Chapter 20 Reading Quiz 1. Genes from two different sources that are combined result in ____. 2. Where are “sticky ends” found? 3. What structures, naturally found in bacteria, cut up foreign DNA? 4. What is the point of the genetic technology “PCR”? 5. Organisms whose genomes carry genes from another species are known as _____.

1. Explain how advances in recombinant DNA technology have helped scientists study the eukaryotic genome. It resulted in the appearance of the biotechnology industry We can now move genes across the species barrier The Human Genome Project  transcribe and translate the entire human genome We hope to improve health and food production

2. Describe the natural function of restriction enzymes. They are major tools in recombinant DNA technology They occur naturally in bacteria where they protect the bacterium against intruding DNA This protection involves restriction in which foreign DNA is cut into small segments

3. Describe how restriction enzymes and gel electrophoresis are used to isolate DNA fragments. Restriction enzymes only recognize short, specific nucleotide sequences called recognition sequences or restriction sites Gel electrophoresis is used to separate either nucleic acids or proteins based upon molecular size, charge, and other physical properties  characteristic banding

4. Explain how the creation of sticky ends by restriction enzymes is useful in producing a recombinant DNA molecule. They are used in the laboratory to join pieces of DNA from different sources (sometimes cells or even different organisms) These are temporary unions  only held by a few hydrogen bonds

5. Outline the procedures for producing plasmid and phage vectors. 1. Isolation of vector and gene-source DNA - bacterial plasmids & foreign DNA with gene of interest 2. Insertion of gene-source DNA into the vector - plasmid DNA cut  mix foreign DNA and plasmids  add DNA ligase to catalyze new covalent bonds 3. Introduction of cloning vector into bacterial cells - naked DNA added to bacterial culture, plasmid DNA taken up by transformation 4. Cloning of cells (and foreign DNA) - reproduced and cloned 5. Identification of cell clones carrying the gene of interest (indicators added)

6. Explain how vectors are used in recombinant DNA technology. Cloning vector  a DNA molecule that can carry foreign DNA into a cell and replicate there Most often used: bacterial plasmids and viruses

7. List and describe the two major sources of genes for cloning. 1. DNA isolated directly from an organism 2. Complementary DNA made in the lab from mRNA templates

8. Describe the function of reverse transcriptase in retroviruses and explain how they are useful in recombinant DNA technology. It is the enzyme that transcribes DNA from an RNA template Viral genomic RNA (reverse transcriptase) Viral DNA Useful in that we can utilize it’s natural function to incorporate genes of interest

9. Describe how bacteria can be induced to produce eukaryotic gene products. Expression vectors allow the synthesis of many eukaryotic proteins in bacterial cells  Expression vectors contain a prokaryotic promoter just upstream of a restriction site where the eukaryotic gene can be inserted  The bacterial host cell recognizes the promoter and proceeds to express the foreign gene that has been linked to it

10. List some advantages for using yeast in the production of gene products. Yeast cells are easy to grow as bacteria and they contain plasmids Some recombinant plasmids combine yeast and bacterial DNA and can replicate in either Posttranslational modifications required to produce eukaryotic proteins can occur

11. List and describe four complementary approaches used to map the human genome. 1. Genetic mapping (linkage)  construct a linkage map with several thousand genetic markers 2. Physical mapping  ordering the DNA fragments - a map made by cutting the DNA of each chromosome into a number of identifiable fragments 3. Sequencing DNA  the complete nucleotide sequence of a genome is the ultimate map 4. PCR amplification  amplify DNA in amounts sufficient for study (polymerase chain reaction) 5. Chromosome walking  start with a known gene and “walk” along the DNA from that locus

12. Explain how RFLP analysis and PCR can be applied to the human genome project. RFLP (restriction fragment length polymorphisms)  differences in restriction fragment length that reflects variations in homologous DNA sequences - used as genetic markers for making linkage maps - are a “genetic fingerprint” PCR (polymerase chain reaction)  allow any piece of DNA to be quickly amplified (copied) in vitro - produces linkage maps without the need for large family pedigree analysis

13. Describe how recombinant DNA technology can have medical applications such as diagnosis of genetic disease, development of gene therapy, vaccine production, and development of pharmaceutical products. Disease  DNA technology to diagnose, early detection, and identification of carriers Gene therapy  traceable genetic disorders may eventually be correctable = normal genes introduced - many social and ethical questions Vaccines  produce specific protein molecules which could cause immune response; gene splicing could make vaccine pathogens safer Pharmaceutical products  make mostly proteins - gene splicing applies to insulin and growth hormone (genetically engineered human insulin)

14. Describe how gene manipulation has practical applications for agriculture. Essentially to improve productivity Animals  bovine growth hormone (made by ecoli); enhances milk production and weight gain  cellulase (ecoli) hydrolyzes cellulose making the whole plant good for feed Plants  easier to engineer – can be made from a single cell - helps with herbicide  resists pathogens and insects - enhanced food value

15. Describe how plant genes can be manipulated using the Ti plasmid carried by Agrobacterium as a vector. Ti plasmid = tumor-inducing Agrobacterium is normally pathogenic  turned the plasmid into a useful vector by eliminating its disease-causing ability without interfering with its potential to move genetic materials into infected plants

16. Explain how foreign DNA may be transferred into monocotyledonous plants. Electroporation  high voltage jolts of electricity to open temporary pores in the cell membrane; foreign DNA can enter the cells through these pores DNA particle gun  the DNA gun shoots tiny DNA-coated metal pellets through the cell walls into the cytoplasm, where foreign DNA becomes integrated into the host cell DNA

17. Describe how recombinant DNA studies and the biotechnology industry are regulated with regards to safety and policy matters. The processes could create hazardous new pathogens Regulation begins with the self-monitoring approach - moves on to governments and regulatory agencies which promote potential industrial, medical, and agricultural benefits but ensure that the new products are safe