Biomedical subjects

Shaojun Yang

Publications and source records attributed to Shaojun Yang.

6 recordsLinked to original sources

PubMed2005

Using DNA microarray to study human cytomegalovirus gene expression.

DNA microarray technology has become one of the most widely used tools for functional genomics and is playing an ever increasing role in the study of viral infections and host-pathogen interactions. This paper describes the development of an oligonucleotide microarray representing all the predicted open reading frames of the human cytomegalovirus (HCMV) and an established protocol for simultaneously measuring the expression of all HCMV genes. To evaluate the performance of the HCMV array, human foreskin fibroblasts were either mock infected or infected with the HCMV AD169 or Toledo strains. Hybridizations were performed to determine the level of detection of HCMV transcripts from both the AD169 and Toledo strains and to assess reproducibility within and between slides. Overall, approximately 95% of the predicted HCMV genes produced detectable levels of mRNA, with median signal to noise and signal to background ratios of 41 and 14, respectively. Scatter plots of samples within an array and between two arrays resulted in average linear regressions above 0.95 and 0.9, respectively, indicating that data from the arrays are highly reproducible. In addition, transcripts from genes found in the Toledo strain but not in AD169 were specifically detected.

Cell Line↗

PubMed2005

Characterization of the elements and proteins responsible for interferon-stimulated gene induction by human cytomegalovirus.

Human cytomegalovirus (HCMV) infection of human fibroblast cells activates a large number of interferon-stimulated genes (ISGs) in a viral envelope-cell membrane fusion-dependent mechanism. In this study, we identified two interferon response elements, the interferon-stimulated response element (ISRE) and the gamma interferon-activated site (GAS), which act as HCMV response sites (VRS). Gel mobility shift assays showed that cellular proteins form specific and identical complexes with ISRE and GAS elements, and the binding of these complexes to ISRE and GAS is stimulated by HCMV infection. Point mutations in the consensus sequences of ISRE and GAS completely abolished their activities in response to HCMV-mediated transactivation, as well as their abilities to interact with HCMV-activated VRS-binding proteins. Interferon regulatory factor 3 does not appear to be present in the VRS-binding complexes or to be involved directly in HCMV-mediated ISG activation. Using ProteinChip technology, four potential proteins were identified, ranging from 20 to 42 kDa, in the VRS-binding complexes. The data suggest that HCMV infection activates VRS-binding proteins, which then bind to the VRS and stimulate ISG expression.

Base Sequence↗

PubMed2005

Two gamma interferon-activated site-like elements in the human cytomegalovirus major immediate-early promoter/enhancer are important for viral replication.

Human cytomegalovirus (HCMV) infection directly initiates a signal transduction pathway that leads to activation of a large number of cellular interferon-stimulated genes (ISGs). Our previous studies demonstrated that two interferon response elements, the interferon-stimulated response element and gamma interferon-activated site (GAS), in the ISG promoters serve as HCMV response sites (VRS). Interestingly, two GAS-like VRS elements (VRS1) were also present in the HCMV major immediate-early promoter-enhancer (MIEP/E). In this study, the importance of these VRS elements in viral replication was investigated. We demonstrate that the expression of the major IE genes, IE1 and IE2, is interferon inducible. To understand the biological significance of this signal transduction pathway in HCMV major IE expression, the two VRS1 in the MIEP/E were mutated. Mutant HCMVs in which the VRS elements were deleted or that contained point mutations grew dramatically more slowly than wild-type virus at a low multiplicity of infection (MOI). Insertion of wild-type VRS1 into the mutant viral genome rescued the slow growth phenotype. Furthermore, the expression levels of major IE RNAs and proteins were greatly reduced during infection with the VRS mutants at a low MOI. HCMV microarray analysis indicated that infection of host cells with the VRS mutant virus resulted in a global reduction in the expression of viral genes. Collectively, these data demonstrate that the two VRS elements in the MIEP/E are necessary for efficient viral gene expression and replication. This study suggests that although the HCMV-initiated signal transduction pathway results in induction of cellular antiviral genes, it also functions to stimulate viral major IE gene expression. This might be a new viral strategy in which the pathway is used to regulate gene expression and play a role in reactivation.

Base Sequence↗

PubMed2004

Coupling generation of cytomegalovirus deletion mutants and amplification of viral BAC clones.

Human cytomegalovirus (HCMV) genome manipulation has always been difficult. Recently, the introduction of full-length HCMV DNA into Escherichia coli as an artificial bacterial chromosome (BAC) clone has allowed reliable targeted mutagenesis. Here, we show the next generation of improvement in designing recombinant HCMV, which will also be applicable to other viral BAC clones. An inducible origin of replication linked with an antibiotic resistance marker was used as a cassette for targeted replacement of sequences within a HCMV BAC clone, TowneBAC. The origin of replication allowed for the induction of increased amounts BAC DNA that improved recovery, ease of use and transfections for mutant viruses. By specific deletion of UL147 and the recombinant GFP gene, we have shown that targeted deletion of a gene and selection for a recombinant genome are coupled with the ability to amplify the BAC clone DNA. These HCMV BAC clones were amplified approximately 10-fold. In the case of the removal of GFP from the clone TowneBAC shown in this study, the resulting BAC DNA preparation following amplification was used for successful primary cell transfection. Both parental and deletion BAC clone transfections gave similar levels of recombinant HCMV, and the GFP deletion virus replicated the same as the TowneBAC in a multi-step growth curve analysis.

Cells, Cultured↗

PubMed2003

Proteome analysis of lens epithelia, fibers, and the HLE B-3 cell line.

PURPOSE: The purpose of this study is to compare the protein composition of the B-3 line of transformed human lens epithelial (HLE) cells to that of freshly dissected HLE cells. This provides baseline data on lens cell proteins from fresh lens cells and from the B-3 cell line, which is often used as a model system for the lens. METHODS: Human lens epithelial cells adherent to the lens capsule were dissected into central (undifferentiated) and peripheral (partially differentiated) populations. Fully differentiated human lens fiber cells were isolated from the outer cortical layers of the lens. HLE B-3 cells were analyzed at several passage levels. Extracts were prepared from each cell type and the proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE). Representative gel patterns were visually compared, spots excised, and trypsin digests prepared. The peptide compositions of the digests were analyzed using either liquid chromatography electrospray ionization tandem mass spectrometry or atmospheric pressure-matrix-assisted laser desorption ionization mass spectrometry, using a liquid chromatography classic ion trap (LCQ) mass spectrometer. RESULTS: Two-DE patterns were obtained for fresh and cultured cell types. Similar patterns were observed between central and peripheral HLE cells, both of which contained high levels of alphaA-, alphaB-, and betaB2-crystallins; alpha-enolase; and aldehyde dehydrogenase. HLE B-3 cultured cells were characterized by a marked loss of crystallins and a relatively higher level of noncrystallin proteins--most notably, high molecular weight, acidic proteins. Whereas subunit d of adenosine triphosphate (ATP) synthase, alphaB-crystallin, galectin, glyceraldehyde-3-phosphate dehydrogenase, alpha-enolase, actin, peptidylprolyl isomerase A, phosphatidylethanolamine-binding protein, and vimentin were present in both fresh and cultured lens epithelium, only the high abundance of alpha-enolase, galectin-1, and vimentin suggested that B-3 cells were lens derived. CONCLUSIONS: Freshly dissected noncultured HLE cells from both central and peripheral regions contain a high concentration of crystallins that mask the detection of less abundant proteins by 2-DE. Transformation and culture of HLE cells causes a loss of these crystallins and an increase in the relative concentration of other proteins. However, most of these noncrystallin proteins were different from those observed in noncultured HLE cells. These results suggest that transformation markedly alters the protein expression pattern in immortalized HLE cells and that caution should be exercised when using them to study properties of HLE cells in vivo.

Aged↗

PubMed2002

Changes in three types of ubiquitin mRNA and ubiquitin-protein conjugate levels during lens development.

Ubiquitin is a small, highly conserved protein that covalently attaches to other proteins to form a unique branched protein structure. The best characterized function of this post-translational modification is to mark the modified protein for degradation by the proteasome. To investigate whether ubiquitin genes are regulated in lens development, the authors analyzed the levels of three ubiquitin mRNAs (UbA(52), UbB and UbC) in freshly dissected fiber and epithelial cells, and in epithelial explants induced to differentiate ex vivo. Explants, comprising the capsule and adherent epithelial cells, were dissected from lenses of 3 day old Sprague Dawley rats and cultured +/-bFGF to induce differentiation. Quantitative competitive RT-PCR was used to determine the mRNA levels in fresh and cultured cells. UbA(52), UbB and UbC mRNAs were 3.2 (P < 0.0001), 5.0 (P < 0.0001) and 6.8 (P < 0.0001) fold higher, respectively, in freshly dissected epithelial cells than in differentiated fiber cells. Immunological spot assays showed that ubiquitin protein is over two fold as high in rat pup lens epithelial cells as in fiber cells. The ubiquitin protein in fiber cells of adult rat is lower than that in adult epithelium and in pup fiber cells, indicating that ubiquitin content further decreased during lens fiber maturation. Western blots showed a greater amount of protein-conjugated ubiquitin (MW > 81 kD) in epithelial cells than in fiber cells, demonstrating a parallel pattern between the expression of ubiquitin mRNA, the level of ubiquitin protein and the level of conjugates in the cells. Epithelial cell explant cultures permit study of cells initiating differentiation. In contrast to fully differentiated fiber cells, explant cultures induced to initiate differentiation underwent differential up-regulation of ubiquitin gene expression. UbA(52) and UbB mRNA levels in +bFGF (differentiating) explant cultures were 2.6 (P < 0.001) and 1.4 (P < 0.001) fold higher, respectively, than those of -bFGF cultures. UbC mRNA content was similar in explants cultured with or without bFGF. Dissection of the isolated epithelial cells into regions representing distinct populations gave results consistent with this observation of the explant results. UbA(52), UbB and UbC mRNAs are 2.0, 2.2 and 1.76 fold higher, respectively, in the peripheral (initiating differentiation) than in the central (undifferentiated) region of epithelial cells. These results together indicate that UbA(52) and UbB mRNAs are transiently increased during the initiation and early stages of differentiation. However, UbC mRNA appears to be relatively unaffected at the earliest stage in this differentiation model and may have a different distribution than UbA(52) and UbB in the anterior lens cells. These data are consistent with an important role for ubiquitin during the early stages of lens differentiation. The selective expression indicates that the three genes have specific differentiation related functions.

Animals↗