mice, Mus domesticus and M. musculus (Appendix A). Allele frequency patterns at 13 molecular markers with known chromosomal positions identify one region with clearly reduced introgression. This piece of the X chromosome may contain genes that confer reproductive barriers between M. domesticus and M. musculus. Expected patterns of hybrid zone introgression for incompatible substitutions between the X chromosome and the autosomes are also investigated using computer simulations (Appendix B). The results indicate that both the locations and shapes of allele frequency clines are distorted by selection against hybrids, with effects related to the dominance of the interacting alleles and inheritance patterns of the X chromosome (hemizygosity). Additionally, loci closely linked to the targets of selection show only weak reductions in introgression, suggesting that neutral gene flow is not strongly impeded by selection against hybrids. Finally, the mouse genome sequence is used to compare patterns of hybrid zone introgression to several genomic attributes (Appendix C). No clear correlates of introgression emerge, suggesting that reproductive isolation between M. domesticus and M. musculus may involve a small number of genes with large effects. Using location in the X-linked region of reduced introgression, a high rate of protein evolution, and restricted expression in the male germ line as criteria, seven candidate genes for reproductive isolation are identified. These results underscore the value of studying natural patterns of introgression in model genetic organisms for understanding the genetic basis of speciation.

Mortality is substantial in alfalfa stands and may be due to random and natural selection. The effects of selection for persistence on single-plant yield are not well understood. This study examined relationships between yield, nonstructural carbohydrates (TNC), and other agronomic characteristics, and persistence in nondormant alfalfa. S₁ progenies were produced on 60 plants dug from a five-yr-old field of 'CUF-101' (Persistent population) in central AZ and 60 greenhouse-grown CUF-101 plants (Random population). Progenies were sown in a replicated field trial at Tucson, AZ in Oct. 1989. Spring and fall forage yield was significantly lower in the Persistent population than in the Random population. Rate of stem regrowth following harvest was also lower in the Persistent population. S₁ progenies from the Persistent population contained approximately 7.1% more TNC in roots and crowns than the Random population, suggesting that TNC and persistence may be positively correlated. These data suggest that plants in the Persistent population exhibited more conservative growth patterns and that a negative genetic correlation may exist between single-plant yield and persistence. Simultaneous selection for traits associated with productivity and persistence may be necessary.

Numerous studies show that plastid DNA is inherited biparentally in alfalfa (Medicago sativa L.). Mitochondrial DNA has been shown to be inherited in a uniparental-maternal fashion in a limited number of sexual crosses. This study investigated the inheritance of mitochondrial DNA in 54 alfalfa progenies from crosses between two cytoplasmic male sterile maternal plants and 54 paternal plants, representing each of the eight basic germplasm groups of M. sativa. Cloned mitochondrial DNA fragments from M. sativa were used as hybridization probes to identify polymorphisms in mitochondrial DNA for Eco RI restriction sites. Polymorphisms were analyzed for patterns of mitochondrial DNA inheritance. All progenies displayed the Eco RI mitochondrial DNA restriction pattern of the maternal parent, indicating that maternal mitochondrial inheritance predominates in alfalfa. The same progenies were also examined for their plastid DNA inheritance pattern. Plastid DNA displayed a mixed inheritance pattern, with both uniparental-maternal and uniparental-paternal inheritance. Variation observed between paternal parents for plastid transmission was absent for mitochondrial transmission.

<p> Breast cancer cell dormancy is a significant clinical problem which contributes to the development of distant metastasis and disease relapse. Currently, no therapies exist which can effectively detect or eradicate dormant cancer cells. </p><p> In this study, we utilized a 3D co-culture dormancy model, recapitulating the inhibitory hematopoietic stem cell niche, which interacts with MDA-MB-231 cells, causing them to enter a state of growth arrest. The knockdown of emerging dormancy regulator gene, p38/MAPK14, in MDA-MB-231 cells allows previously dormant cells to &ldquo;break&rdquo; dormancy and re-enter the cell cycle when grown in the inhibitory niche. Using the newly described in vitro dormancy model, we performed a genomic shRNA library screen, and identified several p38-regulated breast cancer dormancy suppressor gene candidates. Two p38-regulated gene candidates were investigated further. Knockdown of transcription factors and p38 substrates, HBP1 and BHLHB3, in MDA-MB-231 cells lead to re-activation (proliferation) of once indolent cells when cultured in the inhibitory niche. </p><p> The present study illustrates the role of p38 and p38-regulated genes in breast cancer dormancy within the microenvironment of the inhibitory (endosteal) hematopoietic stem cell niche. Additionally, we have identified a list of ~700 breast cancer dormancy suppressor candidate genes. Further analysis and validation experiments are needed to classify novel molecular players and signaling pathways involved in tumor cell dormancy from the list of candidate genes generated in this study.</p>

The heme biosynthetic pathway in Saccharomyces cerevisiae comprises eight enzymatic steps. The HEM6 gene encodes uroporphyrinogen decarboxylase, a cytoplasmic enzyme catalyzing the fifth step of heme biosynthesis. HEM6 was cloned by complementation of a heme auxotrophic yeast strain containing a hem6 mutant allele. An open reading frame of 1086 nucleotides encoding a protein of 362 amino acids was obtained by sequence analysis. Expression of HEM6 was found to be induced two-fold by lactate, a non-fermentable carbon source, but not regulated by heme. Site-directed mutagenesis of a conserved cysteine residue implicated in catalysis demonstrated that cysteine 52 of the yeast enzyme is not essential for enzymatic activity. / The enzyme catalyzing the sixth step in the heme biosynthetic pathway, coproporphyrinogen oxidase, is encoded by the HEM13 gene. Transcription of HEM13 is regulated by the product of the pathway, heme. In the presence of heme, transcription of the ROX1 gene is induced, and the ROX1 protein represses HEM13 transcription. ROX1 is a member of the High Mobility Group (HMG) family of DNA-binding proteins. In order to define the mechanism of repression of HEM13 by ROX1 we synthesized ROX1 protein and derivatives either in vitro or in Escherichia coli as fusions to the glutathione-S-transferase (GST) protein. All ROX1 derivatives containing intact HMG domains were capable of specific binding to DNA sequences from the HEM13 promoter region. In contrast, ROX1 proteins which contained deletions within the HMG domain were no longer capable of binding to DNA. In addition, ROX1 was capable of oligomerization, and the amino-terminal 100 amino acids of ROX1 which constitutes the HMG domain was required for oligomerization as well as for DNA-binding. / The HEM13 promoter contains five possible operator consensus sequences postulated to be binding sites for ROX1. We showed that ROX1 binds to all five sites with differential affinity and that this binding occurs primarily via minor groove contacts. In vivo repression was examined by deletion of the five operator sites, either individually or in combination. Three of the operator sites were shown to act in an additive manner in bringing about repression of HEM13 and a fourth site appeared to be active only when all other operator sites were intact. A random PCR-based selection procedure used to select ROX1 binding sites predicted the optimal ROX1 binding site to consist of the consensus sequence (A/T)TT(T/G)TT. / Finally, a preliminary analysis of the ROX1 protein suggested that the region of ROX1 responsible for repression is located within the carboxy-terminus of the protein. This region of ROX1 may be required for interaction with the general repressor complex SSN6/TUP1. Chromosomal disruption of either of these two genes resulted in derepression of HEM13 expression in a ROX1 wild type strain. Therefore, repression of HEM13 is brought about by multiple regulatory factors interacting at various cis-acting elements within the HEM13 non-coding sequences.

The Saguenay-Lac St. Jean and Charlevoix regions of Quebec are characterized by high incidences of hereditary diseases such as vitamin D-dependent rickets type I (VDD1), cystic fibrosis, and tyrosinemia. This phenomenon may be due to founder effect or genetic drift. Therefore, one may also detect differences in allele frequencies of neutral polymorphisms between these populations and the general population. An assessment of genetic variation was performed utilizing genetic markers spanning chromosome 12q14. Allele frequencies for the genetic markers in the sample populations did not differ significantly from those published for other Caucasian populations. One haplotype, observed in strong linkage disequilibrium with the VDD1 mutation, was found to be infrequent on normal chromosomes. This suggests that all of the VDD1 mutant alleles could be identical by descent, in keeping with a founder effect for VDD1 in northeastern Quebec.