RESUMO
X-arrestin (arrestin-3) is an arrestin present specifically in the outer segments of red-, green-, and blue-cone photoreceptors. The X-arrestin gene is on Xcen-q22, and consists of 17 exons with a promoter containing a TATA box and elements important for photoreceptor expression, including three CRX and one PCE-1-like element. In order to delineate the promoter structure necessary for the pan-cone-specific expression of X-arrestin, the expression of the gene in retinoblastoma cell lines was investigated, and a structure-function analysis of the promoter was conducted in the appropriate cellular substrate. Expression of X-arrestin was detected at a low level in the Y79 retinoblastoma cell line but not in the WERI retinoblastoma cell line. Truncation and expression analysis of the X-arrestin promoter in Y79 showed maximal activity in the proximal 378-bp region containing the CRX and PCE-1-like elements upstream of the TATA and CAAT boxes and a negative regulator in the distal 1-2-kbp region. Mutagenesis of the three CRX and PCE-1-like elements and expression analysis demonstrated complete elimination of the promoter activity. Mutagenesis of the TATA box and PCE-1-like element individually resulted in similar decrease in promoter activity, but the decrease in the promoter activity was greater when the CRX elements were mutagenized with a 5' to 3' spatial gradient in the negative effect, suggesting a cooperative effect of the three CRX elements. The regulation of expression from this promoter may involve the binding of a multi-protein enhanceosome complex at the CRX triplet and the PCE-1-like element, resulting in the recruitment and activation of the RNA polymerase II complex at the downstream TATA box.
Assuntos
Arrestinas/genética , Mutação , Regiões Promotoras Genéticas/genética , Sequência de Bases , Sítios de Ligação/genética , Northern Blotting , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutagênese , Proteínas Recombinantes de Fusão/genética , Retina/metabolismo , Retinoblastoma/genética , Retinoblastoma/patologia , Deleção de Sequência , Transfecção , beta-Galactosidase/genéticaRESUMO
PURPOSE: To detect simultaneously herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), cytomegalovirus (CMV), and varicella-zoster virus (VZV) in ocular specimens suspected of indicating viral infection, and to compare the results of multiplex polymerase chain reaction (PCR) with those of uniplex PCR. METHODS: Forty specimens, collected from 33 patients with clinically suspected herpes virus ocular infection, were tested. DNA was extracted from the specimens and amplified by multiplex and uniplex PCR. RESULTS: Both multiplex PCR and uniplex PCR gave the same results. Nineteen (19/33, 57.6%) patients were PCR-positive, among whom HSV-1 was detected in 13 (13/19, 68.4%) patients, and VZV in 6 (6/19, 31.6%) patients. CONCLUSION: These results demonstrated that multiplex PCR is as reliable as uniplex PCR, and is an accurate and a cost-saving method to identify several agents from a single specimen.
Assuntos
Citomegalovirus/genética , DNA Viral/análise , Infecções Oculares Virais/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Herpesvirus Humano 3/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções por Citomegalovirus/virologia , Primers do DNA/química , Feminino , Herpes Simples/virologia , Herpes Zoster/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos TestesRESUMO
PURPOSE: To examine the efficiency of particle-mediated gene transfer into rabbit corneal endothelium with a Helios gene gun system. METHODS: Using gene gun, plasmid DNA coding for green fluorescent protein(GFP) was transferred into cultured rabbit endothelial cells and rabbit corneal grafts from their endothelial site. Then the transferred corneas were transplanted as autografts. The GFP expression was detected by fluorescence microscopy. Histological examination of corneas was performed by light microscope and scanning electron microscope. RESULTS: In cultured rabbit endothelial cells, the transfection efficiency without damage to cells was about 7% under optimized helium pressure of 120 psi. In ex vivo, the GFP expression was limited to endothelial cells of the grafts at 140 psi. The histological findings from light and scanning electron microscopy of the grafts showed no severe mechanical damage in endothelial cells penetrated by gold particles. CONCLUSIONS: The helios gene gun system successfully transferred genes into corneal endothelial cells. However, further studies will be required to obtain the stable results of the particle mediated gene transfer into the endothelial cells of corneal grafts for clinical practice.
