Dissertations / Theses on the topic 'Genes encoding deduce proteins'

In the course of work aimed at discovering new sigma transcription initiation factors in Escherichia coli, two unknown sigma cross-reacting proteins (SCRP-23 and SCRP-27A) were identified by cross-reaction with antibodies raised against region 2.2 of sigma 70. This thesis describes the mapping, sequencing and characterisation of the corresponding genes. The gene encoding SCRP-23 was located near 652kb on the physical map of the E. coli chromosomes. Its sequence and that of part of a downstream open reading frame were determined, and found to be closely similar to the ahpC and F genes (respectively) of Salmonella typhimurium. These encode the C22 and F52a subunits of the anti-oxidant enzyme, alkylhydroperoxide reductase. The identity of the E. coli genes was further established by their ability when introduced on plasmids into an ahp deletion strain to restore cumene hydroperoxide resistance. Transcription of ahpCF was found to be driven by two promoters: ahpP₁ is dependent on activation by the OxyR transcriptional regulator, whilst ahpP₂ is independent of this factor. Indeed ahpP₂ is located within the OxyR target site, and is repressed when OxyR is activated by oxidation. The gene encoding SCRP-27A, scrP, was located near 3416 kb on the physical map, just 250 bp downstream of arcB. arcB encodes a transmembrane sensor-regulator of respiratory functions.

In order to identify Leishmania proteins which may be immunologically relevant or may play a role in interactions between Leishmania and its mammalian host, a Leishmania major genomic DNA library was constructed in the vector λgt11 and screened with antibodies raised to Leishmania major promastigote membranes. Two recombinant DNA clones were identified which encoded repetitive sequences (Clone 20 and Clone 39). Clone 20 encoded a repetitive peptide of 14 amino acids and clone 39 encoded an unrelated repetitive peptide of 10 amino acids. Analysis of one of these clones, Clone 20, indicated that there were two RNA transcripts of 9500 and 5200 nucleotides expressed which corresponded to this clone in Leishmania major and Leishmania donovani and this expression was not stage-specific. The results of genomic DNA analysis and isolation of additional clones encoding Clone 20 sequences indicated that there were two genes which corresponded to Clone 20 in both Leishmania major and Leishmania donovani and that these genes differed from one another with respect to the number of repeats which they contained. Antibodies against the fusion protein produced by Clone 20 recognized a series of Leishmania major proteins of apparent mol wt 250,000. Analysis of Clone 39 indicated that there was a single transcript of 7500 nucleotides expressed which corresponded to this clone in both Leishmania major and Leishmania donovani and that there was a single gene (or two identical genes) which encoded this transcript. The genomes of many protozoan parasites exhibit a high degree of plasticity with respect to chromosome size and number. The presence of highly repetitive regions within their DNA may be involved in maintaining this plasticity, allowing the parasite to evolve rapidly under selective pressure. Repetitive regions have been identified within many Plasmodia antigens and have been implicated in the ability of this parasite to evade the host immune system. The presence of Leishmania genes encoding proteins containing tandemly repeating peptides may indicate that these proteins play a similar role in evading the host immune system during the course of Leishmania infections. The possible evolution and functions of repetitive proteins in protozoan parasites is discussed.
Medicine, Faculty of
Medical Genetics, Department of
Graduate

Olfaction is one of the most important senses by which insects obtain information about their environment. In the early stages of olfactory perception in insects, odour molecules are carried across the sensillum lymph by small soluble Odorant Binding Proteins (OBPs). This is followed by activation of the appropriate olfactory receptor, resulting in an electrical impulse, and subsequent degradation of the initial signal. OBPs have been studied in a range of insect orders including Lepidoptera, Diptera and Orthoptera, and this study reports the cloning and characterisation of cDNAs with a potential olfactory role in the vetch aphid, Megoura viciae (Buckton, Homoptera: Aphididiae). Construction and sequencing of antennal cDNA libraries identified two cDNAs, MvicOBP1 and Mv164, which were approximately 0.8kb and 1kb respectively. The amino acid sequence of MvicOBP1 has the spacing pattern of six cysteine residues that is characteristic of insect OBPs, and Mv164 shows similarity to insect cytochrome P450 enzymes. RT-PCR showed that these cDNAs have specific or enhanced expression in the chemosensory tissues of M. viciae, and parallel expression patterns suggest a "linked" function. Related sequences are present and expressed in other aphid species, and sequencing of genomic fragments allowed the partial elucidation of the intron/exon organisation of these genes. Subtracted antennal cDNA libraries identified two cDNAs encoding proteins with significant similarity to insect chemosensory proteins (CSPs), cDNAs encoding Juvenile Hormone Binding Proteins (JHBPs), and a tissue-specific cDNA with a potential carrier role. These, coupled with the OBPs, add evidence to the suggestion that there is an insect superfamily of binding proteins. A PBP from Bombyx mori (BmorPBP1) was used as a model system for in vitro expression of an insect OBP and subsequent characterisation of the recombinant protein. Four forms of this protein, identified through their interaction with an anti-BmorPBP antibody, were resolved by isoelectric focusing.

The highly conserved p24 family of proteins are abundant components of the early secretory pathway, although their specific function remains unclear. In the yeast Saccharomyces cerevisiae, deletion of the eight members of this family results in no marked phenotype, whereas in mice, deletion of a single p24 family member, p23, results in embryonic lethality. In addition, in the fly Drosophila melanogaster, two of the nine p24 proteins have been shown to be necessary for dorsoventral patterning of the embryo.
Here, I attempt to determine the function of these proteins by finding new genetic interactors of the p24s using a synthetic genetic array (SGA) analysis in yeast. 44 yeast non-essential genes, when deleted, were identified as being synthetically lethal or sick with the p24s. Many of these interactors are involved in the secretory pathway. To determine the causes of cell death or growth reduction of the double deletion mutants, I studied their ability to secrete the ER protein Kar2p. In many cases of these double deletion mutant strains, Kar2p secretion was exacerbated compared to the p24 deletion alone. This may reflect the loss of fidelity of the transport between the endoplasmic reticulum (ER) and the Golgi apparatus as well as ER retention.

Doctor of Philosophy
Department of Biochemistry
Subbarat Muthukrishnan
The extracellular matrices of cuticle and peritrophic matrix of insects are composed mainly of chitin complexed with proteins, some of which contain chitin-binding domains. This study is focused on the identification and functional characterization of genes encoding proteins that possess one or more copies of the six-cysteine-containing ChtBD2 domain (Peritrophin A motif =CBM_14 =Pfam 01607) in the red flour beetle, Tribolium castaneum. A bioinformatics search of T. castaneum genome yielded previously characterized chitin metabolic enzymes and several additional proteins. Using phylogenetic analyses, the exon-intron organization of the corresponding genes, domain organization of proteins, and temporal and tissue-specificity of expression patterns, these proteins were classified into three large families. The first family includes 11 proteins essentially made up of 1 to 14 repeats of the peritrophin A domain. Transcripts for these proteins are expressed only in the midgut and only during feeding stages of development. We therefore denote these proteins as “Peritrophic Matrix Proteins” or PMPs. The genes of the second and third families are expressed in cuticle-forming tissues throughout all stages of development but not in the midgut. These two families have been denoted as “Cuticular Proteins Analogous to Peritrophins 3” or CPAP3s and “Cuticular Proteins Analogous to Peritophins 1” or CPAP1s based on the number of ChtBD2 domains that they contain. Unlike other cuticular proteins studied so far, TcCPAP1-C protein is localized predominantly in the exocuticle and could contribute to the unique properties of this cuticular layer. RNA interference (RNAi), which down-regulates transcripts for any targeted gene, results in lethal and/or abnormal phenotypes for some, but not all, of these genes. Phenotypes are often unique and are manifested at different developmental stages, including embryonic, pupal and/or adult stages. The experiments presented in this dissertation reveal that while the vast majority of the CPAP3 genes serve distinct and essential functions affecting survival, molting or normal cuticle development. However, a minority of the CPAP1 and PMP family genes are indispensable for survival under laboratory conditions. Some of the non-essential genes may have functional redundancy or may be needed only under special circumstances such as exposure to stress or pathogens.

La régulation post-transcriptionnelle joue un rôle essentiel dans le système nerveux, de l’assemblage à la fonction. Chez les métazoaires, la famille multigénique ELAV / Hu code pour des protéines de liaison à l'ARN produites dans les neurones et impliqués dans cette régulation. Le gène elav (embryonic lethal abnormal visual system) code pour une protéine essentielle produite dans tous les neurones et est présent seulement chez les diptères. L’élucidation des fonctions des plus anciens paralogues de drosophile, fne (found in neurons) et rbp9 (RNA binding protein 9), a fait l'objet de ce travail, à cause de (1) l’étroite relation de ces gènes avec les orthologues de vertébrés, (2) la disponibilité de mutants nuls viables.En collaboration avec le laboratoire de Brian Oliver, NIH, États-Unis, nous avons utilisé le RNA-Seq pour analyser le transcriptome de tête de mutants fne-. Les données ont été analysées ) avec les programmes Cuffdiff et DESeq pour identifier des différences dans les niveaux de transcripts mutants et ceux d’une souche sauvage de référence (WT). L'épissage alternatif a été examiné avec Spanki (SPlicing ANalysis Kit), un suite de programmes que nous avons contribué à développer. Spanki utilise les données de séquence des jonctions exon-exon et compare l’abondance de formes alternatives et mutuellement exclusives de transcrits issus d’un événement d'épissage. Les premières analyses se sont concentrées sur les différences de transcriptomes entre males et femelles sauvages. La détermination sexuelle somatique et le comportement chez la drosophile sont sous le contrôle d'une cascade génétique bien caractérisée et contrôlée par les gènes Sxl, tra, msl2, dsx et fru, mais les cibles en aval de cette voie canonique demeurent largement inconnues. Notre approche nous a permis d’identifier (et de valider par qPCR) de nouveaux gènes exprimés différemment dans les deux sexes. J’ai montré que l’un d’entre eux est le gène fne, que sa régulation est indépendante de tra et tra2, mais dépend de Sxl. Ces propriétés font de fne la première cible identifiée dans une voie alternative de détermination sexuelle prédite par le laboratoire de T. Cline sur la base d'autres études. J'ai aussi montré l’existence d'un rôle précédemment non documenté pour tra et tra2 dans la lignée somatique mâle. Afin d'identifier des réseaux fonctionnels contrôlés par fne, les données du transcriptome de mutants ont été comparées à celles de la souche sauvage de référence. Plusieurs gènes dont le niveau de transcrits ou l’épissage alternatif est altéré en absence du gène fne ont été identifiés et validés par qPCR. Par exemple, l'épissage alternatif de unc-13, essentiel pour l'exocytose des vésicules, est affecté dans les mutants fne. D'autres gènes impliqués dans les fonctions synaptiques ont été identifiés, y compris n-syb, tomosyn, brp et Clc. En outre, il existe des interactions génétiques entre la mutation fne et des mutations affectant les fonctions synaptiques. Ces interactions peuvent causer une létalité synthétique dans les doubles mutants. Finalement, les males mutants fne- ont un comportement de « chaining» également compatible avec une fonction à la synapse. Ainsi, le gène fne établit un lien entre la régulation post-transcriptionnelle, la fonction synaptique et le comportement.Dans la dernière partie de mon travail, j'ai utilisé une approche évolutive pour tenter de distinguer les fonctions spécifiques de fne ou de rbp9, des fonctions ancestrales. Des approches moléculaires, anatomiques et comportementales ont été employées. Les résultats distinguent différentes catégories de gènes, spécifiquement ceux dont l'expression est affectée (1) uniquement par fne (2) uniquement par rbp9 (3) à la fois par fne et rbp9 de manière redondante ou synergique. J’ai mis en évidence une redondance fonctionnelle partielle des deux gènes causant une létalité synthétique à l'âge adulte dans les doubles mutants, mais j’ai aussi identifié des fonctions spécifiques
Post-transcriptional regulation plays pivotal roles from assembly to function of the nervous system. In metazoans, the ELAV/Hu genes constitute a conserved multigene family of pan-neuronal RNA-binding protein involved in post-transcriptional regulation. The elav (embryonic lethal abnormal visual system) gene is the first described family member, it encodes a vital protein and is restricted to dipterans. Elucidating the functions of the more ancient Drosophila paralogs, fne (found in neurons) and rbp9 (RNA-binding-protein-9), has been the focus of this work, because of (1) the close relationship of these genes to the vertebrate orthologs, (2) the availability of viable null mutants.In collaboration with the laboratory of Brian Oliver, NIH, USA, we used RNA-Seq to analyze the head transcriptomes of fne- mutants and a wild type reference strain (WT). The RNA-Seq data was searched for differences in transcript levels using the programs Cuffdiff and DESeq. Alternative splicing was examined using a suite of programs called Spanki (SPlicing ANalysis Kit), whose development we participated in. Spanki utilizes only sequence reads spanning exon-junctions to compare pairs of mutually exclusive alternative splicing events.Initial analyses included sex-specific comparisons in WT transcriptomes. Somatic sexual determination and behavior in Drosophila are under the control of a well characterized genetic cascade initiated by Sxl (Sex lethal), but the targets downstream of this canonical pathway remain largely unknown. As expected, the genes Sxl, tra (transformer), msl2 (male-specific lethal gene), dsx (doublesex) and fru (fruitless), which belong to the canonical sex-determination/dosage compensation pathways were identified by our analyses. In addition, new genes whose transcript expression levels differ between the sexes were identified and validated by qPCR. Further, I obtained evidence for previously undocumented roles of tra and tra2 (transformer 2) in the male soma. Finally, a sex-biased alternative splicing event was identified in fne, whose regulation is independent of tra or tra2, but dependent upon Sxl. This makes of fne the first Sxl-dependent, tra/tra2-independent target identified in a sex determination/differentiation pathway, previously been predicted to exist based upon other studies.The data was analyzed to identify functional networks controlled by fne. I found that it affects the expression of several genes at the level of transcript expression and alternative splicing involved in synaptic functions. They include of unc-13, n-syb (neuronal Synaptobrevin), tomosyn, brp (bruchpilot) and Clc (Clathrin light chain). Further, genetic interactions between fne and shi (shibire) or nrg (neuroglian), which also possess synaptic functions, reveal synthetic lethality in the double mutants. In addition, fne mutant males display a fruitless-like chaining behavior, also consistent with a function at the synapse. Thus the fne gene links post-transcriptional regulation to synaptic function and behavior.Finally, I used an evolutionary approach to distinguish newly evolved functions, i.e. specific to fne or rbp9, from those that are shared and thus more likely to be ancestral. Molecular, anatomical and behavioral approaches in parallel analyses of fne and rbp9, show that they possess both gene-specific and overlapping functions. The latter is evident from synthetic lethality in early adulthood of double mutants. My results distinguish different gene categories with respect to their regulation: genes whose expression is affected (1) only by fne (2) only by rbp9 (3) both by fne and rbp9 in redundant or synergistic ways. Finally, I showed that male-male interactions dramatically differ between fne versus rbp9 mutants, revealing the emergence of a new (or loss of an ancestral) function in behavior

Cyanophages infecting cyanobacteria of the genera Synechococcus and Prochlorococcus are abundant and ubiquitous in aquatic environments. Sequencing of some cyanophage isolates has revealed homologous genes to psbA and psbD that encode key proteins for photosynthesis. Using molecular techniques, this thesis explored the phylogenetic diversity of these genes in cyanophage isolates and natural virus communities. First, I amplified cyanophages psbA and psbD genes fragments from myoviruses infecting marine Synechococcus strain DC2 (=WH7803) and compared them phylogenetically. I demonstrated that psbA was present in all cyanophage genomes, while psbD was presented in only half of them. Moreover, gene-based phylogenies revealed similar tree topologies for both genes. This suggests that psbA and psbD were acquired coordinately but psbD was lost during multiple events. Next, I compared the genetic diversity of viral psbA from a range of environments with the goal of determining if sequences cluster based upon their environments. Phylogenetic reconstruction showed that viral psbA sequences from fresh waters have an evolutionary history distinct from their marine equivalents. Moreover, photosynthesis sequences from cyanophages infecting neither Prochlorococcus nor ' Synechococcus were distinct, as were sequences from different phage families (e.g., podoviruses vs myoviruses). This thesis confirmed that viral psb genes have their own evolutionary history that is distinct from that of their host.
Science, Faculty of
Earth, Ocean and Atmospheric Sciences, Department of
Graduate

The aim of the present study was to isolate gene(s) encoding HMG-I/Y proteins from higher plants and study the regulation of their expression. In order to isolate an HMG-I/Y gene a pea genomic library was screened with a pea HMG-I/Y cDNA resulting in the isolation of three different chimeric clones containing only the 3' end of the HMG-I/Y gene. The coding region (with a single intron of 201 bp) of the pea HMG-I/Y gene was isolated as a 795 bp fragment by PCR on genomic DNA. Several attempts using different approaches to isolate the promoter region of the pea HMG-I/Y gene were unsuccessful. The MHG-I/Y gene was present in a single-copy and expressed in all organs as determined by Southern and northern blot analysis. A cDNA encoding the HMG-I/Y protein from Arabidopsis thaliana was sequenced and characterised. The 903 bp cDNA encodes an HMG-I/Y protein of 204 amino acid residues with a calculated molecular mass of 22 kDa containing 4 copies of the 'AT-hook' motif. Southern blot analysis on Arabidopsis genomic DNA revealed the presence of a single copy of the HMG-I/Y gene. The HMG-I/Y gene was located near the top of chromosome 1 by RFLP analysis of F₈ recombinant inbred lines. Northern blot analysis demonstrated the expression of the HMG-I/Y gene in all organs of Arabidopsis examined with the highest expression in flowers and developing siliques. A genomic clone encoding the HMG-I/Y protein was isolated from an Arabidopsis genomic library using the HMG-I/Y cDNA as a probe. A 6.3 kb genomic fragment was sequenced and includes the coding region (with a single 73 bp intron) of the HMG-I/Y gene and 5209 bp of 5' upstream sequence and 412 bp of 3' downstream sequence.

Sedentary organisms that inhabit the marine intertidal zone must be adapted to withstand forms of environmental stress that are uncommon in other marine environments. Adaptations to these stressors are apparent in the morphologies, behaviors, life histories, and regulation and products of genes of sedentary intertidal organisms (Lent 1969; Garrity 1984; Schmidt et al 2008). However, for those species that have a planktonic larval stage, evolutionary specialization at the molecular level could be constrained by opposing requirements for larvae and adults that are adapted to very different environments. This constraint would have been removed in lineages that lost the ancestral larval stage, and reinstated in lineages that secondarily reacquired a larval stage. A comparative approach using a group in which the planktonic larval stage has been lost and reacquired could be used to test the hypothesis that adaptive evolution of stress-related genes accompanies these life-history transitions.

Genes with known roles in physiological stress tolerance include those that encode the family of 70 kilodalton heat shock proteins (hsp70s ) (Feder and Hofmann 1999). Statistical tests for departures from selective neutrality were applied to sequence variation in hsp70 genes of calyptraeid gastropods. The Calpytraeidae include both intertidal and subtidal species, as well as species that have lost or secondarily reacquired the ancestral planktonic larval stage (Collin 2003a; Collin et al. 2007). I focused on two species: Crepidula fornicata and C. atrasolea. Both species are found in low intertidal and subtidal habitats in the western tlantic region. However, whereas the eggs of C. fornicata hatch into planktonic veligers, the eggs of C. atrasolea develop directly into crawling juveniles. I tested for selection with statistics that characterize the distribution of sequence variation within and between species, comparing values for each hsp70 gene fragment with expectations for selectively neutral loci.

Bacillus thuringiensis subsp. isrealensis (B.t.i.) produces a crystalline endotoxin specific for some larvae of mosquitoes that are vectors of the malaria parasite and other infectious diseases. Fragments were obtained from the 108 kb plasmid from B.t.i. strain 4Q2 which encodes several proteins comprising the delta-endotoxin. These DNA fragments were inserted into the hybrid cyanobacterial cloning vector, pTNTV, downstream from its powerful lambda promoter, and the chimaeras were transformed into Escherichia coli. Ampicillin resistant transformants were screened with radioactively labelled oligonucleotides whose sequences were determined from the published sequences of the B.t.i. 130 kDa polypeptide. Clones showing hybridization were used in bioassays to determine their level of toxicity to the fourth instar larvae of the Aedes aegypti mosquito. Twelve clones were found that demonstrated toxicity which was statistically significantly greater than that observed in controls. Plasmid DNA from some of these clones was isolated, cut with restriction endonucleases, and viewed through agarose gel electrophoresis to confirm that B.t.i. fragments had been inserted into the vector. Future work will investigate the expression of these cloned toxin genes in transformable cyanobacteria and will determine their subsequent activity against the fourth instar larvae of Aedes aegypti and Anopheles quadrimaculatus.
Department of Biology

Thèse doctorat : Sciences du Vivant. Aspects Moléculaires et Cellulaires de la Biologie : Strasbourg 1 : 2006. Thèse doctorat : Sciences du Vivant. Aspects Moléculaires et Cellulaires de la Biologie : Université de Fudan - Shanghai - Chine : 2006.
Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 10 p.

Le nucléosome est l’unité de base de la chromatine dans tous les eucaryotes. L’assemblage du nucléosome est fondamental dans différents processus cellulaires tels que la réplication, la réparation, la recombinaison et la transcription de l’ADN. ‘Nucleosome Assembly Protein1’ (NAP1) est considérée comme un chaperon d’histone facilitant l’assemblage du nucleosome. Nous avons isolé 3 ADNc chez le riz et 4 ADNc chez le tabac codant pour différentes protéines NAP1. Nous avons démontré que les différentes protéines NAP1 se lient in vitro avec différentes classes d'histones avec différentes affinités. De plus, nous avons montré que les protéines NAP1 interagissent avec la cycline B et la tubuline, suggérant que les protéines NAP1 sont impliquées dans des fonctions des microtubules. En utilisant la technique de fusion GFP, nous avons montré que pendant que les protéines OsNAP1;1 et OsNAP1;3 sont localisées toutes les deux dans le cytoplasme et le noyau, les autres protéines NAP1 du riz et du tabac sont localisées plutôt dans le cytoplasme. Par mutagénèse et par l'utilisation d'inhibiteurs d'export nucléaire, nous avons démontré que NtNAP1;1 et OsNAP1;1 font la navette entre le cytoplasme et le noyau. Il apparait donc que la compartimentation constitue un moyen de réguler les activités NAP1 dans la cellule. Par génétique inverse, nous avons démontré que le double mutant nrp1-1nrp2-1 chez Arabidopsis thaliana manifeste un phénotype de racines courtes avec défauts en division et différentiation cellulaire. Ce mutant montre une sensibilité accrue aux dégâts causés à l'ADN et une suppression de l'extinction des gènes au niveau transcriptionnel (TGS) dans l'hétérochromatine. L’ensemble des résultats obtenu par ce travail de thèse constitue une nouvelle avancée pour notre compréhension de la régulation dynamique de la structure de la chromatine dans le développement des plantes.

Secreted phosphoprotein 24 (spp24) is a member of the cystatin superfamily.;This study identifies a rare single-amino acid polymorphism (p.S38F) of human spp24 and its importance has been assessed by comparing the sequence of human spp24 with that of eight other species. The gene encoding spp24 in mouse (Spp2), like its human ortholog, comprises eight exons with an apparently TATA-les promoter. The exon-intron structure is identical in mouse and human and the size and location of intron 1 is conserved between many species. Using several strategies, the gene encoding spp24 in mouse has been mapped to 88832387-88853226 bp of the mouse chromosome 1. An extensive expression study was carried out on the mouse and human genes encoding spp24. These studies indicated that the gene has an expression pattern of a tissue-specific and cell-specific nature, being expressed predominantly in liver and its expression is down-regulated by lipopolysaccharide (LPS) and tumour necrosis factor alpha (TNFalpha). In an attempt to elucidate the function of the spp24 protein in mouse, a pooled-tissues cDNA library was constructed in a yeast two-hybrid vector. Two different constructs comprising the entire spp24 protein and the C-terminal non-cystatin like domain of the protein were used individually as baits in the yeast two-hybrid system to screen the constructed library. Seven potential interacting proteins were identified including granulin precursor also known as acrogranulin/epithelin (Grn), tissue specific transplantation antigen P35B (Tsta3), keratin complex 1, acidic, gene 18 (Krt1-18), keratin complex 1, acidic, gene 13 (Krtl-13 ), vimentin (Vim), similar to protein phosphatase 1, regulatory (inhibitor) subunit 12C (no gene symbol yet assigned) or myosin binding subunit 85 and alpha-actinin-4 (Actn4).

Enterococcus faecalis is a well known nosocomial drug resistant pathogen that is responsible for urinary tract infections, bacteremia, wound infections and endocarditis through the formation of biofilms. It has been shown that 68 genes present within the core genome of E. faecalis are upregulated in biofilm formation. One of those 68 genes is a putative selenium-dependent molybdenum hydroxylase (SDMH). Adjacent to this gene are a series of open reading frames that have been postulated to play a role in the maturation of a labile selenium cofactor. The biosynthesis of this labile cofactor has yet to be studied at either the genetic or biochemical level. The addition of selenium to growth medium caused a significant increase in biofilm density and extracellular hydrogen peroxide by wild type E. faecalis. By site-directed mutagenesis gene products encoded in the SDMH operon were shown to be necessary for the selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. This biofilm and peroxide phenotype is inhibited both by tungsten or auranofin in wild type E. faecalis suggesting the SDMH is a necessary enzyme for selenium-dependent biofilm and peroxide formation. These results show that the gene products encoded within the SDMH operon are necessary for a selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. These mutants will provide the basis for defining the synthesis of the labile selenium cofactor and allow for an expanded understanding of the biological use of selenium.
ID: 030422988; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Thesis (M.S.)--University of Central Florida, 2010.; Includes bibliographical references (p. 38-40).
M.S.
Masters
Department of Biomedical Sciences
Medicine

Many pulses and beans grown for human comsumption are susceptible to insect attack. Winged bean, a high protein crop of the tropics, yield seeds which appear to be immune to infestation by the storage bruchid Callosobruchus maculatus. In this thesis the biochemical basis of this resisitance was investigated. Insect bio-assays were carried out in which protein fractions from seeds of winged bean were incorporated at a range of concentrations into artificial seeds, and their effects upon the development of C.maculatus determined. Both albumin and globulin fractions were toxic to the developing larvae and their toxicity correlated with their haemagglutination activity. Assay of psophocarpin fractions A, B and C found the fraction psophocarpin B to be most insecticidal. On further purification this fraction yielded two lectin fractions and a protease inhibitor fraction. Purified basic lectin was highly insecticidal to C. maculatus larvae with an LC(_50) value of 0.35%. The physiological level of this protein in winged bean seeds is sufficient to account for their resistance to attack by C maculatus. Winged bean trypsin inhibitor was also purified and tested in artificial seeds against C maculatus. However, even at concentrations in excess of twice the physiological concentration it had no deleterious effects upon development. Winged bean protein fractions, incorporated in artificial diets, proved toxic to the Lepidopteran pests Heliothis virescens and Spodoptera littoralis in bio-assays, but it appeared that the basic lectin was not responsible for toxicity towards these insects. Attempts to clone the gene encoding the winged bean basic lectin were made by constructing cDNA and genomic libraries, and heterologous lectin genes from soybean and Phaseolus were investigated as possible probes for the basic lectin gene. Purification of the basic lectin B3 and sequencing of 44% of its primary protein structure, along with comparisons with other legume lectin sequences allowed the synthesis of oligonucleotide primers for use in polymerase chain reaction experiments. However, all the PGR products obtained were shown to be the result of non-specific amplification. Further work needed to obtain the basic lectin gene is discussed.

The extant coelacanths have a close resemblance to the coelacanth fossil records dating back to 230mya. Like their predecessors, the extant coelacanths inhabit rocky caves at a depth of 100-300m below sea level. In the Comoros, the water temperature at these depths is estimated to fluctuate between 14-20°C. High-level adaptation to these environment and lack of competition are thought to have led to the morphological uniformity and slow change throughout the history of the coelacanths. Under stress conditions, proteins unfold or misfold leading to the formation of aggregates. Molecular chaperones facilitate the correct folding of other proteins so that they can attain a stable tertiary structure. In addition, molecular chaperones aid the refolding of denatured proteins and the degradation of terminally misfolded protein after cellular stress. Heat shock proteins form one of the major classes of molecular chaperones. Here we show that, despite high-level adaptation to a unique habitat and slow change, the genome of the coelacanth encodes the major and highly conserved molecular chaperone, Hsp70. Latimeria menadoensis and Latimeria chalumnae contain intronless hsp70 genes encoding Hsp70 proteins archetypal of known Hsp70s. Based on the coelacanth codon usage, we have shown that bacterial protein expression systems, particularly Escherichia coli, may not be appropriate for the overproduction of coelacanth Hsp70s and coelacanth proteins in general. Also interesting, was the discovery that like the rat Hsc70, the L. menadoensis Hsp70 could not reverse thermal sensitivity in a temperate sensitive E. coli DnaK mutant strain, BB2362. We also report the successful isolation of a 1.2 kb region of L. menadoensis hsp70 upstream regulatory region. This region contain three putative heat shock elements, a TATA- box and two CAAT-boxes. This regulatory region resembled the Xenopus, mouse, and particularly tilapia hsp70 promoters, all of which have been shown to drive the expression of reporter genes in a heat dependent manner. Taken together, this data is the first to strongly suggest an inducible Hsp70-base cytoprotection mechanism in the coelacanth. It further provides basis to formulate testable predictions about the regulation, structure and function of Hsp70s in the living fossil, Latimeria.

Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Agriculture worldwide is under great pressure to produce enough food in order to sustain the ever-growing world population. Among the many challenges faced by food producers, crop losses and damage caused by fungal plant pathogens is a major problem. The study of fungal pathogens and the interaction between plants and fungi is therefore essential, and has been carried out for many years. Much has been learned in this time, but the full mechanisms of the various modes of fungal attack and plant defence have still not been elucidated. Many fungi rely on the action of cell-wall degrading enzymes (CWDEs) to breach the plant cell wall and facilitate access to the nutrients within. CWDEs are among the very first enzymes to be secreted at the start of fungal attack, and many of them are considered to be essential pathogenesis factors. Endopolygalacturonases (ePGs) are CWDEs that cleave the homogalacturonan stretches of the plant cell wall and are vital virulence factors for a number of fungi, including Botrytis cinerea. An important defence mechanism of plants involves the inhibition of CWDEs in order to halt or slow down the fungal attack. Plant polygalacturonaseinhibiting proteins (PGIPs) are cell wall associated CWDE-inhibiting proteins that specifically act on fungal ePGs. Many different PGIPs from a number of diverse plant species have been described to date. They are known to have differential inhibition capabilities that often result from only a few key amino acid changes within the leucine-rich repeat (LRR) active domains. Previously, the first grapevine PGIP was isolated and characterised from Vitis vinifera cultivar Pinotage (Vvpgip1). This Vvpgip1 gene was overexpressed in the tobacco species Nicotiana tabacum, and was shown to be very effective in reducing the susceptibility of tobacco towards B. cinerea. The combined results confirmed transgene overexpression, increased PGIP activity and a strong resistance response against Botrytis, leading to the characterisation of these lines as having PGIP-specific resistance phenotypes. In a subsequent transcriptomic analysis of these lines it was found that they display differential expression of cell wall metabolism genes and biochemical characteristics that might indicate possible cell wall strengthening compared to wild-type tobacco under uninfecting conditions. The V. vinifera cultivars are all very susceptible to fungal attack, whereas other grapevine species, specifically the North American Vitis species, are known for their strong resistance and even immunity against many fungal pathogens. Thirty seven PGIPs have previously been isolated from these more resistant species. The amino acid sequences of the active domains of these PGIPs were previously aligned with that of VvPGIP1, and the proteins were found to be highly homologous with each other and with VvPGIP1. The different nonvinifera PGIPs separated into 14 subgroups based on their active domain sequences. For this study, one PGIP from each group was selected for functional analysis in tobacco. The selected PGIP-encoding genes were transformed into tobacco by means of Agrobacterium tumefaciens. Analyses of the putatively transformed plantlets were performed to test for transgene presence, transgene expression, and PGIP activity: final transgenic tobacco populations consisting of three to twelve individually transformed lines of nine different nonvinifera PGIPs were obtained. A subset of the resultant transgenic lines was infected with B. cinerea in two independent whole plant infections over 11-14 days in order to investigate the disease resistance afforded by the various PGIPs towards this fungus. A line from the previously characterised VvPGIP1 population was included as reference; all the infections were contrasted to the WT tobacco. All the infected lines overexpressing the non-vinifera PGIPs displayed very strong disease reduction in comparison to the WT control: after initial primary lesion formation, the spread of fungal infection was contained and halted in these lines, while wild-type tobacco plants were severely affected. Although the VvPGIP1 line displayed the characteristic PGIP-defense response, the non-vinifera PGIP plants displayed smaller lesions, indicating very strong resistance phenotypes. The characterised non-vinifera PGIP overexpressing lines, together with the VvPGIP1 line and the WT control were also used to further evaluate the previous observation that overexpression might lead to changes in expression of cell wall genes. Analysis of the expression of a xyloglucan endotransglycosylase (xth) gene in the transgenic population showed that this gene was down-regulated in healthy uninfected tissue from all the transgenic lines tested. This confirmed previous results and have confirmed in all grapevine PGIP overexpressing lines tested so far that this gene is downregulated. XTH is typically involved in cell wall metabolism and specifically in controlling the strength and elasticity of the plant cell wall. From previous work it is known that downregulation of this gene leads to strengthening of the wall. The results obtained in this study showed that the PGIP-specific resistance phenotype seen for VvPGIP1-overexpressing tobacco could be confirmed in transgenic tobacco overexpressing non-vinifera PGIPs from more resistant grapevine species as well. The fact that these PGIPs lines all performed even better than the VvPGIP1 lines in conferring resistance towards B. cinerea provides an interesting angle for further investigation into the structural differences between the non-vinifera PGIPs and VvPGIP1. The transgenic lines are also excellent material to study the in vivo functions of PGIPs further in the context of plant-pathogen interactions.
AFRIKAANSE OPSOMMING: Die landboubedryf is wêreldwyd onder groot druk om genoeg voedsel te produseer vir die groeiende wêreldbevolking. Een van die grootste probleme wat die bedryf ondervind, is die groot skade wat aan gewasse aangerig word deur patogeniese swamme. Dit is dus noodsaaklik om swamme en die interaksie tussen plante en swamme te bestudeer, en dit word al vir jare gedoen. Hoewel daar al baie geleer is in hierdie tydperk, is die volle meganismes van die verskeie maniere hoe swamme aanval en hoe plante hulleself verdedig, nog nie bekend nie. Verskeie swamme maak staat op die aktiwiteit van selwand-afbrekende ensieme (SWAEe) om deur die plantselwand te breek en sodoende toegang tot voedingstowwe in die plantsel te fasiliteer. SWAEe is van die eerste ensieme wat tydens die begin van patogeniese aanval deur swamme afgeskei word en verskeie SWAEe word as noodsaaklike patogeniese faktore beskou. Endopoligalakturonases (ePGs) is SWAEe wat die homogalakturoniese dele van die plantselwand verteer en is noodsaaklike virulensie faktore vir ‘n aantal swamme, onder andere Botrytis cinerea. ‘n Belangrike weerstandsmeganisme van plante behels die inhibering van swam SWAEe om sodoende die patogeen-aanval te stop of te vertraag. Die poligalakturonase-inhiberende proteïne (PGIPs) van plante is selwand-geassosieerde SWAEinhiberende proteïne wat spesifiek teen swam ePGs optree. Verskeie verskillende PGIPs vanuit verskillende plantspesies is tot dusver beskryf. Dit is bekend dat hulle differensiële inhiberende vermoëns het wat dikwels toegeskryf kan word aan slegs ‘n paar belangrike aminosuurvolgordeverskille in die leusien-ryke herhalende (LRH) aktiewe areas. Die eerste wingerd PGIP is vantevore geïsoleer vanuit Vitis vinifera kultivar Pinotage (Vvpgip1) en gekarakteriseer. Hierdie Vvpgip1 geen is ooruitgedruk in die tabakspesie Nicotiana tabacum en was baie effektief om die weerstand van tabak teen die swam Botrytis cinerea te verhoog. Die ooruitdrukking van die transgeen, verhoogde PGIP aktiwiteit en goeie weerstand teen Botrytis cinerea is bevestig, en het gelei daartoe dat die transgeniese VvPGIP1 plantlyne geklassifiseer is as lyne met PGIP-spesifieke weerstandsfenotipes. ‘n Daaropvolgende transkriptomiese analise van die plantlyne het gewys dat hulle differensiële uitdrukking van selwand-geassosieerde gene het, asook biochemiese eienskappe, wat ‘n moontlike selwandversterking aandui in vergelyking met wilde-tipe tabak in die afwesigheid van infeksie. Die V. vinifera kultivars is hoogs vatbaar vir swamme, terwyl ander wingerdspesies, spesifiek die Noord-Amerikaanse spesies, bekend is vir hoë weerstand en selfs immuniteit teenoor verskeie patogeniese swamme. Sewe-en-dertig PGIPs is vantevore geïsoleer vanuit hierdie meer weerstandbiedende spesies. Die aminosuurvolgordes van die aktiewe areas van hierdie PGIPs is vantevore vergelyk met die van VvPGIP1 en dit is gevind dat hierdie proteïne hoogs homoloog is aan mekaar, sowel as aan VvPGIP1. Die verskillende nie-vinifera PGIPs het in 14 groepe verdeel na aanleiding van die homologie van hulle aktiewe areas. Vir hierdie studie is een PGIP vanuit elkeen van hierdie groepe gekies vir verdere funksionele analise in tabak. Die 14 nie-vinifera PGIP-koderende gene is stabiel oorgedra na tabak deur middel van Agrobacterium tumefaciens. Die vermeende transgeniese plante is geanaliseer vir die teenwoordigheid van die transgeen, die uitdrukking daarvan en PGIP aktiwiteit: bevestigde transgeniese tabak populasies wat wissel van drie tot 12 individuele getransformeerde lyne kon verkry word vir nege van die verskillende nie-vinifera PGIPs. ‘n Aantal van die transgeniese lyne is geïnfekteer met B. cinerea in twee onafhanklike heelplantinfeksies vir 11-14 dae om die siekteweerstand van hierdie PGIPs teenoor die swam te evalueer. ‘n Plantlyn van die VvPGIP1-populasie is as ‘n verwysing ingesluit en al die infeksies is vergelyk met die wilde-tipe tabak. Al die geïnfekteerde lyne wat die nie-vinifera PGIPs ooruitdruk het ‘n baie sterk afname in siektesimptome getoon in vergelyking met die wilde-tipe kontrole: na aanvanklikle primêre lesies gevorm het, is die verspreiding van die infeksie ingeperk en gestop in hierdie lyne, terwyl die wilde-tipe plante baie erg geaffekteer is. Terwyl die VvPGIP1 lyn ook die tipiese PGIPweerstandsrespons getoon het, het die nie-vinifera PGIPe kleiner lesies ontwikkel, wat dui op baie sterk weerstandsfenotipes. Die gekarakteriseerde nie-vinifera PGIP ooruitdrukkende lyne, asook die VvPGIP1 lyn en die wilde-tipe kontrole, is gebruik om die vorige waarneming dat die ooruitdrukking kan lei tot veranderinge in selwandgeen-uitdrukking verder te ondersoek. Analise van die uitdrukking van ‘n xiloglukaan-endotransglikosilase (xth) geen in die transgeniese populasie het getoon dat hierdie geen afgereguleer is in gesonde, oninfekteerde weefsel van al die transgeniese lyne wat getoets is. Dit het vorige resultate bevestig en het ook bevestig dat hierdie geen afgereguleer is in alle wingerd PGIP-ooruitdrukkende lyne wat tot dusver getoets is. XTH is tipies betrokke by selwandmetabolisme, spesifiek by die beheer van selwandsterkte en selwandelastisiteit. Dit is uit vorige werk bekend dat die afregulering van hierdie geen lei tot versterking van die plantselwand. Die resultate verkry tydens hierdie studie het gewys dat die PGIP-spesifieke weerstand fenotipe van VvPGIP1-ooruitdrukkende tabak ook bevestig kon word in transgeniese tabak wat nie-vinifera PGIPs vanuit meer weerstandbiedende wingerdspesies ooruitdruk. Die feit dat hierdie PGIP lyne almal selfs beter weerstand teen B. cinerea bied as VvPGIP1 lyne is ‘n interessante invalshoek vir opvolgende ondersoeke na die belang van strukturele verskille tussen die nie-vinifera PGIPs en VvPGIP1. Hierdie transgeniese lyne is ook uitstekende hulpbronne om die in vivo funksies van PGIPs verder te bestudeer in die konteks van plantpatogeen interaksies.

Thesis (MSc)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: The tolerance of Enterococcus mundtii ST4SA to stressful gastro-intestinal conditions in humans and animals is vital to its success as a probiotic. The need for new effective probiotics with stronger inhibitory (bacteriocin) activity has arisen due to the increasing number of antibiotic resistant pathogens. Enterococci are used in the fermentation of sausages and olives, cheese making and as probiotics. Their role as opportunistic pathogens in humans makes them a controversial probiotic (Moreno et al., 2005). Enterococci occur naturally in the gastro-intestinal tract which renders them intrinsic acid and bile resistance characteristics. E. mundtii ST4SA produces a 3950 Da broad-spectrum antibacterial peptide active against Gram-positive and Gram-negative bacteria, and viruses. The bacteria include Enterococcus faecalis, Streptococcus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae and Staphylococcus aureus. E. mundtii ST4SA inactivates the herpes simplex viruses HSV-1 (strain F) and HSV-2 (strain G), a measles virus (strain MV/BRAZIL/001/91, an attenuated strain of MV), and a polio virus (PV3, strain Sabin). This study focuses on the genetic stability of E. mundtii ST4SA genes when exposed to stress factors in the human and animal gastrointestinal tract. Based on results obtained by real-time PCR, the expression of genes encoding bacST4SA, RecA, GroES and 23S rRNA by E. mundtii ST4SA were not affected when the cells were exposed to acid, bile and pancreatic juice. This suggests that these genes of E. mundtii ST4SA will remain stable in the intestine. This could indicate that other genes of E. mundtii ST4SA could remain stable in the host. Further studies on the stability of genes encoding antibiotic resistance and virulence factors should be conducted to determine their stability and expression in the host in stress conditions. Concluded from this study, E. mundtii ST4SA is an excellent probiotic strain.
AFRIKAANSE OPSOMMING: Enterococcus mundtii ST4SA se weerstandsvermoë teen stresvolle gastrointestinale kondisies is essensieel vir die sukses van hierdie organisme as ‘n probiotikum. Die aanvraag vir nuwe, meer effektiewe probiotika met sterker inhibitoriese (bakteriosien) aktiwiteit is as gevolg van die toename in antibiotikum weerstandbiedende patogene. Enterococci word algemeen gebruik as probiotika, sowel as in die fermentasie van worse, olywe en kaas. Hulle rol as oppertunistiese patogene in mense veroorsaak kontroversie as gevolg van hul toenemende gebruik as probiotika. Enterococci is deel van die natuurlike mikroflora in die gastrointestinale weg van mense en diere. Dit verleen aan hierdie spesies ‘n natuurlike weerstandsvermoë teen maagsure, galsoute en pankreatiese afskeidings. E. mundtii ST4SA produseer ‘n 3950 Da wye spektrum anti-bakteriese peptied, aktief teen Gram positiewe en Gram negatiewe bakterieë sowel as virusse. Hierdie bakterieë sluit Enterococcus faecalis, Streptococcus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae en Staphylococcus aureus in. E. mundtii ST4SA inaktiveer die herpes simpleks virus HSV-1 en HSV-2, ‘n masels virus (MV/BRAZIL/001/91), en ‘n polio virus (PV3, stam Sabin). Hierdie studie fokus op die genetiese stabiliteit van E. mundtii ST4SA gene, wanneer hulle blootgestel word aan stress faktore in die mens en dier gastrointestinale weg. “Intydse” PKR data gebasseer op die uitdrukking van die bacST4SA, RecA, GroES en 23S rRNA gene in stresvolle kondisies dui aan dat E. mundtii ST4SA nie geaffekteer word wanneer die sel blootgestel word aan suur, gal en pankreatiese vloeistowwe nie. Hierdie resultate dui aan dat hierdie gene van E. mundtii ST4SA stabiel sal bly in die intestinale weg van die mens en dier. Dit kan aandui dat ander gene van E. mundtii ST4SA soos die wat kodeer vir virulensie faktore en antibiotikum se weerstandsvermoë stabiel mag bly in die gasheer. Verdere studies wat fokus op die stabiliteit van gene wat kodeer vir antibiotikum weerstandbiedendheid en virulensie faktore moet uitgevoer word om hulle stabiliteit en uitdrukking in die gasheer te bepaal. Bevindings van hierdie studie dui aan dat E. mundtii ST4SA goeie potensiaal het as ‘n probiotikum.

The investigations presented in this thesis represent an effort to understand the regulated expression of cytoskeletal proteins in differentiating cell systems. Vimentin is an intermediate filament protein whose expression is regulated during the differentiation of a variety of cell types. I have isolated DNA probes specific for chicken vimentin and utilized them for the study of vimentin gene regulation. The single chicken vimentin gene encodes multiple mRNAs that differ in the lengths of their 3' untranslated regions. These mRNAs are differentially expressed in a tissue-specific manner. Furthermore, vimentin mRNA increases to high levels during chicken embryonic erythropoiesis, underlying similar changes in vimentin protein accumulation.

Unlike nucleated avian erythrocytes, mammalian erythrocytes are devoid of intermediate filaments. I show that cultured murine erythroleukemia (MEL) cells repress the levels of vimentin mRNA during inducer-mediated differentiation, resulting in a subsequent loss of vimentin filaments. The expression of vimentin in these cells reflects the disappearance of vimentin filaments during mammalian erythropoiesis in vivo. To examine the molecular basis for divergent vimentin gene regulation in avian and mammalian erythropoiesis, I have studied the behavior of chicken and hamster vimentin genes introduced into MEL cells. During MEL cell differentiation, RNA encoded by transfected chicken vimentin genes significantly increases in abundance, whereas RNAs arising from either transfected hamster vimentin genes or the endogenous mouse vimentin gene are repressed. The results suggest that the difference in vimentin expression in avian and mammalian erythropoiesis is due to a divergence of cis-linked vimentin sequences.

Protein 4.1 is an extrinsic membrane protein that facilitates the interaction of spectrin and actin in the erythroid membrane skeleton. Previous studies have shown that chicken protein 4.1 exists as a multiplet of related polypeptides that are differentially expressed during erythropoiesis. I have isolated cloned cDNA probes for chicken protein 4.1, and have found that a single protein 4.1 gene encodes multiple mRNAs by differential processing; the ratios of protein 4.1 mRNAs change during erythroid development. In vitro translation experiments demonstrate that while the expression of protein 4.1 polypeptides is specified initially at the mRNA level by RNA processing, the ultimate expression of protein 4.1 variants is further determined translationally.

Thesis (Ph. D.)--Florida State University, 2005.
Advisor: Dr. Hank W. Bass, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed Sept. 19, 2005). Document formatted into pages; contains viii, 93 pages. Includes bibliographical references.

博士
國立臺灣大學
植物學研究所
88
Late embryogenesis abundant (LEA) proteins are synthesized during the late stages of seed development, and have been widely reported in monocot and dicot plants. In order to understand the developmental regulation and protein function of the soybean GmPM genes encoding LEA proteins, we used the transgenic Arabidopsis as tools in this study. For developmental regulation, a series of fragments of the promoter region were fused to the β-glucuronidase (GUS) reporter gene (uirA), to an Agrobacterium transgenic vector, and transformed the resulting constructs (pZP966, pZP572, pZP510, pZP294 and pZP114) into Arabidopsis thaliana. GUS enzyme activities were detected only in mature seeds, and cotyledons and hypocotyls of seedlings in transgenic Arabidopsis containing any one of the five constructs; they were not detected in other tissues at different developmental stages. During seed development, GUS activity detected at 10 days after flowering (DAF) and increased rapidly to a maximum in the mature seeds at 14 DAF. The longest promoter construct (pZP966) enabled the transgenic plants to exhibit the highest GUS activity, whereas the shortest construct (pZP114) was sufficient to direct the expression of the GUS gene at a detectable level. These findings indicate that the promoter of GmPM9 can be used to express desired genes in seeds during late seed maturation. The expression of the GUS gene could be induced in the leaves of the transgenic plants by salt and desiccation, but not by ABA, cold and wounding treatment. For protein function study, the GmPM1, GmPM2 and GmPM8 are seed maturation proteins of soybean, which were introduced into Arabidopsis by vacuum transformation method independently. Expression of these GmPM genes were regulated by CaMV 35S promoter and the proteins could constitutive accumulate in transgenic Arabidopsis. Cellular localizations of GmPM proteins were detected by immunogold. GmPM1 proteins were found in both cytoplasm and nucleus, GmPM2 proteins were accumulated in cytoplasm and GmPM8 proteins were in cytoplasm and starch grains. The transgenic Arabidopsis were subjected to osmotic and salt stress, there were no difference in the phenotype between transgenic and untransformed plants. These results suggested that GmPM proteins were not sufficient to increase stress tolerance in transgenic Arabidopsis. The untransformed seeds stored at 4℃ for four years loss the germination ability, but the seeds of TAZ2, TAZ16 and TAZ238 stored at the same condition could germinate at high percentage, however, the correlation between GmPM proteins and seed longevity needs to be further examined.

碩士
中臺科技大學
生物科技研究所
103
Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers, a disease causing tremendous loss in agriculture. The virulence of Xcc toward plants depends upon a number of factors, including the ability to produce extracellular enzymes and exopolysaccharide. Clp and RpfF regulate the production of these virulence factors. HD-related output domain (HDOD) is a protein domain of unknown biochemical function. The Xcc genome encodes three proteins (GsmR, HdpA and HdpB) that have an HDOD domain. Among them, only GsmR has been studied. Inactivation of gsmR results in decreased survival under heat shock, high osmotic stress, and SDS stress treatment. GsmR also has a role in cell motility. The expression of gsmR is up-regulated by Clp directly. Domain organization reveals that HdpA has an HDOD domain at the N-terminal and a GGDEF domain at C-terminal, while HdpB is a stand-alone HDOD domain protein. The aim of this study was to functionally characterized the HdpA and HdpB in Xcc, including (1) mutant strain construction and genetic complementation, (2) phenotypic evaluation, and (3) transcriptional regulation. Stress tolerance assay revealed that inactivation of hdpA caused reduction of acrifavine and CuSO4 tolerance. Complementation of full length hdpA can restore these phenotypic changes. The production of extracellular enzymes, bacterial attachment and virulence were not affected in the hdpA mutant strain. HdpA had a minor role in exopolysaccharide production. Cell motility was not reduced following hdpA mutantion, while complementation of full length hdpA but not HDOD domain significantly impacted the cell motility. The complementary strain also showed reduced exopolysaccharide production. Reporter assay indicated that hdpA transcription is positively regulated by RpfF and is subject to catabolite repression. In addition, the expression of hdpA is repressed in the presence of acrifavine, CuSO4 and NaCl, as well as under nitrogen starvation. Phenotypic evaluation of hdpB mutant revealed that none of the phenotypes tested shown significantly differences between the wild type and its hdpB mutant.

碩士
國立臺灣師範大學
生命科學研究所
93
Most edible and medical fungi contain ingradents, such as steroids, triterpenes, nuclear acids, and polysaccharides. These compounds are commonly considered to have pharmaceutical activities or potentials, and therefore function in arresting the growth of tumer cells or stimulating immune systems. Few proteins have recently isolated from Wolfiporia cocos and Taiwanofungus camphorate with immunomodulatory activity, and are described as fungal immunomodulatory proteins (FIPs). Wolfiporia cocos is the most popular fungal ingredient in Chinese prescription, however, knowledge regarding to its bioactive compounds and genomic information is limited. Thus, I would like to explore FIP candidates, and establish a express sequence tag (EST) library from Wolfiporia cocos in this study. A potential FIP gene was characterized from 1-week-old Wolfiporia cocos cDNA library. This gene encodes a protein sharing 50% sequence similarity to Taiwanofungus camphorate FIP (Aca1), and is therefore named as WcFIP1. The recombinant WcFIP1and Aca1 was expressed in E. coli, and their identity were confirmed by western analysis. We concluded that the newly isolated WcFIP1 is one of the fungal immunomodulatory proteins in Wolfiporia cocos.

Symbiosis is the close and protracted interaction between organisms. The molecular interactions that occur during symbiosis are complex with multiple barriers that must be overcome. Many Gram-negative, host-associated bacteria use a type III secretion system to mediate associations with their eukaryotic hosts. This secretion system is a specialized apparatus for the injection of type III effector proteins directly into host cells, which in the case of plant pathogens, are collectively necessary to modulate host defense. The type III secretion system is not a mechanism exclusive to pathogens, however, as many strains of commensal Pseudomonas fluorescens and mutualistic rhizobia demonstrably require a type III secretion system to interact with their host plants. The work presented in this thesis describes genome-enabled approaches for characterizing type III effector genes across the range of plant symbiosis. Using high-throughput sequencing technology, draft genome sequences were generated for the plant pathogen, Xanthomonas hortorum pv. carotae M081, the plant commensal, Pseudomonas fluorescens WH6, and six strains from the plant mutualists Sinorhizobium fredii and Bradyrhizobium japonicum. Analyses of the draft genome sequences and publicly available finished sequences contributed insights into mechanisms of host-association and to increasing the inventory of type III effector sequences as well as developing methods directly applicable for agriculture. Finally, characterization of the genetic diversity of type III effectors from rhizobia shows that collections of type III effectors of mutualists are static, with little diversity in content and sequence variation. This represents the first comprehensive cataloging of type III effector from species of mutualistic bacteria and the first to provide evidence for purifying selection of this important class of genes.
Graduation date: 2012

碩士
國立清華大學
分子醫學研究所
98
The endosymbiotic event leads mitochondrial genes to be translocated into nuclei. However, there are still a few genes conserved in the mitochondrial genome which code for proteins involved in oxidative phosphorylation. These proteins include ND1~ND6 and ND4L subunits of complex I, cytochrome c of complex III, Cox1~Cox3 subunits of complex IV, ATP6 and ATP8 subunits of complex V. Interestingly, the mitochondrial genome in Chlamydomonas reinhardtii lacks nd3, nd4l, cox2, cox3, atp6 and atp8 genes. Previous report suggested that nd3, nd4l, cox2, cox3 and atp6 were transfered to nuclei in C. reinhardtii, and cox2 was further divided into two independent nuclear genes. In this study, allotopic and xenotopic expression strategies were adopted to evaluate the possibilities of applying these methods as treatment concepts for mtDNA diseases. When the mitochondrial targeting signal (MTS) of ND4L, ND3, ATP6, COX3, and COX2a of C. reinhardtii was fused with enhanced green fluorescent proteins (EGFP), the resultant fusion proteins were successfully imported into mitochondria of HeLa and HEK293 cells. However, when the MTSs of the ND4L and ND3 were individually fused with its human homologue, only a limited mitochondrial targeting was observed. To improve the import efficiency, xenotopic expression of several full-length C. reinhardtii nuclear-encoded mitochondrial proteins were directly applied for mitochondrial targeting studies in human cells. By this approach, a significant improvement of mitochondrial import was observed in the targeted complex I ND4L subunit. Nevertheless, when the transactivator of transcription (TAT) region from the HIV was added in front of these constructed C. reinhardtii transgenes, the new designs did not show a significant improvement on mitochondrial targeting. However, when the hydrophilic EGFP protein was added at the C-terminus of the full-length C. reinhardtii ND4L subunit, the process could decrease the hydrophobicity of the importing protein effectively and improve the ability of the mitochondrial targeting. In this report, we proved that the C. reinhardtii ND4L subunit possesses the capability of the mitochondrial targeting in human cells and the hydrophobicity of the importing proteins plays an important role for mitochondrial targeting.

The light-harvesting complexes (LHC) of the unicellular marine chromophyte, Heterosigma carterae, were fractionated by sucrose-density gradient centrifugation, following digitonin solubilization, and by non-denaturing SDS-PAGE. The sucrose gradient allowed for the isolation of a major light-harvesting complex fraction, containing approximately 53% of the total chlorophyll, the majority of the chlorophyll c and a single polypeptide of 19.5 kDa. Up to 12 different polypeptides immunologically related to both the fucoxanthin-Chi a/c complexes (FCPs) and to the chlorophyll a + b-binding proteins (CABs) were detected in thylakoids and in the lower photosystem I (PS I) enriched fractions. Using a modification of the non-denaturing gel system of Allen and Staehelin (1991 Anal. Biochem. 194, 2 14-222) allowed the resolution of a number of large pigment-protein complexes which included several PS I and PS II fractions along with a predominant LHC fraction, an improvement over previously published methods. A Fcp eDNA from Heterosigina carterae has been cloned and sequenced. It encodes a 210 amino acid polypeptide that has similarity to other FCPs and to the CABs of terrestrial plants and green algae. Comparison of the FCP sequence to the recently determined 3- dimensional structure of the pea LHC II complex indicates that many of the key amino acids thought to participate in the binding of chlorophyll and in the formation of complex-stabilizing ionic interactions between hydrophobic regions of the protein are well conserved. In addition, the Fcp genes are part of a large multigene family with greater than 20 related members in Heterosigina. Phylogenetic analyses of the LHC protein sequences shows that the FCPs form a natural group separate from the iPCPs of the dinoflagellates. Though there are obvious similarities between the FCPs and the CABs, the relationships are very distant. Analyses of polypeptides in the red algae Aglaotharnn ion neglectuin and Porphyridium cruentum, in collaboration with Greg Wolfe and Beth Gantt, were the first to demonstrate that polypeptides immunologically related to the CABs and the FCPs are present within the Rhodophyceae. In addition, CAB/FCP-related LHCs have not been detected in a cyanobacterium (Nostoc) and a prochiorophyte (Prochlorothrix). This suggests the CAB/FCP related LHCs arose only once after the establishment of the chioroplast and provides some evidence that suggests chioroplasts evolved from a symbiotic cyanobacterium-like organism only once (monphyletic). The organization of the antennae in Heterosigma carterae is equally as complex as that in the terrestrial plants. This is indicated by the detection of at least 12 LHC-related polypeptides and the presence of a large multigene family encoding the FCPs. In addition, the immunological relatedness and the sequence conservation of the FCPs with the CABs indicates that the structure of the LHCs has been conserved throughout evolution and that these different antennae complexes share a common ancestor.

Despite their complacent appearance, plants are highly perceptive of stimuli in their environment. Responses to these stimuli enable plants to acclimate to the conditions of their local environment. Fluctuations in cellular calcium (Ca2+) have been implicated as critical signals for transducing external stimuli into intracellular information. How Ca 2+ signals are decoded in plants is largely unknown. The full Arabidopsis genome sequence enabled the identification of a family of 50 genes encoding potential Ca2+ sensors that are related to the quintessential Ca2+-binding protein, calmodulin (CaM). These C&barbelow;aM&barbelow;-l&barbelow;ike or CML proteins have the potential to mediate Ca2+ signaling in plant cells. My thesis work is focused on elucidating the potential physiological significance of two related CML proteins, CML23 and CML24 (also known as TCH2). CML23 and CML24 share over 40% amino acid sequence identity with CaM and have 4 EF-hand Ca2+-binding motifs. CML24 is likely a Ca 2+ sensor because it can undergo Ca2+-dependent changes in hydrophobic interaction chromatography and migration rate through denaturing gel electrophoresis, indicating that CML24 binds Ca2+ and, as a consequence, undergoes conformational changes. These characteristics may enable CML24 to interact with, and potentially regulate, targets in a Ca2+-influenced manner. Reverse transcriptase polymerase chain reaction (RT-PCR) and reporter transgenic analysis indicate that CML23 and CML24 expression occurs in all major organs. Quantitative RT-PCR (QRT-PCR) reveals CML24 transcript levels are increased from 2- to 15-fold in plants subjected to touch, darkness, heat, cold, hydrogen peroxide, abscisic acid (ABA) and indole-3-acetic acid (auxin). In addition, CML24 over- and underexpressing transgenics, cml24 point mutants, and cml23/cml24 double mutants have phenotypes which demonstrate these proteins play roles in flowering time regulation, ion sensitivity, and ABA inhibition of germination and seedling growth. Phenotype analysis also suggests CML23 and CML24 may have a role in de-etiolation (light-induced inhibition of hypocotyl elongation), chlorophyll accumulation, and response to pathogens. Furthermore, a yeast two-hybrid screen suggests CML24 interacts with an autophagy protein. Elucidating CML23 and CML24 expression patterns and physiological functions has provided additional insight into the roles of Ca2+ and Ca2+-binding proteins in plant development and response to the environment.