Cloning of E6/E7 Genes of Human Papillomavirus Type 16 DNA

Abstract

To obtain the specific probe which can detect human papillomavirus type 16 (HPV-16)-specific transctipts encoding for E6 and E7 genes in cells or cell lines containing HPV-16 DNA, we cloned 0.57-kbp fragment (nucleotides 198-767) of the HPV-16 DNA into pUC18 and named pHPV-1616-E6E7R. To test the specificity of the cloned probe comparded with whole HPV-16 genome, we cultured normal human oral keratinocytes, and HOK-16B cells, human oral keratinocytes immortalized by transfection with recominant HPV-16 DNA, in koratinocyte growth medium supplemented with pituitary extract, isolated poly(A^(+))RNAs from the cells, and hybridized them with the cloned probe and whole HPV-16 genome. Northern blot analysis showed that multiple poly(A^(+))RNAs hybridized to whole HPV-16 genome were expressed from the HPV-16-immortalized HOK-16B cells, while only two transxripts with sized of 1.9-kb and 1.6-kb hybridized to HPV-16 E6/E7 gene fragment were detected from the cells. However, both probes did not detect the vtral transctipts in normal human oral keratinocytes which are anot infected with HPV-16. These data indicate that 0.57-kbp fragment representing HPV-16 E6/E7 gense can be used to detect HPV-16-specific transctipts encoding for E6 and E7 genes in cells or cell lines contraiing HPV-16 DNA.