Highlights Recombinant DNA (continued)
1. Mutation is a change in the sequence of a DNA molecule. It involves the conversion of one base/nucleotide (say, G) to a different one (say, T).
2. Mutations can have several effects
a. If they do not occur in protein coding regions, they may have no effects (this depends a bit, though)
b. If they occur in protein coding regions, they may change the amino acid sequence of a protein.
c. Change in the amino acid sequence of proteins are factors in evolution.
i. Amino acid changes may help proteins to work better, so they are positive changes
ii. Amino acid changes can inhibit the function of proteins. These are negative changes and may be fatal, in some cases.
3. Another alteration to DNA that can occur in cells is recombination. This involves swaps or exchanges of arms of chromosomes during cellular life. We can also cause recombination to occur in the test tube with sophisticated techniques (see below).
4. Genes are regions of DNA molecules that code for proteins.
5. Plasmids are circular DNA molecules that 1) replicate in bacteria and 2) carry "control sequences" called promoters into which genes can be inserted. Insertion of genes in front of promoters is necessary if one wants to transcribe the gene being inserted. Such plasmids are very useful in biotechnology because they allow users to make proteins in bacteria from almost any source.
6. Getting genes for insertion into plasmids requires isolation of them from the target organism. If this is a eukaryotic cell, one must isolate mRNAs that have had the intervening sequences removed. These are located in the cytoplasm. An enzyme called reverse transcriptasecan convert RNA to DNA and then the DNA can be amplified by PCR and then inserted into a plasmid.
7. Insertion of DNAs into plasmids requires another enzyme called a DNA ligase. DNA ligase links together ends of DNAs and allows the circle to be reformed after it is opened up.
8. Enzymes called restriction enzymes are useful for cutting DNA. They cut it at specific places. The combination of restriction enzymes and DNA ligases allow researchers to create recombinant DNAs in the test tube.
9. The location in the plasmid DNA where the gene is placed is important. Remember that we want a bacterial cell to transcribe (make RNA) and translate (make protein). To make the bacterial cell transcribe, it is necessary to have a "control" sequence called a promoter adjacent to the place in the plasmid where the gene is inserted. Then, when the plasmid is in the cell, RNA will be made of the gene and translation of the RNA will produced the desired protein.
10. Last, we want the plasmid DNA to replicate (make copies of itself) in the bacterial cell. Consequently, we design the plasmid so it can be replicated.
11. Putting the plasmid DNA into bacterial cells is known as transformation. There are several ways to do this. One common way is to electrically shock the bacterial cells very briefly. This causes them to absorb and take in DNAs.
Exam 1 Material Stops HERE
Cells as Factories
1. When we hope for cells to make things for us that we want, we must remember that cells must make many other things in order to be alive. Disturbing the balance of things cells make may kill a cell.