ABSTRACT
<p><b>OBJECTIVE</b>To investigate the function of hepatitis B virus polymerase (HBV Pol) in the viral life cycle by screening the proteins interacting with HBV polymerase.</p><p><b>METHODS</b>The HBV Pol gene was constructed into the pGBKT7 vector. GAL4 yeast two-hybrid system was used to screen the human liver cDNA library to obtain proteins which interacted with HBV Pol. GST-pull down assay was applied to confirm the protein interactions.</p><p><b>RESULTS</b>Ubiquitously expressed transcript (UXT) was selected by the yeast two-hybrid system. GST-pull down assay confirmed the in vitro interaction between HBV Pol and UXT.</p><p><b>CONCLUSIONS</b>UXT is a potential interactor of HBV Pol, and this protein interaction may provide clues of the function of HBV Pol in HBV life cycle.</p>
Subject(s)
Humans , Gene Products, pol , Metabolism , Hepatitis B virus , Neoplasm Proteins , Metabolism , Protein Interaction Mapping , Two-Hybrid System Techniques , Virus ReplicationABSTRACT
<p><b>OBJECTIVE</b>To explore relevant between human cytomegalovirus (HCMV) infection and college students' neurobehaviors.</p><p><b>METHODS</b>87 college students were enlisted. They were tested with Bole. Neurobehavioral evaluation system (B. NES), and HCMV IgG antibody was detected after separation of serum. We analyzed the test results of B. NES by SPSS software.</p><p><b>RESULTS</b>76 college students were infected by HCMV in the past and 11 college students were not infected. The infected group scored 8.89 +/- 6.60 in depression aspect of emotion state test, while control group got 15.73 +/- 9.00. There was Significant difference between infection group and control (P < 0.05). There were no significant differences in other aspects of emotion states, study and memory, perception and mental movement (P > 0.05).</p><p><b>CONCLUSION</b>HCMV infection is associated with depression status.</p>
Subject(s)
Adult , Female , Humans , Male , Cytomegalovirus Infections , Psychology , Emotions , Learning , Memory , Students , Psychology , UniversitiesABSTRACT
<p><b>OBJECTIVE</b>To determine the genes in which exist overlapping ORF in Merlin strains of human cytomegalovirus, and to reveal their structure and functional characteristics.</p><p><b>METHODS</b>We search for overlapping genes of ORF in HCMV Merlin strains' whole genome by Bioinformatics methods, analyzing coding sequence CDS and starting and ending sites of ORF, calculating the length of CDS and ORF, analyzing the molecular weight of encoding protein, overlapping length and coding direction of protein, identifying overlapping sequences and overlapping types, analyzing the expression phase of overlapping genes and the function of proteins.</p><p><b>RESULTS</b>There were 39 overlapping ORF genes in HCMV Merlin strains, accounting for 23% of total genes. Among these 39 genes, there are 13 IE genes, 9 E genes and 17 L genes, which can be divided into 16 contigs. There are 11 contigs when two genes overlap, with 3 contigs in three genes overlapping, and 2 contigs in four genes overlapping. The functions of overlapping genes are widely.</p><p><b>CONCLUSION</b>We found that there are a lot of complex overlapping genes in HCMV Merlin strains, which are basis for further study of the transcription and translation mechanism of overlapping genes.</p>
Subject(s)
Humans , Computational Biology , Contig Mapping , Cytomegalovirus , Genetics , Genes, Duplicate , Genetics , Open Reading Frames , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To identify the mutation of the methylmalonic aciduria (cobalamin deficiency) CblC type, with homocystinuria (MMACHC) gene in a pedigree with methylmalonic aciduria.</p><p><b>METHODS</b>The MMACHC gene mutation was detected using polymerase chain reaction (PCR) and DNA sequencing. The MMACHC gene of 50 healthy people was also sequenced as control.</p><p><b>RESULTS</b>A new mutation of 146_154 del CCTTCCTGG was found in the patient and his father, and was absent in the controls.</p><p><b>CONCLUSION</b>A new mutation (146_154 del CCTTCCTGG) in the MMACHC gene was detected in a Chinese family with methylmalonic aciduria.</p>
Subject(s)
Animals , Child, Preschool , Female , Humans , Male , Pregnancy , Amino Acid Metabolism, Inborn Errors , Genetics , Metabolism , Amino Acid Sequence , Base Sequence , Carrier Proteins , Chemistry , Genetics , Case-Control Studies , DNA Mutational Analysis , Exons , Genetics , Fathers , Methylmalonic Acid , Metabolism , Molecular Sequence Data , Mutation , Pedigree , Polymerase Chain Reaction , Protein Structure, SecondaryABSTRACT
Based on the sequence of BAC (Bacterial Artificial Chromosome) along with the Cre/lox P system, the gene-targeting vectors to multiple loci of the repetitive internal transcribed spacers between rDNA genes in Leghorn chicken were constructed. The key material of multiple loci gene targeting in vivo would be obtained. First, the plasmid of pYLSV-TDN with TK, HRDS2, and Neo genes was constructed. The TK-HRDS2-Neo DNA fragment obtained from the plasmid of pYLSV-TDN was digested by Not I/HindIII and inserted into the upstream of the lox P site of BAC plasmid for obtaining the selective vector of BAC-TDN. The expression vector of pYLVS-GID with EGFP, hIFN genes, and HRDS1 was then obtained. The plasmid of BAC-TDN-VS-GID was obtained by cotransformation of the selective vector of BAC-TDN and the expression vector of pYLVS-GID to E. coli NS3529 through the action of Cre/lox P system. The gene-targeting vector of BAC-TDN-GID to multiple loci of the ITS region in Leghorn chicken was obtained by cleaving the sequence of pYLVS with the homing endonuclease of I -Sce I and ligating with the linker of LS. The insertion and the insert direction of DNA fragments were identified by restriction digestion or PCR and sequencing in each clone. The significance of the technique ofgene-targeting vector to multiple loci are shown as follows. First, the targeting loci were increased to 100 - 300. Second, the problems of unstable expression of inserted genes were partially solved. Third, the need for safety against toxicity integration was resolved. Fourth, the forbidden zone of gene integrating on the repetitive DNA sequences was broken through.
Subject(s)
Animals , Humans , Attachment Sites, Microbiological , Genetics , Chromosomes, Artificial, Bacterial , Genetics , Cloning, Molecular , DNA , Genetics , Metabolism , DNA Restriction Enzymes , Metabolism , DNA, Ribosomal Spacer , Genetics , Escherichia coli , Genetics , Genetic Vectors , Genetics , Green Fluorescent Proteins , Genetics , Integrases , Genetics , Interferon-gamma , Genetics , Polymerase Chain Reaction , Recombinant Fusion Proteins , Genetics , Transformation, GeneticABSTRACT
mPem, a homeobox gene, is expressed in a time and stage specific manner during murine ontogeny. Pem transcripts are abundant in 7- and 8-day mouse embryos, but decrease precipitously thereafter. On Day 9 they become abundant in placenta and yolk sac, persisting there until parturition. Although Pem transcripts are not detectable in most of adult tissues, they are present in reproductive system such as testis, epididymis and ovary. This indicates a important role for Pem during embryogenesis and reproductive development. To study the function of mPem protein, we used a GAL4 based yeast two-hybrid assay to screen a 7-day mouse embryo library with full-length of mPem. 3 proteins were found interacting with mPem protein. One of theses is Mdfic. We confirmed the interaction between mPem and Mdfic in yeast and in vitro. Mdfic, MyoD family inhibitor domain containing, encodes the myoD family inhibitor domain (I-mfa domain). The interaction between mPem and Mdfic suggested they maybe form the transcriptional regulator complex to regulate embryo differentiation.
Subject(s)
Animals , Female , Mice , Pregnancy , Embryo, Mammalian , Embryonic Development , Genes, Homeobox , Genetics , Homeodomain Proteins , Chemistry , Metabolism , Protein Binding , Transcription Factors , Chemistry , Metabolism , Two-Hybrid System TechniquesABSTRACT
Human cytomegalovirus (HCMV) is extremely species specific and does not replicate in experimental animal tissues.To overcome the problem and establish suitable animal models for studying antiviral strategies,the expression of HCMV UL49 gene was explored in mice.UL49-GFP gene was subcloned into the adenovirus shuttle plasmid pDC316,the products(pDC316-UL49-GFP)were co-transfected with helper plasmid pBHGloxE1,3Cre into HEK293 cell lines by liposome reagent,recombinant adenovirus(Ad-UL49-GFP) was generated and confirmed by PCR and Western blot.Ad-UL49-GFP was propagated in 293 cells and purified.The titer of viral stocks was determined by end-point dilution assay.The purified adenoviruses were delivered into mice via the tail vein injection.Fluorescence quantitative PCR and Western blot experiments were used to examine the tissue distribution and duration of UL49 gene expression.The results showed that the recombinant adenovirus were present in vivo.The expression level in tissues arranged in descending order was liver,spleen,kidney,heart and lung.3 days after injection,the liver,spleen,kidney,heart and lung expressed protein UL49 in high lever and then declined gradually.14 days after injection,UL49 protein expression was disappear in some organs except liver and spleen.In conclusion,transgene animal model carrying UL49 gene was successfully established.Therefore,the system may be suitable for selecting anti-HCMV drugs targeting UL49 gene.
ABSTRACT
Human cytomegalovirus (HCMV) is a DNA virus and serious opportunistic pathogen for both newborn and immunocompromised individuals.To research technique for gene silence and antiviral agents, ribozyme M1GS-T6 was constructed from external guide sequences(EGSs)that consist of a sequence complementary to HCMV UL54 gene RNA and M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli. The results showed that M1GS can efficiently cleave the mRNA sequence encoding UL54 protein in vitro.