Detection of simple mutations and polymorphisms in large genomic regions

We have developed a novel technology that makes it possible to detect simple nucleotide polymorphisms directly within a sample of total genomic DNA. It allows, in a single Southern blot experiment, the determination of sequence identity of genomic regions with a combined length of hundreds of kilobases. This technology does not require PCR amplification of the target DNA regions, but exploits preparative size-fractionation of restriction-digested genomic DNA and a newly discovered property of the mismatch-specific endonuclease CEL I to cleave heteroduplex DNA with a very high specificity and sensitivity. We have used this technique to detect various simple mutations directly in the genomic DNA of isogenic pairs of recombinant Pseudomonas aeruginosa, Escherichia coli and Salmonella isolates. Also, by using a cosmid DNA library and genomic fractions as hybridization probes, we have compared total genomic DNA of two clinical P.aeruginosa clones isolated from the same patient, but exhibiting divergent phenotypes. The mutation scan correctly detected a GA insertion in the quorum-sensing regulator gene rhlR and, in addition, identified a novel intragenomic polymorphism in rrn operons, indicating very high stability of the bacterial genomes under natural non-mutator conditions.     

Assay of activity of a soil deaminase

Summary A simple method of assaying activity of a soil deaminase is described. The substrate used in the assay, 1,2-diamino-4-nitrobenzene, is red in colour, intensity decreasing with deamination of the benzene ring. This colour changes was used as the basis for a new assay in which substrate was incubated in a buffered soil slurry for 20 h, after which, the amount of substrate deaminated was determined. The method is simple and precise and, unlike existing soil deaminase assays, does not rely on measurement of the NH ₄ ⁺ product. After demonstration of enzyme kinetics, the assay was used to assess differences in deaminase activities in soils of differing crop-cover and N-status.     

Travellers as sentinels: Assaying the worldwide distribution of polymorphisms associated with artemisinin combination therapy resistance in Plasmodium falciparum using malaria cases imported into Scotland

There is growing evidence that Plasmodium falciparum parasites in southeastern Asia have developed resistance to artemisinin combination therapy. The resistance phenotype has recently been shown to be associated with four single nucleotide polymorphisms in the parasite’s genome. We assessed the prevalence of two of these single nucleotide polymorphisms in P. falciparum parasites imported into Scotland between 2009 and 2012, and in additional field samples from six countries in southeastern Asia. We analysed 28 samples from 11 African countries, and 25 samples from nine countries in Asia/southeastern Asia/Oceania. Single nucleotide polymorphisms associated with artemisinin combination therapy resistance were not observed outside Thailand and Cambodia.     

Molecular cloning and sequencing of the upstream region of the major Bacillus subtilis autolysin gene: a modifier protein exhibiting sequence homology to the major autolysin and the spoIID product.

The upstream region of the N-acetylmuramoyl-L-alanine amidase gene (cwlB; a major Bacillus subtilis autolysin) was cloned into Escherichia coli by chromosome walking. Sequencing of the region showed the presence of two open reading frames, one (designated as cwbA) which starts at a UUG codon and encodes a polypeptide of 705 amino acids with an M(r) of 76,725, and the other (designated as lppX), upstream of cwbA, comprising 102 amino acids and having a signal sequence characteristic of a lipoprotein. Purification of the CwbA protein and determination of its N-terminal amino acid sequence revealed that it contains a presumed signal peptide which is processed after Ala at position 25 from the N-terminal, and that the M(r) of the mature form is 75,000. The amino acid sequences of the N-terminal and C-terminal regions of CwbA were found to be highly homologous with those of the cell wall binding domain of CwlB and the spoIID gene product, respectively. CwbA stimulated the major autolysin activity approximately threefold in vitro. These data indicate that CwbA is the modifier protein of the major autolysin reported by Herbold, D. R. & Glaser, L. (1975; Journal of Biological Chemistry 250, 1676-1682). In-frame fusion between the lppX and lacZ genes demonstrated that lppX is translated in vivo and expressed during the exponential growth phase.     

Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis

Drug resistance in Mycobacterium tuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug‐resistant tuberculosis epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug‐resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug‐resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in Mycobacterium smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A‐site of 16S rRNA that: (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem‐loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug‐resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug‐resistant tuberculosis disease.     

QTL Mapping of Agronomic Waterlogging Tolerance Using Recombinant Inbred Lines Derived from Tropical Maize (Zea mays L) Germplasm

Waterlogging is an important abiotic stress constraint that causes significant yield losses in maize grown throughout south and south-east Asia due to erratic rainfall patterns. The most economic option to offset the damage caused by waterlogging is to genetically incorporate tolerance in cultivars that are grown widely in the target agro-ecologies. We assessed the genetic variation in a population of recombinant inbred lines (RILs) derived from crossing a waterlogging tolerant line (CAWL-46-3-1) to an elite but sensitive line (CML311-2-1-3) and observed significant range of variation for grain yield (GY) under waterlogging stress along with a number of other secondary traits such as brace roots (BR), chlorophyll content (SPAD), % stem and root lodging (S&RL) among the RILs. Significant positive correlation of GY with BR and SPAD and negative correlation with S&RL indicated the potential use of these secondary traits in selection indices under waterlogged conditions. RILs were genotyped with 331 polymorphic single nucleotide polymorphism (SNP) markers using KASP (Kompetitive Allele Specific PCR) Platform. QTL mapping revealed five QTL on chromosomes 1, 3, 5, 7 and 10, which together explained approximately 30% of phenotypic variance for GY based on evaluation of RIL families under waterlogged conditions, with effects ranging from 520 to 640 kg/ha for individual genomic regions. 13 QTL were identified for various secondary traits associated with waterlogging tolerance, each individually explaining from 3 to 14% of phenotypic variance. Of the 22 candidate genes with known functional domains identified within the physical intervals delimited by the flanking markers of the QTL influencing GY and other secondary traits, six have previously been demonstrated to be associated with anaerobic responses in either maize or other model species. A pair of flanking SNP markers has been identified for each of the QTL and high throughput marker assays were developed to facilitate rapid introgression of waterlogging tolerance in tropical maize breeding programs.     

Novel mutation G324C in <Emphasis Type="Italic">WNT1</Emphasis> mapped in a large Pakistani family with severe recessively inherited <Emphasis Type="Italic">Osteogenesis Imperfecta</Emphasis>

Introduction

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disease with skeletal fragility and variable extra-skeletal manifestations. To date several point mutations in 18 different genes causing different types of OI have been identified. Mutations in WNT1 compromise activity of the osteoblasts leading to disturbed bone mass accrual, fragility fractures and progressive skeletal abnormalities. The present study was conducted to determine the underlying genetic cause of an autosomal recessive skeletal dysplasia in a large consanguineous family from Chinute, Pakistan.

Materials and methods

Blood was collected from 24 individuals of affected family along with clinical data. Homozygosity mapping was performed to confirm consanguinity. SNPs were identified, followed by whole exome and Sanger sequencing. In silico characterization of WNT1 mutation was performed using multiple platforms.

Results

Nine affected family members exhibited severe bone deformities, recurrent fractures, short stature and low bone mineral density. SNP array data revealed homozygous segments >?1 Mb in length accounting for 2.1–12.7% of the genome in affected individuals and their siblings and a single 6,344,821 bp homozygous region in all affected individuals on chromosome 12q12-q13. This region includes two potential OI candidate genes WNT1 and VDR. We did whole-exome sequencing for both genes in two patients and identified a novel damaging missense mutation in exon 4 of WNT1: c.1168G?>?T (NM_005430) resulting in p.G324C. Sanger sequencing confirmed segregation of mutation with the disease in family.

Conclusion

We report a novel mutation responsible for OI and our investigation expands the spectrum of disease-causing WNT1 mutations and the resulting OI phenotypes.

     

Patterns of alien plant diversity in the urban landscapes of global biodiversity hotspots: a case study from the Himalayas

In an age of Anthropocene, the urban landscapes are recognised as the ‘hotspots’ of human-mediated alien species introductions. As the cities provide an ideal natural experimental system to investigate the patterns of alien plant diversity in urban landscapes, the present study aimed to unravel the taxonomic, biogeographic and ecological patterns of alien flora of Srinagar—one of the largest urban centres in the Himalayan biodiversity hotspot. The alien flora of Srinagar comprises 325 species, constituting ca.35% of total flora of the city. Out of the 325 alien species documented, 157 species (43%) were recorded to be under cultivation, while 168 species (57%) were growing in the wild (i.e., outside cultivation); those growing in the wild, in turn, comprised 110 cultivation escapes and 58 accidentally introduced plant species. Biogeographically, two-third of the alien plant diversity reported from Srinagar is native to Asia-Temperate. This indicates that climatic similarity between Asia-Temperate and Kashmir Himalayas facilitate in flourishing similar floristic diversity. The study highlights a relatively higher proportion of herbaceous growth form in the aliens growing in the wild (80%) than those under cultivation (43%). Similarly, 82% of the alien species under cultivation had a perennial life span, but those growing in the wild were dominated by annuals (44%). Currently, 45 species are growing as casuals and 124 species are naturalised (including 105 naturalised non-invasive and 19 naturalised invasive). Along the continuum of casual-naturalised-invasive categories, the contribution of cultivation escapes and accidently introduced aliens contrastingly shows decreasing and increasing trends respectively. Interestingly, the results revealed that the human practice of stopping cultivation of alien escapes increased rapidly as we move along the continuum. Thus, the present study has investigated the patterns of alien plant diversity in the urban landscape of Srinagar, and the results obtained offer scientific insights toward better scientific understanding and management of plant invasions in this Himalayan city, with wider policy implications for neighbouring urbanised landscapes in the Himalayas and other mountainous regions across the world.