Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1998-04-13
2000-10-17
Riley, Jezia
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, 435 912, 536 221, 536 231, 536 253, 536 2532, 536 243, 536 2433, C12Q 168, C12P 1934, C07H 1900, C07H 2102, C07H 2104
Patent
active
061329959
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for determining the activity of certain DNA polymerizing enzymes in human or animal body liquids, cell samples or samples from virus infected cultures, these enzymes being caracterized by high processivity. The invention also relates to the use of said method for isoenzyme-typing. The invention also relates to use of said methods for diagnosis and prognosis of tumour deseases or deseases which are caused by or associated with virus infections such as AIDS, T cell leukaemia and herpes virusinfections.
BACKGROUND OF THE INVENTION
Reversed transcriptase, in the following called RT, is a critical enzyme which is responsable for the synthesis of DNA from viral RNA for all retroviruses, including human immune deficiency virus (HIV). Three different enzymatic activities are involved in this process: synthesis of the first DNA strand, degradation of the viral RNA strand in the DNA/RNA hybride and synthesis of the second DNA strand. See e.g. S. P. Goff, (1990) Review, J. AIDS 3, pages 817-831 for an overview.
The RNA and DNA dependent polymerase activities are obviously mediated by the same catalytical group, whereas RNas H activity is mediated through a special part of the molecule. Se e.g. J. Hansen et al. EMBO J. (1988) 7, pages 239-243, M. S. Johnson et al. (1986) PNAS 83, pages 7648-7652. HIV 1 RT appears as a heterodimer in virions consisting of two polypeptides, p66 and p51, with the same N terminals [see e.g. M. M. Lightfoot et al. (1986) J. Virol, 60, pages 771-775, Veronese et al. (1986) Science 231, pages 1289-1291]. p51 is generated by viral protease cleavage of the p66-polypeptides in the dimer of p51 and p15 (see e.g. Mous). In the heterodimer p66 catalyses the polyemerase reaction, but the same sequence in p51 does not [see e.g. Le Grice et al. (1991) EMBO J. 10, pages 3905-3911, Hostomsky et al. (1992), Science 256, pages 1783-1790]. It is assumed that p51 is a part of the binding site for the tRNA primer and a part of the template-primer binding site (see e.g. Kohlstaedt 1992). The RNA-ase H peptid activity in the p66/p51 heterodimer is locatized to the C terminal part of p66 (see e.g. Hansen 1988).
Analysis relating to RT activity has become an accepted technic for detectioning and quantification of retrovirus in cell cultures (see e.g. Pioesz 1980 and Barr-Sinoussi 1983). Together with the p24 antigen test it is used as a confirming test for HIV isolation (see e.g. Jackson 1988, Gupta 1987). RT is also the main target in attempts to find efficient anti-virales against HIV. Many attempts have been made to find selective inhibitors of this enzyme. Although no cure for AIDS not yet has been found, a valuable effect is achieved by treating patients with 3'-azido-3'-deoxy thymidine (AZT). It has, however, been found that treatment with AZT only in a rather short period of treatment (from 3 months to 1 year) produces therapy resistant HIV, with mutated RT. RT tests are presently used for evaluation of reaction mechanisms in potential antivirals. Another great potential use of RT analysis is consequently characterisation of enzymes from therapy resistant mutating viruses.
Conventional measurement of RT activity is carried out with the enzyme in solution, an artificial template/primer construct and tritiated deoxynucleoid triphosphate as the nucleotide substrate (see e.g. Baltimore 1971, Moon & Lee). this system is based on detecting incorporation of radio activity in RNA/DNA hybrides which can be precipitated with trichloro acetic acid (TCA). By using .beta. emitting nucleotides scintillation liquids can be used for detecting radio activity, but this often results in poor reproducability depending on extinction problems. The standard test is coparatibly labor consuming and can not readily be adapted to large scale investigation of a large number of samples. It is also very sensitive to the effects of disturbing factors in the enzyme samples. The latter are often the critical factor for the determination of RT activity in extracts from infected cell cultures o
REFERENCES:
patent: 5503978 (1996-04-01), Schneider et al.
Borukhov et al. "Mapping of trypsin cleavage and antibody-binding sites and delineation of a dispensable domain in the beta subunit of Escherichia coli RNA polymerase" Journal of Biological Chemistry, vol. 266, No. 35, pp. 23921-23926, Dec. 1991.
Porstmann et al. "A sensitive non-isotopic assay specific for HIV-1 associated reverse transcriptase" Journal of Virological Methods, vol. 31, pp. 181-188, 1991.
J. Lennerstrand, A.S. Rytting, C. Orvell, J.S. Gronowitz, and C.F.R. Kallander, "A Method for Combined Immunoaffinity Purification and Assay of HIV-1 Reverse Transcriptase Activity Useful for Crude Samples"-Analytical Biochemistry 235, 141-152 (1995), Article No. 0106.
Gronowitz Jan-Simon
Kallander Clas
Lennerstrand Johan
Cavidi Tech AB
Riley Jezia
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