Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein from Plantago ovata Forsk

Plantago ovata Forsk is a medicinally important plant. Metallothioneins are cysteine rich proteins involved in the detoxification of heavy metals. Molecular cloning and modeling of MT from P. ovata is not reported yet. The present investigation will describe the isolation, structure prediction, characterization, and expression under copper stress of type 2 metallothionein (MT2) from this species. The gene of the protein comprises three exons and two introns. The deduced protein sequence contains 81 amino acids with a calculated molecular weight of about 8.1 kDa and a theoretical pI value of 4.77. The transcript level of this protein was increased in response to copper stress. Homology modeling was used to construct a three-dimensional structure of P. ovata MT2. The 3D structure model of P. ovata MT2 will provide a significant clue for further structural and functional study of this protein.

Identification of Prophages and Prophage Remnants within the Genome of Avibacterium paragallinarum Bacterium

Bacterial whole genome sequencing has delivered an abundance of prophage sequences as a by-product and the analysis of these sequences revealed ways in which phages have affected the genome of their host bacteria in various bacterial species. The aim of this study was to identify the phage-related sequences in the draft assembly of the Avibacterium paragallinarum genome, the causative agent of infectious coryza in poultry. Whole genome assembly was not possible due to the presence of gaps and/or repeats existent on the ends of contigs. However, genome annotation revealed prophage and prophage remnant sequences present in this genome. From the results obtained, a complete Mu-like bacteriophage could be identified that was termed AvpmuC-2M. A complete sequence of HP2-like bacteriophage, named AvpC-2M-HP2, was also identified.

Cost-Effective Paramagnetic Bead Technique for Purification of Cycle Sequencing Products

The quality of sequencing results depends greatly upon the quality and purity of the template as well as the purity of the fluorescently labeled products generated by cycle sequencing. Numerous approaches have been used for purification of cycle sequencing products, including alcohol precipitation, affinity-based chromatography, size exclusion chromatography, commercially-available proprietary methods, and paramagnetic bead technology. In this paper, we describe an affordable paramagnetic technology method using BioMag Carboxyl beads. Compared to other well-established, proprietary methods for purification of cycle sequencing products, this method produced consistently good results, with a very low reagent cost and short procedure time.

Molecular Cloning, Sequencing, and Characterization of a Putative Acetyl-CoA-C-acetyltransferase cDNA from a Highly Fragrant Orchid Hybrid Vanda Mimi Palmer

Vanda Mimi Palmer, a hybrid of Vanda Tan Chay Yan and Vanda tessellata (Roxb.) Hk.f. ex G. Don, is cultivated as a potted ornamental plant mainly for its fragrance rather than its look. Plant acetyl-CoA-C-acetyltransferase (ACA) is involved in the condensation of two acetyl-CoAs to form acetoacetyl-CoA, which condenses with another acetyl-CoA to yield a crucial molecule, 3-hydroxyl-3-methylglutaryl-CoA, at the initial step of the mevalonate (MVA) pathway. An ACA gene from vandaceous orchid has never been reported. We describe the isolation and molecular characterization of an ACA-like gene from V. Mimi Palmer (designated as VMPACA) to facilitate a better understanding of the terpenoid biosynthesis pathway in orchids. The deduced VMPACA encodes a 376-amino-acid protein with a molecular weight of 39 kDa, which comprises an open reading frame of 1128 bp. It is flanked by 87 bp of 5′-untranslated region and 174 bp of 3′-untranslated region including a poly-A tail. Its protein sequence is 81% identical to other plant ACAs and contains a thiolase active site. The fluctuation expression pattern of VMPACA transcript by real-time RT-PCR showed that it is developmentally and temporally regulated with predominant expression in outer and lateral inner tepals compared to vegetative tissues.

Molecular Diversity of miR390-Guided Transacting siRNA Precursor Genes in Lower Land Plants: Experimental Approach and Bioinformatics Analysis

Transacting siRNA loci (TAS3-like) of a particular plant species are usually represented by several gene families. PCR-based approach was used as a phylogenetic profiling tool to probe genomic DNA samples from representatives of evolutionary distant Bryophyta taxa, namely, class Bryopsida (subclasses Bryidae and Dicranidae) and class Sphagnopsida. We found relatives of all four Physcomitrella patens (subclass Funariidae) TAS3-like loci in subclasses Bryidae and Dicranidae. Only representatives of subclass Bryidae encoded TAS3-like genes belonging to P. patens TAS3a and TAS3d families. On the other hand, only the members of order Grimmiales (subclass Dicranidae) encoded gene relatives of P. patens TAS3c family. These data indicate that moss ta-siRNA families have been long conserved during land plant evolution. However, P. patens TAS3-like loci were detected neither in two Sphagnum species from the earliest diverged moss class Sphagnopsida, nor in the Selaginella kraussiana from the earliest extant tracheophyta lineage, Lycopodiopsida.

Gene Expression in Leaves of Susceptible Glycine max during Infection with Phakopsora pachyrhizi Using Next Generation Sequencing

Soybean rust is caused by the obligate biotrophic fungus Phakopsora pachyrhizi, an exotic pathogen causing important yield losses in soybean production. We used an mRNA-Seq strategy to analyze the expression pattern of soybean genes and better understand molecular events occurring in soybean following the infection. cDNA libraries were constructed from RNA isolated from whole infected soybean leaves 10 days after inoculation with P. pachyrhizi and sequenced using an Illumina platform to identify soybean genes that are affected by pathogen growth. We obtained 15 million sequences corresponding to soybean genes. Forty-two percent of the genes were downregulated including genes encoding proteins involved in amino acid metabolism, carbohydrate metabolism, and transport facilitation; 31% were upregulated including genes encoding proteins involved in lipid metabolism, glycan biosynthesis, and signal transduction. Candidate host genes identified in this study will be manipulated to assay their potential to control soybean rust disease.

KRAS Codons 12 and 13 Mutation Analysis: A Comparative Study between Direct Sequencing and a New Sensitive Real-Time PCR Assay

KRAS somatic mutations are found in 30–40% of colorectal cancer (CRC). Seven mutations in codons 12 and 13 of KRAS (95% of the observed human mutations) preclude the efficacy of anti-EGFR therapy for the treatment of CRC. Assessment of KRAS mutational status has become a standard procedure in the management of patients with CRC. Technically, KRAS mutation testing can be performed with different methods, characterized by distinct sensitivities and specificities. The present study analyzed KRAS in 182 CRC histological samples by using direct sequencing and a new kit based on a Real-Time Sequence-Specific Primers-PCR technology. The kit allowed to recover as positive 17 samples that were negative or unclear by sequencing, with a recovery rate equal to 13.82%. This study proposes a fast, sensitive, and high-throughput system to identify such seven described mutations of KRAS gene in CRC samples.

Complete Genome Sequence of Rothia mucilaginosa DY-18: A Clinical Isolate with Dense Meshwork-Like Structures from a Persistent Apical Periodontitis Lesion

Rothia mucilaginosa is an opportunistic pathogen in the human oral cavity and pharynx. We found that R. mucilaginosa DY-18, a clinical isolate from a persistent apical periodontitis lesion, had biofilm-like structures. Similar structures were also observed on R. mucilaginosa ATCC25296. To further study these structures, we determined the complete genome sequence of DY-18 and found it a 2.26-Mb chromosome. Regarding stress responsive systems known to affect biofilm formation in many bacteria, DY-18 genome possessed only two sigma factor genes. One of these encoded an additional sigma factor whose promoter-binding activity may be regulated in response to environmental stimuli. Additionally, several genes assigned to two-component signal transduction systems were presented in this genome. To the best of our knowledge, this is the first complete genome of R. mucilaginosa species and our data raise the possibility that this organism regulates the biofilm phenotype through these stress responsive systems.

Increasing Prevalence of Unique Mutation Patterns in H5N1 Avian Influenza Virus HA and NA Glycoproteins from Human Infections in Egypt

Highly pathogenic avian influenza H5N1 virus (HPAIV) continues to be a candidate of a further influenza virus pandemic. Egypt is the country worst affected by human cases of HPAIV H5N1 infection in 2009. Increased infection of preschool children and decreased mortality rates suggested subtle changes in the epidemiology of the infection. Among other factors, the evolution of several conspicuous viral genetic markers in the HA and NA genes of HPAIV H5N1 viruses of human cases from Egypt and their putative influence on biological virus characteristics described here may contribute to this situation.

Identification and Quantification of Genomic Repeats and Sample Contamination in Assemblies of 454 Pyrosequencing Reads

Contigs assembled from 454 reads from bacterial genomes demonstrate a range of read depths, with a number of contigs having a depth that is far higher than can be expected. For reference genome sequence datasets, there exists a high correlation between the contig specific read depth and the number of copies present in the genome. We developed a sequence of applied statistical analyses, which suggest that the number of copies present can be reliably estimated based on the read depth distribution in de novo genome assemblies. Read depths of contigs of de novo cyanobacterial genome assemblies were determined, and several high read depth contigs were identified. These contigs were shown to mainly contain genes that are known to be present in multiple copies in bacterial genomes. For these assemblies, a correlation between read depth and copy number was experimentally demonstrated using real-time PCR. Copy number estimates, obtained using the statistical analysis developed in this work, are presented. Per-contig read depth analysis of assemblies based on 454 reads therefore enables de novo detection of genomic repeats and estimation of the copy number of these repeats. Additionally, our analysis efficiently identified contigs stemming from sample contamination, allowing for their removal from the assembly.