From Arabidopsis to rice genomics: a survey of French programmes.

During the last ten years, Arabidopsis thaliana has become the most favoured plant system for the study of many aspects of development and adaptation to adverse conditions and diseases. The sequencing of the Arabidopsis thaliana genome is nearly completed with more than 90% of the sequence being released in public databases. This is the first plant genome to be analysed and it has revealed a tremendous amount of information about the nature of the genes it contains and its largely duplicated organisation. French groups have been involved in Arabidopsis genomics at several steps: EST (expressed sequence tags) sequencing, construction and ordering (physical mapping of chromosomes) of a YAC (yeast artificial chromosomes) library, genomic sequencing. In parallel an extensive programme of functional genomics is being undertaken through the systematic analysis of insertional mutants. This information provides a support for analysing other more economically important plant genomes such as the rice genome and constitutes the beginning of a systematic investigation on plant gene functions and will promote new strategies for plant improvement.     

Advanced technologies for genomic analysis in farm animals and its application for QTL mapping

Rapid progress in farm animal breeding has been made in the last few decades. Advanced technologies for genomic analysis in molecular genetics have led to the identification of genes or markers associated with genes that affect economic traits. Molecular markers, large-insert libraries and RH panels have been used to build the genetic linkage maps, physical maps and comparative maps in different farm animals. Moreover, EST sequencing, genome sequencing and SNPs maps are helping us to understand how genomes function in various organisms and further areas will be studied by DNA microarray technologies and proteomics methods. Because most economically important traits in farm animals are controlled by multiple genes and the environment, the main goal of genome research in farm animals is to map and characterize genes determining QTL. There are two main strategies to identify trait loci, candidate gene association tests and genome scan approaches. In recent years, some new concepts, such as RNAi, miRNA and eQTL, have been introduced into farm animal research, especially for QTL mapping and finding QTN. Several genes that influence important traits have already been identified or are close to being identified, and some of them have been applied in farm animal breeding programs by marker-assisted selection.     

Plant genome sequencing: a prelude to the study of its expression

This report illustrates development of plant sequencing programmes. So far Arabidopsis genome has been completely sequenced and a draft of the rice genome is available. The Arabidopsis programmes stimulated sequencing of EST (expressed sequence tags) from numerous cultivated species thus creating an enormous resource. The major challenge is now to correctly annotate all the genes in Arabidopsis and find out a biological and biochemical function for each one. The availability of EST and genome sequence now allows one to analyse the expression of genes at the level of the whole genome.     

A chromosome bin map of 16,000 expressed sequence tag loci and distribution of genes among the three genomes of polyploid wheat

Because of the huge size of the common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) genome of 17,300 Mb, sequencing and mapping of the expressed portion is a logical first step for gene discovery. Here we report mapping of 7104 expressed sequence tag (EST) unigenes by Southern hybridization into a chromosome bin map using a set of wheat aneuploids and deletion stocks. Each EST detected a mean of 4.8 restriction fragments and 2.8 loci. More loci were mapped in the B genome (5774) than in the A (5173) or D (5146) genomes. The EST density was significantly higher for the D genome than for the A or B. In general, EST density increased relative to the physical distance from the centromere. The majority of EST-dense regions are in the distal parts of chromosomes. Most of the agronomically important genes are located in EST-dense regions. The chromosome bin map of ESTs is a unique resource for SNP analysis, comparative mapping, structural and functional analysis, and polyploid evolution, as well as providing a framework for constructing a sequence-ready, BAC-contig map of the wheat genome.     

EST! EST!! EST!!!

A recently published study⁽¹⁾ has identified a set of candidate genes for human diseases based on findings from Drosophila. Each human expressed sequence tag (EST) in a large database was compared with all known Drosophila genes. After eliminating matches between genes of already known function, the remaining sequences were mapped in the human genome. In each region, the phenotypes of all known human diseases were compared with the phenotypes of known Drosophila mutations in order to identify candidate genes for the human diseases. Are the correspondences real or coincidental?     

High-throughput plasmid DNA purification for 3 cents per sample

To accommodate the increasingly rapid rates of DNA sequencing we have developed and implemented an inexpensive, expeditious method for the purification of double-stranded plasmid DNA clones. The robust nature, high throughput, low degree of technical difficulty and extremely low cost have made it the plasmid DNA preparation method of choice in both our expressed sequence tag (EST) and genome sequencing projects. Here we report the details of the method and describe its application in the generation of more than 700 000 ESTs at a rate exceeding 16 000 per week.     

Large Scale Structural Analysis of cDNAs in the Model Legume, Lotus japonicus

Lotus japonicus possesses certain characteristics suited to molecular genetic and genomic analyses and has been adopted as a model species in the study of legume plants. To make a catalogue of genes expressed in L. japonicus and understand biological processes specific to legume plants, large scale EST analyses have been performed. To date, more than 26,000 EST sequences of L. japonicus have been deposited in the public databases. These sequences were developed by five laboratories using different organs. In this review, information obtained from two EST projects carried out in Japan is presented. Some 7137 non-redundant EST groups from young plants and 718 groups from flower buds were generated. A similarity search revealed that homologues of nodulin genes in other legume plants, as well as genes related to secondary metabolism, seed development and the reproductive process, were included in the EST collection, indicating the usefulness of the EST clones in the study of biological phenomena distinctive to legume plant species. Received 23 August 2000/ Accepted in revised form 22 September 2000     

Evaluation of Genome Sequencing Quality in Selected Plant Species Using Expressed Sequence Tags

Background

With the completion of genome sequencing projects for more than 30 plant species, large volumes of genome sequences have been produced and stored in online databases. Advancements in sequencing technologies have reduced the cost and time of whole genome sequencing enabling more and more plants to be subjected to genome sequencing. Despite this, genome sequence qualities of multiple plants have not been evaluated.

Methodology/Principal Finding

Integrity and accuracy were calculated to evaluate the genome sequence quality of 32 plants. The integrity of a genome sequence is presented by the ratio of chromosome size and genome size (or between scaffold size and genome size), which ranged from 55.31% to nearly 100%. The accuracy of genome sequence was presented by the ratio between matched EST and selected ESTs where 52.93% ∼ 98.28% and 89.02% ∼ 98.85% of the randomly selected clean ESTs could be mapped to chromosome and scaffold sequences, respectively. According to the integrity, accuracy and other analysis of each plant species, thirteen plant species were divided into four levels. Arabidopsis thaliana, Oryza sativa and Zea mays had the highest quality, followed by Brachypodium distachyon, Populus trichocarpa, Vitis vinifera and Glycine max, Sorghum bicolor, Solanum lycopersicum and Fragaria vesca, and Lotus japonicus, Medicago truncatula and Malus × domestica in that order. Assembling the scaffold sequences into chromosome sequences should be the primary task for the remaining nineteen species. Low GC content and repeat DNA influences genome sequence assembly.

Conclusion

The quality of plant genome sequences was found to be lower than envisaged and thus the rapid development of genome sequencing projects as well as research on bioinformatics tools and the algorithms of genome sequence assembly should provide increased processing and correction of genome sequences that have already been published.     

Isolation, in silico characterization and chromosomal localization of a group of cDNAs from ciliated epithelial cells after in vitro ciliogenesis

Background

Immotile cilia syndrome (ICS) or primary ciliary dyskinesia (PCD) is an autosomal recessive disorder in humans in which the beating of cilia and sperm flagella is impaired. Ciliated epithelial cell linings are present in many tissues. To understand ciliary assembly and motility, it is important to isolate those genes involved in the process.

Results

Total RNA was isolated from cultured ciliated nasal epithelial cells after in vitro ciliogenesis and expressed sequenced tags (ESTs) were generated. The functions and locations of 63 of these ESTs were derived by BLAST from two public databases. These ESTs are grouped into various classes. One group has high homology not only with the mitochondrial genome but also with one or more chromosomal DNAs, suggesting that very similar genes, or genes with very similar domains, are expressed from both mitochondrial and nuclear DNA. A second class comprises genes with complete homology with part of a known gene, suggesting that they are the same genes. A third group has partial homology with domains of known genes. A fourth group, constituting 33% of the ESTs characterized, has no significant homology with any gene or EST in the database.

Conclusions

We have shown that sufficient information about the location of ESTs could be derived electronically from the recently completed human genome sequences. This strategy of EST localization should be significantly useful for mapping and identification of new genes in the forthcoming human genome sequences with the vast number of ESTs in the dbEST database.     

Characterization and Fine Localization of Two New Genes in Xq28 Using the Genomic Sequence/EST Database Screening Approach

Two new genes were identified and mapped by searching the EST databases with genomic sequences obtained from putative CpG islands of the rodent–human hybrid X3000. Previous mapping of these CpG islands in the proximity of the host cell factor (HCFC1) and GdX genes automatically localized these two new genes to Xq28 in the interval between the L1 cell adhesion molecule (L1CAM) and the glucose-6-phosphate dehydrogenase (G6PD) loci. Both genes are relatively short, contain an ORF of 261 and 105 amino acids, respectively, and are ubiquitously expressed. Combining sequencing of selected CpG islands, derived from hybrids containing small portions of the human genome, with an EST database search is an easy method of identifying and mapping new genes to specific regions of the genome.     

Gene identification in novel eukaryotic genomes by self-training algorithm

Finding new protein-coding genes is one of the most important goals of eukaryotic genome sequencing projects. However, genomic organization of novel eukaryotic genomes is diverse and ab initio gene finding tools tuned up for previously studied species are rarely suitable for efficacious gene hunting in DNA sequences of a new genome. Gene identification methods based on cDNA and expressed sequence tag (EST) mapping to genomic DNA or those using alignments to closely related genomes rely either on existence of abundant cDNA and EST data and/or availability on reference genomes. Conventional statistical ab initio methods require large training sets of validated genes for estimating gene model parameters. In practice, neither one of these types of data may be available in sufficient amount until rather late stages of the novel genome sequencing. Nevertheless, we have shown that gene finding in eukaryotic genomes could be carried out in parallel with statistical models estimation directly from yet anonymous genomic DNA. The suggested method of parallelization of gene prediction with the model parameters estimation follows the path of the iterative Viterbi training. Rounds of genomic sequence labeling into coding and non-coding regions are followed by the rounds of model parameters estimation. Several dynamically changing restrictions on the possible range of model parameters are added to filter out fluctuations in the initial steps of the algorithm that could redirect the iteration process away from the biologically relevant point in parameter space. Tests on well-studied eukaryotic genomes have shown that the new method performs comparably or better than conventional methods where the supervised model training precedes the gene prediction step. Several novel genomes have been analyzed and biologically interesting findings are discussed. Thus, a self-training algorithm that had been assumed feasible only for prokaryotic genomes has now been developed for ab initio eukaryotic gene identification.     

The filarial genome project: analysis of the nuclear, mitochondrial and endosymbiont genomes of Brugia malayi

The Filarial Genome Project (FGP) was initiated in 1994 under the auspices of the World Health Organisation. Brugia malayi was chosen as the model organism due to the availability of all life cycle stages for the construction of cDNA libraries. To date, over 20000 cDNA clones have been partially sequenced and submitted to the EST database (dbEST). These ESTs define approximately 7000 new Brugia genes. Analysis of the EST dataset provides useful information on the expression pattern of the most abundantly expressed Brugia genes. Some highly expressed genes have been identified that are expressed in all stages of the parasite's life cycle, while other highly expressed genes appear to be stage-specific. To elucidate the structure of the Brugia genome and to provide a basis for comparison to the Caenorhabditis elegans genome, the FGP is also constructing a physical map of the Brugia chromosomes and is sequencing genomic BAC clones. In addition to the nuclear genome, B. malayi possesses two other genomes: the mitochondrial genome and the genome of a bacterial endosymbiont. Eighty percent of the mitochondrial genome of B. malayi has been sequenced and is being compared to mitochondrial sequences of other nematodes. The bacterial endosymbiont genome found in B. malayi is closely related to the Wolbachia group of rickettsia-like bacteria that infects many insect species. A set of overlapping BAC clones is being assembled to cover the entire bacterial genome. Currently, half of the bacterial genome has been assembled into four contigs. A consortium has been established to sequence the entire genome of the Brugia endosymbiont. The sequence and mapping data provided by the FGP is being utilised by the nematode research community to develop a better understanding of the biology of filarial parasites and to identify new vaccine candidates and drug targets to aid the elimination of human filariasis.     

Rapid cloning and bioinformatic analysis of spinach Y chromosome-specific EST sequences

The genome of spinach single chromosome complement is about 1000 Mbp, which is the model material to study the molecular mechanisms of plant sex differentiation. The cytological study showed that the biggest spinach chromosome (chromosome 1) was taken as spinach sex chromosome. It had three alleles of sex-related X, X ^m and Y. Many researchers have been trying to clone the sex-determining genes and investigated the molecular mechanism of spinach sex differentiation. However, there are no successful cloned reports about these genes. A new technology combining chromosome microdissection with hybridization-specific amplification (HSA) was adopted. The spinach Y chromosome degenerate oligonucleotide primed-PCR (DOP-PCR) products were hybridized with cDNA of the male spinach flowers in florescence. The female spinach genome was taken as blocker and cDNA library specifically expressed in Y chromosome was constructed. Moreover, expressed sequence tag (EST) sequences in cDNA library were cloned, sequenced and bioinformatics was analysed. There were 63 valid EST sequences obtained in this study. The fragment size was between 53 and 486 bp. BLASTn homologous alignment indicated that 12 EST sequences had homologous sequences of nucleic acids, the rest were new sequences. BLASTx homologous alignment indicated that 16 EST sequences had homologous protein-encoding nucleic acid sequence. The spinach Y chromosome-specific EST sequences laid the foundation for cloning the functional genes, specifically expressed in spinach Y chromosome. Meanwhile, the establishment of the technology system in the research provided a reference for rapid cloning of other biological sex chromosome-specific EST sequences.