Objective To study the genetic basis for biovar conversion of Y. pestis. Methods In silico comparative genomic analysis was conducted and some critical genetic variations of Yersinia pestis were comparatively analyzed by means of PCR and DNA sequencing. Results A 93bp in-frame deletion in glpD gene results in the glycerol negative characteristic of Orientalis strains. A point mutation in the napA gene may cause the negative characteristic of nitrate reduction in Mediaevalis and Microus strains. A 122-bp frameshift deletion in the araC gene may lead to the arabinose negative phenotype of Microus strains. Conclusion In this study, Microtus strains with their unique pathogenic, biochemical and molecular features, were proposed as a novel biovar Microtus. In the light of its differential ability to ferment glycerol and arabinose and to reduce nitrate, Y. pestis can be classified into four biovars-Antiqua(glycerol positive, arabinose positive and nitrate positive), Mediaevalis(glycerol positive, arabinose positive and nitrate negative), Orientalis(glycerol negative, arabinose positive and nitrate positive), and Microtus(glycerol positive, arabinose negative and nitrate negative).

Objective To better understand the pathogenicity and evolution of Yersinia pestis, we carried out the whole genome sequencing of human-avirulent Yersinia pestis strain 91001, which was isolated from a species of rodent-Microtus brandti. Methods We utilized “whole genome shotgun” approach to get the genome sequence of 91001. Based on the finished and annotated genome sequence of 91001, as well as the previously published genome sequences of CO92 and KIM, we performed detailed comparative genomics analysis on their chromosomes and plasmids. Results The genome of 91001 consisted of one chromosome and four plasmids (pPCP1, pCD1, pMT1 and pCRY). The pPCP1 plasmid of 9 609bp was almost identical with its counterparts from reference strains, which possessed 10 CDS. Plasmid pCD1 was found to be a plasmid of the type III secretory apparatus, and its length was 70 159bp. Although its CDS are quite similar to those of the reference plasmids, there were obvious rearrangements which produced certain differences in structure among them. Another plasmid was pMT1, a 106 642bp plasmid, which showed slightly different architecture compared with the reference ones. There was no mutation in virulent-related genes of pMT1 and pMT1 of 91001, which seemed to have retained more fragments of an ancestor plasmid. pCRY was a novel plasmid discovered in this work. It was 21 742bp long and harbored a group of gene encoding type IV secretory system. pCRY seemed to be able to replicate. The length of chromosome of 91001 was 4 595 065bp, and among its 4 037 predicted CDS (coding sequences), 141 were possibly pseudogenes. There were many IS in the chromosome. Due to the rearrangments mediated by IS, the structure of 91001 chromosome showed significant differences compared with CO92 and KIM. According to the results of comparative genomics analysis, we deduced the genetic mechanisms of nitrate reduction, glycerol fermentation, arabinose and milibiose utilization in 91001. Conclusion According to the analysis of plasmids structure, pseudogenes distribution, nitrate reduction negative mechanism, gene comparison and chromosome architecture, we conclude that 91001 and other strains isolated from Microtus brandti and Microtus fuscus evolved from ancestor Y. pestis and then developed into a different lineage. The deletion of large genome fragments from 91001 chromosome and pseuogenes might contribute to its unqiue pathogenicity and host-specificity.

Objective To establish reliable proteomics analysis models for Yersinia pestis and obtain the basic proteomics data of this pathogen. Methods The human-avirulent Y. pestis strain 91001 was cultured as an experimental strain, and the proteins of which were extracted and divided into three parts fractionally according to their solubility. Then three different methods (Shotgun-LC-MS-MS, 1D-LC-MS-MS and 2D-MS) were used to analyze the extracted proteins. The obtained data were compared with the theoretical protein database of strain 91001 so as to identify the expressed protein of Y.pestis strain 91001 in this study. Results 971 proteins were identified by shotgun-LC-MS-MS method, accounting for 23.4% of the predicted proteins of strain 91001 (971/4 143). 915 proteins were identified by 1D-LC-MS-MS method, accounting for 22.1% of the predicted proteins of strain 91001 (915/4 143). However, with 2D-MS only 5.62% of the predicted proteins (233/4 143) were identified. Altogether 1 193 proteins were identified when the results of the 3 methods added together, accounting for 28.7% of all the predicted CDS in 91001. Conclusion The kind and quantity of proteins identified by various proteomics methods differ from each other dramatically, therefore it is necessary to utilize multiple methods to get more reliable protein profiles of Y. pestis.

Objective To study genetic variations of pgm locus and its flanking regions in Yersinia pestis isolated from Chinese natural focus so as to understand differences in virulence between different strains and to improve the prevention of plague. Methods We analysed the sequence variations of pgm locus and its flanking regions by using PCR-sequencing and allele-specific PCR among 260 strains of Y. pestis isolated from different foci and 7 strains of Yersinia pseudotuberculosis. Results For YP1666, only the strains of Xilin Gol grassland type and Microtus fuscus type had intact transmembrane helix, and the T-deletion at base 1406 was unique in strains of Orientalis. Same as Y. pseudotuberculosis, there was no IS100-insertion at the 3′ end of pgm locus of strains from Eastern section of North Tianshan Mountain type, Western section of North Tianshan Mountain type A and B, Xilin Gol grassland type and M. fuscus type. Only the strains of Xilin Gol grassland type and M. fuscus type had IS285-insertion in pigmentation segment and IS100-insertion at its downstream flanking region. pgm locus was deleted entirely from 36 strains, most of which came from Ordos plateau type, Song-liao plain type B , Kunlun Mountain type A and B. Conclusion Strains of Eastern section of North Tianshan Mountain type, Western section of North Tianshan Mountain type A and B, Xilin Gol grassland type and M. fuscus type are the oldest lineage of Chinese isolates. The pgm locus of the strains of these three types is very stable because there is no IS100-insertion at its 3′ terminal. We suggest that the strains of Xilin Gol grassland type and M. fuscus type should be grouped into a fourth biovar: Microtus. pgm locus is highly conserved among strains of different ecotypes, and its variations are well correlated with biovar, focus and ecotype, which indicates that pgm locus has played a role in strains′ adaptation to their environment.

AIM　It is difficult to identify the Chinese crude drug snake gallbladder accurately by morphological and microscopical characteristics or chemical components only. In order to solve the problem, the technique based on DNA molecular marker was introduced into the authentication of snake gallbladder. METHODS　DNA templates were extracted from the membrane or the bile of snake gallbladder, and also from the muscle of the original animal Elaphe schrenckii. About 400 bp DNA fragments of 12S rRNA gene were amplified from the templates and sequenced subsequently. RESULTS　Enough amounts of DNA templates could be extracted from a bit of membrane or bile of snake gallbladder. The sequence of amplicons from the membrane, bile and muscle of the same individual were identical completely. CONCLUSION　The technique of DNA molecular marker could be used for the authentication of snake gallbladder and bile. The results indicate that the technique could be used for the identification of crude drugs from other animal secretion. DNA sequence analysis also demonstrated that the origins of commercial snake gallbladder were complicated and more efficient quality control was necessary for supervising the crude drug in the market.

INTRODUCTIONRetinitis pigmentosa (RP) is the most prevalent group of inherited retinopathies and demonstrates considerable clinical and genetic heterogeneity, with wide variations in disease severity, progression, and gene involvement. We studied a large family with RP to determine the pattern of inheritance and to identify the disease-causing gene/locus.

MATERIALS AND METHODSOphthalmic examination was performed on 35 family members to identify affected individuals and carriers and to characterise the disease phenotype. Genetic linkage analysis was performed using short tandem repeat (STR) polymorphic markers encompassing the known loci for Xlinked RP (xlRP) including RP2, RP3, RP6, RP23, and RP24. Mutation screening was performed by direct sequencing of PCR-amplified genomic DNA of the RP2 and RPGR genes of the affected individuals.

RESULTSA highly penetrant, X-linked form of RP was observed in this family. Age of onset was from 5 to 8 years and visual acuity ranged from 20/25 in children to light perception in older adults. Linkage analysis and direct sequencing showed that no known loci/genes were associated with the phenotype in this kindred.

CONCLUSIONA novel disease gene locus/loci is responsible for the xlRP phenotype in this family.

Adolescent ; Adult ; Age of Onset ; Child ; Child, Preschool ; Chromosome Mapping ; DNA Mutational Analysis ; Eye Proteins ; genetics ; Female ; Genetic Diseases, X-Linked ; genetics ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Lod Score ; Male ; Membrane Proteins ; genetics ; Pedigree ; Phenotype ; Retinitis Pigmentosa ; genetics

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, Viral ; Haplotypes ; Humans ; Mutation ; Open Reading Frames ; Phylogeny ; SARS Virus ; genetics