Analysis of the distribution of messenger RNA during insect development by in situ hybridization with radioactive polyuridylic acid



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The distribution of messenger ribonucleic acid (mRNA) containing polyadenylic acid (poly(A)) sequences during the development of the hemipteran, Oncopeltus fasciatus, and the dipteran, Drosophila hydei, was studied in tissue sections by an in situ hybridization method developed as a part of this thesis. The method involves pre-treatment of the sections with DNase followed by annealling of (3H) - polyuridylic acid (poly(U)) to the poly(A) segment of mRNA in tissue sections, digestion of uncomplexed (3H) - poly(U) with ribonuclease, removal of the hydrolysis products by extraction in cold trichloroacetic acid, and visualization of the position of the poly(U):poly(A) hybrids by autoradiography. The poly(U) in situ hybridization method was concluded to be specific for the detection of poly(A) - containing mRNA sequences in tissue sections. The basis for this conclusion was the absence of silver grain development in sections which had been pre-treated with (1) alkali, which is known to destroy RNA but not DNA, (2) pancreatic ribonuclease A in a buffer of low ionic strength, which can completely hydrolyze poly(A) as well as other polyribonucleotides, and (3) T2 RNase, a ribonuclease showing relative specificity for poly(A). [...]