Gene Encoding Research Articles

OPTIMIZING THE PRODUCTION OF A FUNCTIONAL TYPE A RECOMBINANT ENDOCHITINASE FROM Trichoderma asperellum IN Escherichia coli

Chitinases from the genus Trichoderma fungi are mainly responsible for their anti-fungal activities, which allow them to become the most widely used fungal biocontrol. Therefore, several Trichoderma chitinases have been cloned and expressed to facilitate their production and applications. A previous study of the same authors has characterized an endochitinase from a relatively novel Trichoderma spp., Trichoderma asperellum. To produce this enzyme more economically and efficiently, we reported the synthesis and expression of its synthetic encoding gene in the Escherichia coli M15 strain and established the optimal conditions for preparative scale production of the enzyme in its functional form. By lowering the induction temperatures, we observed substantial improvement in the expression levels of the active enzyme. At 30 oC and 0.5 mM IPTG induction, 1 L of cells yielded approximately 80 - 100 mg of soluble protein, accounting for about 9-11 % of total soluble protein. This figure may be an underestimation of the actual yield, as deduced from the SDS-PAGE data. The recombinant enzyme can be retrieved by simple repeated freezing and thawing cycles and purified to near homogeneity using Ni-NTA chromatography. The purified enzyme showed in vitro colloidal chitin hydrolysis activity. These results could be scaled up to produce soluble 42 kDa chitinase in E. coli. The study demonstrated an economical method to produce chitinases for various agricultural and environmental applications.

Introduction of Modified BglBrick System in Lactococcus lactis for Straightforward Assembly of Multiple Gene Cassettes.

Genetic modification of lactic acid bacteria is an evolving and highly relevant field of research that allows the engineered bacteria to be equipped with the desired functions through the controlled expression of the recombinant protein. Novel genetic engineering techniques offer the advantage of being faster, easier and more efficient in incorporating modifications to the original bacterial strain. Here, we have developed a modified BglBrick system, originally introduced in Escherichia coli and optimized it for the lactic acid bacterium Lactococcus lactis. Six different expression cassettes, encoding model proteins, were assembled in different order as parts of a modified BglBrick system in a novel plasmid pNBBX. All cassettes included nisin promoter, protein encoding gene and transcription terminator. We demonstrated successful intracellular expression of the two fluorescent proteins and display of the four protein binders on the bacterial surface. These were expressed either alone or concomitantly, in combinations of three model proteins. Thus, a modified BglBrick system developed herein enables simple and modular construction of multigene plasmids and controlled simultaneous expression of three proteins in L. lactis.

Unique properties of spacer acquisition by the type III-A CRISPR-Cas system.

Type III CRISPR-Cas systems have a unique mode of interference, involving crRNA-guided recognition of nascent RNA and leading to DNA and RNA degradation. How type III systems acquire new CRISPR spacers is currently not well understood. Here, we characterize CRISPR spacer uptake by a type III-A system within its native host, Streptococcus thermophilus. Adaptation by the type II-A system in the same host provided a basis for comparison. Cas1 and Cas2 proteins were critical for type III adaptation but deletion of genes responsible for crRNA biogenesis or interference did not detectably change spacer uptake patterns, except those related to host counter-selection. Unlike the type II-A system, type III spacers are acquired in a PAM- and orientation-independent manner. Interestingly, certain regions of plasmids and the host genome were particularly well-sampled during type III-A, but not type II-A, spacer uptake. These regions included the single-stranded origins of rolling-circle replicating plasmids, rRNA and tRNA encoding gene clusters, promoter regions of expressed genes and 5′ UTR regions involved in transcription attenuation. These features share the potential to form DNA secondary structures, suggesting a preferred substrate for type III adaptation. Lastly, the type III-A system adapted to and protected host cells from lytic phage infection.

Genome-wide identification and expression analysis of CC-NB-ARC-LRR (NB-ARC) disease-resistant family members from soybean (Glycine max L.) reveal their response to biotic stress

ObjectiveUsing disease-resistant genes is the most effective strategy for protecting crops and ensuring agricultural production, or and protection against infections of different pathogens. Under biotic and abiotic stresses, NB-ARC proteins play a critical role in regulating several critical plant metabolic processes and pathways. MethodsNB-ARC identification and characterization in soybean are still in their infancy, even though R genes have been characterized by various major crop plants. NB-ARC encoding (R) genes in the soybean genome were identified and characterized in silico. ResultsThe 103 NB-ARC genes were computationally identified in the soybean genome, randomly distributed on all soybean chromosomes except 5, 10, and 17. Phylogenetic analysis classified the NB-ARC proteins into nine primary groups. However, synteny analysis results of NB-ARC genes of soybean found the best orthologous hit in the A. thaliana representing sequence conservation up to 80%. Soybean NB-ARC genes displayed a plurality of introns between one to seven among the family members. Although their genomic regions have different sizes, a relatively conserved genetic structure was observed within phylogenetic tree groups. Twenty different domains were kept in a group-specific manner, together with the presence of the NB-ARC signatory. Moreover, the transcriptome based-data expression analysis suggested that NB-ARC genes in between non-pathogens and pathogens after the inoculation of Fusarium oxysporum (biotic stress) in the soybean transcriptome, supporting the conjecture of NB-ARC genes have disease resistance functions in the soybean genome and revealing the potential involvement of these genes in the conserved pathways of the biotic-stress-response. ConclusionThis genome-wide in silico/ computational analysis will be used for accelerating NB-ARC members used for functional characterization, especially under biotic and abiotic stresses.

Clinical and genetic characteristics of Legg-Calve-Perthes disease

Legg-Calve-Perthes disease (LCPD) is a known childhood form of idiopathic femoral head osteonecrosis. It is characterized by a sequence of events involving the capital femoral epiphysis. The disease process is associated with the disruption of the blood supply to the femoral head. In most cases, LCPD appears in a sporadic form. Occurrences of cases in families have also been reported, with some families having more than two affected individuals. The disease etiology is still unknown, however, various factors have been considered for the pathogenesis of LCPD, including very low body weight or short stature at birth, maternal smoking, and secondhand smoke exposure. Interaction of multiple environmental and genetic factors has also been postulated as an underlying player in the development of the disorder. Hypercoagulability may have a major role in LCPD development. Families segregating LCPD largely demonstrate autosomal dominant inheritance. Variants in coagulations genes (Factor 5 and Factor 2) and collagen encoding gene (COL2A1) have been linked to the disease. However, our knowledge of the LCPD pathogenic factors is limited. A better understanding of the association between LCPD and causative factors, for example, the role of hypercoagulability in osteonecrosis development, might lead to the development of improved treatments, to shorten the acute phase of the disease during childhood as well as to possibly reduce the long-term effects of osteoarthritis in adulthood. Detection of large-effect variants underlying LCPD may help in offering extended screening for all first-degree family members. In this review, we would like to discuss the etiological factors underlying LCPD with special emphasis on the role of coagulation factors and mutations in the genes encoding those coagulation factors.

Genetic Polymorphism of ITGA2 C807T Collagen Receptor Encoding Gene of Aspirin Therapy among Javanese-Indonesian Healthy Respondents

BACKGROUND: Aspirin is an antiplatelet drug commonly administered as primary and secondary prophylaxis to prevent thromboembolic events. However, there has been a common incidence of aspirin resistance that leads to a recurrent cerebrovascular disease. One of the causes of such event is the genetic polymorphisms of the integrin alpha-2 (ITGA2) gene that encodes the glycoprotein Ia (GPIa) receptor in the pharmacodynamics of aspirin. AIM: This study analyzed the genetic polymorphism of ITGA2 as the GPIa collagen receptor encoding gene of aspirin therapy among healthy Javanese, the largest ethnic group in Indonesia. METHODS: This cross-sectional study involved 100 respondents who met the inclusion criteria with their blood sample taken for DNA isolation. Identification of genetic polymorphism in the target SNPs was done using the PCR-RFLP method with 5’-CCTTAAAGCTACCGGCCCATGT-3’ forward primer and 5’-TTGGCCTATTAGCACCAAAACTTACC-3’ reverse primer as well as Hpy188Irestriction enzyme to fragment the target at position 244 in the C base. RESULTS: This study found that the dominant genotype and allele were CT (51%) and C (66.5%), respectively. CONCLUSION: The allele frequency of ITGA2 gene in this study was similar to that of the populations in other Asian countries. Further research regarding the effects of ITGA2 C807T polymorphism on the pharmacodynamics of aspirin as an antiplatelet is recommended to minimize atherothrombotic events and examine its interactions as a biomarker of the risk and prognosis of some cancer types.

Glutathione Reductase Encoding Gene (gor) is Associated with Oxidative Stress and Antibiotic Susceptibility in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major causative agent of the hospital- and community-acquired infections. These infections are often antibiotic resistant and difficult to treat. Several intrinsic and acquired resistance mechanisms to antibiotics have reported in P. aeruginosa. Recently, oxidative- stress-scavenging-systems have suggested as a possible intrinsic resistance mechanism to antibiotics because oxidative stresses induced by bactericidal antibiotics contribute to bacterial killing effects. However, this remains controversial such that further clarification is required. Glutathione reductase is a key enzyme in the maintenance of the optimum level of intracellular glutathione-redox potential to ensure normal functioning of cellular processes including the detoxification of oxidative stress. In this study, the role of a glutathione-reductase-encoding gene (gor) in oxidative stress and antibiotic susceptibility was determined in P. aeruginosa. Results showed that a gor-mutant strain was more susceptible to hydrogen peroxide (but not superoxide) than the parental strain and 100% of cells were killed with 0.01% hydrogen peroxide while the parental strain survived at the same concentration of hydrogen peroxide. The gor-mutant strain was also more susceptible to carbenicillin, chloramphenicol, ciprofloxacin, and tetracycline than the parental strain, which was confirmed by bacterial killing-kinetics. These results suggest that the gor gene is associated with oxidative stress and susceptibility to bactericidal as well as bacteriostatic antibiotics and that the oxidative-stress-scavenging-systems may be a possible drug-target for multidrug resistant P. aeruginosa.

Identification of the tail assembly chaperone genes of T4-Like phages suggests a mechanism other than translational frameshifting for biogenesis of their encoded proteins

Tape measure (TM) proteins are essential for the formation of long-tailed phages. TM protein assembly into tails requires the action of tail assembly chaperones (TACs). TACs (e.g. gpG and gpT of E. coli phage lambda) are usually produced in a short (TAC-N) and long form (TAC-NC) with the latter comprised of TAC-N with an additional C-terminal domain (TAC-C). TAC-NC is generally synthesized through a ribosomal frameshifting mechanism. TAC encoding genes have never been identified in the intensively studied Escherichia coli phage T4, or any related phages. Here, we have bioinformatically identified putative TAC encoding genes in diverse T4-like phage genomes. The frameshifting mechanism for producing TAC-NC appears to be conserved in several T4-like phage groups. However, the group including phage T4 itself likely employs a different strategy whereby TAC-N and TAC-NC are encoded by separate genes (26 and 51 in phage T4).

The G505A Nonsynonymous Single-Nucleotide Polymorphism (SNP) in TAFI, the Gene Encoding Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) Is Pleiotropically Associated with TAFI Antigen Levels and Coronary Heart Disease (CHD) in Mexican Americans of South Texas

Studies have reported that the G505A nonsynonymous (ns)-SNP encoding the Ala147Thr amino acid substitution in TAFI, is associated with a reduction in risk of venous thrombosis and myocardial infarction. Interestingly, homozygosity for the A-allele (AA) of G505A is associated with increased TAFI antigen (TAFI:Ag) levels in plasma. In our study of the genetic determinants of cardiovascular disease (CVD) in Mexican Americans of South Texas, we performed an exome-wide scan in relation to plasma TAFI antigen levels in 784 individuals. While accounting for age, sex, and their interactions as confounders in linear mixed model, we found a heritability of 59% for TAFI:Ag levels (p=1.4E-19), and that ns-SNP G505A was the only variant showing exome-wide significance (p=3.5E-14; Figure A). Figure B shows the quantile-quantile distribution of the p-values from all the exome-wide tests. Clearly, the p-value distribution reveals that there is no systematic bias that may act to skew the p-values, and that the lone exception¾in agreement between observed and expected quantiles¾is due to this TAFI SNP, which strongly suggests a truly significant effect. Consistent with previous reports, the regression coefficient for G505A as a predictor of TAFI:Ag levels showed them to be increasing from the homozygous for the major G-allele (GG), to the heterozygous individuals (GA), to the individuals homozygous for the minor A-allele (AA) (Figure B). We also investigated if G505A is associated with our CHD variable. Using a statistical genetic model for the liability to disease conditional on a threshold, which is equivalent to a probit mixed model, we found that the G505A ns-SNP in TAFI, encoding TAFI Ala147Thr, is significantly associated with a reduction in the risk of CHD (p=0.002). As observed in existing literature, potential limitations of this study include the ELISA assay used for the quantification of TAFI levels. Some ELISA assays measure proTAFI, TAFIa, and TAFIi. However, recent evidence suggests that there may be cross reactivity between TAFIa and TAFIi. This can result in measuring ongoing TAFI activation peptides and elevated levels of TAFIa which ultimately goes on to make resistant fibrin. This is likely the marker that results in the increased risk of venous thromboembolism. To mitigate this confounding factor, an ELISA assay specific to measuring TAFI antigen is needed. In conclusion, we found that TAFI G505A is pleiotropically associated with TAFI:Ag levels and risk of CHD. [Display omitted] DisclosuresDeFronzo: AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Intarcia: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding.

Molecular Detection of Cellulase -Encoding Gene in Thermophilic Streptomyces and Its Cloning in Escherichia coli Origami strain

Molecular Detection of Cellulase -Encoding Gene in Thermophilic Streptomyces and Its Cloning in Escherichia coli Origami strain

Identification of Polymorphisms of the CSN2 Gene Encoding β-Casein in Greek Local Breeds of Cattle.

This e research focused on the detection and identification of genetic polymorphisms in exon 7 of the β-casein CSN2 gene in blood samples from Greek Holstein cows and from local breeds of cattle, such as Vrachykeratiki, Katerinis, and Sykias. For this purpose, DNA was isolated from 780 blood samples obtained from Greek Holstein cows, 86 from three local breeds of cattle, namely Brachyceros, Katerinis, and Sykias, and 14 from Greek buffalo. The desired region of exon 7 was amplified by PCR, resulting in 121 and 251 bp products in bovine and buffalo samples. The PCR product was digested with restriction fragment length polymorphism (RFLP) on agarose gels. The restriction enzymes DdeI and TaqI were used. All of the blood samples had the amplified size. The results showed that 74.4% of the Greek Holstein cows had the A2A2 β-casein genotype, the three native breads Vrachykeratiki had 57.7%, and the other two had 100% of the A2A2 β-casein. From the 14 Greek buffalo, 100% had the A2A2 β-casein.

Phytophthora infestans Ago1-associated miRNA promotes potato late blight disease.

Phytophthora spp. cause serious damage to plants by exploiting a large number of effector proteins and small RNAs (sRNAs). Several reports have described modulation of host RNA biogenesis and defence gene expression. Here, we analysed Phytophthora infestans Argonaute (Ago) 1 associated small RNAs during potato leaf infection. Small RNAs were co-immunoprecipitated, deep sequenced and analysed against the P. infestans and potato genomes, followed by transcript analyses and transgenic assays on a predicted target. Extensive targeting of potato and pathogen-derived sRNAs to a range of mRNAs was observed, including 638 sequences coding for resistance (R) proteins in the host genome. The single miRNA encoded by P. infestans (miR8788) was found to target a potato alpha/beta hydrolase-type encoding gene (StABH1), a protein localized to the plasma membrane. Analyses of stable transgenic potato lines harbouring overexpressed StABH1 or artificial miRNA gene constructs demonstrated the importance of StABH1 during infection by P. infestans. miR8788 knock-down strains showed reduced growth on potato, and elevated StABH1 expression levels were observed when plants were inoculated with the two knock-down strains compared to the wild-type strain 88069. The findings of our study suggest that sRNA encoded by P. infestans can affect potato mRNA, thereby expanding our knowledge of the multifaceted strategies this species uses to facilitate infection.

Cloning, Characterization, Expression Analysis, and Agglutination Studies of Novel Gene Encoding β-d-Galactose, N-Acetyl-d-Glucosamine and Lactose-Binding Lectin from Rice Bean (Vigna umbellata)

Lectins are glycoproteins and known for their peculiar carbohydrate-binding activity and their insect-pest-resistant properties. Earlier we have published our research finding on novel gene encoding Bowman-Birk type protease inhibitor with insecticidal properties from rice bean. This paper presents first report on cloning, sequencing, and expression of RbL ORF of 843bp encoding 280 amino acids long lectin precursor from rice bean (Vigna umbellata) seeds. Blast analysis revealed more than 90% similarity of RbL protein with Vigna aconitifolia and Vigna angularis lectins. Phylogenetic analysis also revealed a close relationship between RbL and other legume lectins. Sequence analysis of genomic DNA revealed intronless nature of RbL gene (GenBank accession No. MT043160). The isolated RbL ORF was expressed in E. coli BL-21(DE3) cells and maximum expression was recorded with 0.5mM IPTG after 4h incubation at 37°C. Western blotting confirmed RbL protein expression in E. coli. Recombinant protein (His6-RbL) of ~ 35kDam.wt was purified using Ni-NTA affinity chromatography to the extent of 0.26mg/ml. In silico analysis characterized RbL protein as acidic, stable, hydrophobic, and secretary protein with one signal peptide cleavage site (A26-A27) and four N-glycosylation sites. Template-based 3D model of RbL was structured using MODELLER tool and validated as good quality model. Structural analysis revealed dominance of β-pleated sheets and β-turns in RbL protein structure. β-D-galactose, N-acetyl-D-glucosamine, and lactose were predicted as putative ligands for RbL protein. Hydrogen bonding and hydrophobic forces were the major interactions between the predicted ligands and RbL protein. Agglutination and agglutination inhibition assays confirmed the binding specificity of RbL protein with the trypsinized rabbit erythrocytes and with the predicted ligands, respectively. Gene ontology analysis functionally annotated RbL protein as a plant defense protein. The novel information generated in the study is not mere pre-experimental findings but could also lay foundation for future research on exploring RbL gene and encoding protein for different biomedical and biotechnological applications.

Overexpression of the Gene Encoding Neurosecretory Protein GL Precursor Prevents Excessive Fat Accumulation in the Adipose Tissue of Mice Fed a Long-Term High-Fat Diet.

We previously identified a novel small hypothalamic protein, neurosecretory protein GL (NPGL), which induces feeding behavior and fat accumulation in rodents depending on their diet. In the present study, we explored the effects of NPGL on feeding behavior and energy metabolism in mice placed on a long-term high-fat diet with 60% calories from fat (HFD 60). Overexpression of the NPGL precursor gene (Npgl) over 18 weeks increased food intake and weight. The weekly weight gain of Npgl-overexpressing mice was higher than that of controls until 7 weeks from induction of overexpression, after which it ceased to be so. Oral glucose tolerance tests showed that Npgl overexpression maintained glucose tolerance and increased blood insulin levels, and intraperitoneal insulin tolerance tests showed that it maintained insulin sensitivity. At the experimental endpoint, Npgl overexpression was associated with increased mass of the perirenal white adipose tissue (WAT) and decreased mass of the epididymal WAT (eWAT), resulting in little effect on the total WAT mass. These results suggest that under long-term HFD 60 feeding, Npgl overexpression may play a role in avoiding metabolic disturbance both by accelerating energy storage and by suppressing excess fat accumulation in certain tissues, such as the eWAT.

Hypoxic effects on the mitochondrial content and functions of the placenta in fetal growth restriction.

In this study we examined the hypothesis that a hypoxic intrauterine environment causes mitochondrial dysfunction of trophoblasts in fetal growth restriction (FGR). The mtDNA content, mRNA levels of mitochondrial encoded genes (ND6, COX I), mitochondrial membrane proteins (COX I, COX IV and VDAC), HIF-1α and BINP3 (mitophagy receptor) protein levels were examined in FGR placentas and normal placentas. The mitochondrial function (ATP production and mitochondrial membrane potential-ΔΨm) and above related proteins were further examined in hypoxic HTR-8/SVneo cells induced by cobalt chloride (CoCl2). Mitophagy and its regulating mechanism under hypoxia in FGR was also investigated. Compared with normal controls, both FGR placentas and CoCl2-treated trophoblast cells demonstrated statistically lower mtDNA content, reduced mRNAs of mitochondrial encoding genes, and decreased mitochondrial membrane proteins, accompanied by increased HIF-1α. Mitochondrial functions were impaired as demonstrated by decreased ATP production, and, reduced ΔΨm in CoCl2-treated cells. Meanwhile, mitophagy was markedly enhanced as indicated by increased LC3 fluorescent puncta in mitochondria of hypoxic trophoblastic cells. The upregulated BINP3 expression was demonstrated in FGR placentas as well as in hypoxic trophoblastic cells. We demonstrated that hypoxic conditions lead to impaired mitochondrial function in trophoblasts in FGR. Reduced mtDNA may be associated with enhanced mitophagy via activating HIF-1α/BINP3 signalling pathway, that may, in turn, affect nutrition and energy transfer to the growth-restricted fetus.

Downregulation of the ypdA Gene Encoding an Intermediate of His-Asp Phosphorelay Signaling in Aspergillus nidulans Induces the Same Cellular Effects as the Phenylpyrrole Fungicide Fludioxonil.

Many eukaryotic histidine-to-aspartate (His-Asp) phosphorelay systems consist of three types of signal transducers: a His-kinase (HK), a response regulator (RR), and a histidine-containing phosphotransfer intermediate (HPt). In general, the HPt acts as an intermediate between the HK and the RR and is indispensable for inducing appropriate responses to environmental stresses. In a previous study, we attempted but were unable to obtain deletion mutants of the ypdA gene in order to characterize its function in the filamentous fungus Aspergillus nidulans. In the present study, we constructed the CypdA strain in which ypdA expression is conditionally regulated by the A. nidulans alcA promoter. We constructed CypdA strains with RR gene disruptions (CypdA-sskAΔ, CypdA-srrAΔ, and CypdA-sskAΔsrrAΔ). Suppression of YpdA induced by ypdA downregulation activated the downstream HogA mitogen-activated protein kinase cascade. YpdA suppression caused severe growth defects and abnormal hyphae, with features such as enhanced septation, a decrease in number of nuclei, nuclear fragmentation, and hypertrophy of vacuoles, both regulated in an SskA-dependent manner. Fludioxonil treatment caused the same cellular responses as ypdA suppression. The growth-inhibitory effects of fludioxonil and the lethality caused by ypdA downregulation may be caused by the same or similar mechanisms and to be dependent on both the SskA and SrrA pathways.

Polycyclic aromatic hydrocarbons modulate the activity of Atlantic cod (Gadus morhua) vitamin D receptor paralogs in vitro.

Vitamin D receptor (VDR) mediates the biological function of the steroid hormone calcitriol, which is the metabolically active version of vitamin D. Calcitriol is important for a wide array of physiological functions, including calcium and phosphate homeostasis. In contrast to mammals, which harbor one VDR encoding gene, teleosts possess two orthologous vdr genes encoding Vdr alpha (Vdra) and Vdr beta (Vdrb). Genome mining identified the vdra and vdrb paralogs in the Atlantic cod (Gadus morhua) genome, which were further characterized regarding their phylogeny, tissue-specific expression, and transactivational properties induced by calcitriol. In addition, a selected set of polycyclic aromatic hydrocarbons (PAHs), including naphthalene, phenanthrene, fluorene, pyrene, chrysene, benzo[a]pyrene (BaP), and 7-methylbenzo[a]pyrene, were assessed for their ability to modulate the transcriptional activity of gmVdra and gmVdrb in vitro. Both gmVdra and gmVdrb were activated by calcitriol with similar potencies, but gmVdra produced significantly higher maximal fold activation. Notably, none of the tested PAHs showed agonistic properties towards the Atlantic cod Vdrs. However, binary exposures of calcitriol together with phenanthrene, fluorene, or pyrene, antagonized the activation of gmVdra, while chrysene and BaP significantly potentiated the calcitriol-mediated activity of both receptors. Homology modeling, solvent mapping, and docking analyses complemented the experimental data, and revealed a putative secondary binding site in addition to the canonical ligand-binding pocket (LBP). Calcitriol was predicted to interact with both binding sites, whereas PAHs docked primarily to the LBP. Importantly, our in vitro data suggest that PAHs can interact with the paralogous gmVdrs and interfere with their transcriptional activities, and thus potentially modulate the vitamin D signaling pathway and contribute to adverse effects of crude oil and PAH exposures on cardiac development and bone deformities in fish.

Alpha Carbonic Anhydrase 5 Mediates Stimulation of ATP Synthesis by Bicarbonate in Isolated Arabidopsis Thylakoids

We studied bicarbonate-induced stimulation of photophosphorylation in thylakoids isolated from leaves of Arabidopsis thaliana plants. This stimulation was not observed in thylakoids of wild-type in the presence of mafenide, a soluble carbonic anhydrase inhibitor, and was absent in thylakoids of two mutant lines lacking the gene encoding alpha carbonic anhydrase 5 (αCA5). Using mass spectrometry, we revealed the presence of αCA5 in stromal thylakoid membranes of wild-type plants. A possible mechanism of the photophosphorylation stimulation by bicarbonate that involves αCA5 is proposed.

Detection of Class 1 Integron Encoding Gene in Multidrug Resistance (MDR) Citrobacter freundii Isolated from Healthy Broiler Chicken

This study was aimed to find out that broiler chicken farms have problems with antibiotic resistance Citrobacter freundii and determined the prevalence and class 1 integron encoding gene. Multidrug resistance Citrobacter freundii was collected from broiler chicken among one hundred and sixty cloacal swab samples from 32 farms in Blitar for 3 months. The method of bacterial inoculation used MacConkey agar and biochemical test was conducted by IMViC and TSIA test. Citrobacter freundii for antibiotic sensitivity pattern was tested by disk diffusion, and the multidrug resistance encoding gene was tested by PCR. This study exposed 160 samples, and 13.75% (22/160) samples were positive of Citrobacter freundii. The antibiotic sensitivity pattern showed high resistances against ampicillin and erythromycin (77.27%), tetracycline (59.09%), trimetropim-sulfamethoxazole (50%), and streptomycin (22.72%). Isolates that were detected as multidrug resistance were continued with PCR testing to prove the existence of a class 1 integron encoding gene. Multidrug resistance Citrobacter freundii isolated from broiler chicken farms in Blitar were 81.82% (18/22), and were indicated that five were positive Class 1 Integron encoding gene. The results of this study showed that the prevalence and distribution of multidrug resistance Citrobacter freundii were high, so it can cause the spread of antimicrobial resistance to public health. Class 1 integron encoding gene was found 22.72% from multidrug resistance Citrobacter freundii by PCR. It was concluded that broiler chicken farms need assessment management to reduce and avoid multidrug resistance bacteria in animals and human. Therefore, the use of appropriate antibiotics is a good step to reduce the incidence of MDR in poultry.

SDG712, a Putative H3K9-Specific Methyltransferase Encoding Gene, Delays Flowering through Repressing the Expression of Florigen Genes in Rice

SET domain group (SDG) proteins have been identified to be involved in histone modification and participate in diverse biological processes. Rice contains 41 SDG genes, however, most of which have not been functionally characterized. Here, we report the identification and functional investigation of rice SDG712 gene. Phylogenic analysis revealed that SDG712 belongs to the H3K9-specific SDG subclade. SDG712 is highly expressed in leaves during reproductive growth stage with obvious circadian rhythmic pattern. Mutation of SDG712 promotes rice flowering, while overexpression of SDG712 delays rice flowering. Gene expression analysis suggested that SDG712 acts downstream of Hd1, while acts upstream of Ehd1, Hd3a and RFT1. Subcellular localization assay demonstrated that SDG712 is localized in the nucleus. Chromatin immunoprecipitation (ChIP) assay showed that the H3K9me2 levels at Hd3a and RFT1 loci were increased in SDG712 overexpression transgenic plants, indicating that SDG712 may mediate the H3K9 di-methylation on these loci to repress rice flowering. Taken together, our findings demonstrated that SDG712 is a negative flowering regulatory gene in rice, and it delays flowering through repressing key flowering regulator gene Ehd1 and the florigen genes Hd3a and RFT1.