Putative Structural Genes Research Articles

Unique regulatory network of dragon fruit simultaneously mitigates the effect of vanadium pollutant and environmental factors.

Under changing climatic conditions, plants are simultaneously facing conflicting stresses in nature. Plants can sense different stresses, induce systematic ROS signals, and regulate transcriptomic, hormonal, and stomatal responses. We performed transcriptome analysis to reveal the integrative stress response regulatory mechanism underlying heavy metal stress alone or in combination with heat and drought conditions in pitaya (dragon fruit). A total of 70 genes were identified from 31,130 transcripts with conserved differential expression. Furthermore, weighted gene co-expression network analysis (WGCNA) identified trait-associated modules. By integrating information from three modules and protein-protein interaction (PPI) networks, we identified 10 interconnected genes associated with the multifaceted defense mechanism employed by pitaya against co-occurring stresses. To further confirm the reliability of the results, we performed a comparative analysis of 350 genes identified by three trait modules and 70 conserved genes exhibiting their dynamic expression under all treatments. Differential expression pattern of genes and comparative analysis, have proven instrumental in identifying ten putative structural genes. These ten genes were annotated as PLAT/LH2, CAT, MLP, HSP, PB1, PLA, NAC, HMA, and CER1 transcription factors involved in antioxidant activity, defense response, MAPK signaling, detoxification of metals and regulating the crosstalk between the complex pathways. Predictive analysis of putative candidate genes, potentially governing single, double, and multifactorial stress response, by several signaling systems and molecular patterns. These findings represent a valuable resource for pitaya breeding programs, offering the potential to develop resilient "super pitaya" plants.

Genome-Transcriptome Transition Approaches to Characterize Anthocyanin Biosynthesis Pathway Genes in Blue, Black and Purple Wheat.

Colored wheat has gained enormous attention from the scientific community, but the information available on the anthocyanin biosynthetic genes is very minimal. The study involved their genome-wide identification, in silico characterization and differential expression analysis among purple, blue, black and white wheat lines. The recently released wheat genome mining putatively identified eight structural genes in the anthocyanin biosynthesis pathway with a total of 1194 isoforms. Genes showed distinct exon architecture, domain profile, regulatory elements, chromosome emplacement, tissue localization, phylogeny and synteny, indicative of their unique function. RNA sequencing of developing seeds from colored (black, blue and purple) and white wheats identified differential expressions in 97 isoforms. The F3H on group two chromosomes and F3'5'H on 1D chromosomes could be significant influencers in purple and blue color development, respectively. Apart from a role in anthocyanin biosynthesis, these putative structural genes also played an important role in light, drought, low temperature and other defense responses. The information can assist in targeted anthocyanin production in the wheat seed endosperm.

Identification and characterization of bacteriocin biosynthetic gene clusters found in multiple bacteriocins producing Lactiplantibacillus plantarum PUK6

The multiple bacteriocins produced by Lactiplantibacillus plantarum PUK6 isolated from misozuke-tofu (tofu pickled in miso) were identified as plantaricins A, EF, and NC8. The pln locus (21,847 bp) containing the three plantaricin structural genes and another newly found putative bacteriocin structural genes (orf1 and orf2) were determined, and a biosynthesis mechanism was proposed. Reverse transcription-PCR analysis revealed that orf1 and orf2, the putative two-peptide bacteriocin structural genes, were expressed after 8h (logarithmic growth phase) and 20h (stationary growth phase) of cultivation of the PUK6 strain. Additionally, the growth inhibition profile obtained using the chemically synthesized mature peptides of Orf1 and Orf2 (referred to as mOrf1 and mOrf2) showed that the equimolar mixture of mOrf1 and mOrf2 exhibited bactericidal effect against the indicator strain Latilactobacillus sakei subsp. sakei JCM 1157T. Furthermore, fluorescence microscopic analysis revealed disruption of the cell membranes. These findings indicate that orf1 and orf2 are structural genes encoding class IIb bacteriocins consisting of two peptides. Therefore, the novel bacteriocin encoded by plnPUK6α (orf1) and plnPUK6β (orf2) genes was named plantaricin PUK6. Since the PUK6 strain produces multiple bacteriocins, when used as a starter culture, it could contribute to improving the shelf life of fermented foods and preventing the appearance of bacteriocin-resistant bacteria.

A Rare, Virulent Clostridium perfringens Bacteriophage Susfortuna Is the First Isolated Bacteriophage in a New Viral Genus.

Background: Clostridium perfringens is a well known swine pathogen. C. perfringens type A is considered the causative agent of enteric diseases in neonatal and weaned piglets. Phage therapy using C. perfringens phages in vivo has previously proved effective. Materials and Methods: Pig fecal samples were used to isolate phages, with Clostridium perfringens type A as host. Complete genome sequencing, comparative genomics, a proteome analysis and electron microscopy were used to characterize the phage. Results: Clostridium phage Susfortuna has a double-stranded DNA genome of 19,046 bp with a G+C% content of 29.2, inverted terminal repeats and 28 predicted coding sequences (CDSs). Putative functions could not be assigned to most of the CDSs (64.3%). Transmission electron microscopy of phage Susfortuna revealed an isometric head and a short protruding tail stub resembling the structure of the Podoviridae family. A proteome analysis of phage Susfortuna identified seven structural proteins, but only one could be assigned with a putative function. Conclusions: Based on the morphology, the inverted terminal repeats and the small genome size, phage Susfortuna belongs to subfamily Picovirinae within the Podoviridae family in the order Caudovirales. Together with C. perfringens bacteriophage CPD7, phage Susfortuna represent a new genus of bacteriophages with very limited DNA sequence similarity to other known C. perfringens phages. Despite the limited DNA sequence similarity, the gene synteny among putative structural genes of phage Susfortuna is conserved among several C. perfringens bacteriophages belonging to the Podoviridae family indicating a common ancestor.

Transcriptomic analysis reveals flavonoid biosynthesis of Syringa oblata Lindl. in response to different light intensity

BackgroundHazy weather significantly increase air pollution and affect light intensity which may also affect medicinal plants growth. Syringa oblata Lindl. (S. oblata), an effective anti-biofilm medicinal plants, is also vulnerable to changes in plant photoperiods and other abiotic stress responses. Rutin, one of the flavonoids, is the main bioactive ingredient in S. oblata that inhibits Streptococcus suis biofilm formation. Thus, the present study aims to explore the biosynthesis and molecular basis of flavonoids in S. oblata in response to different light intensity.ResultsIn this study, it was shown that compared with natural (Z0) and 25% ~ 35% (Z2) light intensities, the rutin content of S. oblata under 50% ~ 60% (Z1) light intensity increased significantly. In addition, an integrated analysis of metabolome and transcriptome was performed using light intensity stress conditions from two kinds of light intensities which S. oblata was subjected to: Z0 and Z1. The results revealed that differential metabolites and genes were mainly related to the flavonoid biosynthetic pathway. We found out that 13 putative structural genes and a transcription factor bHLH were significantly up-regulated in Z1. Among them, integration analysis showed that 3 putative structural genes including 4CL1, CYP73A and CYP75B1 significantly up-regulated the rutin biosynthesis, suggesting that these putative genes may be involved in regulating the flavonoid biosynthetic pathway, thereby making them key target genes in the whole metabolic process.ConclusionsThe present study provided helpful information to search for the novel putative genes that are potential targets for S. oblata in response to light intensity.

An RNAi Screen for Genes Involved in Nanoscale Protrusion Formation on Corneal Lens in Drosophila melanogaster

The "moth-eye" structure, which is observed on the surface of corneal lens in several insects, supports anti-reflective and self-cleaning functions due to nanoscale protrusions known as corneal nipples. Although the morphology and function of the "moth-eye" structure, are relatively well studied, the mechanism of protrusion formation from cell-secreted substances is unknown. In Drosophila melanogaster, a compound eye consists of approximately 800 facets, the surface of which is formed by the corneal lens with nanoscale protrusions. In the present study, we sought to identify genes involved in "moth-eye" structure, formation in order to elucidate the developmental mechanism of the protrusions in Drosophila. We re-examined the aberrant patterns in classical glossy-eye mutants by scanning electron microscope and classified the aberrant patterns into groups. Next, we screened genes encoding putative structural cuticular proteins and genes involved in cuticular formation using eye specific RNAi silencing methods combined with the Gal4/UAS expression system. We identified 12 of 100 candidate genes, such as cuticular proteins family genes (Cuticular protein 23B and Cuticular protein 49Ah), cuticle secretion-related genes (Syntaxin 1A and Sec61 ββ subunit), ecdysone signaling and biosynthesis-related genes (Ecdysone receptor, Blimp-1, and shroud), and genes involved in cell polarity/cell architecture (Actin 5C, shotgun, armadillo, discs large1, and coracle). Although some of the genes we identified may affect corneal protrusion formation indirectly through general patterning defects in eye formation, these initial findings have encouraged us to more systematically explore the precise mechanisms underlying the formation of nanoscale protrusions in Drosophila.

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Isolation and expression analysis of four putative structural genes involved in anthocyanin biosynthesis in Begonia semperflorens

SummaryAs the major colour pigments in plants, anthocyanins play a positive photoprotective role in plant resistance to abiotic stress. In this work, variations in anthocyanin concentration were determined in the leaves of Begonia semperflorens growing under high light (HL; 900 ?mol m-2 s-1) or low temperature (LT; 15°C day/6°C night) conditions. Partial cDNA sequences of four structural genes associated with the biosynthesis of anthocyanins were isolated from leaves. These genes encoded phenylalanine ammonia-lyase (BsPAL), chalcone synthase (BsCHS), flavonone-3-hydroxylase (BsF3H), and anthocyanidin synthase (BsANS). The patterns of expression of these genes after HL or LT treatment were investigated using real-time reverse transcription-quantitative PCR (RT-qPCR). Anthocyanins, which remained at low concentrations in untreated leaves, increased significantly after HL or LT treatment.Transcript levels of all four genes corresponded, in general, with these changing patterns, but their expression profiles were significantly different between the HL and LT treatments. After LT treatment, the expression of BsPAL and BsF3H increased significantly in the first 2 d, then decreased, while transcript levels of BsCHS and BsANS increased gradually and reached their maximum level by day-6. During HL treatment, the expression of BsCHS and BsF3H responded almost immediately 0.5 d after treatment, then BsF3H expression decreased sharply to a level only slightly higher than in the untreated controls. However, expression of BsCHS remained at high levels. Expression of BsANS increased sharply 1 d after HL treatment, then decreased until day-4. Transcription of BsPAL increased rapidly 2 d after treatment, then decreased rapidly. Understanding the different regulatory mechanisms of anthocyanin biosynthesis under HL or LT stress would improve our understanding of the genetic and physiological mechanisms of adaption of B. semperflorens to abiotic stresses.

Isolation and expression analysis of six putative structural genes involved in anthocyanin biosynthesis in Tulipa fosteriana

Getting basic molecular information of pigment formation in tulip flower is a prerequisite for enhancing the range of flower colors. In this study, six cDNA clones encoding putative anthocyanin biosynthesis enzymes were isolated from petals of tulip (Tulipa fosteriana) ‘Shangnong Zaoxia’ by rapid-amplification of cDNA ends (RACE): chalcone synthase (TfCHS1), chalcone isomerase (TfCHI1), flavanone 3-hydroxylase (TfF3H1), flavonoid 3′-hydroxylase (TfF3′H1), dihydroflavonol 4-reductase (TfDFR1) and anthocyanidin synthase (TfANS1). Southern blot analysis showed they all present as multiple copies in the tulip genome. In parallel, the expression profiles of these genes in different tulip tissues as well as in petals during flower development were measured by real-time quantitative PCR. The expression levels of TfCHS1, TfCHI1 and TfF3H1 were higher in stems and leaves than those in floral tissues (stamens, pistils, petals), the expression of TfF3′H1, TfDFR1 and TfANS1 showed highest levels in petals compared with other tissues. During flower development, the transcript levels of TfCHS1 and TfF3H1 were continuously increased, which were in general constant with the changing pattern of anthocyanin accumulation; the mRNA levels of TfDFR1 and TfANS1 and anthocyanin accumulation were both significantly increased; a low level transcript of TfF3′H1 was detected in petals throughout pigmentation process; TfCHI1 expression level firstly dramatically decreased and then gently increased. These results suggest that the red pigment accumulation in petals of T. fosteriana was positively related to the expression levels of TfCHS1, TfF3H1, TfDFR1 and TfANS1.

Activation of Arabidopsis Seed Hair Development by Cotton Fiber-Related Genes

Each cotton fiber is a single-celled seed trichome or hair, and over 20,000 fibers may develop semi-synchronously on each seed. The molecular basis for seed hair development is unknown but is likely to share many similarities with leaf trichome development in Arabidopsis. Leaf trichome initiation in Arabidopsis thaliana is activated by GLABROUS1 (GL1) that is negatively regulated by TRIPTYCHON (TRY). Using laser capture microdissection and microarray analysis, we found that many putative MYB transcription factor and structural protein genes were differentially expressed in fiber and non-fiber tissues. Gossypium hirsutum MYB2 (GhMYB2), a putative GL1 homolog, and its downstream gene, GhRDL1, were highly expressed during fiber cell initiation. GhRDL1, a fiber-related gene with unknown function, was predominately localized around cell walls in stems, sepals, seed coats, and pollen grains. GFP:GhRDL1 and GhMYB2:YFP were co-localized in the nuclei of ectopic trichomes in siliques. Overexpressing GhRDL1 or GhMYB2 in A. thaliana Columbia-0 (Col-0) activated fiber-like hair production in 4–6% of seeds and had on obvious effects on trichome development in leaves or siliques. Co-overexpressing GhRDL1 and GhMYB2 in A. thaliana Col-0 plants increased hair formation in ∼8% of seeds. Overexpressing both GhRDL1 and GhMYB2 in A. thaliana Col-0 try mutant plants produced seed hair in ∼10% of seeds as well as dense trichomes inside and outside siliques, suggesting synergistic effects of GhRDL1 and GhMYB2 with try on development of trichomes inside and outside of siliques and seed hair in A. thaliana. These data suggest that a different combination of factors is required for the full development of trichomes (hairs) in leaves, siliques, and seeds. A. thaliana can be developed as a model a system for discovering additional genes that control seed hair development in general and cotton fiber in particular.

Genomic and Experimental Evidence Suggests that Verrucomicrobium spinosum Interacts with Eukaryotes.

Our knowledge of pathogens and symbionts is heavily biased toward phyla containing species that are straightforward to isolate in pure culture. Novel bacterial phyla are often represented by a handful of strains, and the number of species interacting with eukaryotes is likely underestimated. Identification of predicted pathogenesis and symbiosis determinants such as the Type III Secretion System (T3SS) in the genomes of “free-living” bacteria suggests that these microbes participate in uncharacterized interactions with eukaryotes. Our study aimed to test this hypothesis on Verrucomicrobium spinosum (phylum Verrucomicrobia) and to begin characterization of its predicted T3SS. We showed the putative T3SS structural genes to be transcriptionally active, and that expression of predicted effector proteins was toxic to yeast in an established functional screen. Our results suggest that the predicted T3SS genes of V. spinosum could encode a functional T3SS, although further work is needed to determine whether V. spinosum produces a T3SS injectisome that delivers the predicted effectors. In the absence of a known eukaryotic host, we made use of invertebrate infection models. The injection or feeding of V. spinosum to Drosophila melanogaster and Caenorhabditis elegans, respectively, was shown to result in increased mortality rates relative to controls, a phenomenon exaggerated in C. elegans mutants hypersensitive to pathogen infection. This finding, although not conclusively demonstrating pathogenesis, suggests that V. spinosum is capable of pathogenic activity toward an invertebrate host. Symbiotic interactions with a natural host provide an alternative explanation for the results seen in the invertebrate models. Further work is needed to determine whether V. spinosum can establish and maintain interactions with eukaryotic species found in its natural habitat, and whether the predicted T3SS is directly involved in pathogenic or symbiotic activity.

The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic acid, a side-chain precursor for cephem antibiotics

Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the β-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-aminopenicillanic acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins.

Genes, Enzymes, and Regulation of para -Cresol Metabolism in Geobacter metallireducens

In aerobic and facultatively anaerobic bacteria, the degradation of para-cresol (p-cresol) involves the initial hydroxylation to p-hydroxybenzyl alcohol by water catalyzed by the soluble, periplasmatic flavocytochrome p-cresol methylhydroxylase (PCMH; alpha(2)beta(2) composition). In denitrifying bacteria the further metabolism proceeds via oxidation to p-hydroxybenzoate, the formation of p-hydroxybenzoyl-coenzyme A (CoA), and the subsequent dehydroxylation of the latter to benzoyl-CoA by reduction. In contrast, the strictly anaerobic Desulfobacterium cetonicum degrades p-cresol by addition to fumarate, yielding p-hydroxybenzylsuccinate. In this work, in vitro enzyme activity measurements revealed that the obligately anaerobic Geobacter metallireducens uses the p-cresol degradation pathway of denitrifying bacteria. Surprisingly, PCMH, which is supposed to catalyze both p-cresol hydroxylation and p-hydroxybenzyl alcohol oxidation to the corresponding aldehyde, was located in the membrane fraction. The alpha subunit of the enzyme was present in two isoforms, suggesting an alphaalpha'beta(2) composition. We propose that the unusual asymmetric architecture and the membrane association of PCMH might be important for alternative electron transfer routes to either cytochrome c (in the case of p-cresol oxidation) or to menaquinone (in the case of p-hydroxybenzyl alcohol oxidation). Unusual properties of further enzymes of p-cresol metabolism, p-hydroxybenzoate-CoA ligase, and p-hydroxybenzoyl-CoA reductase were identified and are discussed. A proteomic approach identified a gene cluster comprising most of the putative structural genes for enzymes involved in p-cresol metabolism (pcm genes). Reverse transcription-PCR studies revealed a different regulation of transcription of pcm genes and the corresponding enzyme activities, suggesting the presence of posttranscriptional regulatory elements.

Reducing haziness in white wine by overexpression of Saccharomyces cerevisiae genes YOL155c and YDR055w

Grape proteins aggregate in white wine to form haze. A novel method to prevent haze in wine is the use of haze protective factors (Hpfs), specific mannoproteins from Saccharomyces cerevisiae, which reduce the particle size of the aggregated proteins. Hpf1p was isolated from white wine and Hpf2p from a synthetic grape juice fermentation. Putative structural genes, YOL155c and YDR055w, for these proteins were identified from partial amino acid sequences of Hpf1p and Hpf2p, respectively. YOL155c also has a homologue, YIL169c, in S. cerevisiae. Comparison of the partial amino acid sequence of deglycosylated-Hpf2p with the deduced protein sequence of YDR055w, confirmed five of the 15 potential N-linked glycosylation sites in this sequence were occupied. Methylation analysis of the carbohydrate moieties of Hpf2p indicated that this protein contained both N- and O-linked mannose chains. Material from fermentation supernatant of deletion strains had significantly less activity than the wild type. Moreover, YOL155c and YIL169c overexpressing strains and a strain overexpressing 6xHis-tagged Hpf2p produced greater haze protective activity than the wild type strains. A storage trial demonstrated the short to midterm stability of 6xHis-tagged Hpf2p in wine.

Molecular genetic analyses of human endogenous retroviral elements belonging to the HERV-P family in primates, human tissues, and cancer cells

Human endogenous retroviral sequences family P (HERV-P) proviral sequences have been located within the human genome. Here, we identify and analyze novel putative structural genes of HERV-P in primates, human tissues, and cancer cells with an aim toward better understanding their evolutionary relationships and transcriptional potential. The expression pattern of HERV-P structural genes indicates that they are actively amplified in human tissues and widely expressed in cancer cells, suggesting a potential role in carcinogenesis. Phylogenetic analyses suggest that the HERV-P family may be divided into two distinct categories that arose during primate evolution via active gene duplication. Taken together, our data provide a better understanding of the dynamic evolutionary features and potential functional roles of the HERV-P gene family.

BAGEL: a web-based bacteriocin genome mining tool

A common problem in the annotation of open reading frames (ORFs) is the identification of genes that are functionally similar but have limited or no sequence homology. This is particularly the case for bacteriocins, a very diverse group of antimicrobial peptides produced by bacteria and usually encoded by small, poorly conserved ORFs. ORFs surrounding bacteriocin genes are often biosynthetic genes. This information can be used to locate putative structural bacteriocin genes. Here, we describe BAGEL, a web server that identifies putative bacteriocin ORFs in a DNA sequence using novel, knowledge-based bacteriocin databases and motif databases. Many bacteriocins are encoded by small genes that are often omitted in the annotation process of bacterial genomes. Thus, we have implemented ORF detection using a number of published ORF prediction tools. In addition, BAGEL takes into account the genomic context, i.e. for each potential bacteriocin-encoding ORF, the sequence of the surrounding region on the genome is analyzed for genes that might encode proteins involved in biosynthesis, transport, regulation and/or immunity. These innovations make BAGEL unique in its ability to detect putative bacteriocin gene clusters in (new) bacterial genomes. BAGEL is freely accessible at: .

Identification and transcriptional analysis of nitrate assimilation genes in the halophilic archaeon Haloferax mediterranei

Sequencing a 6,720-bp segment of the extreme halophilic archaeon Haloferax mediterranei genome has revealed the genomic organization of the putative structural genes for nitrate assimilation. We report a gene operon containing nasABC and nasD gene. nasA encodes an assimilatory nitrate reductase, nasB codes for a membrane protein with similarity to the NarK transporter, nasC encodes a protein with similarity to MobA; and nasD codes for an assimilatory ferredoxin-dependent nitrite reductase. Reverse transcription-PCR and primer extension experiments have demonstrated the existence of one polycistronic messenger nasABC and one monocistronic nasD initiated from a different promoter. The gene order and the grouping in two adjacent transcriptional units constitutes a novel organization of nas genes. The promoter regions harbor direct palindromes reminiscent of target sites for binding of a hypotetical regulatory protein(s). Transcription of the nasABC and nasD regions was found to be repressed by the presence of ammonium as nitrogen source.

Complete nucleotide sequence of the circular megaplasmid pHCG3 of Oligotropha carboxidovorans: function in the chemolithoautotrophic utilization of CO, H 2 and CO 2

Oligotropha carboxidovorans harbors the low-copy-number, circular, 133,058-bp DNA megaplasmid pHCG3, which is essential in the chemolithoautotrophic utilization of CO (carboxidotrophy), H 2 (hydrogenotrophy) and CO 2 under aerobic conditions. The complete nucleotide sequence of pHCG3 revealed 125 open reading frames. Of these, 95 were identified as putative structural genes. The plasmid carries the four gene clusters cox (14.54 kb, 12 genes), cbb (13.33 kb, 13 genes), hox (23.35 kb, 19 genes plus one ORF) and tra/trb (25.01 kb, 22 genes plus 2 ORFs), which assemble the functions required for the utilization of CO, CO 2 or H 2, and the conjugal transfer of the plasmid, respectively. The gene clusters cox, cbb and hox form a 51.2-kb chemolithoautotrophy module. The tra/trb cluster on the plasmid pHCG3 of O. carboxidovorans has a similar architecture as the Ti-plasmid of Agrobacterium tumefaciens. The tra/trb cluster is separated from the chemolithoautotrophy module by two regions (25.2 and 29.6 kb) with miscellaneous or mostly unknown functions. These regions carry a number of single genes coding for replication and stabilization of pHCG3 as well as the components of a putative system of global regulation of plasmid replication in O. carboxidovorans. An oriV encodes the replication proteins RepABC. Sequence comparisons of pHCG3-encoded genes suggest that major genetic exchange between O. carboxidovorans and the proteobacteria has occurred.

Use of putative QTLs and structural genes in marker assisted selection for diastatic power in malting barley (Hordeum vulgare L.)

The usefulness of marker assisted selection (MAS) to improve diastatic power was demonstrated by selecting quantitative trait loci (QTLs) and structural gene alleles involved in enhanced diastatic power and activity of its component hydrolytic enzymes from Alexis, Amagi Nijo, Harrington, Haruna Nijo, and Sloop. Six unmapped breeders' populations involving these donor sources of malting quality were used for MAS. For each population, individual lines were pooled into classes separated on the basis of either the presence or absence of malting quality parent marker alleles at each of 9 identified loci (QTLs or structural genes). Diastatic power, β-amylase, and α-amylase activities were determined for each line, and used to compare alternative marker allele class means. Lines carrying malting parent marker alleles at a chromosome 5H locus abg463 were associated with 21–44% higher α-amylase activity levels, depending on the cross. The malting parent alleles at the chromosome 4H Bmy1 locus were associated with increased diastatic power and β-amylase activity. A simple PCR marker detecting the Bmy1 locus was found to be effective in screening for improved diastatic power, β-amylase activity, and thermostability. Lines carrying malting parent alleles at the chromosome 2H Bmy2 locus produced differences in diastatic power and β-amylase activity that, after adjusting for the correlated effect of malt protein, became non-significant. The Alexis allele of the chromosome 1H EBmac501 locus was associated with significant differences in all traits for a population carrying this source. The implication of these results to the improvement of diastatic power through MAS is discussed.