※ 引述《pop909 (....)》之銘言： 4. Gene expression of eukaryotic cells are reflected in forms of RNA(hnRNA) whose molecular weight is much higher than that of mRNA. Biochemical study of hnRNA splicing is processed by the participation of small nuclear RNA. Dr. Chow and her adviser approached RNA splicing with electron microscopic technique and cell fractionation. Could you figure out a general scheme of their study? hnRNA is a nuclear precursor to the cytoplasmic mRNA, now we know hnRNA experienced "RNA process",which delete intron and conjugate exon. So the molecular weight of hnRNA is much higher than that of mRNA. Dr. Chow's experiment, which was based on hybridization and electron microscopy (EM), went as follows. First they would form an R-loop between Ad2 mRNAs and the Ad2 genomic DNA. An R-loop is formed when an mRNA hybridizes to its coding region in double-stranded DNA and forms a bubble in which one side contains the DNA-RNA hybrid and the other side contains the displaced single strand of DNA. They knew that at the 3-end of the mRNA there would be a poly-A tail that would not hybridize to the DNA, but whose presence could be detected by hybridization to a single-stranded DNA probe. Their interest lay not in the 3-terminal poly-A tails, but rather in the 5-terminal sequences. They guess was that the hypothetical "transcription primers" that gave rise to the 5-terminal sequences were actually encoded as all or part of the VA RNA sequences (two small viral RNAs of no known function). These were known to map around 28% on the genome and so they chose to use a long single-stranded fragment of Ad2 DNA containing this region as a probe. Thus if the two regions of the mRNA really were encoded at disparate locations in the genomic DNA then the resulting R-loops should have a single loop in the single stranded DNA probe attached to one end of the R-loops. Reference: http://www.bioinfo.org.cn/book/Great%20Experments/great7.htm 大概講解一下 hnRNA就現在的理解是直接由DNA Transcription 未經RNA Splicing 的 pre-mRNA 所以保留了Intron與Exon 因此分子量比只有Exon的mRNA大了許多倍 這題問如何用電顯的方法"看"到這個現象 所用的方法 就是利用被某病毒(應該是Ritrovirus類的)感染的細胞 此細胞自己的GENE Expression 被surpress 而病毒的gene在細胞內大量表現 細胞核內出現大量病毒的hnRNA與反轉錄的cDNA 細胞質內則有病毒的mRNA 如果用病毒的DNA當作probe(即做處理使此段DNA能被電顯看見) 利用此DNA probe 與mRNA Hybridization(註一) 則用電顯看到的結果 如普生課本p309 fig15.15(b) Intron會形成一個"R-loop" 註一 DNA-RNA hybridization When a mixture of DNA and RNA is heated to denaturation temperatures to form single strands and then cooled RNA can hybridize (form a double helix) with DNA that has a complementary nucleotide sequence. Useful for determining relationships between DNAs and RNAs. --

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