Reverse transcriptase problems

A solution to the problem of introns is to isolate mRNA extracted from the human pancreas cells that make insulin. These cells are rich in insulin mRNA from which introns have already been spliced out. Using the enzyme reverse transcriptase it is possible to convert this spliced mRNA into a DNA copy. This copy DNA (cDNA), which carries the uninterrupted genetic information for insulin can be cloned. Although yeast cells (Saccharomyces) can splice out introns it is normal practice to eliminate them anyway by cDNA cloning. 

The human retrovirus HIV can be controlled using chemotherapy directed at the reverse transcriptase and aspartyl protease encoded by the viral genome as with other microbial pathogens, however, resistance to drug therapy becomes a major problem. Figure 7.3 shows a crystal structure (PDB 1HXW) of the HIV protease, where mutated amino acids (shown in cyan) lead to disrupted binding of the clinically effective inhibitor ritonavir [24]. 

Indinavir (Crixivan) is a potent inhibitor of HIV reverse transcriptase. It produces the side effects common to aU protease inhibitors and also may produce nephrolithiasis, urolithiasis, and possibly renal insufficiency or renal failure. This problem occurs more fre-... 

As an example ddl (2,3-dideoxyinosine), an inhibitor of the HIV reverse transcriptase (RT-HIV), has an only 30-s half-life at pH 1 (at 37°C). Some formulations allow circumventing the problem of the consequent inefficient oral administration. A chemical alternative consists in the introduction of a fluorine atom in 2j] to disfavour the generation of the oxonium ion involved in the proteolysis (Fig. 16) [57,58], Indeed, the fluorinated analogue is stable in acidic medium while keeping the in vitro anti-viral activity. However, it seems that other problems prevented the clinical development [5,57]. 

Since eukaryotic chromosomes are linear, the ends of these chromosomes require a special solution to ensure complete replication. This can be seen in figure 26.26. At the very end of a linear duplex a primer is necessary to initiate DNA replication. After RNA primer removal there is bound to be a gap at the 5 end of the newly synthesized DNA chains. Since DNA synthesis always requires a primer the usual way of filling this gap is not going to solve the problem. This dilemma is overcome by a special structure at the ends (telomeres) of eukaryotic chromosomes and a special type of reverse transcriptase (telomerase) that synthesizes telomeric DNA. In many eukaryotes the telomeres contain short sequences (frequently hexamers) that are tan-demly repeated many times. Telomerase contains an RNA that binds to the 3 ends and also serves as a template for the extension of these ends. Prior to replication, the 3 ends of the chromosome are extended with additional tandemly repeated hexamers. The 3 ends are extended sufficiently so that there is room to accommodate an RNA primer. In this way there is no net loss of DNA from the 5 ends as a result of replication. After replication the 3 end is somewhat... 

To secure a gene from a eukaryotic genome, the most efficient method among several is to obtain cDNA (complementary DNA) from mRNA via reverse transcriptase. Use of cDNA avoids dealing with the problem of introns, where a complete gene can stretch over thousands of base pairs but is divided into several small exons, alternating with introns of sometimes remarkable size [several thousand base pairs (kB) for one intron]. As, however, only a few eukaryotic genes are fully sequenced and deposited in one of the databases, one has to rely on ESTs (expressed... 

Some viruses have secondary structure, which can prevent the production of cDNA detectable in a PCR assay by early termination of the synthesis reaction. To overcome this problem one can raise the temperature of incubation used in first-strand synthesis to 42°C or higher. This will reduce some secondary structures, but will also reduce the half-life of the reverse transcriptase. AMV reverse transcriptase may be used instead, because it has an optimal temperature of 42°C. Unfortunately, AMV RT has more endogenous RNaseH activity than M-MLV RT, thus on average AMV RT produces shorter cDNA fragments. RNaseH deficient RT enzymes are also available (e.g., the Superscript enzymes from Invitrogen), and there is some evidence that these may be the most sensitive type of RT enzymes for PCR assays. The RT conditions required for the efficient detection of individual viruses can only be determined empirically. 

Obviously, an adaptation of Loeb s scheme to the selection of RNA polymerases (DNA-dependent transcriptases, or RNA-dependent replicases), reverse transcriptases, or DNA polymerases with altered substrate tolerance is difficult because these enzymes hardly enable the conferring of a growth advantage to host cells. In an exceptional approach to this problem, Andrew Ellington and colleagues focused... 

One strategy to address these problems is to develop antiretroviral drugs with targets other than reverse transcriptase and protease. In the collaboration of our laboratories at the University of North Carolina and Panacos Pharmaceuticals, we have taken advantage of the huge molecular diversity found in natural products. Plants are a major source of biologically active compounds and can provide good leads that are structurally unique and/or have new mechanisms of action. 

An enzyme that catalyzes the formation of DNA from an RNA template. This is an enzyme critical for the replication of RNA viruses such as HIV and is a major drag target for AIDS and other RNA viral diseases. See O Conner, T.E., Reverse transcriptase-progress, problems, and prospects, Bibl. Haematol. 39, 1165-1181, 1973 Wu, A.M. and Gallo, R.C., Reverse transcriptase, CRC Crit. Rev. Biochem. 3, 289-347, 1975 Verma, I.M., The... 

As discussed in Chapter 1, polymorphism is a common problem encountered in the synthesis of pharmaceuticals. In the process development for a drug candidate of reverse transcriptase inhibitor, six crystal forms were identified. The first pilot plant batch produced all Form III, not the desired Form I. The purpose of this example is to illustrate the development of a robust crystallization process to consistently grow the desired crystal form. 

Although this amplification technique is very sensitive and has tremendous application potential, it is not without problems. The powerful amplification procedure may yield false-positive results when samples are contaminated by nucleic acid left over from previously amplified DNA. Other problems include primer artifact formation and nonspecific hybridization of primers to DNA samples. Several modifications to the original PCR technology have been made over the years to improve the sensitivity and application potential for PCR, including the use of multiple sets of amplification primers, multiplex PCR, PCR amplification of RNA by converting targeted RNA with reverse transcriptase to complementary DNA templates (which are then suitable for DNA amplification by traditional PCR techniques), and real-time quantitative PCR. 

Telomeres, the physical ends of linear chromosomes, consist of tandem arrays of a short DNA sequence, TTAGGG In vertebrates. Telomeres provide the solution to the end-repllcatlon problem—the Inability of DNA polymerases to completely replicate the end of a double-stranded DNA molecule. Telomerase, a reverse transcriptase that contains an RNA template, adds TTAGGG repeats to chromosome ends to lengthen or maintain the 5- to 20-kb regions of repeats that decorate the ends of human chromosomes (see Figure... 

Ribonucleic acid is a difficult nucleic acid to work with because it is very sensitive to RNases that are prevalent in the lab (your hands are a good source of RNases ) and the techniques for sequencing RNA are indirect. To circumvent these problems, mRNA can be converted into cDNA using an enzyme called reverse transcriptase (Chapter 30). Reverse transcriptase is an RNA-dependent DNA polymerase that uses an RNA template to synthesize cDNA. The single-stranded cDNA... 

Another problem that has arisen with prolonged use of AZT is that AZT-resistant mutants of the virus appear. It is well known that HIV is a virus that mutates rapidly. Some of these mutant forms of the virus have an altered reverse transcriptase that wiU no longer use AZT. When these mutants appear, AZT is no longer useful in treating the infection. 

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