Ribonuclease cDNA synthesis

Drought also has a profound effect on protein synthesis. In many plant tissues, a reduced water potential causes a reduction of total protein synthesis and a rapid dissociation of polyribosomes. The latter has been shown not to be the consequence of increase in ribonuclease activity (Hsiao, 1973 Dhindsa Bewley, 1976). For a specific protein, Jacobsen, Hanson Chandler (1986) have shown in barley leaves that water stress enhances the synthesis of one of the a-amylase isozymes. Using a cDNA probe they found that water-stressed leaves contained much more a-amylase mRNA than unstressed plants. 

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. 

In the ribosubstitution method these problems are circumvented by the addition of one or more ribonucleotides between the DNA primer and the radioactive cDNA. This site is susceptible to cleavage with ribonuclease or alkali. This method can also be used in conjunction with other primed synthesis methods for DNA sequencing (Barnes, 1978 Sanger, Nicklen and Coulson, 1977). 

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