Gene expression profile of Helicobacter pylori in response to growth temperature variation.

A Helicobacter pylori whole-genome DNA microarray was constructed to study expression profiles of H. pylori in response to a sudden temperature transfer from 37 degrees C to 20 degrees C. The expression level of the genome at each of four time points (15, 30, 60, and 120 min) after temperature downshift was compared with that just before cold treatment. Globally, 10.2 % (n=167) of the total predicted H. pylori genes (n=1636) represented on the microarray were significantly differentially expressed (p<0.05) over a 120 min period after shift to low temperature. The expression profiles of the differentially expressed genes were grouped, and their expression patterns were validated by quantitative real-time PCR. Up-regulated genes mainly included genes involved in energy metabolism and substance metabolism, cellular processes, protein fate, ribosomal protein genes, and hypothetical protein genes, which indicate the compensational responses of H. pylori to temperature downshift. Those genes play important roles in adaption to temperature downshift of H. pylori. Down-regulation of DNA metabolism genes and cell envelope genes and cellular processes genes may reflect damaged functions under low temperature, which is unfavorable to bacterial infection and propagation. Overall, this time-course study provides new insights into the primary response of H. pylori to a sudden temperature downshift, which allow the bacteria to survive and adapt to the new host environment.

Genetic diversity among major endemic strains of Leptospira interrogans in China.

BACKGROUND: Leptospirosis is a world-widely distributed zoonosis. Humans become infected via exposure to pathogenic Leptospira spp. from contaminated water or soil. The availability of genomic sequences of Leptospira interrogans serovar Lai and serovar Copenhageni opened up opportunities to identify genetic diversity among different pathogenic strains of L. interrogans representing various kinds of serotypes (serogroups and serovars). RESULTS: Comparative genomic hybridization (CGH) analysis was used to compare the gene content of L. interrogans serovar Lai strain Lai with that of other 10 L. interrogans strains prevailed in China and one identified from Brazil using a microarray spotted with 3,528 protein coding sequences (CDSs) of strain Lai. The cutoff ratio of sample/reference (S/R) hybridization for detecting the absence of genes from one tested strain was set by comparing the ratio of S/R hybridization and the in silico sequence similarities of strain Lai and serovar Copenhageni strain Fiocruz L1-130. Among the 11 strains tested, 275 CDSs were found absent from at least one strain. The common backbone of the L. interrogans genome was estimated to contain about 2,917 CDSs. The genes encoding fundamental cellular functions such as translation, energy production and conversion were conserved. While strain-specific genes include those that encode proteins related to either cell surface structures or carbohydrate transport and metabolism. We also found two genomic islands (GIs) in strain Lai containing genes divergently absent in other strains. Because genes encoding proteins with potential pathogenic functions are located within GIs, these elements might contribute to the variations in disease manifestation. Differences in genes involved in O-antigen biosynthesis were also identified for strains belonging to different serogroups, which offers an opportunity for future development of genomic typing tools for serological classification. CONCLUSION: CGH analyses for pathogenic leptospiral strains prevailed in China against the L. interrogans serovar Lai strain Lai CDS-spotted microarrays revealed 2,917 common backbone CDSs and strain specific genes encoding proteins mainly related to cell surface structures and carbohydrated transport/metabolism. Of the 275 CDSs considered absent from at least one of the L. interrogans strains tested, most of them were clustered in the rfb gene cluster and two putative genomic islands (GI A and B) in strain Lai. The strain-specific genes detected via this work will provide a knowledge base for further investigating the pathogenesis of L interrogans and/or for the development of effective vaccines and/or diagnostic tools.

Comparative genomics profiling of clinical isolates of Helicobacter pylori in Chinese populations using DNA microarray.

In order to search for specific genotypes related to this unique phenotype, we used whole genomic DNA microarray to characterize the genomic diversity of Helicobacter pylori (H. pylori) strains isolated from clinical patients in China. The open reading frame (ORF) fragments on our microarray were generated by PCR using gene-specific primers. Genomic DNA of H. pylori 26695 and J99 were used as templates. Thirty-four H. pylori isolates were obtained from patients in Shanghai. Results were judged based on ln(x) transformed and normalized Cy3/Cy5 ratios. Our microarray included 1882 DNA fragments corresponding to 1636 ORFs of both sequenced H. pylori strains. Cluster analysis, revealed two diverse regions in the H. pylori genome that were not present in other isolates. Among the 1636 genes, 1091 (66.7%) were common to all H. pylori strains, representing the functional core of the genome. Most of the genes found in the H. pylori functional core were responsible for metabolism, cellular processes, transcription and biosynthesis of amino acids, functions that are essential to H. pylori's growth and colonization in its host. In contrast, 522 (31.9%) genes were strain-specific genes that were missing from at least one strain of H. pylori. Strain-specific genes primarily included restriction modification system components, transposase genes, hypothetical proteins and outer membrane proteins. These strain-specific genes may aid the bacteria under specific circumstances during their long-term infection in genetically diverse hosts. Our results suggest 34 H. pylori clinical strains have extensive genomic diversity. Core genes and strain-specific genes both play essential roles in H. pylori propagation and pathogenesis. Our microarray experiment may help select relatively significant genes for further research on the pathogenicity of H. pylori and development of a vaccine for H. pylori.

A single C to T transition in intron 5 of LMBR1 gene is associated with triphalangeal thumb-polysyndactyly syndrome in a Chinese family.

Triphalangeal thumb-polysyndactyly syndrome (TPT-PS) is a type of human hand-foot malformation. In this study, we collected data from a Chinese family with TPT-PS and mapped the disease region to chromosome 7q36. By using a fine mapping study and a haplotype analysis, we narrowed the affected region to 1.7cM between markers D7S2465 and D7S2423, which contains four candidate genes: HLXB9, LMBR1, NOM1, and RNF32. By sequence analysis, we found no sequence alterations, which are specific to the patients in the transcribed regions and in the intron-exon boundaries among the four genes. After closely examining intron 5 of the LMBR1 gene, we discovered a single C to T transition in the affected TPT-PS individuals of the Chinese subject family. The position of this C to T transition is located close to other sequence alterations involved in several preaxial polydactyly (PPD) families, supporting the notion that intron 5 of LMBR1 contains a cis-acting regulator of limb-specific Sonic Hedgehog (SHH). We postulate that the disruption of this cis-regulator via a single C to T transition results in the dysregulation of SHH, which leads to the TPT-PS found in this case.

Genome-wide transcriptional analysis of temperature shift in L. interrogans serovar lai strain 56601.

BACKGROUND: Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptation to a range of new environmental conditions in the organs and tissues of the infected host. Several studies have shown that a shift in culture temperature from 28 degrees C to 37 degrees C, similar to that encountered during infection of a host from an environmental source, is associated with differential synthesis of several proteins of the outer membrane, periplasm and cytoplasm. The whole genome of the Leptospira interrogans serogroup Icterohaemorrhagiae serovar lai type strain #56601 was sequenced in 2003 and microarrays were constructed to compare differential transcription of the whole genome at 37 degrees C and 28 degrees C. RESULTS: DNA microarray analyses were used to investigate the influence of temperature on global gene expression in L. interrogans grown to mid-exponential phase at 28 degrees C and 37 degrees C. Expression of 106 genes differed significantly at the two temperatures. The differentially expressed genes belonged to nine functional categories: Cell wall/membrane biogenesis genes, hemolysin genes, heat shock proteins genes, intracellular trafficking and secretion genes, two-component system and transcriptional regulator genes, information storage and processing genes, chemotaxis and flagellar genes, metabolism genes and genes with no known homologue. Real-time reverse transcription-PCR assays confirmed the microarray data. CONCLUSION: Microarray analyses demonstrated that L. interrogans responds globally to temperature alteration. The data delineate the spectrum of temperature-regulated gene expression in an important human pathogen and provide many new insights into its pathogenesis.

Simple sequence repeat-based consensus linkage map of Bombyx mori.

We established a genetic linkage map employing 518 simple sequence repeat (SSR, or microsatellite) markers for Bombyx mori (silkworm), the economically and culturally important lepidopteran insect, as part of an international genomics program. A survey of six representative silkworm strains using 2,500 (CA)n- and (CT)n-based SSR markers revealed 17-24% polymorphism, indicating a high degree of homozygosity resulting from a long history of inbreeding. Twenty-nine SSR linkage groups were established in well characterized Dazao and C108 strains based on genotyping of 189 backcross progeny derived from an F(1) male mated with a C108 female. The clustering was further focused to 28 groups by genotyping 22 backcross progeny derived from an F(1) female mated with a C108 male. This set of SSR linkage groups was further assigned to the 28 chromosomes (established linkage groups) of silkworm aided by visible mutations and cleaved amplified polymorphic sequence markers developed from previously mapped genes, cDNA sequences, and cloned random amplified polymorphic DNAs. By integrating a visible mutation p (plain, larval marking) and 29 well conserved genes of insects onto this SSR-based linkage map, a second generation consensus silkworm genetic map with a range of 7-40 markers per linkage group and a total map length of approximately 3431.9 cM was constructed and its high efficiency for genotyping and potential application for synteny studies of Lepidoptera and other insects was demonstrated.

A novel mutation in the second half of the keratin 17 1A domain in a large pedigree with delayed-onset pachyonychia congenita type 2.

Pachyonychia congenita type 2 (PC-2), also known as Jackson-Lawler type PC, is an autosomal dominant disorder characterized by hypertrophic nail dystrophy associated with focal keratoderma and multiple pilosebaceous cysts. We report a large Chinese pedigree of typical delayed-onset PC-2 that includes 19 affected members. Direct sequencing of PCR products revealed a novel heterozygous 325A-->G mutation in the affected members. This mutation predicts the substitution of asparagine by aspartic acid in codon 109 (N109D) located in the second half of the keratin 17 1A domain, where similar mutation in keratin 5 is associated with the mild Weber-Cockayne form of epidermolysis bullosa simplex.

[A novel keratin 17 gene mutation in a Chinese pedigree of delayed-onset pachyonychia congenita type II].

OBJECTIVE: To detect the keratin 17 gene mutation in a Chinese pedigree of typical delayed-onset pachyonychia congenita type II (PC-II) and to explore the relationship between the genetic mutation and the phenotype of PC-II. METHODS: The DNA was extracted from the blood samples of 19 patients with PC-II in four generations in the pedigree, 1 unaffected member of the pedigree, and 50 un-related normal persons. Nested PCR was used to amplify the mutation hot spot in the exon 1 of keratin 17 gene. The PCR products were directly sequenced to detect the mutation. RESULTS: Sequencing of the PCR products revealed that the codon 109 (AAC) was mutated as GAC in the nine affected members of the pedigree, causing the substitution of asparagine by aspartic acid in codon 109 (N109D) located in the 1A domain of keratin 17 gene. No such mutation was found in the unaffected member of the pedigree and the 50 unrelated controls. CONCLUSION: The novel missense mutation (N109D) located in the second half of 1A domain of keratin 17 gene underlies the affected members' phenotype, delayed-onset pachyonychia congenita type II.

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes.

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

A high-resolution genetic and physical map of a mouse coat abnormality locus (Uncv).

More than a hundred of loci (genes) affect the development of mouse and human hair. A locus of Uncv (uncovered) has been confirmed to be involved in hairlessness for homozygote and sparse hair for heterozygote. Except hairlessness(or called uncovered coat), the homozygote was also accompanied by growth retard and puberty delay. Identification of the mutation in the gene will be important for understanding the related diseases in human. Although the uncovered locus (Uncv) has been mapped to the mouse distal chromosome 11(Chr11), the high-resolution genetic map and physical map of the locus has not been created. In this study, 2074 F2 mouse populations from backcross [BALB/c (Uncv/Uncv) x C3H (+/+)]x BALB/c (Uncv/Uncv) and [BALB/c (Uncv/Uncv) x C57BL/6 (+/+)] x BALB/c(Uncv/ Uncv) were genotyped using 16 polymorphic markers with an approximately 20 cM interval on mouse distal Chr11. By genetic linkage analysis, Uncv locus was mapped to an approximately 1.4 cM interval between markers D11Mit337 and D11Mit338 with the following order: proximal D11Mit338-D11Mit203 (Uncv)-D11Mit103 -D11Mit337 distal on mouse Chr11. And then, a contig of 35 BACs representing the Uncv-containing region was constructed. The contig covered 800-1000 kb region flanked by 189K10-SP6 and D11Mit103. Together, we have constructed the high-resolution genetic map and detailed physical map of the Uncv region. This will facilitate the identification of the Uncv loci.