Transcription is the process by which the genetic information in DNA is converted into RNA. The mechanism of transcription can be described simply in 6 steps (5 in prokaryotes): The section of DNA to be transcribed is recognized by proteins involved in transcription. Hydrogen bonds between base pairs of DNA are broken and the DNA double helix "unzips." Complimentary RNA bases bind to the now exposed DNA bases RNA Polymerase binds the RNA bases together to form a strand of mRNA Hydrogen bonds formed between the original DNA strand and the new RNA strand are broken in eukaryotes, the new RNA strand is further processed and moves to the cytoplasm. The segment of DNA to be transcribed is called a transcription unit, and contains at least one gene. If this gene codes for a protein, the RNA transcription product is called Messenger RNA, or mRNA. The gene may also code for Rybosomal RNA (rRNA), transfer RNA (tRNA), or a ribozyme.Next-> Developmental-Genetics
SplicingRNA splicing is the removal of introns or intervening sequences (parts that do not code for anything and lay between coding regions). The coding regions are known as exons (expressed sequences). At each end of an intron, there is short sequence that "small nuclear ribonucleoproteins" (snRNPs) recognize and together with other proteins that form an assembly known as a spliceosome, cut out introns and then joined the exons. Note: Only eukaryotes contain introns in the precursor of messenger RNA (mRNA). prokaryotes, such as bacteria, do not.
PolyadenylationPolyadenylation occurs in Eukaryotes to prevent RNA trasncript degradation. A number of enzymes are involved, which add hundreds to thousands of adenines to the 3' end of an mRNA transcript. Many of these adenines will be lost before translation - but enough are added to prevent degradation of the script prior to translation.
Recombinant DNA Cloning TechnologyThis term refers to the process of transferring DNA fragment of interests from one organism to a self-replicating genetic element e.g. a bacterial plasmid. The DNA of interest can then be propagated in a foreign host cell. This technology has been instigated from the 1970s, since then it has become widely used and is a common practice in molecular biology labs to this very day.
TranspositionTransposition is the integration of transposable elements into the genome. Transposable elements are DNA segments that jump around the genome and integrate themselves into different regions.
DiscoveryThe first description of mobile genetic elements in a genome was made by Barbara McClintock working at Cold Spring Harbor in the 1950s. While attempting to explain the odd phenotypic behavior of mosaic color striations on corn kernels, she came to the conclusion that there were genetic elements in corn that could move among the chromosomes. Although her experimental support was strong, her conclusions was so far from the mainstream understanding of the nature of chromosomes, that she was politely ignored. In the late 1970s the discovery of bacterial transposons directed renewed attention on her pioneering work, and her efforts were resoundingly accepted when she was awarded an unshared Nobel prize in 1983.
Uses in GeneticsTransposable elements are very useful in studying the genome. They allow researchers to search for genes and enhancers and find interesting relationships between phenotypes and genotypes. By using p-elements and transposase, DNA constructs can be formed to randomly jump around the genome. P-elements flank the DNA sequence you want to jump around and transposase is used to cut the squence out and reinsert it elsewhere. On method for using transposable elements to find various enhancer sites is to construct a transposable element that contains the Gal-4 gene with a weak promoter with a p-element upstream and downstream. Another construct with UAS site (when the gal-4 protein binds to the UAS site, anything downstream is expressed) and a marker gene downstream that can, for example, encode for a fluorescent protein is also created. By inserting these constructs into 2 strains of flies, you will have 1 strain with the transposable Gal-4 gene and another strain with the stationary UAS construct. A third mouse strain containing transposase gene is needed. Strain 1: Transposable Gal-4 strain Strain 2: UAS marker Strain Strain 3: Transposase Strain First, strains 1 and 3 are crossed to produce flies with both the p-elements and the transposase. This allows the construct with gal-4 to jump around to random locations in the genome and depending on the location of gal-4, the amount of gal-4 expression with be changed. Afterwards, the gal-4 construct is stabilized by crossing it with normal flies and genotyped to find the flies with only the gal-4 gene. These flies are then crossed brother to sister to produce specific lines of homozygous gal-4 mutants. These new lines are then crossed with the UAS marker strain 2 and the effects on phenotype are observed. If for example a gal-4 construct lands next to a tissue specific enhancer for the eye, the gal-4 protein will bind to the UAS site and the marker gene will be expressed causing, for example, the tissue color to be green. Using probes for the known gal-4 sequence, the region of DNA is isolated and sequenced to find the enhancer site. This method can be used to find various enhancers that can be used in other experiments.