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Chapter 20

Chapter 20. Biotechnology. Fig. 20-2. Cell containing gene of interest. Bacterium. 1. Gene inserted into plasmid. Bacterial chromosome. Plasmid. Gene of interest. Recombinant DNA ( plasmid ). DNA of chromosome. genetic engineering. 2. Plasmid put into bacterial cell.

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Chapter 20

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  1. Chapter 20 Biotechnology

  2. Fig. 20-2 Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome genetic engineering 2 Plasmid put intobacterial cell ---recombinant DNA --- Recombinantbacterium 3 Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest Gene ofInterest Protein expressedby gene of interest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene Gene used to alter bacteria for cleaning up toxic waste Gene for pest resistance inserted into plants Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth

  3. 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 TECHNIQUE Agrobacterium tumefaciens Tiplasmid Site whererestrictionenzyme cuts T DNA RESULTS DNA withthe geneof interest RecombinantTi plasmid Plant with new trait

  5. Fig. 20-23

  6. Fig. 20-22 Clonedgene Insert RNA version of normal alleleinto retrovirus. 1 Viral RNA Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured. 2 Retroviruscapsid Viral DNA carrying the normalallele inserts into chromosome. 3 Bonemarrowcell frompatient Bonemarrow Inject engineeredcells into patient. 4

  7. Fig. 20-10 Restriction fragment lanalysis:RFLP (restriction fragment length polymorphism Normal -globin allele Normalallele Sickle-cellallele 175 bp Large fragment 201 bp DdeI DdeI DdeI DdeI Largefragment Sickle-cell mutant -globin allele 376 bp 201 bp175 bp Large fragment 376 bp DdeI DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

  8. Fig. 20-14 • In situ hybridization uses fluorescent dyes • attached to probes to identify the location of specific mRNAs • in place in the intact organism 50 µm

  9. Fig. 20-21 • Short tandem repeats (STRs), • which are variations in the number of repeats of specific DNA sequences DNA T Normal allele SNP C Disease-causingallele

  10. Fig. 20-24 (a) This photo shows EarlWashington just before his release in 2001,after 17 years in prison. Source of sample STRmarker 1 STRmarker 2 STRmarker 3 Semen on victim 17, 19 13, 16 12, 12 Earl Washington 16, 18 14, 15 11, 12 17, 19 13, 16 12, 12 Kenneth Tinsley (b) These and other STR data exonerated Washington andled Tinsley to plead guilty to the murder.

  11. Fig. 20-1

  12. Fig. 20-3-3 Restriction site DNA cloning 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 2 One possible combination DNA ligaseseals strands. 3 Recombinant DNA molecule

  13. 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. DNA cloning Fig. 20-4-1 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments

  15. Fig. 20-4-2 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids

  16. Fig. 20-4-3 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids

  17. Fig. 20-4-4 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids RESULTS Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non-recombinant plasmidwith intact lacZ gene One of manybacterial clones

  18. 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 Foreign genomecut up withrestrictionenzyme Large insertwith many genes Large plasmid or BACclone Recombinantphage DNA Bacterial clones Recombinantplasmids Phageclones (a) Plasmid library (b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones

  20. Fig. 20-5a Foreign genomecut up withrestrictionenzyme or Recombinantphage DNA Bacterial clones Recombinantplasmids Phageclones (a) Plasmid library (b) Phage library

  21. 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 Large insertwith many genes Large plasmid BACclone (c) A library of bacterial artificial chromosome (BAC) clones

  23. 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 innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA DNA polymerase cDNA

  25. 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. 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 … … 5 G G C T A A C T T A G C 3 C C G A T T G A A T C G 5 3

  27. Fig. 20-7 • TECHNIQUE Radioactivelylabeled probemolecules ProbeDNA Gene ofinterest Multiwell platesholding library clones Single-strandedDNA from cell Film Nylon membrane Nylonmembrane Location ofDNA with thecomplementarysequence

  28. 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. 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 3 5 TECHNIQUE Targetsequence 3 5 Genomic DNA 1 5 3 Denaturation 5 3 2 Annealing Cycle 1yields 2 molecules Primers 3 Extension Newnucleo-tides Cycle 2yields 4 molecules Cycle 3yields 8 molecules;2 molecules(in whiteboxes)match targetsequence

  31. Fig. 20-9 TECHNIQUE Powersource Gel Electrophoresis and Southern Blotting Mixture ofDNA mol-ecules ofdifferentsizes – Cathode Anode + Gel 1 Powersource – + Longermolecules 2 Shortermolecules RESULTS

  32. Southern blotting • 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

  33. Fig. 20-11 TECHNIQUE Heavyweight Restrictionfragments I II III Nitrocellulosemembrane (blot) DNA + restriction enzyme Gel Sponge I Normal-globinallele II Sickle-cellallele III Heterozygote Papertowels Alkalinesolution 2 1 3 Preparation of restriction fragments DNA transfer (blotting) Gel electrophoresis Radioactively labeledprobe for -globin gene Probe base-pairswith fragments I II III I II III Fragment fromsickle-cell-globin allele Film overblot Fragment fromnormal -globin allele Nitrocellulose blot 4 5 Probe detection Hybridization with radioactive probe

  34. 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 TECHNIQUE Primer Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged) DNA(template strand) dATP ddATP dCTP ddCTP dTTP ddTTP DNA polymerase dGTP ddGTP DNA (template strand) Labeled strands Shortest Longest Directionof movementof strands Longest labeled strand Detector Laser Shortest labeled strand RESULTS Last baseof longestlabeledstrand Last baseof shortestlabeledstrand

  36. 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 1 cDNA synthesis mRNAs cDNAs Primers 2 PCR amplification -globingene 3 Gel electrophoresis Embryonic stages RESULTS 1 2 3 4 5 6

  38. 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 Tissue sample 1 Isolate mRNA. 2 Make cDNA by reversetranscription, usingfluorescently labelednucleotides. mRNA molecules Labeled cDNA molecules(single strands) DNA fragmentsrepresentingspecific genes 3 Apply the cDNA mixture to amicroarray, a different gene ineach spot. The cDNA hybridizeswith any complementary DNA onthe microarray. DNA microarray DNA microarraywith 2,400human genes 4 Rinse off excess cDNA; scanmicroarray for fluorescence.Each fluorescent spot represents agene expressed in the tissue sample.

  40. Determining Gene Functionin 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. Concept 20.3: Cloning organisms may lead to production of stem cells for research and other applications • Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell

  42. 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 RESULTS Transversesection ofcarrot root 2-mgfragments A singlesomaticcarrot celldevelopedinto a maturecarrot plant. Fragments werecultured in nu-trient medium;stirring causedsingle cells toshear off intothe liquid. Singlecellsfree insuspensionbegan todivide. Embryonicplant developedfrom a culturedsingle cell. Plantlet wascultured onagar medium. Later it wasplantedin soil.

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

  46. 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 Mammarycell donor Egg celldonor 2 1 Egg cellfrom ovary Nucleusremoved Cells fused 3 Culturedmammary cells 3 Nucleus frommammary cell Grown inculture 4 Early embryo Implantedin uterusof a thirdsheep 5 Surrogatemother Embryonicdevelopment 6 Lamb (“Dolly”)genetically identical tomammary cell donor RESULTS

  48. 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. Problems Associated with Animal Cloning • In most nuclear transplantation studies, only asmall 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

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