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Suppression subtractive hybridization method for the identification of a new strain of murine hepatitis virus from xenografted SCID mice.

ABSTRACT: During attempts to clone retroviral determinants associated with a mouse model of Langerhans cell histiocytosis (LCH), suppression subtractive hybridization (SSH) was used to identify unique viruses in the liver of severe combined immunodeficiency (SCID) mice transplanted with LCH tissues. A partial genomic sequence of a murine coronavirus was identified, and the whole genome (31428 bp) of the coronavirus was subsequently sequenced using PCR cloning techniques. Nucleotide sequence comparisons revealed that the genome sequence of the new virus was 91-93% identical to those of known murine hepatitis viruses (MHVs). The predicted open reading frame from the nucleotide sequence encoded all known proteins of MHVs. Analysis at the protein level showed that the virus was closely related to the highly virulent MHV-JHM strain. The virus strain was named MHV-MI. No type D retroviruses were found. Degenerate PCR targeting of type D retrovirus and 5'-RACE targeting of other types of retroviruses confirmed the absence of any retroviral association with the LCH xenografted SCID mice.

SUBMITTER: Islam MM

PROVIDER: S-EPMC4635179 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Suppression subtractive hybridization method for the identification of a new strain of murine hepatitis virus from xenografted SCID mice.

Islam Mohammed M MM Toohey Brendan B Purcell Damian F J DF Kannourakis George G

Archives of virology 20150908 12

During attempts to clone retroviral determinants associated with a mouse model of Langerhans cell histiocytosis (LCH), suppression subtractive hybridization (SSH) was used to identify unique viruses in the liver of severe combined immunodeficiency (SCID) mice transplanted with LCH tissues. A partial genomic sequence of a murine coronavirus was identified, and the whole genome (31428 bp) of the coronavirus was subsequently sequenced using PCR cloning techniques. Nucleotide sequence comparisons re ...[more]

PMID: 26347284

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Project description:AimTo clone and identify human genes transactivated by PS1TP5 by constructing a cDNA subtractive library with suppression subtractive hybridization (SSH) technique.MethodsSSH and bioinformatics techniques were used for screening and cloning of the target genes transactivated by PS1TP5 protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-myc-his(A)-PS1TP5 and pcDNA3.1(-)-myc-his(A) empty vector, respectively, and SSH technique was employed to analyze the differentially expressed DNA sequence between the two groups. After digestion with restriction enzyme Rsa I, small size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. The tester cDNA was hybridized with driver cDNA twice and subjected to nested PCR for two times, and then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5alpha. The cDNA was sequenced and analyzed in GenBank with Vector NTI 9.1 and NCBI BLAST software after PCR amplification.ResultsThe subtractive library of genes transactivated by PS1TP5 was constructed successfully. The amplified library contained 90 positive clones. Colony PCR showed that 70 clones contained 200-1000-bp inserts. Sequence analysis was performed in 30 clones randomly, and the full-length sequences were obtained by bioinformatics technique. Altogether 24 coding sequences were obtained, which consisted of 23 known and 1 unknown. One novel gene with unknown functions was found and named as PS1TP5TP1 after being electronically spliced, and deposited in GenBank (accession number: DQ487761).ConclusionPS1TP5 is closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, formation mechanism of hepatic fibrosis, and occurrence and development of tumor. Understanding PS1TP5 transactive proteins may help to bring some new clues for further studying the biological functions of pre-S1 protein.

Project description:Infection of humans with Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, can cause hepatitis of various levels of severity. When the three human isolates of E. chaffeensis, each belonging to a different genogroup, are inoculated into severe combined immunodeficiency mice, the order of severity of clinical signs and bacterial burden detected in the liver is as follows (from greatest to least severity and highest to lowest burden): strain Wakulla, followed by strain Liberty, followed by strain Arkansas. In this article, we used microarray analysis to define transcriptional profiles characteristic of the histopathological features in the mouse liver. Cytokine and chemokine profiles and their receptor profiles were strikingly different among the three strains of E. chaffeensis: gamma interferon, CCL5, CXCL1, CXCL2, CXCL7, CXCL9, interleukin 2 receptor gamma (IL2Rgamma), IL21R, CCR2, and CXCR6 were highly upregulated with strain Arkansas; and tumor necrosis factor (TNF), CCL2, CCL3, CCL5, CCL6, CCL12, CCL20, CXCL2, CXCL7, CXCL9, CXCL13, TNF receptor superfamily 9 (TNFRSF9), TNFRSF13beta, IL1R2, IL2Rgamma, IL20Rbeta, IL21R, CCR1, CCR2, and CXCR4 were highly upregulated with strain Wakulla. With strain Liberty, only CXCL13 was highly upregulated, and IL13Ralpha2 was downregulated. In livers infected with the Arkansas strain, monocytes/macrophages and NK cells were enriched in the granulomas and an increase in NK cell marker mRNAs was detected. Livers infected with the Wakulla strain displayed infiltration of significantly more neutrophils and an increase in neutrophil marker mRNAs. Genes commonly upregulated in liver tissue infected with the three strains are other host innate immune and inflammatory response genes, including those encoding several acute-phase proteins. Genes downregulated commonly are related to host physiologic functions. The results suggest that marked modulation of host cytokine and chemokine profiles by E. chaffeensis strains underlies the distinct host liver disease.

Project description:BackgroundMicroalgae have been extensively investigated and exploited because of their competitive nutritive bioproducts and biofuel production ability. Chlorella are green algae that can grow well heterotrophically and photoautotrophically. Previous studies proved that shifting from heterotrophy to photoautotrophy in light-induced environments causes photooxidative damage as well as distinct physiologic features that lead to dynamic changes in Chlorella intracellular components, which have great potential in algal health food and biofuel production. However, the molecular mechanisms underlying the trophic transition remain unclear.Methodology/principal findingsIn this study, suppression subtractive hybridization strategy was employed to screen and characterize genes that are differentially expressed in response to the light-induced shift from heterotrophy to photoautotrophy. Expressed sequence tags (ESTs) were obtained from 770 and 803 randomly selected clones among the forward and reverse libraries, respectively. Sequence analysis identified 544 unique genes in the two libraries. The functional annotation of the assembled unigenes demonstrated that 164 (63.1%) from the forward library and 62 (21.8%) from the reverse showed significant similarities with the sequences in the NCBI non-redundant database. The time-course expression patterns of 38 selected differentially expressed genes further confirmed their responsiveness to a diverse trophic status. The majority of the genes enriched in the subtracted libraries were associated with energy metabolism, amino acid metabolism, protein synthesis, carbohydrate metabolism, and stress defense.Conclusions/significanceThe data presented here offer the first insights into the molecular foundation underlying the diverse microalgal trophic niche. In addition, the results can be used as a reference for unraveling candidate genes associated with the transition of Chlorella from heterotrophy to photoautotrophy, which holds great potential for further improving its lipid and nutrient production.

Project description:BACKGROUND: Insect ovarioles are classified into two categories: panoistic and meroistic, the later having apparently evolved from an ancestral panoistic type. Molecular data on oogenesis is practically restricted to meroistic ovaries. If we aim at studying the evolutionary transition from panoistic to meroistic, data on panoistic ovaries should be gathered. To this end, we planned the construction of a Suppression Subtractive Hybridization (SSH) library to identify genes involved in panoistic choriogenesis, using the cockroach Blattella germanica as model. RESULTS: We constructed a post-vitellogenic ovary library by SSH to isolate genes involved in choriogenesis in B. germanica. The tester library was prepared with an ovary pool from 6- to 7-day-old females, whereas the driver library was prepared with an ovary pool from 3- to 4-day-old females. From the SSH library, we obtained 258 high quality sequences which clustered into 34 unique sequences grouped in 19 contigs and 15 singlets. The sequences were compared against non-redundant NCBI databases using BLAST. We found that 44% of the unique sequences had homologous sequences in known genes of other organisms, whereas 56% had no significant similarity to any of the databases entries. A Gene Ontology analysis was carried out, classifying the 34 sequences into different functional categories. Seven of these gene sequences, representative of different categories and processes, were chosen to perform expression studies during the first gonadotrophic cycle by real-time PCR. Results showed that they were mainly expressed during post-vitellogenesis, which validates the SSH technique. In two of them corresponding to novel genes, we demonstrated that they are specifically expressed in the cytoplasm of follicular cells in basal oocytes at the time of choriogenesis. CONCLUSION: The SSH approach has proven to be useful in identifying ovarian genes expressed after vitellogenesis in B. germanica. For most of the genes, functions related to choriogenesis are postulated. The relatively high percentage of novel genes obtained and the practical absence of chorion genes typical of meroistic ovaries suggest that mechanisms regulating chorion formation in panoistic ovaries are significantly different from those of meroistic ones.

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Suppression subtractive hybridization method for the identification of a new strain of murine hepatitis virus from xenografted SCID mice.

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Suppression subtractive hybridization method for the identification of a new strain of murine hepatitis virus from xenografted SCID mice.

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