Ribonuclease gene synthesis

Synthesis of cDNA, usually in radiolabeled form is accomplished with reverse transcriptase, the enzyme from retroviruses that synthesize a DNA-RNA hybrid from ssRNA.570 572 A short oligo (dT) primer is usually hybridized to the 3 poly (A) tail to initiate synthesis. Reverse transcriptase also has ribonuclease (RNase H) activity and will digest away the RNA. If desired, synthesis of the second strand can be carried out by a DNA polymerase to give a complete DNA duplex. Many gene sequences have been deduced from cDNA copies. 

After synthesis, the pre-tRNA molecule folds up into the characteristic stem-loops structures (Fig. 1) and non-tRNA sequence is cleaved from the 5 and 3 ends by ribonucleases. In prokaryotes, the CCA sequence at the 3 end of the tRNA (which is the site of bonding to the amino acid) is enclosed by the tRNA gene but this is not the case in eukaryotes. Instead, the CCA is added to the 3 end after the trimming reactions by tRNA nucleotidyl transferase. Another difference between prokaryotes and eukaryotes is that eukaryotic pre-tRNA molecules often contain a short intron in the loop of the anticodon arm (Fig. 4). 

Nambiar KP, Stackhouse J, Stauffer DM, Kennedy WP, Eldredge JK, Benner SA. Total synthesis and cloning of a gene coding for the ribonuclease S protein. Science 1984 223(4642) 1299-1301. 

Johnson PA, MacLean C, Marsden HS, Dalziel RG, Everett RD (1986) The product of gene USll of herpes simplex virus type 1 is expressed as a true late gene. J Gen Virol 67 871 883 Kerr IM, Brown RE (1978) pppA2 p5 A2 p5 A an inhibitor of protein synthesis synthesized with an enzyme fraction from interferon-treated cells. Proc Natl Acad Sci USA 75 256 260 Khabar KS, Dhalla M, Siddiqui Y, Zhou A, Al-Ahdal MN, Der SD, Silverman RH. Williams BR (2000) Effect of deficiency of the double-stranded RNA-dependent protein kinase, PKR, on antiviral resistance in the presence or absence of ribonuclease L HSV-1 replication is particularly sensitive to deficiency of the major IFN-mediated enzymes [In Process Citation]. J Interferon Cytokine Res 20 653-659... 

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