Jianfei Hu

A draft sequence of the rice genome (Oryza sativa L. ssp indica)

The genome of the japonica subspecies of rice, an important cereal and model monocot, was sequenced and assembled by whole-genome shotgun sequencing. The assembled sequence covers 93% of the 420-megabase genome. Gene predictions on the assembled sequence suggest that the genome contains 32,000 to 50,000 genes. Homologs of 98% of the known maize, wheat, and barley proteins are found in rice. Synteny and gene homology between rice and the other cereal genomes are extensive, whereas synteny with Arabidopsis is limited. Assignment of candidate rice orthologs to Arabidopsis genes is possible in many cases. The rice genome sequence provides a foundation for the improvement of cereals, our most important crops.

Comparative analysis of eubacterial DNA polymerase III alpha subunits.

DNA polymerase III is one of the five eubacterial DNA polymerases that is responsible for the replication of DNA duplex. Among the ten subunits of the DNA polymerase III core enzyme, the alpha subunit catalyzes the reaction for polymerizing both DNA strands. In this study, we extracted genomic sequences of the alpha subunit from 159 sequenced eubacterial genomes, and carried out sequence-based phylogenetic and structural analyses. We found that all eubacterial genomes have one or more alpha subunits, which form either homodimers or heterodimers. Phylogenetic and domain structural analyses as well as copy number variations of the alpha subunit in each bacterium indicate the classification of alpha subunit into four basic groups: polC, dnaE1, dnaE2, and dnaE3. This classification is of essence in genome composition analysis. We also consolidated the naming convention to avoid further confusion in gene annotations.

A draft sequence of the rice (Oryza sativa ssp.indica) genome

The sequence of the rice genome holds fundamental information for its biology, including physiology, genetics, development, and evolution, as well as information on many beneficial phenotypes of economic significance. Using a “whole genome shotgun” approach, we have produced a draft rice genome sequence ofOryza sativa ssp.indica, the major crop rice subspecies in China and many other regions of Asia. The draft genome sequence is constructed from over 4.3 million successful sequencing traces with an accumulative total length of 2214.9 Mb. The initial assembly of the non-redundant sequences reached 409.76 Mb in length, based on 3.30 million successful sequencing traces with a total length of 1797.4 Mb from anindica variant cultivar93-11, giving an estimated coverage of 95.29% of the rice genome with an average base accuracy of higher than 99%. The coverage of the draft sequence, the randomness of the sequence distribution, and the consistency of BIG-ASSEMBLER, a custom-designed software package used for the initial assembly, were verified rigorously by comparisons against finished BAC clone sequences from bothindica andjapanica strains, available from the public databases. Over all, 96.3% of full-length cDNAs, 96.4% of STS, STR, RFLP markers, 94.0% of ESTs and 94.9% unigene clusters were identified from the draft sequence. Our preliminary analysis on the data set shows that our rice draft sequence is consistent with the comman standard accepted by the genome sequencing community. The unconditional release of the draft to the public also undoubtedly provides a fundamental resource to the international scientific communities to facilitate genomic and genetic studies on rice biology.

A Genome Sequence of Novel SARS-CoV Isolates: the Genotype, GD-Ins29, Leads to a Hypothesis of Viral Transmission in South China

We report a complete genomic sequence of rare isolates (minor genotype) of the SARS-CoV from SARS patients in Guangdong, China, where the first few cases emerged. The most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 (referred to the complete sequence of BJ01) within an overlapped region composed of BGI-PUP5 (BGI-postulated uncharacterized protein 5) and BGI-PUP6 upstream of the N (nucleocapsid) protein. The discovery of this minor genotype, GD-Ins29, suggests a significant genetic event and differentiates it from the previously reported genotype, the dominant form among all sequenced SARS-CoV isolates. A 17-nt segment of this extra sequence is identical to a segment of the same size in two human mRNA sequences that may interfere with viral genome replication and transcription in the cytosol of the infected cells. It provides a new avenue for the exploration of the virus-host interaction in viral evolution, host pathogenesis, and vaccine development.

The structure analysis and antigenicity study of the N protein of SARS-CoV.

The Coronaviridae family is characterized by a nucleocapsid that is composed of the genome RNA molecule in combination with the nucleoprotein (N protein) within a virion. The most striking physiochemical feature of the N protein of SARS-CoV is that it is a typical basic protein with a high predicted pI and high hydrophilicity, which is consistent with its function of binding to the ribophosphate backbone of the RNA molecule. The predicted high extent of phosphorylation of the N protein on multiple candidate phosphorylation sites demonstrates that it would be related to important functions, such as RNA-binding and localization to the nucleolus of host cells. Subsequent study shows that there is an SR-rich region in the N protein and this region might be involved in the protein-protein interaction. The abundant antigenic sites predicted in the N protein, as well as experimental evidence with synthesized polypeptides, indicate that the N protein is one of the major antigens of the SARS-CoV. Compared with other viral structural proteins, the low variation rate of the N protein with regards to its size suggests its importance to the survival of the virus.

Complete Genome Sequences of the SARS-CoV: the BJ Group (Isolates BJ01-BJ04)

Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city’s hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.

Genome Organization of the SARS-CoV

Annotation of the genome sequence of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) is indispensable to understand its evolution and pathogenesis. We have performed a full annotation of the SARS-CoV genome sequences by using annotation programs publicly available or developed by ourselves. Totally, 21 open reading frames (ORFs) of genes or putative uncharacterized proteins (PUPs) were predicted. Seven PUPs had not been reported previously, and two of them were predicted to contain transmembrane regions. Eight ORFs partially overlapped with or embedded into those of known genes, revealing that the SARS-CoV genome is a small and compact one with overlapped coding regions. The most striking discovery is that an ORF locates on the minus strand. We have also annotated non-coding regions and identified the transcription regulating sequences (TRS) in the intergenic regions. The analysis of TRS supports the minus strand extending transcription mechanism of coronavirus. The SNP analysis of different isolates reveals that mutations of the sequences do not affect the prediction results of ORFs.

The E Protein Is a Multifunctional Membrane Protein of SARS-CoV

The E (envelope) protein is the smallest structural protein in all coronaviruses and is the only viral structural protein in which no variation has been detected. We conducted genome sequencing and phylogenetic analyses of SARS-CoV. Based on genome sequencing, we predicted the E protein is a transmembrane (TM) protein characterized by a TM region with strong hydrophobicity and α-helix conformation. We identified a segment (NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH) in the carboxyl-terminal region of the E protein that appears to form three disulfide bonds with another segment of corresponding cysteines in the carboxyl-terminus of the S (spike) protein. These bonds point to a possible structural association between the E and S proteins. Our phylogenetic analyses of the E protein sequences in all published coronaviruses place SARS-CoV in an independent group in Coronaviridae and suggest a non-human animal origin.

Evolution and variation of the SARS-CoV genome.

Knowledge of the evolution of pathogens is of great medical and biological significance to the prevention, diagnosis, and therapy of infectious diseases. In order to understand the origin and evolution of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus), we collected complete genome sequences of all viruses available in GenBank, and made comparative analyses with the SARS-CoV. Genomic signature analysis demonstrates that the coronaviruses all take the TGTT as their richest tetranucleotide except the SARS-CoV. A detailed analysis of the forty-two complete SARS-CoV genome sequences revealed the existence of two distinct genotypes, and showed that these isolates could be classified into four groups. Our manual analysis of the BLASTN results demonstrates that the HE (hemagglutinin-esterase) gene exists in the SARS-CoV, and many mutations made it unfamiliar to us.

Applications of the double-barreled data in whole-genome shotgun sequence assembly and analysis.

Double-barreled (DB) data have been widely used for the assembly of large genomes. Based on the experience of building the whole-genome working draft ofOryza sativa L. ssp.Indica, we present here the prevailing and improved uses of DB data in the assembly procedure and report on novel applications during the following data-mining processes such as acquiring precise insert fragment information of each clone across the genome, and a new kind of low-cost whole-genome microarray. With the increasing number of organisms being sequenced, we believe that DB data will play an important role both in other assembly procedures and in future genomic studies.