12.4 and 12.6 Flashcards
Note 1 —-»
Researchers often wish to clone eukaryotic genes—which usually contain stretches of non-coding introns—in prokaryotic cells, which lack the machinery to remove introns. To overcome this problem, a researcher can use mRNA as the starting material for cloning. The chosen cells transcribe their genes within the nucleus, producing mRNA. The researcher isolates the mRNA in a test tube. Single-stranded DNA transcripts are made from the mRNA using reverse transcriptase, a viral enzyme that can synthesize DNA from an RNA template. Another enzyme is added to break down the mRNA, and DNA polymerase (the enzyme that replicates DNA) is used to synthesize a second DNA strand.
Reverse Template
An enzyme encoded and used by retroviruses catalyzes the synthesis of DNA on an RNA template.
Complementary DNA
A DNA molecule made in vitro using mRNA as a template and the enzyme reverse transcriptase.
Why is the use of a viral enzyme critical to producing cDNAs?
The viral enzyme reverse transcriptase produces DNA from RNA; most cells lack such an enzyme.
Note 2 —-»
Recombinant cells and organisms are used to manufacture gene products, chiefly proteins. By transferring the gene for the desired protein into a bacterium, yeast, or other kinds of cell that is easy to grow in culture, a molecular biologist can produce large quantities of useful proteins that are otherwise difficult to obtain.
Note 2 —-»
Mammals provide the cells of choice for making some gene products. Many proteins that mammalian cells normally secrete are glycoproteins, proteins with chains of sugars attached. Because only mammalian cells can attach the sugars correctly, mammalian cells must be used for making these products. For example, recombinant mammalian cells growing in laboratory cultures are currently used to produce tissue plasminogen factor (TPA), a drug administered after heart attacks. Mammalian cells are also used to produce human erythropoietin (EPO), a hormone that stimulates the production of red blood cells. EPO can save lives as a treatment for anemia.
Note 3 —-»
A whole animal can be produced with recombinant gene transfer when doing so is the only or optimal way to manufacture a needed pharmaceutical. Whole animals that have been produced using recombinant DNA are referred to as genetically engineered animals. Although useful, genetically engineered animals are difficult and costly to produce. Typically, a biotechnology company starts by injecting the desired DNA into a large number of animal embryos, which are then implanted into surrogate mothers. Once a genetically engineered animal is successfully produced, it may be cloned. The result can be a genetically identical herd—a grazing pharmaceutical “factory” of “pharm” animals that produce otherwise rare biological substances for medical use.
Why can’t all human proteins be synthesized in E. coli?
Because bacteria cannot correctly produce some proteins, such as ones that require the attachment of sugar groups
