Hybridization Of Genomic DNA Research Articles

Hereditary Colorectal Cancer Diagnosis by Next-Generation Sequencing.

Pathogenic germline variants causally contribute to the etiology of colorectal cancer (CRC) and polyposis. The era of massively parallel sequencing, also known as next-generation sequencing (NGS), make it highly possible, effective, and efficient to offer rapid and cost-effective diagnosis for CRC. To aid clinical laboratories in testing the most clinically significant genes, along with the published ACMG CRC technical standard guidelines, this protocol aims to provide a step-by-step technical workflow for carrying out the NGS-panel based CRC molecular diagnosis focusing on the wet lab portion of library preparation and massively parallel sequencing. Using the most popular pull-down-based target enrichment, the chapter particularly encompasses genomic DNA (gDNA) fragmentation, adapter ligation, indexing, hybridization, and capture, which is the most variable and technically challenging part of NGS testing involving at least 3 quality control (QC) checkpoints plus the pre- and post-capture PCR. The gDNA extraction and sequencing is less covered because they are relatively standard technologies with little variations and choices. Although this protocol also introduces pertinent testing algorithms and a brief guideline for pre- and post-testing genetic counselling, the audiences are required to refer to National Comprehensive Cancer Network (NCCN) clinical practice guidelines to determine the most appropriate testing strategies. Since NGS panel-based testing is a highly complex and dynamic platform with multiple choices from different technology and commercial resources, this technical benchtop-based protocol also aims to cover some of the key ramification points for decision-making by each laboratory at the discretion of the directors. © 2023 Wiley Periodicals LLC. Basic Protocol: Hereditary colorectal cancer (CRC) diagnosis by next-generation sequencing.

A novel approach to improving hybrid capture sequencing targeting efficiency

At present, approaches to hybrid capture sequencing have many limitations, from their significant complexity and labor requirements, to their low enrichment efficiency, resulting in their limited utilization. In an effort to overcome these drawbacks, we have developed a novel method that relied upon direct genomic DNA hybridization and single-stranded DNA library preparation. Using this novel protocol, we were able to achieve a targeting efficiency as high as 75%, and we found this approach to overall be an efficient and simple approach to DNA library construction.

Prevalence and molecular characteristics of mcr-1 gene in Salmonella typhimurium in a tertiary hospital of Zhejiang Province.

Objectivesmcr-1 gene has been widely reported in the world. This study aimed to analyze the prevalence and molecular characteristics of mcr-1 gene in Salmonella typhimurium from Quzhou People’s Hospital.Materials and methodsA total of 62 S. typhimurium isolates were isolated and preserved in our laboratory from 2007 to 2016. PCR was used to screen plasmid-mediated colistin resistance gene, mcr-1. For mcr-1-positive isolates, susceptibilities to colistin and other antibiotics were assessed using broth microdilution or agar dilution methods. The genetic location of mcr-1 was determined by analysis of pulsed-field gel electrophoresis profiles of S1-digested genomic DNA and subsequent Southern blot hybridization. The multi-locus sequence type and other drug resistance genes found in the mcr-1-positive isolates were analyzed by performing whole genome sequencing. Genetic environment of mcr-1 gene was analyzed by RAST and Easyfig.ResultsA total of three S. typhimurium isolates were identified to be mcr-1 positive, with the prevalence rate of 4.8% (3/62). The minimum inhibitory concentration values of colistin for all these isolates were 8 µg/mL. The three mcr-1-positive isolates carried mcr-1 gene on two different types of plasmids having the sizes of ~54.7–78.2 kb and 310.1 kb, respectively. All the three isolates belonged to ST34 and carried various resistant genes.ConclusionColistin-resistant, mcr-1-positive S. typhimurium isolates belonging to ST34 have been isolated from Quzhou People’s Hospital. Surveillance needs to be strengthened to identify colistin resistance and prevent the spread of drug-resistant bacteria in the hospital.

Development of direct assays for Toxoplasma gondii and its use in genomic DNA sample

This work describes an approach for the selection and detection of specific DNA probes related to Toxoplasma gondii, a protozoan parasite responsible for toxoplasmosis. The detection system was developed on graphite carbon electrode modified with poly(3-hydroxybenzoic acid) sensitized with ToxG1 probe. The hybridization of the specific genomic DNA related to T. gondii showed good response by direct detection of guanine residue oxidation using differential pulse voltammetry (DPV). The biosensor was able to distinguish both the complementary and non-complementary targets and detect up to 100ngμL−1 of the T. gondii genomic DNA. The hybridization (ToxG1: T. gondii genomic DNA) was confirmed by optical measurement. Optical assays using gold nanoparticles:ToxG1 probe showed a significant change in the absorbance peak in the presence of the T. gondii genomic DNA according to the electrochemical results. This novel biosensor shows potential as electrochemical transducer and was successfully applied in the biological sample.

A Transcriptomic Comparison of Two Bambara Groundnut Landraces under Dehydration Stress.

The ability to grow crops under low-water conditions is a significant advantage in relation to global food security. Bambara groundnut is an underutilised crop grown by subsistence farmers in Africa and is known to survive in regions of water deficit. This study focuses on the analysis of the transcriptomic changes in two bambara groundnut landraces in response to dehydration stress. A cross-species hybridisation approach based on the Soybean Affymetrix GeneChip array has been employed. The differential gene expression analysis of a water-limited treatment, however, showed that the two landraces responded with almost completely different sets of genes. Hence, both landraces with very similar genotypes (as assessed by the hybridisation of genomic DNA onto the Soybean Affymetrix GeneChip) showed contrasting transcriptional behaviour in response to dehydration stress. In addition, both genotypes showed a high expression of dehydration-associated genes, even under water-sufficient conditions. Several gene regulators were identified as potentially important. Some are already known, such as WRKY40, but others may also be considered, namely PRR7, ATAUX2-11, CONSTANS-like 1, MYB60, AGL-83, and a Zinc-finger protein. These data provide a basis for drought trait research in the bambara groundnut, which will facilitate functional genomics studies. An analysis of this dataset has identified that both genotypes appear to be in a dehydration-ready state, even in the absence of dehydration stress, and may have adapted in different ways to achieve drought resistance. This will help in understanding the mechanisms underlying the ability of crops to produce viable yields under drought conditions. In addition, cross-species hybridisation to the soybean microarray has been shown to be informative for investigating the bambara groundnut transcriptome.

Lymantria mathura nucleopolyhedrovirus: Identification, occurrence and genetic diversity in Iwate Prefecture, Japan

A high prevalence of nuclear polyhedrosis has been observed in larval outbreaks of Lymantria mathura in Iwate Prefecture, Japan. However, the virus responsible has not been identified. Here the virus was designated Lymantria mathura nucleopolyhedrovirus (LymaNPV), based on partial sequence results of the lef-8, lef-9, and polh genes and transmission electron microscopic observations. Diagnosis by polymerase chain reaction targeting of the partial polh gene and a subsequent restriction fragment length polymorphism (RFLP) analysis indicated that LymaNPV was an exclusive causative agent for the nuclear polyhedrosis of the L. mathura larvae. LymaNPV was also detected from a very small fraction of L. dispar larvae that co-occurred with L. mathura larvae where the prevalence of LymaNPV-caused disease was high. A bioassay using LymaNPV against L. dispar larvae produced infection with the inoculated virus. This finding was consistent with the results of the field sample analysis. LymaNPV contained six genome types in the L. mathura populations sampled, as determined by RFLP of LymaNPV genomic DNA and southern blot hybridization analyses. None of the genome types was unique to any sampling site, indicating that some flow had occurred among sites. However, genome-type composition seemed to differ among sites. This study provides basic information about the interaction between L. mathura and LymaNPV.

Horizontal Acquisition of a Multidrug-Resistance Module (R-type ASSuT) Is Responsible for the Monophasic Phenotype in a Widespread Clone of Salmonella Serovar 4,[5],12:i:.

Salmonella enterica serovar 4,[5],12:i:- is a monophasic variant of S. Typhimurium incapable of expressing the second-phase flagellar antigen (fljAB operon), and it is recognized to be one of the most prevalent serovars causing human infections. A clonal lineage characterized by phage type DT193, PulseNet PFGE profile STYMXB.0131 and multidrug resistance to ampicillin, streptomycin, sulphonamides and tetracycline (R-type ASSuT) is commonly circulating in Europe. In this study we determined the deletions affecting the fljAB operon and the resistance region responsible for the R-type ASSuT in a strain of Salmonella enterica serovar 4,5,12:i:- DT193/STYMXB.0131, through an approach based on PCRs and Southern blot hybridization of genomic DNA. Using a set of nine specific PCRs, the prevalence of the resistance region was assessed in a collection of 144 S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 strains isolated from Germany, Switzerland and Italy. A 28 kb-region is embedded between the loci STM2759 and iroB, replacing the DNA located in between, including the fljAB operon. It encompasses the genes blaTEM−1, strA-strB, sul2 and tet(B) responsible for the R-type ASSuT together with genes involved in plasmid replication and orfs of unknown function characteristically located on IncH1 plasmids. Its location and internal structure is fairly conserved in S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 strains regardless of the isolation source or country. Hence, in the S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 clonal lineage widespread in Germany, Switzerland and Italy, a resistance region derived from IncH1 plasmids has replaced the chromosomal region encoding the second flagellar phase and is an example of the stabilization of new plasmid-derived genetic material due to integration into the bacterial chromosome.

Abstract 4574: Genomic copy number variations characterize prognosis of oropharyngeal squamous cell carcinoma, including p16 positive subset, after surgery-based treatment

Abstract Background High-risk human papillomavirus (HPV)-related (+) oropharyngeal squamous cell carcinoma (OSCC) exhibits unique clinical and molecular characteristics, compared to HPV-unrelated - OSCCs. As different patterns of genomic instability is expected in HPV-driven carcinogenesis, we questioned if patterns of genomic copy number variations (CNVs) differ depending on HPV association, and predict prognosis, after surgery-based treatment for OSCCs in Korean population. Methods Fifty-eight OSCCs patients, who underwent surgery-based treatment and followed for 42 months on median, were enrolled. HPV status was determined by p16 immunohistochemistry (IHC), and HPV DNA PCR. Genome-wide CNV analysis was performed using array-based comparative genomic hybridization of genomic DNA from paraffin-embedded specimen. Clinical association and survival analyses were followed. Results p16 predicted the overall survival (HR = 0.27, CI: 0.39-0.80, p = 0.0006), better than HPV DNA PCR (HR = 0.83, CI: 0.66-1.29, p = 0.64) or smoking history (HR = 2.18, CI: 0.92-5.18, p = 0.071). In overall, number of CNVs did not significantly differed between p16+/- groups. However, 30 locus showed different CNV pattern between these groups. PRDM2 was amplified only in p16+ group, while EGFR and 11q13.3 was more amplified in p16- patients. Loss of FGF18 lead to worse prognosis, and gain of CDK10 and RAD18 was linked to better prognosis in all patients. Amplification of HRAS and loss of KDR lead to better OS in p16+ group. Conclusions After surgery-based treatment, p16+ OSCCs exhibits different CNV patterns at some loci, compared with p16- counterpart. Specific patterns of CNVs do predict better survival, especially in p16+ OSCCs. In the future, clinical trials should stratify patients based on these patterns to apply escalated treatment to obtain better survival. Citation Format: Yuh-S. Jung, Arang Rhie, Jong-Il Kim, Jin Soo Lee, Junsun Ryu, Weon Seo Park. Genomic copy number variations characterize prognosis of oropharyngeal squamous cell carcinoma, including p16 positive subset, after surgery-based treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4574. doi:10.1158/1538-7445.AM2015-4574

Root transcript profiling of two Rorippa species reveals gene clusters associated with extreme submergence tolerance.

Complete submergence represses photosynthesis and aerobic respiration, causing rapid mortality in most terrestrial plants. However, some plants have evolved traits allowing them to survive prolonged flooding, such as species of the genus Rorippa, close relatives of Arabidopsis (Arabidopsis thaliana). We studied plant survival, changes in carbohydrate and metabolite concentrations, and transcriptome responses to submergence of two species, Rorippa sylvestris and Rorippa amphibia. We exploited the close relationship between Rorippa species and the model species Arabidopsis by using Arabidopsis GeneChip microarrays for whole-genome transcript profiling of roots of young plants exposed to a 24-h submergence treatment or air. A probe mask was used based on hybridization of genomic DNA of both species to the arrays, so that weak probe signals due to Rorippa species/Arabidopsis mismatches were removed. Furthermore, we compared Rorippa species microarray results with those obtained for roots of submerged Arabidopsis plants. Both Rorippa species could tolerate deep submergence, with R. sylvestris surviving much longer than R. amphibia. Submergence resulted in the induction of genes involved in glycolysis and fermentation and the repression of many energy-consuming pathways, similar to the low-oxygen and submergence response of Arabidopsis and rice (Oryza sativa). The qualitative responses of both Rorippa species to submergence appeared roughly similar but differed quantitatively. Notably, glycolysis and fermentation genes and a gene encoding sucrose synthase were more strongly induced in the less tolerant R. amphibia than in R. sylvestris. A comparison with Arabidopsis microarray studies on submerged roots revealed some interesting differences and potential tolerance-related genes in Rorippa species.

Molecular cloning and expression analysis of PDR1-like gene in ginseng subjected to salt and cold stresses or hormonal treatment

The plant pleiotropic drug resistance (PDR) family of ATP-binding cassette (ABC) transporters is potentially involved in diverse biological processes. Currently, little is known about their actual physiological functions. A Panax ginseng PDR transporter gene (PgPDR1) was cloned and the cDNA has an open reading frame of 4344 bp. The deduced amino acid sequence contained the characteristic domains of PDR transporters: Walker A, Walker B, and ABC signature. Genomic DNA hybridization analysis indicated that one copy of PgPDR1 gene was present in P. ginseng. Subcellular localization showed that PgPDR1-GFP fusion protein was specifically localized in the cell membrane. Promoter region analysis revealed the presence of cis-acting elements, some of which are putatively involved in response to hormone, light and stress. To understand the functional roles of PgPDR1, we investigated the expression patterns of PgPDR1 in different tissues and under various conditions. Quantitative real-time PCR (qRT-PCR) and Western blotting analysis showed that PgPDR1 was expressed at a high level in the roots and leaves compared to seeds and stems. The expression of PgPDR1 was up-regulated by salicylic acid (SA) or chilling, down-regulated by ABA, and regulated differently at transcript and protein levels by MeJA. These results suggest that PgPDR1 might be involved in responding to environmental stresses and hormones.

Complex Genetic Architecture of Cardiac Disease in a Wild Type Inbred Strain of Drosophila melanogaster

Natural populations of the fruit fly, Drosophila melanogaster, segregate genetic variation that leads to cardiac disease phenotypes. One nearly isogenic line from a North Carolina peach orchard, WE70, is shown to harbor two genetically distinct heart phenotypes: elevated incidence of arrhythmias, and a dramatically constricted heart diameter in both diastole and systole, with resemblance to restrictive cardiomyopathy in humans. Assuming the source to be rare variants of large effect, we performed Bulked Segregant Analysis using genomic DNA hybridization to Affymetrix chips to detect single feature polymorphisms, but found that the mutant phenotypes are more likely to have a polygenic basis. Further mapping efforts revealed a complex architecture wherein the constricted cardiomyopathy phenotype was observed in individual whole chromosome substitution lines, implying that variants on both major autosomes are sufficient to produce the phenotype. A panel of 170 Recombinant Inbred Lines (RIL) was generated, and a small subset of mutant lines selected, but these each complemented both whole chromosome substitutions, implying a non-additive (epistatic) contribution to the “disease” phenotype. Low coverage whole genome sequencing was also used to attempt to map chromosomal regions contributing to both the cardiomyopathy and arrhythmia, but a polygenic architecture had to be again inferred to be most likely. These results show that an apparently simple rare phenotype can have a complex genetic basis that would be refractory to mapping by deep sequencing in pedigrees. We present this as a cautionary tale regarding assumptions related to attempts to map new disease mutations on the assumption that probands carry a single causal mutation.

Stable germ line transformation of a leafy vegetable crop amaranth (Amaranthus tricolor L.) mediated by Agrobacterium tumefaciens

We have optimized a procedure for genetic transformation of a major leafy vegetable crop, Amaranthus tricolor L., using epicotyl explant co-cultivation with Agrobacterium tumefaciens. Two disarmed A. tumefaciens strains EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT harboring the neomycin phosphotransferase II gene (nptII) and the β-glucuronidase gene (gus), were evaluated as vector systems. The former displayed a higher transforming efficiency. Several key factors influencing the transformation events were optimized. The highest percentage of transformed shoots (24.24%) was achieved using hand-pricked epicotyl explants, a 10-min infection period, with 100 μM acetosyringone-pretreated Agrobacterium culture corresponding to OD600 ≅ 0.6 and diluted to 109 cells ml−1, followed by 4 d co-cultivation in the regeneration medium. Putative transformed explants capable of forming shoots were selected on medium supplemented with 75 μg ml−1 kanamycin, and transient as well as stable glucuronidase expression was determined by histochemical analysis. From a total of 48 selected shoot lines derived from independent transformation events with epicotyl explants co-cultivated with EHA 105, 32 showed positive PCR amplification for both the nptII and gus genes. Germ line transformation and transgene stability were evident in progeny of primary transformed plants (T0). Among T1 seedlings of 12 selected transgenic plant lines, kanamycin-resistant and kanamycin-sensitive seedlings segregated in a ratio typical of the Mendelian monohybrid pattern (3:1) as verified by the chi-square (χ 2) test. Southern hybridization of genomic DNA from kanamycin-resistant T1 transgenic segregants to an nptII probe substantiated stable integration of the transgene. Neomycin phosphotransferase (NPTII) activity was detected in leaf protein extracts of selected T1 transgenic plants, thereby confirming stable expression of the nptII gene.

Characterization of Capsicum annuum Genetic Diversity and Population Structure Based on Parallel Polymorphism Discovery with a 30K Unigene Pepper GeneChip

The widely cultivated pepper, Capsicum spp., important as a vegetable and spice crop world-wide, is one of the most diverse crops. To enhance breeding programs, a detailed characterization of Capsicum diversity including morphological, geographical and molecular data is required. Currently, molecular data characterizing Capsicum genetic diversity is limited. The development and application of high-throughput genome-wide markers in Capsicum will facilitate more detailed molecular characterization of germplasm collections, genetic relationships, and the generation of ultra-high density maps. We have developed the Pepper GeneChip® array from Affymetrix for polymorphism detection and expression analysis in Capsicum. Probes on the array were designed from 30,815 unigenes assembled from expressed sequence tags (ESTs). Our array design provides a maximum redundancy of 13 probes per base pair position allowing integration of multiple hybridization values per position to detect single position polymorphism (SPP). Hybridization of genomic DNA from 40 diverse C. annuum lines, used in breeding and research programs, and a representative from three additional cultivated species (C. frutescens, C. chinense and C. pubescens) detected 33,401 SPP markers within 13,323 unigenes. Among the C. annuum lines, 6,426 SPPs covering 3,818 unigenes were identified. An estimated three-fold reduction in diversity was detected in non-pungent compared with pungent lines, however, we were able to detect 251 highly informative markers across these C. annuum lines. In addition, an 8.7 cM region without polymorphism was detected around Pun1 in non-pungent C. annuum. An analysis of genetic relatedness and diversity using the software Structure revealed clustering of the germplasm which was confirmed with statistical support by principle components analysis (PCA) and phylogenetic analysis. This research demonstrates the effectiveness of parallel high-throughput discovery and application of genome-wide transcript-based markers to assess genetic and genomic features among Capsicum annuum.

Development and application of a 6.5 million feature Affymetrix Genechip® for massively parallel discovery of single position polymorphisms in lettuce (Lactuca spp.).

BackgroundHigh-resolution genetic maps are needed in many crops to help characterize the genetic diversity that determines agriculturally important traits. Hybridization to microarrays to detect single feature polymorphisms is a powerful technique for marker discovery and genotyping because of its highly parallel nature. However, microarrays designed for gene expression analysis rarely provide sufficient gene coverage for optimal detection of nucleotide polymorphisms, which limits utility in species with low rates of polymorphism such as lettuce (Lactuca sativa).ResultsWe developed a 6.5 million feature Affymetrix GeneChip® for efficient polymorphism discovery and genotyping, as well as for analysis of gene expression in lettuce. Probes on the microarray were designed from 26,809 unigenes from cultivated lettuce and an additional 8,819 unigenes from four related species (L. serriola, L. saligna, L. virosa and L. perennis). Where possible, probes were tiled with a 2 bp stagger, alternating on each DNA strand; providing an average of 187 probes covering approximately 600 bp for each of over 35,000 unigenes; resulting in up to 13 fold redundancy in coverage per nucleotide. We developed protocols for hybridization of genomic DNA to the GeneChip® and refined custom algorithms that utilized coverage from multiple, high quality probes to detect single position polymorphisms in 2 bp sliding windows across each unigene. This allowed us to detect greater than 18,000 polymorphisms between the parental lines of our core mapping population, as well as numerous polymorphisms between cultivated lettuce and wild species in the lettuce genepool. Using marker data from our diversity panel comprised of 52 accessions from the five species listed above, we were able to separate accessions by species using both phylogenetic and principal component analyses. Additionally, we estimated the diversity between different types of cultivated lettuce and distinguished morphological types.ConclusionBy hybridizing genomic DNA to a custom oligonucleotide array designed for maximum gene coverage, we were able to identify polymorphisms using two approaches for pair-wise comparisons, as well as a highly parallel method that compared all 52 genotypes simultaneously.

Sphingobium limneticum sp. nov. and Sphingobium boeckii sp. nov., two freshwater planktonic members of the family Sphingomonadaceae , and reclassification of Sphingomonas suberifaciens as Sphingobium suberifaciens comb. nov.

Two novel chemo-organoheterotrophic members of the Sphingomonadaceae were isolated from alpine and pre-alpine lakes. Cells stained gram-negative, were motile and rod-shaped, and formed yellow, circular, convex colonies on different agar media. Strains 301(T) and 469(T) were strictly aerobic, catalase- and oxidase-positive, and grew at temperatures between 10 and 40 °C (optimum, 28 °C), and at pH values between 5 and 10 (optimum, pH 7). Both strains contained Q-10 as the dominant quinone, sphingoglycolipids and 2-hydroxymyristic acid, whereas 3-hydroxy fatty acids were absent. Major fatty acids of strain 301(T) were C(18 : 1)ω7c (53.3 %) and C(16 : 1)ω7c (22.9 %), with C(14 : 0) 2-OH (10.8 %) as the major 2-hydroxy fatty acid. Fatty acids of strain 469(T) were dominated by C(18 : 1)ω7c (34.4 %), C(16 : 1)ω7c (32.0 %) and C(14 : 0) 2-OH (15.2 %) as the major 2-hydroxy fatty acid. The genomic DNA G+C contents of strains 301(T) and 469(T) were 63.4 and 64.6 mol%, respectively. 16S rRNA gene sequence comparison indicated that both strains belonged to the genus Sphingobium. This classification was supported by the presence of spermidine as the major polyamine. The phylogenetically closest relatives of strain 301(T) were Sphingobium amiense DSM 16289(T), Sphingobium vermicomposti DSM 21299(T), Sphingobium yanoikuyae DSM 7462(T) and Sphingobium scionense DSM 19371(T) (98.8, 98.0, 97.9 and 97.4 % sequence similarity, respectively). DNA-DNA hybridization of genomic DNA yielded similarities in the range 43.2-12.1 % between strain 301(T) and the type strains of these four Sphingobium species. Closest relatives of strain 469(T) were Sphingomonas suberifaciens DSM 7465(T) and Sphingobium scionense DSM 19371(T) (97.1 and 96.5 % 16S rRNA gene sequence similarity, respectively). The degree of DNA-DNA hybridization between strain 469(T) and Sphingomonas suberifaciens DSM 7465(T) was 17.9 %. Based on the results of the molecular analyses and their phenotypic characteristics, strains 301(T) and 469(T) represent two novel species of the genus Sphingobium. The name Sphingobium limneticum sp. nov. is proposed for strain 301(T)( = DSM25076(T) = LMG 26659(T)). The name Sphingobium boeckii sp. nov. is proposed for strain 469(T) ( = DSM 25079(T) = LMG 26901(T)). The polyphasic analysis also suggests that Sphingomonas suberifaciens should be reclassified as Sphingobium suberifaciens comb. nov. with Ca1(T) ( = EY 2404(T) = ATCC 49355(T) = CIP 105429(T) = DSM 7465(T) = ICMP 12535(T) = NBRC 15211(T) = JCM 8521(T) = LMG 17323(T) = NCPPB 3629(T)) as the type strain.

Highly efficient sorghum transformation

A highly efficient microprojectile transformation system for sorghum (Sorghum bicolor L.) has been developed by using immature embryos (IEs) of inbred line Tx430. Co-bombardment was performed with the neomycin phosphotransferase II (nptII) gene and the green fluorescent protein (gfp) gene, both under the control of the maize ubiquitin1 (ubi1) promoter. After optimization of both tissue culture media and parameters of microprojectile transformation, 25 independent transgenic events were obtained from 121 bombarded IEs. The average transformation frequency (the total number of independent transgenic events divided by the total number of bombarded IEs) was 20.7% in three independent experiments. Transgenic events were confirmed by both PCR screening and Southern hybridization of genomic DNA from primary transgenics (T0). More than 90% of transformants were fertile and displayed normal morphology in a containment glasshouse. Co-transformation rate of the nptII and gfp genes was 72% in these experiments. The segregation of nptII and gfp in T1 progenies was observed utilizing fluorescence microscopy and geneticin selection of seedlings indicating both were inherited in the T1 generation. The transformation procedure, from initiating IEs to planting putative transgenic plantlets in the glasshouse, was completed within 11–16 weeks, and was approximately threefold more efficient than the previously reported best sorghum transformation system.

Detection of Alicyclobacillus species in fruit juice using a random genomic DNA microarray chip.

This study describes a method using a DNA microarray chip to rapidly and simultaneously detect Alicyclobacillus species in orange juice based on the hybridization of genomic DNA with random probes. Three food spoilage bacteria were used in this study: Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus. The three Alicyclobacillus species were adjusted to 2 × 10(3) CFU/ml and inoculated into pasteurized 100% pure orange juice. Cy5-dCTP labeling was used for reference signals, and Cy3-dCTP was labeled for target genomic DNA. The molar ratio of 1:1 of Cy3-dCTP and Cy5-dCTP was used. DNA microarray chips were fabricated using randomly fragmented DNA of Alicyclobacillus spp. and were hybridized with genomic DNA extracted from Bacillus spp. Genomic DNA extracted from Alicyclobacillus spp. showed a significantly higher hybridization rate compared with DNA of Bacillus spp., thereby distinguishing Alicyclobacillus spp. from Bacillus spp. The results showed that the microarray DNA chip containing randomly fragmented genomic DNA was specific and clearly identified specific food spoilage bacteria. This microarray system is a good tool for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., and is useful and applicable to the fruit juice industry.

Abstract 4967: Array-based genomic resequencing of acute myeloid leukemia

Abstract Somatic mutations within cancer-related genes play a central role in the pathogenesis of many human malignancies, as clearly demonstrated for the cases with mutated RAS and EGFR. Although a new generation of sequencing technologies is now available, whole-genome resequencing of many samples remains a demanding task. DNA microarray-based sequencing is suitable for analysis of multiple samples. However, currently available platforms are limited in the number of nucleotides that each array is able to probe. To overcome such limitations, we have now developed extra-large DNA arrays (“wafers”) for resequencing the human genome, in a collaborative study with Perlegen Sciences, Inc. With its capacity to assay ∼9 Mbp, we could place, on the array, oligonucleotides to interrogate sequence alterations at protein-coding exons and exon-intron boundaries for 5648 human genes that are possibly involved in malignant transformation mechanism. To identify transforming mutations in acute myeloid leukemia (AML), hybridization of genomic DNA from CD34-positive blasts of AML (n = 19) or myeloproliferative disorder (MPD) (n = 1) to the arrays revealed a total of 9148 nonsynonymous nucleotide changes on 3403 independent genes in this cohort (Phase I). To discriminate somatic changes from germ-line polymorphisms, we developed a second array for a direct analysis of the 9148 nucleotide changes, which was hybridized with genomic DNA from the leukemic blasts as well as with that from CD4-positive T cell fractions (paired controls) from the same cohort (Phase II). Analysis of CD4-positive fractions revealed that most of these nucleotide changes in Phase I were also present in the paired control genome, leaving only 11 somatic, nonsynonymous mutations among 11 genes. One of these somatic changes results in a Met-to-Ile substitution at amino-acid position 511 of Janus kinase 3 (JAK3), and the JAK3(M511I) protein exhibited transforming potential both in vitro and in vivo. Further screening for JAK3 mutations showed novel and known transforming changes in 9 out of 286 cases with leukemia. Our experiments also showed a somatic change responsible for the Arg-to-His substitution at amino-acid position 882 of DNA methyltransferase 3A, which results in a loss of >50% of the catalytic activity. Our data have thus revealed a unique profile of gene mutations in human leukemias. Given that only a small number of sequence alterations were proved to be “somatic” after Phase II selection, it would be important to use appropriate paired control samples in the search for somatic mutations in the cancer genome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4967. doi:10.1158/1538-7445.AM2011-4967

Emergence of NDM-1-producing Acinetobacter baumannii in China

To investigate the prevalence of bla(NDM-1) in Gram-negative bacilli in China. A total of 11 298 clinical Gram-negative bacilli, covering Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, were collected for PCR-based surveillance of bla(NDM-1) from 57 hospitals representing 18 provinces in China. For bla(NDM-1)-positive isolates, antibiotic susceptibilities were assessed and molecular typing was performed using PFGE. The genetic location of bla(NDM-1) was determined by analysis of PFGE profiles of S1 nuclease-digested genomic DNA and Southern blot hybridization. Plasmid transfer to E. coli recipients was investigated using filter mating and electroporation. Four A. baumannii isolates with bla(NDM-1) were identified in four different provinces in China: no positive isolates were detected among E. coli, K. pneumoniae and P. aeruginosa. These bla(NDM-1)-positive A. baumannii were resistant to all carbapenems and cephalosporins, and three remained susceptible to fluoroquinolones, aminoglycosides and colistin. The four NDM-1-producing A. baumannii were clonally diverse and carried bla(NDM-1) on different plasmids. Plasmids carrying bla(NDM-1) were successfully transferred from three of the four isolates to E. coli recipients, although the transconjugants and transformants were prone to lose the transferred plasmids after passage in the absence of ampicillin selection. We describe the emergence of A. baumannii producing NDM-1 in China. Systemic surveillance network should be established for monitoring these resistant bacteria.

A Combination of Biochemical and Proteomic Analyses Reveals Bx-LEC-1 as an Antigenic Target for the Monoclonal Antibody 3-2A7-2H5-D9-F10 Specific to the Pine Wood Nematode

Pine wilt disease (PWD) is one of the most devastating forest diseases in Asia and Europe. The pine wood nematode, Bursaphelenchus xylophilus, has been identified as the pathogen underlying PWD, although the pathology is not completely understood. At present, diagnosis and confirmation of PWD are time consuming tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for B. xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically, but not those from other closely related nematodes. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions, or bands isolated from SDS-PAGE, two-dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two nonidentical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to D-(+)-raffinose and N-acetyllactosamine were much higher than that to L-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.