Genome-assisted analysis of dissimilatory metal-reducing bacteria

The availability of whole genome sequences for Shewanella oneidensis and Geobacter sulfurreducens has provided numerous new biological insights into the function of these model dissimilatory metal-reducing bacteria. Many of these findings, including the identification of a high number of c-type cytochromes in both organisms, have resulted from comparative genomic analyses, and several have been experimentally confirmed. These genome sequences have also aided the identification of genes important for the reduction of metal ions and other electron acceptors utilized during anaerobic growth, by facilitating the identification of genes disrupted by random insertions. Technologies for assaying global expression patterns for genes and proteins have also been employed, but their application has been limited mainly to the analysis of the role of global regulatory genes and to identifying genes expressed or repressed in response to specific electron acceptors. It is anticipated that details of the mechanisms of metal ion respiration, and metabolism in general, will eventually be revealed by comprehensive, systems-level analyses enabled by functional genomics data.     

泛基因组及其在植物功能基因组学研究中的应用

参考基因组是现代功能基因组学的核心框架,以此为基础的现代基因组学技术在过去20年对植物遗传变异发掘、功能基因克隆等研究起了巨大的推动作用.然而,越来越多的研究发现,单一或少数参考基因组不能完整代表和呈现物种或特定群体内的所有基因组变异,因此其在功能基因组学研究中应用存在很大的局限性,甚至会导致错误的结果.泛基因组是指物...     

植物外源基因组成分鉴定方法的研究进展

具外源基因组成分(外源染色体/染色体片段/基因)植株是目前进行基因组学研究以及作物改良的重要材料。迄今为止,已建立了基于性状观测、染色体分析、特异蛋白、DNA序列4种鉴定外源基因组成分的策略。其中,基于DNA序列的分子标记技术是当前鉴定外源基因组成分的主要手段,文中归纳了用于小麦、甘蓝等重要作物外源基因组成分的分子标记,且对简单重复序列(Simple sequence repeat, SSR)、插入缺失(Insertion-deletion,InDel)、单核苷酸多态性(Single nucleotide polymorphism,SNP)等9种标记进行了系统的比较。相比单一的鉴定方法,组合法更全面精准,文中对各组合法的应用情况进行统计和分析,提供了小麦族、芸薹族等作物的最佳鉴定组合。新一代分子标记InDel、SNP易实现高通量检测,对于外源渗入基因的精细定位展现了一定的优越性。此外,可以考虑一些新鉴定方法的加入,如微阵列比较基因组杂交(Microarray-based comparative genomic hybridization,array-CGH)、抑制差减杂交(Suppression subtractive hybridization,SSH)。     

Cloning of a novel pilin-like gene from Bordetella pertussis: homology to the fim2 gene

A search for pilin genes in a Bordetella pertussis (Bp) genomic library has led to the identification of several clones which hybridize to synthetic oligonucleotides with sequences derived from amino acid sequences of Bp fimbrial subunits. One of these clones (corresponding to a gene we have named fimX) contains an open reading frame encoding a protein with a molecular weight of about 20 kD and a sequence similar but not identical to the fimbrial subunit fim2 and to other fimbrial protein sequences. In this communication we present the cloning and nucleotide sequence of the fimX gene and its homology to the fim2 gene. A genomic analysis on the positional relationship between the two genes is also presented.     

Identifying potential tRNA genes in genomic DNA sequences.

We have developed an algorithm that automatically and reproducibly identifies potential tRNA genes in genomic DNA sequences, and we present a general strategy for testing the sensitivity of such algorithms. This algorithm is useful for the flagging and characterization of long genomic sequences that have not been experimentally analyzed for identification of functional regions, and for the scanning of nucleotide sequence databases for errors in the sequences and the functional assignments associated with them. In an exhaustive scan of the GenBank database, 97.5% of the 744 known tRNA genes were correctly identified (true-positives), and 42 previously unidentified sequences were predicted to be tRNAs. A detailed analysis of these latter predictions reveals that 16 of the 42 are very similar to known tRNA genes, and we predict that they do, in fact, code for tRNA, yielding a false-positive rate for the algorithm of 0.003%. The new algorithm and testing strategy are a considerable improvement over any previously described strategies for recognizing tRNA genes, and they allow detections of genes (including introns) embedded in long genomic sequences.     

Genetic approaches for understanding virulence in Toxoplasma gondii

Virulence of the protozoan parasite Toxoplasma gondii is highly variable and dependent upon the genotype of the parasite. The application of forward and reverse genetic approaches for understanding the genetic basis of virulence has resulted in the identification of several members of the ROP family as key mediators of virulence. More recently, modern genomic techniques have been used to address strain differences in virulence and have also identified additional members of the ROP family as likely mediators. The development of forward and reverse genetic, as well as modern genomic techniques, and the path to the discovery of the ROP genes as virulence factors is reviewed here.     

A novel genomics approach for the identification of drug targets in pathogens, with special reference to Pseudomonas aeruginosa

Complete genome sequences of several pathogenic bacteria have been determined, and many more such projects are currently under way. While these data potentially contain all the determinants of host-pathogen interactions and possible drug targets, computational tools for selecting suitable candidates for further experimental analyses are currently limited. Detection of bacterial genes that are non-homologous to human genes, and are essential for the survival of the pathogen represents a promising means of identifying novel drug targets. We have used three-way genome comparisons to identify essential genes from Pseudomonas aeruginosa. Our approach identified 306 essential genes that may be considered as potential drug targets. The resultant analyses are in good agreement with the results of systematic gene deletion experiments. This approach enables rapid potential drug target identification, thereby greatly facilitating the search for new antibiotics. These results underscore the utility of large genomic databases for in silico systematic drug target identification in the post-genomic era.     

Analysis of distal flanking regions of maize 19-kDa zein genes

Two genomic fragments from maize, each containing a 19-kDa zein gene with extensive flanking regions, have been sequenced and examined by computer-aided analysis and Southern blotting techniques. Sequence analysis of the distal flanking sequences has revealed interesting sequence motifs, some not seen before. In particular, four nearly identical, G + C-rich, 17 to 21-bp perfect palindromes were found clustered in a 133-bp stretch lying 2 kb upstream from the zein-coding region in the genomic clone pMS2. These palindromic sequences exhibit other interesting features, including a precise spatial organization with respect to each other, and their proximity to several other repeated motifs in the same region. Southern blot analysis indicates that these palindromes, or closely related sequences, are found frequently in the maize genome. Possible secondary structures for the palindrome units are presented, which resemble functionally important sequences found upstream from other eukaryotic genes.     

Future tools for association mapping in crop plants

Association mapping currently relies on the identification of genetic markers. Several technologies have been adopted for genetic marker analysis, with single nucleotide polymorphisms (SNPs) being the most popular where a reasonable quantity of genome sequence data are available. We describe several tools we have developed for the discovery, annotation, and visualization of molecular markers for association mapping. These include autoSNPdb for SNP discovery from assembled sequence data; TAGdb for the identification of gene specific paired read Illumina GAII data; CMap3D for the comparison of mapped genetic and physical markers; and BAC and Gene Annotator for the online annotation of genes and genomic sequences.     

Genomic DNA macroarrays as a tool for analysis of gene expression in Leishmania

Gene-array technologies have been applied in a wide number of organisms to study gene expression profiling under several physiological and experimental conditions. Gene-array implementations combined with the information arising from emerging genome sequencing projects are expected to be in the near future a major tool to characterize genes involved in different processes. So far, gene expression profile studies in trypanosomatids have been performed in microarrays that use a glass support to immobilize fragments of genomic DNA followed by fluorescent detection. Here, we wanted to test the potential of genomic DNA macroarrays of Leishmania infantum using nylon membranes and radioactive detection. Nylon macroarrays present a number of advantages since the processing of the membranes is based on standard Southern blotting protocols familiar to molecular biologists, and the data acquisition equipment is available to most research institutions. Nylon macroarrays were employed to search for genes showing increased mRNA abundance during an axenic differentiation of L. infantum promastigotes to amastigotes. Several clones were rescued and, after validation by Northern blot assays, these L. infantum sequences were used to screen the Leishmania major gene database. The L. major contigs with high homology to the L. infantum sequences allowed a consistent identification of the regulated genes.     

FISH技术在贝类分子生物学研究中的应用

在牡蛎和其它的海产贝类中,基因组研究的许多重要领域,如：利用非整倍体在牡蛎种间进行基因转移,三体牡的分离,牡蛎和其它贝类的连锁图的建立,三倍体的基因组稳定性和染色体缺失的分析等在缺少可靠的方法鉴定染色体而受到了限制,传统的带型技术很难鉴定牡的染色体。一种新的生物学技术－荧光原位杂交（FISH）为其提供了新的机遇。通过将DNA序列直接杂交到染色体上,FISH不仅是鉴定染色体的一个有力的工具,也是许多基因组研究如基因定位的一种有效的方法,结合最新研究成果,概述了FISH技术在贝类中的应用背景、现状和展望。     

Ecology and genomics of Bacillus subtilis

Bacillus subtilis is a remarkably diverse bacterial species that is capable of growth within many environments. Recent microarray-based comparative genomic analyses have revealed that members of this species also exhibit considerable genomic diversity. The identification of strain-specific genes might explain how B. subtilis has become so broadly adapted. The goal of identifying ecologically adaptive genes could soon be realized with the imminent release of several new B. subtilis genome sequences. As we embark upon this exciting new era of B. subtilis comparative genomics we review what is currently known about the ecology and evolution of this species.     

Genomic and microarray approaches to coral reef conservation biology

New technologies based on DNA microarrays and comparative genomics hold great promise for providing the background biological information necessary for effective coral reef conservation and management. Microarray analysis has been used in a wide range of applications across the biological sciences, most frequently to examine simultaneous changes in the expression of large numbers of genes in response to experimental manipulation or environmental variation. Other applications of microarray methods include the assessment of divergence in gene sequences between species and the identification of fast-evolving genes. Arrays are presently available for only a limited range of species, but with appropriate controls they can be used for related species, thus avoiding the considerable costs associated with development of a system de novo. Arrays are in use or preparation to study stress responses, early development, and symbiosis in Acropora and Montastraea. Ongoing projects on several corals are making available large numbers of expressed gene sequences, enabling the identification of candidate genes for studies on gamete specificity, allorecognition and symbiont interactions. Over the next few years, microarray and comparative genomic approaches are likely to assume increasingly important and widespread use to study many aspects of the biology of coral reef organisms. Application of these genomic approaches to enhance our understanding of genetic and physiological correlates during stress, environmental disturbance and disease bears direct relevance to the conservation of coral reef ecosystems. S. Forêt and K.S. Kassahn contributed equally.     

From genomics to functional markers in the era of next-generation sequencing

The availability of complete genome sequences, along with other genomic resources for Arabidopsis, rice, pigeon pea, soybean and other crops, has revolutionized our understanding of the genetic make-up of plants. Next-generation DNA sequencing (NGS) has facilitated single nucleotide polymorphism discovery in plants. Functionally-characterized sequences can be identified and functional markers (FMs) for important traits can be developed at an ever-increasing ease. FMs are derived from sequence polymorphisms found in allelic variants of a functional gene. Linkage disequilibrium-based association mapping and homologous recombinants have been developed for identification of “perfect” markers for their use in crop improvement practices. Compared with many other molecular markers, FMs derived from the functionally characterized sequence genes using NGS techniques and their use provide opportunities to develop high-yielding plant genotypes resistant to various stresses at a fast pace.