Rapid and Efficient Protocols for Throughput Extraction of High Quality Plasmid DNA from Strains of Xanthomonas axonopodis pv malvacearum and Escherichia coli

Efficient protocols developed to isolate low copy plasmid DNA from Xanthomonas axonopodis pv malvacearum (Xam) and high copy recombinant plasmid DNA from Escherichia coli are described. The protocol for extraction of low copy plasmid DNA from strains of Xam yielded high concentrations of plasmid DNA and used easily available and inexpensive chemicals in simple steps. The protocol for plasmid extraction from E. coli was rapid, cost-effective and yet yielded high concentrations of plasmid DNA. The procedures are simple and can be used to process several samples at one time. The plasmid DNA extracted by two methods was sufficiently pure, free from protein and other cellular contaminants and amenable to various molecular manipulations.     

Characterization of Variability in Some Pathogenic Species of Alternaria Based on the Nucleotide Sequence of Ribosomal DNA

The internal transcribed spacer (ITS) sequences within the ribosomal DNA (rDNA) region were targeted to delineate genetic variability among eight Alternaria species that cause economically important diseases in crops. The rDNA regions of Alternaria species comprising of rRNA genes and the ITS regions were cloned and sequenced. Phylogenetic relationship based on the rDNA sequences and PCR-RFLP of amplified rDNA sequences clustered eight species of Alternaria into three major groups. A. macrospora and A. helianthi accumulated wide genetic variations and are distantly related to rest of the six species which formed two major groups. Group I comprised of three species viz., A. dianthicola, A. brassicae and A. citri, while group II had A. longipes, A. porri and A. alternata. Incorporation of unique stretches of nucleotides and single nucleotide substitutions within relatively conserved ITS1 and ITS2 regions led to clustering of the members of Alternaria species in each group. The divergent sequences within the ITS regions can be employed to design species-specific PCR primer for use in molecular diagnostics.     

Real-time PCR assay for rapid,efficient and accurate detection of <Emphasis Type="Italic">Paramyrothecium roridum</Emphasis> a leaf spot pathogen of <Emphasis Type="Italic">Gossypium</Emphasis> species

Here we report a highly sensitive real-time PCR (qPCR) assay to detect Paramyrothecium roridum from pure culture and infected samples of cotton plants. A specific set of primer pair pMyro F/R is designed to target the 185 bp ITS region of rDNA of Paramyrothecium roridum species and validated using qPCR. The fluorescence signals were detected above the baseline threshold from samples containing Paramyrothecium roridum DNA, whereas other samples did not produce any fluorescence or produced fluorescence which did not reach detection threshold values. A single dissociation peak of increased fluorescence was obtained for the specific primers at 92.2 °C melting temperature. The limit of detection using SYBR Green dye in this assay was up to 0.1 pg per µL of DNA from pure culture of P. roridum. The assay is accurate, sensitive, less laborious and time saving for detection of P. roridum in infected tissues of cotton.     

Sequence analysis of coat protein and molecular profiling of sunflower necrosis virus (SNV) strains from Indian subcontinent

Sunflower is one of the leading edible oilseed crops of the world and is an important oil-producing crop of India. The sunflower necrosis disease caused by sunflower necrosis virus (SNV) has become a major hurdle for cultivation of sunflower in India. However, there is lack of genetic information and  standard methods for detection and identification of the SNV. To address this issue, we have developed an application using coat protein (CP) to perform molecular profiling of SNV strains. The nucleic acid and amino acid sequence analysis of CP of SNV strains collected from different regions of Maharashtra and Karnataka showed high percent homology (96.89–98.87%). However, 3D structural analysis generated eleven distinct groups of SNV strains.  Comparative bioinformatic analyses of nucleic acid and amino acid sequences with different genera of positive stranded (+) ssRNA viruses established their phylogentic relationship with ~25 (+) ssRNA viruses viz., Ilarvirus, Bromovirus, Cucumovirus, Alfamovirus, Comovirus, Nepovirus, Sequivirus, Potyvirus and Closterovirus. Additionally, the phylogenetic analysis revealed three distinct clusters, wherein major cluster I comprised SNV strains and Tobacco streak virus together showing 99% sequence homology and established closer phylogenetic relationship among all member viruses.     

Identification of an Arabidopsis locus required for resistance to turnip crinkle virus

Inoculation of turnip crinkle virus (TCV) into a (TCV)-resistant line of Arabidopsis thaliana , Di-17, results in the development of a hypersensitive response (HR) on the inoculated leaves. In contrast, an HR does not occur when leaves of the TCV-susceptible Di-3 line or the susceptible ecotypes Columbia (Col-0), or Landsberg erecta ( Ler ) are inoculated. Genetic analysis of progeny from crosses between Di-17 and either Di-3, Col-0 or Ler demonstrates that the development of an HR is regulated by a single dominant nuclear locus, herein designated HRT . Using progeny from a Di-17 X Col-0 cross, HRT was mapped to chromosome 5, where it is tightly linked to the DFR locus. We also demonstrate that a variety of resistance-associated phenomena, including the TCV-induced accumulation of salicylic acid, camalexin and autofluorescent cell-wall material, correlate with the HR, suggesting the possibility that HRT is required for their activation.     

Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N₂ fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.     

Escherichia coli populations in unpredictably fluctuating environments evolve to face novel stresses through enhanced efflux activity

There is considerable understanding about how laboratory populations respond to predictable (constant or deteriorating environment) selection for single environmental variables such as temperature or pH. However, such insights may not apply when selection environments comprise multiple variables that fluctuate unpredictably, as is common in nature. To address this issue, we grew replicate laboratory populations of Escherichia coli in nutrient broth whose pH and concentrations of salt (NaCl) and hydrogen peroxide (H₂O₂) were randomly changed daily. After ~170 generations, the fitness of the selected populations had not increased in any of the three selection environments. However, these selected populations had significantly greater fitness in four novel environments which have no known fitness‐correlation with tolerance to pH, NaCl or H₂O₂. Interestingly, contrary to expectations, hypermutators did not evolve. Instead, the selected populations evolved an increased ability for energy‐dependent efflux activity that might enable them to throw out toxins, including antibiotics, from the cell at a faster rate. This provides an alternate mechanism for how evolvability can evolve in bacteria and potentially lead to broad‐spectrum antibiotic resistance, even in the absence of prior antibiotic exposure. Given that environmental variability is increasing in nature, this might have serious consequences for public health.     

Distribution of CC-chemokine receptor-5-?32 allele among the tribal and caste population of Vidarbha region of Maharashtra state

BACKGROUND:

Genetic relationships among the ethnic groups are not uniform across the geographical region. Considering this assumption, we analyzed the frequency of the CC-chemokine receptor-5 (CCR5)-∆32 allele of the CCR5 chemokine receptor, which is considered a Caucasian marker, in Bhil tribal and Brahmin caste sample sets from the population.

MATERIALS AND METHODS:

108 blood samples were collected from 6 tribe''s populations and a caste population from the district of Vidarbha region.

RESULTS AND DISCUSSION:

The presence of low frequencies of CCR5-Δ32 in an individual of Bhil tribe (0.034, χ² value 0.017) in the present study implies that these communities may have a better resistance toward human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) than the other studied tribe sample, as non-show such mutation.

CONCLUSION:

The marginal presence of the allele seen in the studied tribal population could be due to gene flow from the people of European descent. However, lack of the homozygous CCR5-Δ32 mutation and the low prevalence of heterozygous CCR5-Δ32 mutations suggest that the Indians are highly susceptible to HIV/AIDS, and this correlates with the highest number of HIV/AIDS infected individuals in India.