Burkholderia sp. MBA4 was identified from soil for its ability to grow on monobromoacetic acid. A dehalogenase, Deh4a, confers a dehalogenation function in MBA4. A permease gene, deh4p, forms a haloacid operon with deh4a. Deh4a has been well characterized but the regulatory mechanism of the haloacid operon was unknown. Electrophoretic mobility shift assay shows that at least one regulatory protein exists and binds with the promoter of deh4a. A DNA-affinity column purified several DNA-binding proteins and two proteins were identified by tandem mass spectroscopy as putative transcriptional regulators of B. xenovorans LB400. One of these proteins was named IclR1 and subjected to further analysis in this study. Here I report the cloning of the iclR1 gene and the functional study of this protein. The iclR1 gene was cloned by means of chromosome walking. The iclR1 gene has 837 bases and encodes 278 amino acids. The putative protein is classified as a member of the IclR family. Recombinant IclR1 was produced in E. coli and purified by Ni-NTA column. The experimental size of IclR1 is 27.5 kD and a dimer of 52.3 kD can be identified in vitro with cross-linking reagent. Purified IclR1 failed to bind the deh4a promoter, and mutants with a disrupted iclR1 gene or over-expressing IclR1 has no effect on the deh4a expression. It is likely that IclR1 is not a regulator of the haloacid operon. EMSA shows that IclR1 binds to its own promoter which contains a palindrome sequence and a pair of inverted repeats upstream of the start codon. The transcription start site of iclR1 was determined to be a G 110 bp upstream of the start codon by 5’ RACE. The iclR1 promoter region was ligated with a lacZ reporter gene, and transformed into wild type MBA4, a disruptant and an over-producer mutant. ONPG assay shows that the expression of the reporter is induced by NaCl. The transcript level of iclR1 is also higher in NaCl-containing medium. Over-expression of IclR1 inhibits the expression of the reporter, indicating that IclR1 is a self-regulated repressor. The growth of an iclR1 disruptant is more sensitive to salt. These results suggest that IclR1 is beneficial for the survival of the cell in NaCl stress, but excessive IclR1 prevent the responding system from overworking. Since MBA4 is very sensitive to NaCl, understanding the NaCl-related physiology of MBA4 is important. The gene(s) under direct control of IclR1 is unknown and the specific function of IclR1 awaits further study. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Leptin, the protein product of obese gene, is a 16-kD adipokine with regulatory functions on food intake and energy metabolism. At present, limited information is available on leptin functions and regulation in lower vertebrates mainly due to the fact that the primary structure of leptin is highly diversified from fish to mammals. Leptin in teleost fish is even more complicated as leptin isoforms have been reported presumably as a result of whole-genome duplication that occurred during the evolution of modern-day bony fish. Using grass carp (Ctenopharyngodon idella) as a model, I sought to investigate the physiological functions and endocrine regulation of leptin in bony fishes. As a first step, the structural identities of two leptins, namely leptin A and B, were established by 5’/3’RACE. The two isoforms share low levels of amino acid sequence homology with mammalian leptins but their deduced 3D-protein models are highly comparable to that of the human counterpart. In grass carp, leptin A and B are widely expressed with highest levels of expression detected in the liver with leptin A as the dominant form. To study the biological actions of grass carp leptins, recombinant proteins of leptin A and B were produced in Escherichia coli and found to inhibit both basal and NPY-stimulated food consumption and feeding behavior in goldfish by both intraperitoneal and intracerebroventricular injection. In addition to the anorexic effects observed, the effects of leptin on pituitary hormone secretion and synthesis were also examined in primary culture of carp pituitary cells. Using reverse transcription-polymerase chain reaction coupled to laser captured microdissection, leptin receptor expression was detected in somatotrophs, gonadotrophs and lactotrophs. Furthermore, leptin A and B were both effective in increasing basal secretion, cell content and transcript expression of growth hormone, luteinizing hormone and prolactin in carp pituitary cells. In the same study, parallel rises in somatolactin α and β mRNA levels without major changes in transcript expression of other pituitary hormones were also noted. These stimulatory effects were mediated by differential coupling with Janus kinase-2 (JAK2)/ signal transducers and activators of transcription (STATs), mitogen-activated protein kinase (MAPK) and/or phosphoinositide 3-kinase (PI3K)/Akt pathways. Although leptin A and B exhibited similar effects on feeding and pituitary hormone expression, their endocrine regulation appears to be quite different. In primary culture of carp hepatocytes, insulin could reduce leptin A but not leptin B mRNA levels through MAPK but not PI3K/Akt pathway. Glucagon, in contrast, could trigger leptin A but not leptin B mRNA expression via the cAMP/PKA cascades and this stimulatory effect could be negated by co-treatment with insulin. At the hepatic level, SLα could also induce leptin A but not leptin B mRNA expression via JAK2 activation of PI3K/Akt cascades. Parallel treatment with SLβ, however, was found to up-regulate leptin B but not leptin A transcription by MAPK coupling to JAK2. These results suggest that the two leptin isoforms identified in grass carp are responsible for similar biological functions but under differential regulation by various endocrine factors coupled to a number of post-receptor signaling mechanisms. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

No description available.

Le role des contributions epigenetiques lors du developpement d'un zygote en un organisme adulte a ete etudie a travers la caracterisation genetique et moleculaire de deux genes a effet maternel, mau-2 et clk-2, chez le nematode C. elegans. Des mutations dans ces deux genes produisent des patrons d'heritabilite differents, ainsi que des phenotypes distincts. Les mutants mau-2 sont secourus partiellement par un effet maternel, mais sont normaux quand un allele de type sauvage est exprime chez le zygote. Le gene mau-2 participe au guidage de nombreuses migrations de cellules et axones lors du developpement. Ce gene code pour une proteine de fonction inconnue, qui a ete conservee lors de l'evolution. mau-2 fonctionne dans les cellules qui migrent pendant le developpement du systeme nerveux. D'autre part, les mutants clk-2 sont completement secourus maternellement, excepte pour leurs competences reproductrices. De plus, ils dependent strictement de la presence d'un allele de type sauvage chez la mere pour leur developpement embryonnaire. clk-2 est requis pour la regulation des rythmes du developpement et du comportement. clk-2 est un gene essentiel, dont la fonction est requise entre la fin de la maturation des oocytes et le stade embryonnaire de deux cellules. clk-2 code pour une proteine qui est similaire a Tel2p, une proteine chez la levure, et est requis pour le maintien d'une longeur normale des telomeres chez le ver. Bien que mau-2 et clk-2 fonctionne differemment pendant le developpement du ver, la base de l'effet maternel semble etre tres similaire dans les deux cas. Une quantite considerable de transcrit est accumule dans les oocytes et est probablement transferee au zygote. Le transcrit ainsi fourni au zygote permet la traduction de proteine de type sauvage en quantite suffisante pour mener la plupart des fonctions de ces deux genes lors du developpement.

Traditional screens for female sterile mutants have revealed only two genes which when mutant, give a fully penetrant 16 nurse cell phenotype. One way to gain a better understanding of the function of these genes in oocyte determination, is to identify genes which interact with them. Using P-element mutagenesis, I have isolated one dominant suppressor and five dominant enhancers of Bicaudal-D, and begun the phenotypic and molecular characterization of three of these genes. By deficiency screening, I have identified two different loci which act as dominant enhancers of Bic-D, and eight different loci which act as dominant suppressors of Bic-D. Further work on defining the locus responsible for the strong suppression phenotype associated with one of these deficiencies, revealed betaH-spectrin to be a strong dominant suppressor of loss-of-function Bic-D alleles, and a strong dominant enhancer of Bic-D gain-of-function alleles.

Prolidase (E.C.3.4.13.9) hydrolyzes imidodipeptides. Prolidase deficiency (PD) (McKusick no. 170100) is an autosomal recessive disorder characterized by a highly variable clinical phenotype. Mutation analysis was performed on a panel of 10 PD cell lines. Single-stranded conformation polymorphism analysis (SSCP) analysis on four overlapping cDNA-PCR products covering the entire coding region of the prolidase gene revealed seven novel mutations: G $ to$ A,551 (R184Q); G $ to$ A,833 (G278D); G $ to$ A, 1342 (G448R); G $ to$ A, 1354 (E452K); delGAG, 1354-1356 (delE452); a deletion of exon 5; and a deletion of exon 7. We used inverse PCR to clone intronic regions flanking exons 5 and 7 and designed primers for conventional PCR of these regions of the genome in patients expressing mRNAs with deleted exons. Two splice acceptor site mutations were identified: a G $ to$ C, nt $-$1 of intron 4 and an A $ to$ G, nt $-$2 of intron 6. To assess the biochemical phenotypes of four of these mutations (R184Q, G278D, G448R, and delE452), we have designed a transient expression system for prolidase in COS-1 cells. The enzyme was expressed as a fusion protein carrying the HA1 epitope of influenza hemagglutinin, allowing its immunological discrimination from the endogenous enzyme. Expression of the R184Q mutation produced 7.4% of control enzymatic activity while the expression of the G278D, G448R and delE452 produced inactive enzymes. Western analysis of the R184Q, G278D and G448R prolidases revealed stable immunoreactive material whereas the delE452 prolidase was not detectable. Pulse-chase experiments revealed that the delE452 mutant protein was synthesized but unstable. / The R184Q allele is carried by an asymptomatic individual, suggesting that its residual activity may be sufficient to prevent the development of symptoms. The other alleles, G278D, G448R, and delE452 which completely abolish enzyme activity associate with the symptomatic form of the disorder. Interestingly, these substitutions are all located at or very close to the putative metal binding residues of prolidase. / We have cloned and sequenced the mouse prolidase cDNA. We have cloned a genomic DNA fragment which carries exons 2, 3, and 4 of the mouse prolidase gene. We constructed a vector for the targeted disruption of the prolidase gene in mouse embryonic stem cells, to create a mouse model for prolidase deficiency.

Many traits of economic importance in crop plants are quantitative, complicating the selection for desirable characteristics. Recent studies suggest a complex relationship between genotype and phenotype, with genetic variability often appearing as differences in gene expression rather than structural changes in proteins. In oat (Avena sativa L.), lipid and protein content are economically important traits. In the first of four studies reported here, partial sequences for eight genes involved in lipid or protein biosynthesis were obtained from ten oat cultivars with varying lipid and protein content. Phylogenetic analysis showed that these sequences clustered into families possibly corresponding to homeologous genes. Some cultivar- and family-specific polymorphisms were identified. In the second study, we surveyed differential gene expression between developing kernels of cultivars Kanota and Ogle by constructing reciprocal subtractive libraries. Of the 195 contig sequences obtained, only a minority had homology to characterized sequences. Grouping these sequences in categories based on gene ontology of their BLAST hits showed different profiles of expression for each cultivar. In the third study, we tested a method for transforming macroarray data consisting of dividing spot signal by the median array background. This reduced variation due to array exposure time. In the fourth study, gene expression levels were considered as quantitative traits in the Kanota x Ogle mapping population. Macroarrays featuring oat clones differentially expressed between both parents were hybridized with cDNA from the population lines. Among the 33 significant expression quantitative trait loci detected, most clustered to linkage group 29--43, a possible "hot-spot" of gene expression regulation.

Four Sinorhizobium meliloti mutants with IPTG-dependent phenotypes were isolated via random Tn5tac1 mutagenesis. The growth phenotypes of Rm30192 (pheT), Rm30193 (ffh ), and Rm300196 (unmapped) were conditional on the presence of IPTG, while that of Rm30194 was conditional on its absence. The insertion in Rm30194 is upstream of tacA, which appears to be under conditional control of the Tn5tac1 Ptac. While all four strains formed root nodules on alfalfa, Rm300192 and Rm30193 attained only 53% and 36% respectively, of the wild-type shoot dry weight value. / Five previously isolated Tn5tac1 mutants were also characterized. Significantly, Rm30044 (bhbA) was able to use arabinose but not glutamate as sole carbon source, a novel finding. Finally, Rm30047 (gpsA), which lacks NADPH-dependent glycerol-3-phosphate dehydrogenase (G3PDH) activity, was found to have an NADH-dependent G3PDH activity that was repressed by IPTG, possibly due to conditional expression by the Tn5tac1 P tac of genes downstream of gpsA.

Bicaudal-C, a Drosophila gene, is required for centripetal follicle cell migration in the egg chamber and anterior patterning of the embryo; its exact functions are unknown. BICAUDAL-C carries five KH domains required for in vitroRNA-binding and for in vivo activity. Here, I show that Bicaudal-C functions, through RNA-binding, to regulate oskar expression during mid-oogenesis. Females carrying mutations in Bicaudal-C in the regions encoding its KH domains display premature, ectopic translation of OSKAR at the anterior of their stages 8--10 oocytes; this likely accounts for the disruption of anterior patterning in embryos of Bicaudal-C/+ females. Also here, I propose that oskar and gurken share common regulators, including Bicaudal-C. This is based on the discovery of dorsalized progeny of Bicaudal-C/+ females, and on the dominant enhancement of the Bicaudal-C/+ phenotype by specific gurken and cornichon alleles. Finally, I describe a yeast two-hybrid screen used to investigate BICAUDAL-C-protein interaction.

Flax (Linum usitatissimum L.) peroxidase cDNAs (FLXPER1--4) were isolated from a lambdagt10 shoot library using probes encoding the amino termini of class III plant peroxidases. These probes were obtained by PCR amplification of the library with lambda primers flanking the EcoRI cloning site, and a mixed oligonucleotide corresponding to the catalytic domain (HFHDCFV) found in secretory plant peroxidases. The transcriptional expressions of FLXPER1 and FLXPER2 appear to be specific to stem based on northern blot analyses. FLXPER1 and FLXPER2 encode acidic peroxidases of calculated isoelectric points (pIc) 4.6 and 4.7, respectively. However, FLXPER1 differs from FLXPER2 in amino acid sequence and in the possession of additional amino acids at its carboxy terminus containing motifs found in membrane-anchored proteins. The FLXPER1 anchoring motifs show striking similarity to those found in a blue copper-type protein (LP18) whose expression in pea (Pisum sativum) has been correlated with lignin deposition. FLXPER3 is expressed in leaf, root, and stein. Partial genomic sequences of FLXPER3 and a highly homologous FLXPER5 were also obtained. FLXPER3 and FLXPER5 do not have the typical class HI plant peroxidase third intron, located within the highly conserved VALSGAHT haem-binding motif. FLXPER3 and FLXPER5 do have, however, an intron downstream of this motif at a location not found in any other class III plant peroxidase gene. FLXPER4 encodes a basic (pIc 8.5) extracellular peroxidase. A modified version of the restriction fragment length polymorphism-coupled domain-directed differential display (RC4D) technique was used to evaluate class III peroxidase transcriptional expression in flax organs. The method revealed the expression of 20 to 30 peroxidase genes in each plant organ examined. It also confirmed the stem-specific nature of FLXPER2 and the ubiquitous character of FLXPER3. FLXPER1 was found in all organs investigated in contrast to results observed by northern b