Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mouse mammary tumor virus long terminal repeat (MMTV LTR) has been introduced into cultured murine cells, using the 69% transforming fragment of bovine papilloma virus type 1 (BPV). Transformed cells contain up to 200 copies of the chimeric molecules per diploid genome. The restriction endonuclease map of the acquired recombinants, as well as the physical structure of the DNA, indicates that the LTR-BPV molecules present in these cells occur exclusively as unintegrated, extrachromosomal episome. When a 72-base pair direct repeat "enhancer" element (derived from the Harvey sarcoma retrovirus) was included in the MMTV LTR-BPV chimeric plasmids, DNA acquired through transfection, with a single exception, was integrated or rearranged or both. The transcriptional potential of the episomal MMTV promoter present in these cells was tested in two ways. First, steady-state levels of MMTV-initiated RNA were measured by quantitative S1 mapping. Second, the relative number of transcription complexes initiated in vivo was determined by using a subnuclear fraction highly enriched for MMTV-BPV minichromosomes in an in vitro transcription extension assay. Both approaches showed that the MMTV LTR present in the episomal state was capable of supporting glucocorticoid hormone-regulated transcription. We have therefore demonstrated the hormone response for the first time in a totally defined primary sequence environment. Significant differences both in the basal level of MMTV-initiated transcription and in the extent of glucocorticoid induction were observed in individual cell lines with similar episomal copy numbers. These phenotypic variations suggest that epigenetic structure is an important component of the mechanism of regulation.
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PMID:Glucocorticoid regulation of transcription at an amplified, episomal promoter. 631 79

To date over 60 different human papilloma virus (HPV) types have been described and novel HPV genomes are still being identified. The identification and taxonomy of papilloma viruses has become increasingly complex However, some types, especially HPV-16, -18 and to a lesser extent HPV-31 and -33, which are found in a high proportion of invasive cervical cancers and their metastases, are classified as high risk types For preventive reasons it is important to identify and classify the different HPV types in clinical specimens. Many of the methods used until recently are cumbersome. In this paper we use molecular biology techniques which permit a rapid screening and typing of high risk HPVs in clinical specimens. The screening procedure is based on the very sensitive method of polymerase chain reaction. Using a set of general primers derived from the E1 open reading frame, which anneal to a large variety of human papilloma virus DNA, we can classify samples into positive or negative for the presence of HPV sequences in a single step. The typing of the high risk HPV types is achieved by restriction enzyme analysis using the endonuclease Alu I which cleaves each high risk HPV type at different sites, thus permitting the easy identification of each type after agarose gel electrophoresis.
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PMID:A rapid method for the screening and typing of high risk HPVs using molecular biology techniques. 857

The fragile histidine triad (FHIT) gene encompasses the common chromosomal fragile site FRA3B. Human papilloma virus (HPV), which is the main aetiological agent in cervical cancers, has been found to be able to integrate its genes into the chromosome 3 fragile site of cultured cells, deleting a piece of DNA which includes the FHIT gene. Eighty-six microdissected archival cervical LLETZ biopsies comprising cases of cervical intraepithelial neoplasia (CIN) 1 (n=27), CIN3 (n=30) and microinvasive carcinoma (n=29) were evaluated for HPV infection and FHIT gene loss of heterozygosity (LOH). FHIT gene LOH was detected by polymerase chain reaction (PCR) using fluorescently labelled intragenic microsatellite markers D3S1300 and D3S4103. PCR products were analysed on a semi-automated DNA sequencer using Fragment Manager(trade mark) software to determine allele loss. The HPV status of the lesions was determined by PCR using generic and type-specific primers in conjunction with restriction endonuclease digestion. The results were analysed using Epi-Info and SPSS-PC statistical analysis software. Haematoxylin and eosin-stained sections from the 86 cases were profiled for six histopathological features, some of which have been previously shown to be associated with microinvasive cancer. FHIT gene LOH was found in 36% of CIN1 cases, 52% of CIN3 cases and 73% of microinvasive cases (p=0.029). HPV 16 DNA was found in 68% of CIN3 cases and 93% of microinvasive cases (p<0.001). The second most prevalent HPV type found was HPV 31, which was present in only four lesions, three of which had FHIT gene LOH. When FHIT gene LOH was evaluated versus HPV 16 and 31 infection using the chi-square test, a statistically significant relationship was found (p=0.014). FHIT gene LOH was found to be independent of the histopathological features evaluated. The finding of a statistically significant relationship between FHIT gene LOH and oncogenic HPV infection suggests a link between the integration of viral DNA and subsequent gene deletion in the progression of cervical cancer. FHIT gene anomalies may prove to be excellent markers of progression in early uterine cervical cancers.
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PMID:Deletion of the FHIT gene in neoplastic and invasive cervical lesions is related to high-risk HPV infection but is independent of histopathological features. 1111 68

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) has high specificity to its target DNA as a gene editing tool. This characteristic makes it useful for DNA detection. Combining the advantages of CRISPR/Cas9 and PCR, this study establishes a novel CRISPR/Cas9-based DNA detection method, named CRISPR/Cas9-typing PCR version 4.0 (ctPCR4.0). This method can detect target DNA in one pot with high specificity and sensitivity. In a homogenous reaction, the target DNA is first cleaved by a pair of Cas9- single-guide RNA complexes and thus releases two single strands with free 3' ends, allowing a pair of oligonucleotides to anneal with the strands. The annealed oligonucleotides provide templates for DNA polymerization from the free 3' ends. A universal primer annealing site is thus produced at the end of two single strands. The target DNA is then amplified by PCR using a universal primer. This method was first verified by accurately detecting the cloned L1 fragments of 10 genotypes of high-risk human papilloma viruses (HPVs). This method was then validated by detecting the L1 fragments of two highest-risk HPVs, HPV 16 and HPV 18, in the genomic DNA of two HPV-positive cervical carcinoma cells, HeLa and SiHa. Finally, this method was further validated by accurately detecting 10 high-risk HPVs in 30 clinical samples.
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PMID:A One-Pot CRISPR/Cas9-Typing PCR for DNA Detection and Genotyping. 3312 24


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