1. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Chapter 20 Biotechnology

2. Fig. 20-2 DNA of chromosome Cell containing gene of interest Gene inserted into plasmid Plasmid put into bacterial cell Recombinant DNA ( plasmid ) Recombinant bacterium Bacterial chromosome Bacterium Gene of interest Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Plasmid Gene of Interest Protein expressed by gene of interest Basic research and various applications Copies of gene Protein harvested Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth 2 4 1 3 genetic engineering ---recombinant DNA ---

3. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Concept 20.2: DNA technology allows us to study the sequence, expression, and function of a gene DNA cloning allows researchers to – Compare genes and alleles between individuals – Locate gene expression in a body – Determine the role of a gene in an organism Several techniques are used to analyze the DNA of genes

4. Fig. 20-25 Site where restriction enzyme cuts T DNA Plant with new trait Ti plasmid Agrobacterium tumefaciens DNA with the gene of interest Recombinant Ti plasmid TECHNIQUE RESULTS

5. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-23

6. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-22 Bone marrow Cloned gene Bone marrow cell from patient Insert RNA version of normal allele into retrovirus. Retrovirus capsid Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Inject engineered cells into patient. 1 2 3 4

7. Fig. 20-10 Restriction fragment lanalysis: RFLP (restriction fragment length polymorphism Normal allele Sickle-cell allele Large fragment (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp (a) Dde I restriction sites in normal and sickle-cell alleles of  -globin gene Normal  -globin allele Sickle-cell mutant  -globin allele Dde I Large fragment 376 bp 201 bp 175 bp Dde I

8. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-14 50 µm In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism

9. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-21 Disease-causing allele DNA SNP Normal allele T C Short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences

10. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-24 This photo shows Earl Washington just before his release in 2001, after 17 years in prison. These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder. (a) Semen on victim Earl Washington Source of sample Kenneth Tinsley STR marker 1 STR marker 2 STR marker 3 (b) 17, 19 16, 18 17, 19 13, 1612, 12 14, 1511, 12 13, 1612, 12

11. Fig. 20-1

12. Fig. 20-3-3 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5353 3535 1 One possible combination Recombinant DNA molecule DNA ligase seals strands. 3 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 2 DNA cloning

13. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Cloning a Eukaryotic Gene in a Bacterial Plasmid In gene cloning, the original plasmid is called a cloning vector A cloning vector is a DNA molecule that can carry foreign DNA into a host cell and replicate there

14. Fig. 20-4-1 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE DNA cloning

15. Fig. 20-4-2 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid

16. Fig. 20-4-3 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids

17. Fig. 20-4-4 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids RESULTS Colony carrying non- recombinant plasmid with intact lacZ gene One of many bacterial clones Colony carrying recombinant plasmid with disrupted lacZ gene

18. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Storing Cloned Genes in DNA Libraries A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome A genomic library that is made using bacteriophages is stored as a collection of phage clones

19. Fig. 20-5 Bacterial clones Recombinant plasmids Recombinant phage DNA or Foreign genome cut up with restriction enzyme (a) Plasmid library(b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones Phage clones Large plasmid Large insert with many genes BAC clone

20. Fig. 20-5a Bacterial clones Recombinant plasmids Recombinant phage DNA or Foreign genome cut up with restriction enzyme (a) Plasmid library(b) Phage library Phage clones

21. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert BACs are another type of vector used in DNA library construction

22. Fig. 20-5b (c) A library of bacterial artificial chromosome (BAC) clones Large plasmid Large insert with many genes BAC clone

23. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells

24. Fig. 20-6-5 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA Degraded mRNA DNA polymerase cDNA

25. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Screening a Library for Clones Carrying a Gene of Interest A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene This process is called nucleic acid hybridization

26. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings A probe can be synthesized that is complementary to the gene of interest For example, if the desired gene is – Then we would synthesize this probe G 5 3 …… GGCCCTTTAAA C 3 5 CCGGGAAATTT

27. Fig. 20-7 Probe DNA Radioactively labeled probe molecules Film Nylon membrane Multiwell plates holding library clones Location of DNA with the complementary sequence Gene of interest Single-stranded DNA from cell Nylon membrane TECHNIQUE

28. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Expressing Cloned Eukaryotic Genes After a gene has been cloned, its protein product can be produced in larger amounts for research Cloned genes can be expressed as protein in either bacterial or eukaryotic cells The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems – YACs behave normally in mitosis and can carry more DNA than a plasmid – Eukaryotic hosts can provide the post-translational modifications that many proteins require

29. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

30. Fig. 20-8 5 Genomic DNA TECHNIQUE Cycle 1 yields 2 molecules Denaturation Annealing Extension Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Target sequence Primers New nucleo- tides 3 3 3 3 5 5 5 1 2 3

31. Fig. 20-9 Mixture of DNA mol- ecules of different sizes Power source Longer molecules Shorter molecules Gel Anode Cathode TECHNIQUE RESULTS 1 2 + + – – Gel Electrophoresis and Southern Blotting

32. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel Southern blotting

33. Fig. 20-11 TECHNIQUE Nitrocellulose membrane (blot) Restriction fragments Alkaline solution DNA transfer (blotting) Sponge Gel Heavy weight Paper towels Preparation of restriction fragments Gel electrophoresis I II III Radioactively labeled probe for  -globin gene DNA + restriction enzyme III Heterozygote II Sickle-cell allele I Normal  -globin allele Film over blot Probe detection Hybridization with radioactive probe Fragment from sickle-cell  -globin allele Fragment from normal  -globin allele Probe base-pairs with fragments Nitrocellulose blot 1 4 5 3 2

34. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings DNA Sequencing --- dideoxy chain termination method Relatively short DNA fragments can be sequenced by the dideoxy chain termination method dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment The DNA sequence can be read from the resulting spectrogram

35. Fig. 20-12 DNA (template strand) TECHNIQUE RESULTS DNA (template strand) DNA polymerase Primer Deoxyribonucleotides Shortest Dideoxyribonucleotides (fluorescently tagged) Labeled strands Longest Shortest labeled strand Longest labeled strand Laser Direction of movement of strands Detector Last base of longest labeled strand Last base of shortest labeled strand dATP dCTP dTTP dGTP ddATP ddCTP ddTTP ddGTP

36. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Analyzing Gene Expression Nucleic acid probes can hybridize with mRNAs transcribed from a gene Probes can be used to identify where or when a gene is transcribed in an organism mRNA – Northern blotting – Reverse transcriptase-polymerase chain reaction (RT-PCR)

37. Fig. 20-13 TECHNIQUE RESULTS Gel electrophoresis cDNAs  -globin gene PCR amplification Embryonic stages Primers 1 2 3 4 5 6 mRNAs cDNA synthesis 1 2 3

38. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Studying the Expression of Interacting Groups of Genes Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays DNA microarray assays – compare patterns of gene expression in different tissues, at different times, or under different conditions

39. Fig. 20-15 TECHNIQUE Isolate mRNA. Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample. Tissue sample mRNA molecules Labeled cDNA molecules (single strands) DNA fragments representing specific genes DNA microarray with 2,400 human genes DNA microarray 1 2 3 4

40. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Determining Gene Function in vitro mutagenesis and RNA interference (RNAi) One way to determine function is to disable the gene and observe the consequences Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype

41. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell Concept 20.3: Cloning organisms may lead to production of stem cells for research and other applications

42. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Cloning Plants: Single-Cell Cultures One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism A totipotent cell is one that can generate a complete new organism

43. Fig. 20-16 EXPERIMENT Transverse section of carrot root 2-mg fragments Fragments were cultured in nu- trient medium; stirring caused single cells to shear off into the liquid. Single cells free in suspension began to divide. Embryonic plant developed from a cultured single cell. Plantlet was cultured on agar medium. Later it was planted in soil. A single somatic carrot cell developed into a mature carrot plant. RESULTS

44. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Cloning Animals: Nuclear Transplantation In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg However, the older the donor nucleus, the lower the percentage of normally developing tadpoles

45. Fig. 20-17 EXPERIMENT Less differ- entiated cell RESULTS Frog embryo Frog egg cell UV Donor nucleus trans- planted Frog tadpole Enucleated egg cell Egg with donor nucleus activated to begin development Fully differ- entiated (intestinal) cell Donor nucleus trans- planted Most develop into tadpoles Most stop developing before tadpole stage

46. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Reproductive Cloning of Mammals In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus

47. Fig. 20-18 TECHNIQUE Mammary cell donor RESULTS Surrogate mother Nucleus from mammary cell Cultured mammary cells Implanted in uterus of a third sheep Early embryo Nucleus removed Egg cell donor Embryonic development Lamb (“Dolly”) genetically identical to mammary cell donor Egg cell from ovary Cells fused Grown in culture 1 3 3 4 5 6 2

48. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent”

49. Fig. 20-19

50. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Problems Associated with Animal Cloning In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth Many epigenetic changes, – such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development

51. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Stem Cells of Animals A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types embryonic stem cells :at the blastocyst stage, to differentiate into all cell types The aim of stem cell research is to supply cells for the repair of damaged or diseased organs