Design and cloning of hammerhead ribozymes targeted to NL43 HIV-1 Vif MRNA.HIV HIV (Human Immunodeficiency Virus), either of two closely related retroviruses that invade T-helper lymphocytes and are responsible for AIDS. There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for the vast majority of AIDS in the United States. , the etiological agent of the Aquired Immunodeficiency Syndrome (AIDS) poses a worldwide threat to public health. The ability of HIV to adapt to novel antiviral drugs makes gene therapy and the use of ribozymes a promising avenue of research in the battle against AIDS. Hammerhead ribozymes are RNA RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic molecules that act as enzymes, catalyzing the substrate-specific cleavage of substrate RNA. Ribozyme Ribozyme A ribonucleic acid (RNA) molecule that, like a protein, can catalyze specific biochemical reactions. Examples include self-splicing rRNA and RNase P, both involved in catalyzing RNA processing reactions (that is, the biochemical reactions that convert targeting HIV RNAs could potentially inhibit viral replication, reducing HIV expression. Vif (Virion virion Entire virus particle, consisting of an outer protein shell (called a capsid) and an inner core of nucleic acid (either RNA or DNA). The core gives the virus infectivity, and the capsid provides specificity (i.e., determines which organisms the virus can infect). Infectivity Factor), one of fifteen distinct proteins encoded by the HIV genome, enhances viral infectivity by blocking a host antiviral defense pathway that relies on the host protein APOBEC3G. Vif's ability to inhibit this cellular retroviral defense mechanism makes it a potentially advantageous target for ribozyme-mediated down-regulation. A catalytic and a non-catalytic(A) hammerhead ribozyme targeted to a GUU at nucleotide 5154 of the Vif sequence were designed based on the model of Haseloff and Gerlach (Nature, 1988). These ribozymes differed by a single nucleotide in the catalytic core. These ribozymes were synthesized and used as templates in PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) reactions to generate double-stranded DNA DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. . Each was cloned into pPCR-Script by blunt-end ligation and transformed into XL-10 Gold Ultracompetent cells. Several colonies were analyzed for the presence of ribozyme-positive plasmid DNA by PCR using an M13 primer mix. Ribozyme orientation was determined by PCR using M13 and ribozyme-specific primers. Two clones were subsequently sequenced to verify ribozyme presence. These plasmids, pVif5154 Rz and pVif5154A Rz will be used in in vitro cleavage reactions to measure ribozyme kinetics. Pending the outcome of these tests, future studies may also include tissue culture testing of these ribozymes * Supported by NIH Grant 1R15 GM66678-01 |
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