Exploring the Antarctic soil metagenome as a source of novel cold-adapted enzymes and genetic mobile elements / Explorando el metagenoma del suelo antártico como fuente de nuevas enzimas adaptadas al frío y elementos génicos móviles

Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications, and for the detection of genetic elements associated with gene mobilization, respectively. Fourteen lipase/esterase-, 14 amylase-, 3 protease-, and 11 cellulase-producing clones were detected by activity-driven screening, with apparent maximum activities around 35 °C for both amylolytic and lipolytic enzymes, and 35-55 °C for cellulases, as observed for other cold-adapted enzymes. However, the behavior of at least one of the studied cellulases is more compatible to that observed for mesophilic enzymes. These enzymes are usually still active at temperatures above 60 °C, probably resulting in a psychrotolerant behavior in Antarctic soils. Metagenomics allows to access novel genes encoding for enzymatic and biophysic properties from almost every environment with potential benefits for biotechnological and industrial applications. Only intI- and tnp-like genes were detected by PCR, encoding for proteins with 58-86 %, and 58-73 % amino acid identity with known entries, respectively. Two clones, BAC 27A-9 and BAC 14A-5, seem to present unique syntenic organizations, suggesting the occurrence of gene rearrangements that were probably due to evolutionary divergences within the genus or facilitated by the association with transposable elements. The evidence for genetic elements related to recruitment and mobilization of genes (transposons/integrons) in an extreme environment like Antarctica reinforces the hypothesis of the origin of some of the genes disseminated by mobile elements among "human-associated" microorganisms.

A partir de muestras de suelo antártico se obtuvo la metagenoteca PP1. Esta fue sometida a análisis funcionales y genotípicos para el aislamiento de nuevas enzimas adaptadas al frío con potenciales aplicaciones, y para la detección de elementos génicos asociados a la movilización de genes, respectivamente. Por tamizaje fenotípico se detectaron 14, 14, 3 y 11 clones productores de lipasas/esterasas, proteasas, amilasas y celulasas, respectivamente, con actividades máximas aparentes de 35 °C para las amilasas y lipasas, y de 35-55 °C para las celulasas, tal como se observó para otras enzimas adaptadas al frío. Sin embargo, una celulasa parece ser compatible con enzimas mesófilas, las que usualmente se mantienen activas hasta por sobre 60 °C. Este hecho probablemente esté asociado a un comportamiento psicrotolerante en los suelos antárticos. La metagenómica permite acceder a una nueva miríada de productos metabólicos con potenciales beneficios para aplicaciones biotecnológicas e industriales. Se detectaron los genes tipo intI y tnp por PCR, y sus productos génicos deducidos tuvieron identidades del 58 al 86 % y del 58 al 73 % con secuencias conocidas, respectivamente. Dos clones, BAC 27A-9 y BAC 14A-5, parecen presentar organizaciones sintéticas únicas, lo cual sugiere la existencia de rearreglos génicos probablemente debidos a divergencias evolutivas dentro del género o facilitados por la asociación de elementos de transposición. La evidencia de elementos génicos relacionados con el reclutamiento y la movilización de genes en ambientes extremos como la Antártida refuerza la hipótesis sobre el origen de algunos genes diseminados por elementos móviles entre los microorganismos asociados al ser humano.

Detection of single copy sequences using BAC-FISH and C-PRINS techniques in sunflower chromosomes

Bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and cycling-primed in situ labeling (C-PRINS) techniques were evaluated for integration of physical and genetic maps of sunflower (Helianthus annuus L.). Single-site SSR markers were selected from three linkage groups of a high-density sunflower genetic map. This selection was based on previously identified QTL associated to S. sclerotiorum. These markers were used to select BACs contaning single copy sequences for BAC-FISH aplication. Blocking of highly dispersed repetitive sunflower sequences reduced unspecific hybridization, and allowed the detection of specific signals for BACs containing SSR markers HA4222 and HA2600, anchored to LG 16 and LG 10, respectively. Single-site FISH signal detection was optimized by adjusting the relative quantity and quality of unlabelled repetitive sequences present in the blocking DNA. The SSR marker ORS1247 anchored to the LG 17 was detected by C-PRINS, which yielded fluorescence signals that were specific and intense. This progress in localizing single-copy sequences using BAC-FISH and indirect C-PRINS strategies in sunflower will facilitate the integration of genetic and physical maps, allowing the identification of chromosomes containing key genes and/or QTL associated to agronomic important traits in sunflower.

Construction and partial characterization of a Corynebacterium pseudotuberculosis bacterial artificial chromosome library through genomic survey sequencing

Corynebacterium pseudotuberculosis is a gram-positive bacterium that causes caseous lymphadenitis in sheep and goats. However, despite the economic losses caused by caseous lymphadenitis, there is little information about the molecular mechanisms of pathogenesis of this bacterium. Genomic libraries constructed in bacterial artificial chromosome (BAC) vectors have become the method of choice for clone development in high-throughput genomic-sequencing projects. Large-insert DNA libraries are useful for isolation and characterization of important genomic regions and genes. In order to identify targets that might be useful for genome sequencing, we constructed a C. pseudotuberculosis BAC library in the vector pBeloBAC11. This library contains about 18,000 BAC clones, with inserts ranging in size from 25 to 120 kb, theoretically representing a 390-fold coverage of the C. pseudotuberculosis genome (estimated to be 2.5-3.1 Mb). Many genomic survey sequences (GSSs) with homology to C. diphtheriae, C. glutamicum, C. efficiens, and C. jeikeium proteins were observed within a sample of 215 sequenced clones, confirming their close phylogenetic relationship. Computer analyses of GSSs did not detect chimeric, deleted, or rearranged BAC clones, showing that this library has low redundancy. This GSSs collection is now available for further genetic and physical analysis of the C. pseudotuberculosis genome. The GSS strategy that we used to develop our library proved to be efficient for the identification of genes and will be an important tool for mapping, assembly, comparative, and functional genomic studies in a C. pseudotuberculosis genome sequencing project that will begin this year.

Cytogenetics and fluorescence in-situ hybridization in detection of hematological malignancies.

BACKGROUND: The technique of Fluorescence In-Situ Hybridization (FISH), a hybrid of cytogenetics and molecular biology has increased the resolution and application of cytogenetics in various neoplastic processes. In various types of leukemias, primary investigation by conventional cytogenetic [CC] technique followed by FISH has increased our understanding of the abnormal clonal formation involving different gene region. AIMS: Present study is aimed to use different kinds of in-house FISH probes in various hematological malignancies and its correlation with conventional cytogenetic finding. MATERIAL AND METHODS: Cytogenetic study was carried out in 360 patients either from peripheral blood or from bone marrow cells suspected for various types of leukemias. Four of 360 cases were further selected for FISH study by using different types of in-house probes, such as BAC [Bacterial Artificial Chromosome], PAC [Phague Artificial Chromosome], alphoid, PCP [Partial Chromosome Paint] and WCP [Whole Chromosome paint]. RESULTS: The results confirmed breakpoints of inversion 16 and del 16 in case 2 and 3 respectively. Whereas, case 1 did not confirm the cytogenetic findings of t(15;17) by PML/RARa fusion signals as multiple cell lines were involved in the patients. PCP and WCP were helpful in the identification of the marker chromosome in case 1. Telomeric and centromeric probes confirmed the cytogenetic findings of t(5;7) in case 4. CONCLUSION: We observe from this study that, in addition to the conventional cytogenetic study, FISH study provide further confirmation of chromosomal rearrangements. This facilitates our understanding of the neoplastic process more precisely for the better prognostication of the patient.

Molecular cytogenetic assignment of genes to bovine chromosome 5

Seven genes were assigned by molecular cytogenetic methods to bovine chromosome 5. To accomplish this, specific primers were either publicly available or were designed from highly conserved regions of the publicly available mammalian gene sequences. The identity of the amplified segments was verified by sequencing and alignment with the published sequences. The optimized primers that amplified the desired bovine genes were used for screening a bovine bacterial artificial chromosome library. The positive clones were localized to a specific band of bovine chromosome 5 by fluorescence in situ hybridization. The genes HOXC4, SP1 and IGFBP6 were localized to band q21, COL2A1 was localized to bands q21-q23, IGF1 was localized to band q26, MB to band q31 and the gene CYP2D6 was localized to band q35. The cytogenetic assignment of SP1, IGFBP6, COL2A1, IGF1, MB and CYP2D6 is first reported here and the assignment of HOXC4 refines the previous assignment of this gene. The identification and localization of these genes further support the development of the human to bovine comparative map through characterizing the homologous segments conserved in the evolution of these species. This information will be useful for the future localization of genes that affect economically important traits in bovines

New genomic resources for the honey bee(Apis mellifera L): development of a deep-coverage BAC library and a preliminary STC database

We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning enzyme HindIII in order to develop resources for structural genomics research. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and 2 empty vectors. Based on an estimated genome size of 270 Mb, this library provides approximately 15 haploid genome equivalents, allowing >99 probability of recovering any specific sequence of interest. High-density colony filters were gridded robotically using a Genetix Q-BOT in a 4 x 4 double-spotted array on 22.5-cm2 filters. Screening of the library with four mapped honey bee genomic clones and two bee cDNA probes identified an average of 21 positive signals per probe, with a range of 7-38 positive signals per probe. An additional screening was performed with nine aphid gene fragments and one Drosophila gene fragment resulting in seven of the nine aphid probes and the Drosophila probe producing positive signals with a range of 1 to 122 positive signals per probe (average of 45). To evaluate the utility of the library for sequence tagged connector analysis, 1152 BAC clones were end sequenced in both forward and reverse directions, giving a total of 2061 successful reads of high quality. End sequences were queried against SWISS-PROT, insect genomic sequence GSS, insect EST, and insect transposable element databases. Results in spreadsheet format from these searches are publicly available at the Clemson University Genomics Institute (CUGI) website in a searchable format (http://www.genome.clemson.edu/projects/stc/bee/AM__Ba/)

Annotation and BAC/PAC localization of nonredundant ESTs from drought-stressed seedlings of an indica rice.

To decipher the genes associated with drought stress response and to identify novel genes in rice, we utilized 1540 high-quality expressed sequence tags (ESTs) for functional annotation and mapping to rice genomic sequences. These ESTs were generated earlier by 3'-end single-pass sequencing of 2000 cDNA clones from normalized cDNA libraries constructed form drought-stressed seedlings of an indica rice. A rice UniGene set of 1025 transcripts was constructed from this collection through the BLASTN algorithm. Putative functions of 559 nonredundant ESTs were identified by BLAST similarity search against public databases. Putative functions were assigned at a stringency E value of 10(-6) in BLASTN and BLASTX algorithms. To understand the gene structure and function further, we have utilized the publicly available finished and unfinished rice BAC/PAC (BAC, bacterial artificial chromosome; PAC, P1 artificial chromosome) sequences for similarity search using the BLASTN algorithm. Further, 603 nonredundant ESTs have been mapped to BAC/PAC clones. BAC clones were assigned by a homology of above 95% identity along 90% of EST sequence length in the aligned region. In all, 700 ESTs showed rice EST hits in GenBank. Of the 325 novel ESTs, 128 were localized to BAC clones. In addition, 127 ESTs with identified putative functions but with no homology in IRGSP (International Rice Genome Sequencing Program) BAC/PAC sequences were mapped to the Chinese WGS (whole genome shotgun contigs) draft sequence of the rice genome. Functional annotation uncovered about a hundred candidate ESTs associated with abiotic stress in rice and Arabidopsis that were previously reported based on microarray analysis and other studies. This study is a major effort in identifying genes associated with drought stress response and will serve as a resource to rice geneticists and molecular biologists.