Analyzing and Engineering Genes

1. Analyzing and Engineering Genes Chapter 19

2. Analyzing and Engineering DNA • Genetic engineering- Manipulation of DNA sequences in organisms • Recombinant DNA technology used to engineer genes • Biotechnology-the manipulation of organisms to create products or cure diseases • Goals of genetic engineering • Improve our understanding of how genes work • Advance biotechnology

3. An Example:Using Recombinant DNA Techniques to Manufacture Proteins: The Effort to Cure Pituitary Dwarfism

4. Pituitary Dwarfism • Pituitary dwarfism results from the abnormal production of growth hormone, encoded by the GH1 gene • Humans affected by pituitary dwarfism grow slowly • maximum adult height of about 4 feet • Could be treated successfully with growth hormone therapy, but only if the protein came from humans

5. Pituitary Dwarfism • Using recombinant DNA technology to produce a safe supply of growth hormone • Involved cloning the human gene, introducing the gene into bacteria (or yeast), and having these microbes synthesize the hormone • Reverse transcriptase was used to make complementary DNA (cDNA) from mRNA isolated from pituitary cells

7. Cloning the Gene • Genetic cloning—the process of producing many identical copies of a gene—was used to clone the cDNAs for analysis to determine which encoded the growth hormone protein

9. DNA enterscell Fragment ofDNA from anotherbacterial cell Bacterial chromosome(DNA) Bacteria as a Tool for Manipulating DNA • In nature, bacteria can transfer DNA in three ways • Transformation, the taking up of DNA from the fluid surrounding the cell • Can even take up DNA from dead cells

10. Conjugation, the union of cells and the DNA transfer between them Transduction, the transfer of bacterial genes by a phage Mating bridge Phage Fragment ofDNA from anotherbacterial cell(former phagehost) Donor cell(“male”) Recipient cell(“female”) Figure 12.1B Bacteria as a Tool for Manipulating DNA

11. Donated DNA Degraded DNA Crossovers Recipient cell’schromosome Recombinantchromosome • The transferred DNA is then integrated into the recipient cell’s chromosome

12. Bacterial plasmids can serve as carriers for gene transfer An F factor is a DNA segment in bacteria that enables conjugation and contains an origin of replication The F factor starts replication and transfers part of the chromosome F factor (integrated) Male (donor) cell Origin of F replication Bacterial chromosome F factor startsreplication andtransfer of chromosome Recipient cell Only part of thechromosome transfers Recombination can occur Bacterial Phages as Carriers

13. An F factor can exist as a plasmid, a small circular DNA molecule separate from the bacterial chromosome R plasmids carry genes for resistance of antibiotics and that is how bacteria can become resistant F factor (plasmid) Male (donor) cell Bacterial chromosome F factor startsreplication andtransfer Plasmids Plasmid completestransfer andcircularizes Cell now male

14. Plasmids are used to customize bacteria • Plasmids are key tools for DNA technology • Researchers use plasmids to insert genes into bacteria • Plasmids are obtained from other bacteria • Desired DNA inserted into plasmid • Bacteria takes up DNA • Can be used for several applications

15. What are Plasmids? • Small, circular DNA molecules that replicate independently of the chromosome • Can be used to carry recombinant genes in bacteria. • Restriction endonucleases are enzymes that cut DNA at specific base sequences called recognition sites • Often make staggered cuts in the DNA, resulting in sticky ends. • Plasmids and cDNAs cut with the same restriction endonuclease can be spliced together at their sticky ends

17. Transformation: Introducing Recombinant Plasmids into Bacterial Cells

18. Transformation • Plasmids serve as a vector—a vehicle for transferring recombinant genes to a new host. • Plasmids can be introduced into bacteria by transformation • Can be used to replicate and make more DNA • Can be used to make proteins

19. Cell containing geneof interest 1 Bacterium Plasmidisolated 2 DNA isolated 3 Gene inserted into plasmid Bacterialchromosome Plasmid Gene ofinterest Recombinant DNA(plasmid) DNA 4 Plasmid put intobacterial cell Recombinantbacterium 5 Cell multiplies withgene of interest Copies of gene Copies of protein Gene for pestresistanceinserted intoplants Clones of cell Protein used to make snow format highertemperature Gene used to alter bacteriafor cleaning up toxic waste Protein used to dissolve bloodclots in heart attack therapy

20. Genome cut up with restriction enzyme Recombinantplasmid Recombinantphage DNA OR Phage clone Bacterialclone Phage library Plasmid library Genomic Libraries • Recombinant DNA technology allows the construction of genomic libraries • Genomic libraries are sets of DNA fragments containing all of an organism’s genes • Copies of DNA fragments can be stored in a cloned bacterial plasmid or phage

21. Using Nucleic Acid Hybridization to Finda Target Gene

22. Nucleic Acid Probes • A probe is a single-stranded fragment of a labeled, known gene • Binds to a complementary sequence in the sample being analyzed • Can be used to screen for bacterial colonies containing a plasmid with the growth hormone gene

25. Mass Producing the Gene

26. Mass Producing the Growth Hormone • The human growth hormone cDNA was cloned in a plasmid under the control of a bacterial promoter • Bacteria carrying these plasmids can make a large quantity of human growth hormone • Hormone can be purified and used to treat patients

27. Ethical Issues • Hormone used for kids who did not suffer from pituitary dieases • Approved use only for children projected to reach adult heights of less than 5'3" for males and less than 4'11" for females. • Also problems with these hormones being used as popular performance-enhancing drug for athletes

28. Sequencing DNA

29. DNA Sequencing • Can be used to infer the amino acid sequence of the protein product • Known sequence can be compared to the sequences of genes that have the same function in various species • Can be used to infer the function of the protein • Huge libraries of information about DNA sequences, GenBank

30. PCR • Polymerase chain reaction (PCR) is an in vitro DNA synthesis reaction in which a section of DNA is amplified millions of times

31. PCR • PCR ingredients: a DNA template, two primers that bracket the region to be amplified, dNTPs, buffer, and DNA polymerase. • Requires about 30 cycles, with each cycle containing three steps • Denaturation to separate the DNA strands, • Annealing to allow the flanking primers to anneal to the denatured DNA • Extension step for synthesis of the complementary strand.

32. Gel Electrophoresis • Physically sorts out macromolecules (DNA, RNA) on the basis of their charge and size • Current is run through the gel and since DNA is negatively charged it moves through the gel • The longer the DNA molecules are. The slower they move • Bands are made, each consisting of DNA molecules of one size

33. Mixture of DNAmolecules ofdifferent sizes Longermolecules Powersource Gel Shortermolecules Glassplates Completed gel Gel Electrophoresis • Restriction fragments of DNA can be sorted by size

34. Restriction Fragment Analysis • Everyone’s DNA sequence is different • Scientists can compare DNA sequences of different individuals based on the size of the fragments created by restriction enzymes • They can only use DNA that varies from person to person • When run on a gel it makes a distinct pattern

35. 1 2 Allele 1 Allele 2 w Longer fragments Cut z x Shorter fragments Cut Cut y y DNA from chromosomes Restriction Fragment Analysis

36. Restriction fragmentpreparation 1 Restrictionfragments Gel electrophoresis 2 Filter paper Blotting 3 Radioactive probe 4 Probe Detection of radioactivity(autoradiography) 5 Film Detecting Harmful Alleles • Radioactive single-stranded DNA complimentary strands are used to verify the presence of certain nucleic acid sequences known to code for harmful alleles

37. Gene Therapy • Gene therapy is the introduction of a gene to replace or augment a mutant gene that is causing an abnormal phenotype • The two primary vectors for introducing therapeutic genes into human cells are retroviruses and adenoviruses • Adenoviruses only work for a short time • Retroviruses integrate and are better

38. Gene Therapy • Gene therapy is highly experimental, extremely expensive, and very controversial. • Holds great promise for the treatment of a wide variety of inherited diseases • Require many years of additional research and testing, as well as the refinement of legal and ethical guidelines.

39. Biotechnology • Genetic engineering in agriculture focuses on • Reducing herbivory • Making crops herbicide resistant • Improving the quality of the food product

40. Golden Rice • Half the world's population depends on rice as a staple food • Contains no vitamin A. • Lack of vitamin A in the diet leads to blindness, diarrhea, respiratory infections, and childhood diseases such as measles.

41. Agrobacterium tumefaciens • Agrobacterium tumefaciens is often used for genetic transformation of plants through transfer of its Ti (tumor-inducing) plasmid • Can be disarmed and used to insert DNA

44. Golden Rice • β-carotene is a precursor of vitamin A. • “Golden rice” has been genetically modified to contain beta-carotene • This rice could help prevent vitamin A deficiency

45. GMO’s and the Environment • Genetic engineering involves some risks • Possible ecological damage from pollen transfer between GM and wild crops • Pollen from a transgenic variety of corn that contains a pesticide may stunt or kill monarch caterpillars