BIOLOGY CONCEPTS & CONNECTIONS Fourth Edition Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Neil A. Campbell Jane B. Reece Lawrence G. Mitchell Martha R. Taylor From PowerPoint ® Lectures for Biology: Concepts & Connections CHAPTER 12 DNA Technology and the Human Genome Modules 12.1 – 12.6

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Research on E. coli revealed that these bacteria have a sexual mechanism that can bring about the combining of genes from two different cells This discovery led to the development of recombinant DNA technology –a set of techniques for combining genes from different sources From E.Coli to a Map of Our Genes

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings DNA technology has many useful applications –The Human Genome Project –The production of vaccines, cancer drugs, and pesticides –Engineered bacteria that can clean up toxic wastes

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Transformation, the taking up of DNA from the fluid surrounding the cell 12.1 In nature, bacteria can transfer DNA in three ways BACTERIA AS TOOLS FOR MANIPULATING DNA Figure 12.1A DNA enters cell Fragment of DNA from another bacterial cell Bacterial chromosome (DNA)

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Transduction, the transfer of bacterial genes by a phage Conjugation, the union of cells and the DNA transfer between them Fragment of DNA from another bacterial cell (former phage host) Phage Sex pili Mating bridge Donor cell (“male”) Recipient cell (“female”) Figure 12.1CFigure 12.1B

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings The transferred DNA is then integrated into the recipient cell’s chromosome Figure 12.1D Donated DNA Recipient cell’s chromosome Crossovers Degraded DNA Recombinant chromosome

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings An F factor is a DNA segment in bacteria that enables conjugation and contains an origin of replication 12.2 Bacterial plasmids can serve as carriers for gene transfer Figure 12.2A F factor (integrated) Male (donor) cell Origin of F replication Bacterial chromosome F factor starts replication and transfer of chromosome Only part of the chromosome transfers Recipient cell Recombination can occur

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings An F factor can exist as a plasmid, a small circular DNA molecule separate from the bacterial chromosome Figure 12.2B, C F factor (plasmid) Male (donor) cell Bacterial chromosome F factor starts replication and transfer Plasmid completes transfer and circularizes Cell now male Plasmids

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Plasmids are key tools for DNA technology –Researchers use plasmids to insert genes into bacteria 12.3 Plasmids are used to customize bacteria: An overview

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Figure 12.3 Plasmid isolated 1 Bacterium Bacterial chromosome Plasmid 2 DNA isolated Cell containing gene of interest DNA Gene of interest 3 Gene inserted into plasmid Recombinant DNA (plasmid) 4 Plasmid put into bacterial cell Recombinant bacterium 5 Copies of geneCopies of protein Clones of cellGene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy Protein used to make snow form at higher temperature Cell multiplies with gene of interest

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Restriction enzymes cut DNA at specific points DNA ligase “pastes” the DNA fragments together The result is recombinant DNA 12.4 Enzymes are used to “cut and paste” DNA Figure 12.4 DNA 1 Restriction enzyme recognition sequence Restriction enzyme cuts the DNA into fragments Sticky end Restriction enzyme cuts the DNA into fragments Addition of a DNA fragment from another source Two (or more) fragments stick together by base-pairing DNA ligase pastes the strand Recombinant DNA molecule

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Bacteria take the recombinant plasmids and reproduce This clones the plasmids and the genes they carry –Products of the gene can then be harvested 12.5 Genes can be cloned in recombinant plasmids: A closer look

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Figure 12.5 Isolate DNA from two sources 1 E. coli Cut both DNAs with the same restriction enzyme 2 Plasmid Human cell DNA Gene V Sticky ends Mix the DNAs; they join by base-pairing 3 Add DNA ligase to bond the DNA covalently 4 Recombinant DNA plasmid Gene V Put plasmid into bacterium by transformation 5 Clone the bacterium 6 Bacterial clone carrying many copies of the human gene

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 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 12.6 Cloned genes can be stored in genomic libraries Figure 12.6 Genome cut up with restriction enzyme Recombinant plasmid OR Recombinant phage DNA Bacterial clone Phage clone Phage library Plasmid library

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Reverse transcriptase can be used to make smaller cDNA libraries –These contain only the genes that are transcribed by a particular type of cell 12.7 Reverse transcriptase helps make genes for cloning OTHER TOOLS OF DNA TECHNOLOGY

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Figure 12.7 Transcription 1 CELL NUCLEUS DNA of eukaryotic gene RNA transcript mRNA ExonIntronExonIntronExon TEST TUBE Reverse transcriptase cDNA strand cDNA of gene (no introns) RNA splicing (removes introns) 2 Isolation of mRNA from cell and addition of reverse transcriptase; synthesis of DNA strand 3 Breakdown of RNA 4 Synthesis of second DNA strand 5

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings A nucleic acid probe can tag a desired gene in a library 12.8 Nucleic acid probes identify clones carrying specific genes Figure 12.8A Radioactive probe (DNA) Mix with single- stranded DNA from various bacterial (or phage) clones Single-stranded DNA Base pairing indicates the gene of interest

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings DNA probes can identify a bacterial clone carrying a specific gene Figure 12.8B Bacterial colonies containing cloned segments of foreign DNA Radioactive DNA Transfer cells to filter 1 Solution containing probe Treat cells on filter to separate DNA strands 2 Add probe to filter 3 Probe DNA Gene of interest Single-stranded DNA from cell Hydrogen-bonding Autoradiography 4 Developed film Colonies of living cells containing gene of interest Compare autoradiograph with master plate 5 Master plate Filter paper

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings A labeled probe can reveal patterns of gene expression in different kinds of cells This technique may revolutionize the diagnosis and treatment of cancer 12.9 Connection: DNA microarrays test for the expression of many genes at once Figure 12.9 cDNA DNA of gene DNA microarray, actual size (6,400 genes)

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Restriction fragments of DNA can be sorted by size Gel electrophoresis sorts DNA molecules by size Figure Mixture of DNA molecules of different sizes Power source Gel Glass plates Longer molecules Shorter molecules Completed gel

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Scientists can compare DNA sequences of different individuals based on the size of the fragments Restriction fragment analysis is a powerful method that detects differences in DNA sequences Figure 12.11A Allele 1Allele 2 w x y Cut z y DNA from chromosomes

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Figure 12.11B 12 Longer fragments Shorter fragments

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Radioactive probes are also used to make comparisons Figure 12.11C Radioactive, single- stranded DNA (probe) Restriction fragment preparation 1 Restriction fragments Gel electrophoresis 2 Blotting 3 Filter paper Probe Radioactive probe 4 Detection of radioactivity (autoradiography) 5 Film

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings The polymerase chain reaction (PCR) can quickly clone a small sample of DNA in a test tube The PCR method is used to amplify DNA sequences Figure Initial DNA segment 1248 Number of DNA molecules

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings The 23 chromosomes in the haploid human genome contain about 3 billion nucleotide pairs –This DNA is believed to include about 35,000 genes and a huge amount of noncoding DNA Most of the human genome does not consist of genes THE CHALLENGE OF THE HUMAN GENOME

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Much of the noncoding DNA consists of repetitive nucleotide sequences –One example includes telomeres at the end of the chromosomes Figure 12.13A End of DNA molecule Repeated unit NUCLEOTIDE SEQUENCE OF A HUMAN TELOMERE

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Barbara McClintock discovered that segments of DNA called transposons can move about within a cell’s genome Figure 12.13B, C

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings The Human Genome Project involves: –genetic and physical mapping of chromosomes –DNA sequencing –comparison of human genes with those of other species Connection: The Human Genome Project is unlocking the secrets of our genes Figure 12.14

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings DNA fingerprinting can help solve crimes Connection: DNA technology is used in courts of law OTHER APPLICATIONS OF DNA TECHNOLOGY Figure 12.15A, B Defendant’s blood Blood from defendant’s clothes Victim’s blood

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Recombinant cells and organisms are used to manufacture useful proteins Connection: Recombinant cells and organisms can mass-produce gene products Table 12.16

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings These sheep carry a gene for a human blood protein that is a potential treatment for cystic fibrosis Figure 12.16

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Hormones, cancer-fighting drugs, and new vaccines are being produced using DNA technology –This lab equipment is used to produce a vaccine against hepatitis B Connection: DNA technology is changing the pharmaceutical industry and medicine Figure 12.17

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings New genetic varieties of animals and plants are being produced –A plant with a new trait can be created using the Ti plasmid Connection: Genetically modified organisms are transforming agriculture

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Figure 12.18A Insertion of gene into plasmid using restriction enzyme and DNA ligase 1 Agrobacterium tumefaciens Ti plasmid T DNA Restriction site Introduction into plant cells in culture 2 Recombinant Ti plasmid Plant cell T DNA carrying new gene within plant chromosome Regeneration of plant 3 Plant with new trait DNA containing gene for desired trait

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings “Golden rice” has been genetically modified to contain beta-carotene –This rice could help prevent vitamin A deficiency Figure 12.18B

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Techniques for manipulating DNA have potential for treating disease by altering an afflicted individual’s genes –Progress is slow, however –There are also ethical questions related to gene therapy Connection: Gene therapy may someday help treat a variety of diseases Figure Cloned gene (normal allele) 1 Insert normal gene into virus Viral nucleic acid Retrovirus 2 Infect bone marrow cell with virus 3 Viral DNA inserts into chromosome Bone marrow cell from patient Bone marrow 4 Inject cells into patient

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 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 Connection: Could GM organisms harm human health or the environment? RISKS AND ETHICAL QUESTIONS Figure 12.20A, B

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Our new genetic knowledge will affect our lives in many ways The deciphering of the human genome, in particular, raises profound ethical issues –Many scientists have counseled that we must use the information wisely Connection: DNA technology raises important ethical questions Figure 12.21A-C