Capsule gene regulation in Bacillus anthracis and implications for virulence

The poly-D-glutamic acid capsule of Bacillus anthracis is considered essential for lethal anthrax disease. Yet investigations of capsule function have been limited primarily to attenuated B. anthracis strains lacking certain genetic elements. In work presented in this thesis, I constructed and characterized a genetically complete (pXO1 + pXO2+) B. anthracis strain (UT500) and isogenic mutants deleted for two previously identified capsule gene regulators, atxA and acpA, and a newly-identified regulator, acpB. Results of transcriptional analysis and microscopy revealed that atxA controls expression of the first gene of the capsule biosynthesis operon, capB, via positive transcriptional regulation of acpA and acpB. acpA and acpB appear to be partial functional homologs. Deletion of either gene alone has little effect on capsule synthesis. However, a mutant deleted for both acpA and acpB is noncapsulated. Thus, in contrast to previously published models, my results suggest that atxA is the master regulator of cap gene expression in a genetically complete strain. A detailed transcriptional analysis of capB and the regulatory genes was performed to establish the effects of the regulators and CO2/bicarbonate on specific mRNAs of target genes. CO2/bicarbonate is a well-established signal for B. anthracis capsule synthesis in culture. Taqman RT-PCR results indicated that growth in the presence of elevated CO2 greatly increased expression of acpA, acpB and capB but not atxA. 5′ end mapping of capB and acpA revealed atxA-regulated and atxA-independent transcriptional start sites for both genes. All atxA-regulated start sites were also CO2-regulated. A single atxA-independent start site was identified 5 ′ of acpB. However, RT-PCR analysis indicated that capD and acpB are co-transcribed. Thus, it is likely that atxA-mediated control of acpB expression occurs via transcriptional activation of the atxA-regulated start sites of capB. Finally, I examined the contribution of the B. anthracis capsule to virulence. The virulence of the parent strain, mutants deleted for the capsule biosynthesis genes ( capBCAD), and mutants missing the capsule regulator genes was compared using a mouse model for inhalation anthrax. The data indicate that in this model, capsule is essential for virulence. Mice survived infection with the noncapsulated capBCAD and acpA acpB mutants. These mutants initiated germination in the lung, but did not disseminate to the spleen. The acpA mutant had an LD50 value similar to the parent strain and was able to disseminate and cause lethal infection. Unexpectedly, the acpB mutant had a higher LD 50 and a reduced ability to disseminate. During in vitro culture, the acpB single mutant produces capsule and toxin similar to the parent strain. It is likely that acpB regulates the expression of downstream genes that contribute to the virulence of B. anthracis. ^

Proteomic identification and functional characterization of prohibitin 1 and 2 in T cell activation, expansion, survival and disease

Several immune pathologies are the result of aberrant regulation of T lymphocytes. Pronounced T cell proliferation can result in autoimmunity or hematologic malignancy, whereas loss of T cell activity can manifest as immunodeficiency. Thus, there is a critical need to characterize the signal transduction pathways that mediate T cell activation so that novel and rational strategies to detect and effectively control T cell mediated disease can be achieved. ^ The first objective of this dissertation was to identify and characterize novel T cell regulatory proteins that are differentially expressed upon antigen induced activation. Using a functional proteomics approach, two members of the prohibitin (Phb) family of proteins, Phb1 and Phb2, were determined to be upregulated upon activation of primary human T cells. Furthermore, their regulated expression was dependent upon CD3 and CD28 signaling pathways which synergistically increased their expression. In contrast to previous reports of Phb nuclear localization, both proteins were determined to localize to the mitochondrial inner membrane of human T cells. Additionally, novel Phb phosphorylation sites were identified and characterized using mass spectrometry, phosphospecific antibodies and site directed mutagenesis. ^ Prohibitins have been proposed to play important roles in cancer development however the mechanism of action has not been elucidated. The second objective of this dissertation was to define the functional role of Phbs in T cell activity, survival and disease. Compared to levels in normal human T cells, Phb expression was higher in the human tumor T cell line Kit225 and subcellularly localized to the mitochondrion. Ablation of Phb expression by siRNA treatment of Kit225 cells resulted in disruption of mitochondrial membrane potential and significantly enhanced their sensitivity to cell death, suggesting they serve a protective function in T cells. Furthermore, Q-RT-PCR analysis of human oncology cDNA expression libraries indicated the Phbs may represent hematological cancer biomarkers. Indeed, Phb1 and Phb2 protein levels were 6-10 fold higher in peripheral blood mononuclear cells isolated from malignant lymphoma and multiple myeloma patients compared to healthy individuals. ^ Taken together, Phb1 and Phb2 are novel phosphoproteins upregulated during T cell activation and transformation to function in the maintenance of mitochondrial integrity and perhaps energy metabolism, thus representing previously unrecognized intracellular biomarkers and therapeutic targets for regulating T cell activation and hematologic malignancies. ^

Transcriptional regulation of lung tumor suppressor GPRC5A in malignant and normal cells

Chronic exposure of the airways to cigarette smoke induces inflammatory response and genomic instability that play important roles in lung cancer development. Nuclear factor kappa B (NF-κB), the major intracellular mediator of inflammatory signals, is frequently activated in preneoplastic and malignant lung lesions. ^ Previously, we had shown that a lung tumor suppressor GPRC5A is frequently repressed in human non-small cell lung cancers (NSCLC) cells and lung tumor specimens. Recently, other groups have shown that human GPRC5A transcript levels are higher in bronchial samples of former than of current smokers. These results suggested that smoking represses GPRC5A expression and thus promotes the occurrence of lung cancer. We hypothesized that cigarette smoking or associated inflammatory response repressed GPRC5A expression through NF-κB signaling. ^ To determine the effect of inflammation, we examined GPRC5A protein expression in several lung cell lines following by TNF-α treatment. TNF-α significantly suppressed GPRC5A expression in normal small airway epithelial cells (SAEC) as well as in Calu-1 cells. Real-time PCR analysis indicated that TNF-α inhibits GPRC5A expression at the transcriptional level. NF-κB, the major downstream effectors of TNF-α signaling, mediates TNF-α-induced repression of GPRC5A because over-expression of NF-κB suppressed GPRC5A. To determine the region in the GPRC5A promoter through which NF-κB acts, we examined the ability of TNF-α to inhibit a series of reporter constructs with different deletions of GPRC5A promoter. The luciferase assay showed that the potential NF-κB binding sites containing region are irresponsible for TNF-α-induced suppression. Further analysis using constructs with different deletions in p65 revealed that NF-κB-mediated repression of GPRC5A is transcription-independent. Co-immunoprecipitation assays revealed that NF-κB could form a complex with RAR/RXR heterodimer. Moreover, the inhibitory effect of NF-κB has been found to be proportional to NF-κB/RAR ratio in luciferase assay. Finally, Chromatin IP demonstrated that NF-κB/p65 bound to GPRC5A promoter as well as RAR/RXR and suppressed transcription. Taken together, we propose that inflammation-induced NF-κB activation disrupts the RA signaling and suppresses GPRC5A expression and thus contributes to the oncogenesis of lung cancer. Our studies shed new light on the pathogenesis of lung cancer and potentially provide novel interventions for preventing and treating this disease. ^

Identification, purification, sequencing and characterization of human lung fibroblast motility -stimulating factor for human soft tissue sarcoma cells

Paracrine motogenic factors, including motility cytokines and extracellular matrix molecules secreted by normal cells, can stimulate metastatic cell invasion. For extracellular matrix molecules, both the intact molecules and the degradative products may exhibit these activities, which in some cases are not shared by the intact molecules. We found that human peritumoral and lung fibroblasts secrete motility-stimulating activity for several recently established human sarcoma cell strains. The motility of lung metastasis-derived human SYN-1 sarcoma cells was preferentially stimulated by human lung and peritumoral fibroblast motility-stimulating factors (FMSFs). FMSFs were nondialyzable, susceptible to trypsin, and sensitive to dithiothreitol. Cycloheximide inhibited accumulation of FMSF activity in conditioned medium; however, addition of cycloheximide to the migration assay did not significantly affect motility-stimulating activity. Purified hepatocyte growth factor/scatter factor (HGF/SF), rabbit anti-hHGF, and RT-PCR analysis of peritumoral and lung fibroblast HGF/SF mRNA expression indicated that FMSF activity was unrelated to HGF/SF. Partial purification of FMSF by gel exclusion chromatography revealed several peaks of activity, suggesting multiple FMSF molecules or complexes.^ We purified the fibroblast motility-stimulating factor from human lung fibroblast-conditioned medium to apparent homogeneity by sequential heparin affinity chromatography and DEAE anion exchange chromatography. Lysylendopeptidase C digestion of FMSF and sequencing of peptides purified by reverse phase HPLC after digestion identified it as an N-terminal fragment of human fibronectin. Purified FMSF stimulated predominantly chemotaxis but chemokinesis as well of SYN-1 sarcoma cells and was chemotactic for a variety of human sarcoma cells, including fibrosarcoma, leiomyosarcoma, liposarcoma, synovial sarcoma and neurofibrosarcoma cells. The motility-stimulating activity present in HLF-CM was completely eliminated by either neutralization or immunodepletion with a rabbit anti-human-fibronectin antibody, thus further confirming that the fibronectin fragment was the FMSF responsible for the motility stimulation of human soft tissue sarcoma cells. Since human soft tissue sarcomas have a distinctive hematogenous metastatic pattern (predominantly lung), FMSF may play a role in this process. ^

IL -12 inhibits the metastatic potential of osteosarcoma cells and induces tumor regression via a mechanism involving Fas /Fas ligand pathway

Despite multiple changes in the adjuvant chemotherapy regimens used to treat osteosarcoma (OS), the 2-year metastasis-free survival has remained at 65–70% for the past 10 years. Characterizing the molecular determinants that permit metastatic spread of tumor cells is a crucial element in developing new approaches for the treatment of osteosarcoma. Since OS metastasizes almost exclusively to the lung, an organ with constitutive Fas ligand (FasL) expression, we hypothesized that the expression of Fas (CD95, APO-1) by OS cells may play a role in the ability of these cells to form lung metastases. Fas expression was quantified in human SAOS-2 OS cells and selected variants (LM2, LM4, LM5, LM6, LM7). Using northern blot, FACS and RT-PCR analysis, low Fas expression was found to correlate with higher metastatic potential in these cell lines. The highly metastatic LM7 cell line was transfected with the full-length human Fas gene and injected into athymic nude mice. The median number of metastatic nodules per mouse fell from over 200 to 1.1 and the size of the nodules decreased from a range of 0.5–9.0 mm to less than 0.5 mm in the Fas-transfected cell line compared to the native LM7 cell line. Additionally, the subsequent incidence of lung metastases was lower in the Fas-expressing cell line. IL-12 was seen to upregulate Fas expression in the highly metastatic LM sublines in vitro. To visualize the effects of IL-12 in vivo, nude mice were injected with LM7 cells and treated biweekly for 4 weeks with Ad.mIL-12, saline control or Ad.βgal. Lung sections were analyzed via immunchistochemistry for Fas expression. A higher expression of Fas was found in tumors from mice receiving IL-12. To study the mechanism by which IL-12 upregulates Fas, LM7 cells were transfected with a luciferase reporter gene construct containing the full-length human fas promoter. Treatment with IL-12 increased luciferase activity. We therefore conclude that IL-12 influences the metastatic potential of OS cells by upregulating the fas promoter, resulting in increased cell surface Fas expression and susceptibility to Fas-induced cell death. ^

Genome -wide and locus -specific instability

The discovery of expanded simple repeated sequences causing or associated with human disease has lead to a new area of research involved in the elucidation of how the expanded repeat causes disease and how the repeat becomes unstable. ^ To study the genetic basis of the (CTG)n repeat instability in the DMPK gene in myotonic dystrophy (DM1) patients, somatic cell hybrids were constructed between the lymphocytes of DM1 patients and a variety of Chinese hamster ovary (CHO) cell DNA repair gene deficient mutants. By using small pool PCR (SP-PCR), the instability of the (CTG)n can be quantitated for both the frequency and sizes of length change mutations. ^ Additional SP-PCR analysis on 2/11 subclones generated from this original hybrid showed a marked increase in large repeat deletions, ∼50%. A bimodal distribution of repeats was seen around the progenitor allele and at a large deleted product (within the normal range) with no intermediate products present. ^ To determine if the repair capacity of the CHO cell led to a mutator phenotype in the hamster and hybrid clones, SP-PCR was also done on 3 hamster microsatellites in a variety of hamster cell backgrounds. No variant alleles were seen in over 2500 genome equivalents screened. ^ Human-hamster hybrids have long been shown to be chromosomally unstable, yet information about the stability of repeated sequences was not known. To test if repeat instability was associated with either intact or non-intact human chromosomes, more than 300 microsatellite repeats on 13 human chromosomes (intact and non-intact) were analyzed in eight hybrid cells. No variants were seen between the hybrid and patient alleles in the hybrids. ^ To identify whether DM1 patients have a previously undetected level of genome wide instability or if the instability is truly locus specific, SP-PCR was done on 6 human microsatellites within the patient used to make the hybrid cells. No variants were seen in over 1000 genomes screened. ^ These studies show that the somatic cell hybrid approach is a genetically stable system that allows for the determination of factors that could lead to changes in microsatellite instability. It also shows that there is something inherent about the DM1 expanded (CTG)n repeat that it is solely targeted by, as of yet, and unknown mechanism that causes the repeat to be unstable. (Abstract shortened by UMI.)^

Molecular and biochemical analyses of wounding in mesocarp of peach (Prunus persica L. Batsch) ripe fruits

The physiological and molecular responses of ripe fruit to wounding were evaluated in two peach (Prunus persica) varieties ('Glohaven', GH, melting and 'BigTop', BT, slow melting nectarine) by comparing mesocarp samples from wedges (as in minimal processing) and whole fruit as the control. Slight differences between the two varieties were detected in terms of ethylene production, whereas total phenol and flavonoid concentrations, and PPO and POD enzyme activities showed a general increase in wounded GH but not in BT. This was associated with the better appearance of the BT wedges at the end of the experimental period (72 h). Microarray (genome-wide ?PEACH3.0) analysis revealed that a total number of 2218 genes were differentially expressed (p < 0.01, log2 fold change expression ratio >1 or <-1) in GH 24 h after wounding compared to the control. This number was much lower (1208) in BT. According to the enrichment analysis, cell wall, plasma membrane, response to stress, secondary metabolic processes, oxygen binding were the GO categories over-represented among the GH up-regulated genes, whereas plasma membrane and response to endogenous stimulus were the categories over-represented among the down-regulated genes. Only 32 genes showed a common expression trend in the two varieties 24 h after wounding, whereas a total of 512 genes (with highly represented transcription factors), displayed opposite behavior. Quantitative RT-PCR analysis confirmed the microarray data for 18 out of a total of 20 genes selected. Specific WRKY, AP2/ERF and HSP20 genes were markedly up-regulated in wounded GH, indicating the activation of regulatory and signaling mechanisms probably related to different hormone categories. Compared to BT, the expression of specific genes involved in phenylpropanoid and triterpenoid biosynthetic pathways showed a more pronounced induction in GH, highlighting the difference between the two peach varieties in terms of molecular responses to wounding in the mesocarp tissue.

Seawater carbonate chemistry and processes during experiments with amphipod Gammarus locusta, 2009

We report an investigation of the effects of increases in pCO2 on the survival, growth and molecular physiology of the neritic amphipod Gammarus locusta which has a cosmopolitan distribution in estuaries. Amphipods were reared from juvenile to mature adult in laboratory microcosms at three different levels of pH in nominal range 8.1-7.6. Growth rate was estimated from weekly measures of body length. At sexual maturity the amphipods were sacrificed and assayed for changes in the expression of genes coding for a heat shock protein (hsp70 gene) and the metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase (gapdh gene). The data show that the growth and survival of this species is not significantly impacted by a decrease in sea water pH of up to 0.5 units. Quantitative real-time PCR analysis indicated that there was no significant effect of growth in acidified sea water on the sustained expression of the hsp70 gene. There was a consistent and significant increase in the expression of the gapdh gene at a pH of ~7.5 which, when combined with observations from other workers, suggests that metabolic changes may occur in response to acidification. It is concluded that sensitive assays of tissue physiology and molecular biology should be routinely employed in future studies of the impacts of sea water acidification as subtle effects on the physiology and metabolism of coastal marine species may be overlooked in conventional gross "end-point" studies of organism growth or mortality.

Estudio molecular de gluteninas de alto y bajo peso molecular en Triticum aestivum ssp. vulgare L. y su relación con la calidad panadera

El trigo blando (Triticum aestivum ssp vulgare L., AABBDD, 2n=6x=42) presenta propiedades viscoélasticas únicas debidas a la presencia en la harina de las prolaminas: gluteninas y gliadinas. Ambos tipos de proteínas forman parte de la red de gluten. Basándose en la movilidad en SDS-PAGE, las gluteninas se clasifican en dos grupos: gluteninas de alto peso molecular (HMW-GS) y gluteninas de bajo peso molecular (LMW-GS). Los genes que codifican para las HMW-GS se encuentran en tres loci del grupo 1 de cromosomas: Glu-A1, Glu-B1 y Glu-D1. Cada locus codifica para uno o dos polipéptidos o subunidades. La variación alélica de las HMW-GS es el principal determinante de de la calidad harino-panadera y ha sido ampliamente estudiado tanto a nivel de proteína como de ADN. El conocimiento de estas proteínas ha contribuido sustancialmente al progreso de los programas de mejora para la calidad del trigo. Comparadas con las HMW-GS, las LMW-GS forman una familia proteica mucho más compleja. La mayoría de los genes LMW se localizan en el grupo 1 de cromosomas en tres loci: Glu-A3, Glu-B3 y Glu-D3 que se encuentran estrechamente ligados a los loci que codifican para gliadinas. El número de copias de estos genes ha sido estimado entre 10-40 en trigo hexaploide, pero el número exacto aún se desconoce debido a la ausencia de un método eficiente para diferenciar los miembros de esta familia multigénica. La nomenclatura de los alelos LMW-GS por electroforesis convencional es complicada, y diferentes autores asignan distintos alelos a la misma variedad lo que dificulta aún más el estudio de esta compleja familia. El uso de marcadores moleculares para la discriminación de genes LMW, aunque es una tarea dificil, puede ser muy útil para los programas de mejora. El objetivo de este trabajo ha sido profundizar en la relación entre las gluteninas y la calidad panadera y desarrollar marcadores moleculares que permitan ayudar en la correcta clasificación de HMW-GS y LMW-GS. Se han obtenido dos poblaciones de líneas avanzadas F4:6 a partir de los cruzamientos entre las variedades ‘Tigre’ x ‘Gazul’ y ‘Fiel’ x ‘Taber’, seleccionándose para los análisis de calidad las líneas homogéneas para HMW-GS, LMW-GS y gliadinas. La determinación alélica de HMW-GS se llevó a cabo por SDS-PAGE, y se complementó con análisis moleculares, desarrollándose un nuevo marcador de PCR para diferenciar entre las subunidades Bx7 y Bx7*del locus Glu-B1. Resumen 2 La determinación alélica para LMW-GS se llevó a cabo mediante SDS-PAGE siguiendo distintas nomenclaturas y utilizando variedades testigo para cada alelo. El resultado no fue concluyente para el locus Glu-B3, así que se recurrió a marcadores moleculares. El ADN de los parentales y de los testigos se amplificó usando cebadores diseñados en regiones conservadas de los genes LMW y fue posteriormente analizado mediante electroforesis capilar. Los patrones de amplificación obtenidos fueron comparados entre las distintas muestras y permitieron establecer una relación con los alelos de LMW-GS. Con este método se pudo aclarar la determinación alélica de este locus para los cuatro parentales La calidad de la harina fue testada mediante porcentaje de contenido en proteína, prueba de sedimentación (SDSS) y alveógrafo de Chopin (parámetros P, L, P/L y W). Los valores fueron analizados en relación a la composición en gluteninas. Las líneas del cruzamiento ‘Fiel’ x ‘Taber’ mostraron una clara influencia del locus Glu-A3 en la variación de los valores de SDSS. Las líneas que llevaban el nuevo alelo Glu-A3b’ presentaron valores significativamente mayores que los de las líneas con el alelo Glu-A3f. En las líneas procedentes del cruzamiento ‘Tigre ’x ‘Gazul’, los loci Glu-B1 y Glu-B3 loci mostraron ambos influencia en los parámetros de calidad. Los resultados indicaron que: para los valores de SDSS y P, las líneas con las HMW-GS Bx7OE+By8 fueron significativamente mejores que las líneas con Bx17+By18; y las líneas que llevaban el alelo Glu-B3ac presentaban valores de P significativamente superiores que las líneas con el alelo Glu-B3ad y significativamente menores para los valores de L . El análisis de los valores de calidad en relación a los fragmentos LMW amplificados, reveló un efecto significativo entre dos fragmentos (2-616 y 2-636) con los valores de P. La presencia del fragmento 2-636 estaba asociada a valores de P mayores. Estos fragmentos fueron clonados y secuenciados, confirmándose que correspondían a genes del locus Glu-B3. El estudio de la secuencia reveló que la diferencia entre ambos se hallaba en algunos SNPs y en una deleción de 21 nucleótidos que en la proteína correspondería a un InDel de un heptapéptido en la región repetida de la proteína. En este trabajo, la utilización de líneas que difieren en el locus Glu-B3 ha permitido el análisis de la influencia de este locus (el peor caracterizado hasta la fecha) en la calidad panadera. Además, se ha validado el uso de marcadores moleculares en la determinación alélica de las LMW-GS y su relación con la calidad panadera. Summary 3 Bread wheat (Triticum aestivum ssp vulgare L., AABBDD, 2n=6x=42) flour has unique dough viscoelastic properties conferred by prolamins: glutenins and gliadins. Both types of proteins are cross-linked to form gluten polymers. On the basis of their mobility in SDS-PAGE, glutenins can be classified in two groups: high molecular weight glutenins (HMW-GS) and low molecular weight glutenins (LMW-GS). Genes encoding HMW-GS are located on group 1 chromosomes in three loci: Glu-A1, Glu-B1 and Glu-D1, each one encoding two polypeptides, named subunits. Allelic variation of HMW-GS is the most important determinant for bread making quality, and has been exhaustively studied at protein and DNA level. The knowledge of these proteins has substantially contributed to genetic improvement of bread quality in breeding programs. Compared to HMW-GS, LMW-GS are a much more complex family. Most genes encoded LMW-GS are located on group 1 chromosomes. Glu-A3, Glu-B3 and Glu-D3 loci are closely linked to the gliadin loci. The total gene copy number has been estimated to vary from 10–40 in hexaploid wheat. However, the exact copy number of LMW-GS genes is still unknown, mostly due to lack of efficient methods to distinguish members of this multigene family. Nomenclature of LMW-GS alleles is also unclear, and different authors can assign different alleles to the same variety increasing confusion in the study of this complex family. The use of molecular markers for the discrimination of LMW-GS genes might be very useful in breeding programs, but their wide application is not easy. The objective of this work is to gain insight into the relationship between glutenins and bread quality, and the developing of molecular markers that help in the allele classification of HMW-GS and LMW-GS. Two populations of advanced lines F4:6 were obtained from the cross ‘Tigre’ x ‘Gazul’ and ‘Fiel’ x ‘Taber’. Lines homogeneous for HMW-GS, LMW-GS and gliadins pattern were selected for quality analysis. The allele classification of HMW-GS was performed by SDS-PAGE, and then complemented by PCR analysis. A new PCR marker was developed to undoubtedly differentiate between two similar subunits from Glu-B1 locus, Bx7 and Bx7*. The allele classification of LMW-GS was initially performed by SDS-PAGE following different established nomenclatures and using standard varieties. The results were not completely concluding for Glu-B3 locus, so a molecular marker system was applied. DNA from parental lines and standard varieties was amplified using primers designed in conserved domains of LMW genes and analyzed by capillary electrophoresis. The pattern of amplification products obtained was compared among samples and related to the protein allele classification. It was possible to establish a correspondence between specific amplification products and almost all LMW alleles analyzed. With this method, the allele classification of the four parental lines was clarified. Flour quality of F4:6 advanced lines were tested by protein content, sedimentation test (SDSS) and alveograph (P, L, P/L and W). The values were analyzed in relation to the lines prolamin composition. In the ‘Fiel’ x ‘Taber’ population, Glu-A3 locus showed an influence in SDSS values. Lines carrying new allele Glu-A3b’, presented a significantly higher SDSS value than lines with Glu-A3f allele. In the ‘Tigre ’x ‘Gazul’ population, the Glu-B1 and Glu-B3 loci also showed an effect in quality parameters, in SDSS, and P and L values. Results indicated that: for SDSS and P, lines with Bx7OE+By8 were significantly better than lines with Bx17+By18; lines carrying Glu-B3ac allele had a significantly higher P values than Glu-B3ad allele values. lines with and lower L The analysis of quality parameters and amplified LMW fragments revealed a significant influence of two peaks (2-616 y 2-636) in P values. The presence of 2-636 peak gave higher P values than 2-616. These fragments had been cloned and sequenced and identified as Glu-B3 genes. The sequence analysis revealed that the molecular difference between them was some SNPs and a small deletion of 21 nucleotides that in the protein would produce an InDel of a heptapeptide in the repetitive region. In this work, the analysis of two crosses with differences in Glu-3 composition has made possible to study the influence of LMG-GS in quality parameters. Specifically, the influence of Glu-B3, the most interesting and less studied loci has been possible. The results have shown that Glu-B3 allele composition influences the alveograph parameter P (tenacity). The existence of different molecular variants of Glu-B3 alleles have been assessed by using a molecular marker method. This work supports the use of molecular approaches in the study of the very complex LMW-GS family, and validates their application in the analysis of advanced recombinant lines for quality studies.

Functional identification and reconstitution of an odorant receptor in single olfactory neurons

The olfactory system is remarkable in its capacity to discriminate a wide range of odorants through a series of transduction events initiated in olfactory receptor neurons. Each olfactory neuron is expected to express only a single odorant receptor gene that belongs to the G protein coupled receptor family. The ligand–receptor interaction, however, has not been clearly characterized. This study demonstrates the functional identification of olfactory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca2+-imaging and reverse transcription–coupled PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printed olfactory neuron that displayed odorant-induced Ca2+ increase. Next, recombinant adenovirus-mediated expression of the isolated receptor gene was established in the olfactory epithelium by using green fluorescent protein as a marker. The infected neurons elicited external Ca2+ entry when exposed to the odorant that originally was used to identify the receptor gene. Experiments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. The odorant receptor-mediated signal transduction appears to be reconstituted by this two-step approach: the receptor screening for given odorant(s) from single neurons and the functional expression of the receptor via recombinant adenovirus. The present approach should enable us to examine not only ligand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest.

Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae

A quantitative and selective genetic assay was developed to monitor expansions of trinucleotide repeats (TNRs) in yeast. A promoter containing 25 repeats allows expression of a URA3 reporter gene and yields sensitivity to the drug 5-fluoroorotic acid. Expansion of the TNR to 30 or more repeats turns off URA3 and provides drug resistance. When integrated at either of two chromosomal loci, expansion rates were 1 × 10−5 to 4 × 10−5 per generation if CTG repeats were replicated on the lagging daughter strand. PCR analysis indicated that 5–28 additional repeats were present in 95% of the expanded alleles. No significant changes in CTG expansion rates occurred in strains deficient in the mismatch repair gene MSH2 or the recombination gene RAD52. The frequent nature of CTG expansions suggests that the threshold number for this repeat is below 25 in this system. In contrast, expansions of the complementary repeat CAG occurred at 500- to 1,000-fold lower rates, similar to a randomized (C,A,G) control sequence. When the reporter plasmid was inverted within the chromosome, switching the leading and lagging strands of replication, frequent expansions were observed only when CTG repeats resided on the lagging daughter strand. Among the rare CAG expansions, the largest gain in tract size was 38 repeats. The control repeats CTA and TAG showed no detectable rate of expansions. The orientation-dependence and sequence-specificity data support the model that expansions of CTG and CAG tracts result from aberrant DNA replication via hairpin-containing Okazaki fragments.

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease α-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of α-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH2, RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.

The active digestion of uniparental chloroplast DNA in a single zygote of Chlamydomonas reinhardtii is revealed by using the optical tweezer

The non-Mendelian inheritance of organelle genes is a phenomenon common to almost all eukaryotes, and in the isogamous alga Chlamydomonas reinhardtii, chloroplast (cp) genes are transmitted from the mating type positive (mt+) parent. In this study, the preferential disappearance of the fluorescent cp nucleoids of the mating type negative (mt−) parent was observed in living young zygotes. To study the change in cpDNA molecules during the preferential disappearance, the cpDNA of mt+ or mt− origin was labeled separately with bacterial aadA gene sequences. Then, a single zygote with or without cp nucleoids was isolated under direct observation by using optical tweezers and investigated by nested PCR analysis of the aadA sequences. This demonstrated that cpDNA molecules are digested completely during the preferential disappearance of mt− cp nucleoids within 10 min, whereas mt+ cpDNA and mitochondrial DNA are protected from the digestion. These results indicate that the non-Mendelian transmission pattern of organelle genes is determined immediately after zygote formation.

Short-term correction of factor VIII deficiency in a murine model of hemophilia A after delivery of adenovirus murine factor VIII in utero

Development of in utero gene transfer approaches may provide therapies for genetic disorders with perinatal morbidity. In hemophilia A, prenatal and postnatal bleeding may be catastrophic, and modest increments in factor VIII (FVIII) activity are therapeutic. We performed transuterine i.p. gene transfer at day 15 of gestation in a murine model of hemophilia A. Normal, carrier (XHX), and FVIII-deficient (XHY and XHXH) fetuses injected with adenoviral vectors carrying luciferase or β-galactosidase reporter genes showed high-level gene expression with 91% fetal survival. The live-born rates of normal and FVIII-deficient animals injected in utero with adenovirus murine FVIII (3.3 × 105 plaque-forming units) was 87%. FVIII activity in plasma was 50.7 ± 10.5% of normal levels at day 2 of life, 7.2 ± 2.2% by day 15 of life, and no longer detectable at day 21 of life in hemophilic animals. Injection of higher doses of murine FVIII adenovirus at embryonic day 15 produced supranormal levels of FVIII activity in the neonatal period. PCR analysis identified viral genomes primarily in the liver, intestine, and spleen, although adenoviral DNA was detected in distal tissues when higher doses of adenovirus were administered. These studies show that transuterine i.p. injection of adenoviral vectors produces therapeutic levels of circulating FVIII throughout the neonatal period. The future development of efficient and persisting vectors that produce long-term gene expression may allow for in utero correction of genetic diseases originating in the fetal liver, hematopoietic stem cells, as well as other tissues.

Overexpression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain

Biochemically active wheat thioredoxin h has been overexpressed in the endosperm of transgenic barley grain. Two DNA constructs containing the wheat thioredoxin h gene (wtrxh) were used for transformation; each contained wtrxh fused to an endosperm-specific B1-hordein promoter either with or without a signal peptide sequence for targeting to the protein body. Twenty-two stable, independently transformed regenerable lines were obtained by selecting with the herbicide bialaphos to test for the presence of the bar herbicide resistance gene on a cotransformed plasmid; all were positive for this gene. The presence of wtrxh was confirmed in 20 lines by PCR analysis, and the identity and level of expression of wheat thioredoxin h was assessed by immunoblots. Although levels varied among the different transgenic events, wheat thioredoxin h was consistently highly expressed (up to 30-fold) in the transgenic grain. Transgenic lines transformed with the B1-hordein promoter with a signal peptide sequence produced a higher level of wheat thioredoxin h on average than those without a signal sequence. The overexpression of thioredoxin h in the endosperm of germinated grain effected up to a 4-fold increase in the activity of the starch debranching enzyme, pullulanase (limit dextrinase), the enzyme that specifically cleaves α-1,6 linkages in starch. These results raise the question of how thioredoxin h enhances the activity of pullulanase because it was found that the inhibitor had become inactive before the enzyme showed appreciable activity.