Outline and Chapter notes to accompany chapter 4 GENETIC ENGINEERING AND GENOMICS Dec., 2003 A. GENETIC ENGINEERING CHANGES THE WAY THAT GENES ARE TRANSFERRED. METHODS OF GENETIC ENGINEERING Requires restriction endonucleases, also called RESTRICTION ENZYMES Cutting several pieces of DNA with the same enzyme results in MATCHING "STICKY ENDS" GENETICALLY ENGINEERED INSULIN (same technique can be used to produce other useful gene products): 1. Take cells that make a useful product from a human or animal. 2. Cut the DNA with a restriction enzyme into RESTRICTION FRAGMENTS. 3. Isolate the DNA fragment containing the gene. 4. Also isolaste a bacterial PLASMID and cut it with the same restriction enzyme; the plasmid must also have a gene that can be used to select bacteria that have incorporated the plasmid, such as ability to survive on a medium deficient in a particular amino acid or other nutrient. 5. Mix the human DNA fragments with the plasmid; some plasmids will recombine with the human DNA fragments. 6. Allow bacteria to take up the new plasmid, and select for those bacteria that have done so. 7. Test the bacteria for the presence of the human gene; isolate any bacteria possessing the gene. 8. Grow the bacteria in large numbers (called CLONING); allow the bacteria to produce the (medically or commercially useful) protein product of the introduced gene. RELATED SPIN-OFF TECHNOLOGIES: Making other human gene products in other species Growing other species with human genetic traits, in the hope that their tissues, if transplanted into human patients, will not be rejected Making spider silk proteins in goats' milk GENE THERAPY (genetic engineering used to fix a human gene defect): 1. Isolate human cells containing the normal version of the gene. 2. Grow these cells in tissue culture; isolate DNA from them. 3. Use a restriction enzyme to cut the DNA into fragments with sticky ends. 4. Isolate DNA from a virus (such as LASN) and cut this DNA with the same restriction enzyme. 5. Mix the DNA fragments and allow new viruses to take up the recombinant DNA. 6. Obtain cells from a patient who is incapable of making an important enzyme because their DNA lacks the normal gene. 7. Combine these cells with the virus containing the recombinant DNA. (The virus thus acts as a VECTOR for inserting the recombinant DNA into human cells.) 8. Grow the human cells in tissue culture and isolate those which make the proper enzyme. 9. Inject the genetically engineered cells back into the patient who donated the cells; hopefully, the cells will proliferate in sufficient numbers to produce adequate amounts of the previously missing enzyme in the patient. B. MOLECULAR TECHNIQUES HAVE LED TO NEW USES FOR GENETIC INFORMATION. THE FIRST DNA MARKER: RESTRICTION-FRAGMENT LENGTH POLYMORPHISMS The fragments cut by restriction enzymes differ in length among different individuals, and these differences are inherited. The RFLP technique (available since 1980) has allowed rapid advances in gene mapping: Cut DNA with restriction enzymes. Use electrophoresis to separate RESTRICTION FRAGMENTS differing in length; variation in the lengths of particular fragments is a RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP). Try to find a family with a genetic condition and a RFLP that accompanies the condition; the responsible gene and the RFLP will therefore be nearby on the same chromosome. USING DNA MARKERS TO IDENTIFY INDIVIDUALS (forensic uses): Matching DNA samples between suspects and evidence from a crime scene Identifying the father in paternity disputes Using DNA from relatives to identify dead bodies from crime scenes, wars, etc. USING DNA TESTING IN HISTORICAL CONTROVERSIES Using DNA to confirm relationships (as in the Thomas Jefferson case) C. THE HUMAN GENOME PROJECT HAS CHANGED BIOLOGY SEQUENCING THE HUMAN GENOME A GENOME is the complete hereditary material of an organism. One goal of the Human Genome Project was to map the human DNA sequence. A common method of sequencing is the dideoxy method, using fluorescent dyes to identify the last base in each of many fragments. Another goal was to map the location of all functional genes. Many DNA sequences have not yet been matched to any known genetic function. THE HUMAN GENOTYPE DRAFT SEQUENCE was published in 2001. Less than 5% of the sequences seems to code for functional genes. The remaining 95% consists of repetetive sequences. Very little is known with any certainty about the function of this 95%. The total number of human genes is just over 30,000, much fewer than had previously been estimated. Over 99.9% of the sequence is the same in humans tested from all over the world. Less than 1/10 of 1% of the genome is responsible for all known genetic variation among human beings. Many genes and their products have not yet had their functions identified. Most human genes are closely similar in sequence to the genes of other organisms as distant as yeasts and bacteria. MAPPING THE HUMAN GENOME requires: sequencing of long fragments, commonly by the dideoxy method; assembling the fragment sequences into an overall sequence. ETHICAL ISSUES RAISED by the Human Genome Project include: privacy; misuse of medical information; knowing one's fate (actually one's predispositions) early in life; doctors and insurers "playing God". D. GENOMICS IS A NEW FIELD OF BIOLOGY DEVELOPED AS A RESULT OF THE HUMAN GENOME PROJECT BIOINFORMATICS is the use of computer methods to search genome sequences and find matches and near-matches: among different fragments (for assembly into a complete sequence); among different genes, to study gene duplication and its consequences; among different organisms, to discover evolutionary history. COMPARATIVE GENOMICS is the comparison of the genomes of different species. It is used to study their evolution. FUNCTIONAL GENOMICS is the study of how genes and their products lead to important phenotypic conditions such as those of medical interest. Scientists hope that revealing these causative pathways will help us to better understand diseases and their possible treatments. PROTEOMICS is the study of protein sequences, including comparative and functional aspects. ---------------------------------- Dec., 2003 PERMISSION IS HEREBY GRANTED to instructors who have adopted the book BIOLOGY TODAY for classroom use to download, modify, and use these notes as needed to aid them in in their teaching. Students of such instructors may likewise use and modify these notes as study aids.