Reverse transcriptase nucleotide sequencing

Abbreviation Bp, nucleotide base pairs cDNA, complementary DNA ChIP, chromatin Immunoprecipi-tation Cy5, cyanine 5-dCTP Cy3, cyanine 3-dCTP ESTs, expressed sequence tags FDR, false discovery rate MIAME, minimum information about a microarray experiment mRNA, RNA, messenger NIA, National Institutes of Aging RFUs, relative fluorescence units RT-PCR, reverse transcriptase polymerase chain reaction SAGE, serial analysis of gene expression SAM, significance analysis of microarrays... 

Initiation of reverse transcription in HIV-infected cells relies on a critical RNA-RNA interaction between tRNA y s, which is preferentially packaged into the viral particle, and a specific viral RNA seqnence. The 3 -terminaI 18 nucleotides of tRNA y are complementary to the primer binding site (PBS) sequence located in the 5 -Iong terminal repeat (LTR) of the viral RNA genome (Figure 10.3). The UUU anticodon of the tRNA is complementary to and binds to an adenosine rich loop located 8 nucleotides upstream (5 ) of the PBS. This RNA-RNA duplex which is formed when tRNA y s binds to the PBS fits within the active site of HIV-1 reverse transcriptase, bnt mnitiple interactions between the viral RNA and tRNA y are necessary for efficient initiation of reverse transcription. This interaction nucleates the reverse transcription complex which contains viral RNA, reverse transcriptase, tRNA y pl , nncleocapsid p7, and Vpr (Viral protein R), as well as multiple host factors." ... 

Figure 1 illustrates how SELEX is used to select an RNA species that binds tightly to ATR In step (1), a random mixture of RNA polymers is subjected to unnatural selection by passing it through a resin to which ATP is attached. The practical limit for the complexity of an RNA mixture in SELEX is about 1015 different sequences, which allows for the complete randomization of 25 nucleotides (425 = 1015). When longer RNAs are used, the RNA pool used to initiate the search does not include all possible sequences. RNA polymers that pass through the column are discarded those that bind to ATP are washed from the column with salt solution and collected. The collected RNA polymers are amplified by reverse transcriptase to make many DNA complements to the selected RNAs then an RNA polymerase makes many RNA complements of the resulting DNA molecules. This new pool of RNA is subjected to the same selection procedure, and the cycle is repeated a dozen or more times. At the end, only a few aptamers, in this... 

About 45% of the sequence of the RNA polymerase encoded by phage T7, which transcribes RNA from the phage DNA, is also similar to that of the Klenow fragment. Sequences of these DNA polymerases are distantly related to those of reverse transcriptases.279280 The 136-kDa polymerase y functions in mitochondria but is encoded in a nuclear gene. It is the only DNA polymerase that is inhibited by antiviral nucleotide analogs such as AZT (Box 28-C).280a b... 

Telomerase is a reverse transcriptase that copies the DNA sequence of the telomeric repeats from a small guide RNA that is part of the enzyme. Tire first telomerase studied was the relatively abundant enzyme from Tetrahymena. It contains a 159-nucleotide RNA with the sequence 5 -CAACCCCAA-3 at positions 43-51. This sequence is complementary to the 5 -TTGGGG-3 repeat sequence of the Tetrahymena telomeres.450 451 A 127-kDa human protein contains a similar guide RNA with the sequence 5 -CUAACCCUACC-3, which is complementary to the human telomere repeat sequence as is illustrated in Fig. 27-21.452-454 Telomerases455 456 evidently allow the cell to elongate the telomere 5 -ends using the... 

Determining amino acid sequences of proteins by classic procedures is a tedious process. In addition, proteins are often insoluble (e.g., membrane proteins) or cannot be easily purified. However, if the protein s gene, mRNA, or cDNA of the mRNA are available, the amino acid sequence of the protein can be determined from nucleotide sequences rapidly and unambiguously using the universal genetic code. In this code, each amino acid in correlated with one or more nucleotide triplets in mRNA (see Chapter 12). mRNA is "transcribed" from DNA and can be used to synthesize complementary (cDNA) via an enzyme called reverse transcriptase. In many cases it is easier to isolate the mRNA or cDNA of a protein than the protein itself. 

Telomerase is a ribonucleoprotein complex that exists in eukaryotic cells for the apparently sole purpose of synthesizing telomeric DNA, which consists of tandemly repeated sequences that contain clusters of G-residues and forms the ends of chromosomes. Telomerase comprises two essential core components, a protein subunit that has reverse transcriptase (RT) activity and an RNA sequence (hTR) that contains clusters of C-residues and serves as the template substrate for the RT (6). The G-rich DNA and C-rich RNA anneal to form a partial duplex with DNA as the primer. RT-mediated polymerization of dGTP and other complementary triphosphate substrates produces a DNA terminus that has been extended by around six nucleotides. The new end can become a substrate for either another round of telomerase-mediated elongation or primase/polymerase-mediated lagging-strand synthesis. 

Enzymatically produce mRNA/DNA hybrids The mRNAs in the extract are used as a template, or guide, for the production of complementary DNA. Reverse transcriptase uses mRNA as a template and catalyzes the sjmthesis of a strand of DNA bearing a sequence of nucleotide bases complementary to that of the mRNA. The reaction is carried out in a test tube. At this point, one has a hybrid structure containing a strand of mRNA and a strand of DNA. The cloning procedure, on the other hand, requires the use of dsDNA, not the mRNA/DNA hybrid. The reaction mixture is then made mildly alkaline and incubated. Ibis procedure destroys (hydrolyzes) the mRNAbut not the DNA. 

The identification of the modified residue is established when the sample is co-electrophoresed with a sequence (Section 4.4.3.1.3). Since the reverse transcriptase stops or pauses 3 of the modified band, the dideoxy sequencing bands are displaced by one base relative to the modified base, i.e. if a residue is modified the corresponding band will appear as one nucleotide shorter than the base will appear in the sequence lane. 

Fig. 5.8. Primer extension analysis of a modification that does not affect reverse transcriptase extension directly, (a) 2 -0-methylation, which renders the G insensitive to RNase T1 digestion, is used as an example. Analysis of a methylated G where there are no G s for 12 nucleotides 3 of the methylated G. (b) Analysis of a G modification in a sequence without other G s for at least 12 nucleotides around the modified G. The latter situation only detects the presence of the modification but does not allow quantification.

---