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October, 2006
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Adenine Nucleotides:

Complementary DNA

JoVE 10818

Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to study gene expression in different cells and tissues. The scientist converts mRNA into complementary DNA (cDNA) via reverse transcription. Because mRNA does not contain introns (non-coding regions) and other regulatory sequences, cDNA—unlike genomic DNA—also allows researchers to directly determine the amino acid sequence of the peptide encoded by the gene. cDNA can be generated by several methods, but a common way is to first extract total RNA from cells, and then isolate the mRNA from the more predominant types—transfer RNA (tRNA) and ribosomal (rRNA). Mature eukaryotic mRNA has a poly(A) tail—a string of adenine nucleotides—added to its 3’ end, while other types of RNA do not. Therefore, a string of thymine nucleotides (oligo-dTs) can be attached to a substrate such as a column or magnetic beads, to specifically base-pair with the poly(A) tails of mRNA. While mRNA with a poly(A) tail is captured, the other types of RNA are washed away. Next, reverse transcriptase—a DNA polymerase enzyme from retroviruses—is used to generate cDNA from the mRNA. Since, like most DNA polymerases, reverse transcriptase can add nucleotides only to the 3’ end of a chain, a pol

 Core: Biotechnology

pre-mRNA processing

JoVE 11003

In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA, to distinguish it from mature mRNA.

Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it. This 5’ cap helps the cell distinguish mRNA from other types of RNA in the cell and plays a role in subsequent translation. During or shortly after transcription, a large complex called the spliceosome cuts out various parts of the pre-mRNA transcript, rejoining the remaining sequences. RNA sequences that remain in the transcript are called “exons” (expressed sequences) while portions removed are called “introns”. Interestingly, a single RNA segment can be an exon in one cell type and an intron in another. Similarly, a single cell can contain multiple variants of a gene transcript that has been alternatively spliced, enabling the production of multiple proteins from a single gene. When transcription is completed, an enzyme adds approximately 30-200 adenine nucleotides to the 3’ end of the pre-mRNA molecule. This poly-A tail protects the mRNA fr

 Core: Gene Expression
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