Viruses. CAMPBELL BIOLOGY Reece Urry Cain Wasserman Minorsky Jackson. Lecture Presentation by Nicole Tunbridge and Kathleen Fitzpatrick TENTH EDITION

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2 CAMPBELL BIOLOGY Reece Urry Cain Wasserman Minorsky Jackson TENTH EDITION 19 Viruses Lecture Presentation by Nicole Tunbridge and Kathleen Fitzpatrick 2014 Pearson Education, Inc.

3 A Borrowed Life A virus is an infectious particle consisting of genes packaged in a protein coat Viruses are much simpler in structure than even prokaryotic cells Viruses cannot reproduce or carry out metabolism outside of a host cell

4 Figure 19.1

5 Figure 19.1a

6 Concept 19.1: A virus consists of a nucleic acid surrounded by a protein coat Viruses were detected indirectly long before they were actually seen

7 The Discovery of Viruses: Scientific Inquiry Tobacco mosaic disease stunts growth of tobacco plants and gives their leaves a mosaic coloration In the late 1800s, some researchers hypothesized that a particle smaller than bacteria caused the disease In 1935, Wendell Stanley confirmed this hypothesis by crystallizing the infectious particle, now known as tobacco mosaic virus (TMV)

8 Experiment Figure Extracted sap from tobacco plant with tobacco mosaic disease 2 Passed sap through a porcelain filter known to trap bacteria 3 Rubbed filtered sap on healthy tobacco plants 4 Healthy plants became infected

9 Figure 19.2a

10 Figure 19.2b

11 Figure 19.2c

12 Structure of Viruses Viruses are not cells A virus is a very small infectious particle consisting of nucleic acid enclosed in a protein coat and, in some cases, a membranous envelope

13 Viral Genomes Viral genomes may consist of either Double- or single-stranded DNA, or Double- or single-stranded RNA Depending on its type of nucleic acid, a virus is called a DNA virus or an RNA virus The genome is either a single linear or circular molecule of the nucleic acid Viruses have between three and several thousand genes in their genome

14 Capsids and Envelopes A capsid is the protein shell that encloses the viral genome Capsids are built from protein subunits called capsomeres A capsid can have a variety of structures

15 RNA Capsomere Figure 19.3 Capsomere of capsid DNA Membranous envelope RNA Capsid Head DNA Tail sheath Tail fiber Glycoprotein Glycoproteins nm nm (diameter) nm (diameter) nm 20 nm 50 nm 50 nm 50 nm (a) Tobacco mosaic (b) Adenoviruses (c) Influenza viruses (d) Bacteriophage T4 virus

16 RNA Capsomere DNA Figure 19.3a Capsomere of capsid Glycoprotein nm nm (diameter) 20 nm (a) Tobacco mosaic virus 50 nm (b) Adenoviruses

17 Figure 19.3aa 20 nm (a) Tobacco mosaic virus

18 Figure 19.3ab 50 nm (b) Adenoviruses

19 Figure 19.3b Membranous envelope RNA Capsid Head DNA Tail sheath Tail fiber Glycoproteins nm (diameter) nm 50 nm 50 nm (c) Influenza viruses (d) Bacteriophage T4

20 Figure 19.3ba 50 nm (c) Influenza viruses

21 Figure 19.3bb 50 nm (d) Bacteriophage T4

22 Some viruses have accessory structures that help them infect hosts Viral envelopes (derived from membranes of host cells) surround the capsids of influenza viruses and many other viruses found in animals Viral envelopes contain a combination of viral and host cell molecules

23 Bacteriophages, also called phages, are viruses that infect bacteria They have the most complex capsids found among viruses Phages have an elongated capsid head that encloses their DNA A protein tail piece attaches the phage to the host and injects the phage DNA inside

24 Concept 19.2: Viruses replicate only in host cells Viruses are obligate intracellular parasites, which means they can replicate only within a host cell Each virus has a host range, a limited number of host cells that it can infect

25 General Features of Viral Replicative Cycles Once a viral genome has entered a cell, the cell begins to manufacture viral proteins The virus makes use of host enzymes, ribosomes, trnas, amino acids, ATP, and other molecules Viral nucleic acid molecules and capsomeres spontaneously selfassemble into new viruses

26 Figure Entry and uncoating DNA VIRUS Capsid 3 Transcription and manufacture of capsid proteins 2 Replication HOST CELL Viral DNA mrna Viral DNA Capsid proteins 4 Self-assembly of new virus particles and their exit from the cell

27 Animation: Simplified Viral Reproductive Cycle

28 Replicative Cycles of Phages Phages are the best understood of all viruses Phages have two alternative reproductive mechanisms: the lytic cycle and the lysogenic cycle

29 The Lytic Cycle The lytic cycle is a phage replicative cycle that culminates in the death of the host cell The lytic cycle produces new phages and lyses (breaks open) the host s cell wall, releasing the progeny viruses A phage that reproduces only by the lytic cycle is called a virulent phage Bacteria have defenses against phages, including restriction enzymes that recognize and cut up certain phage DNA

30 Figure Attachment

31 1 Attachment Figure Entry of phage DNA and degradation of host DNA

32 1 Attachment Figure Entry of phage DNA and degradation of host DNA 3 Synthesis of viral genomes and proteins

33 1 Attachment Figure Phage assembly 2 Entry of phage DNA and degradation of host DNA Head Tail Tail fibers 4 Self-assembly 3 Synthesis of viral genomes and proteins

34 1 Attachment Figure Phage assembly 5 Release 2 Entry of phage DNA and degradation of host DNA Head Tail Tail fibers 4 Self-assembly 3 Synthesis of viral genomes and proteins

35 Animation: Phage T4 Lytic Cycle

36 The Lysogenic Cycle The lysogenic cycle replicates the phage genome without destroying the host The viral DNA molecule is incorporated into the host cell s chromosome This integrated viral DNA is known as a prophage Every time the host divides, it copies the phage DNA and passes the copies to daughter cells

37 Figure 19.6 Phage DNA Phage Tail fiber The phage injects its DNA. Bacterial chromosome Phage DNA circularizes. Daughter cell with prophage Prophage exits chromosome. Many cell divisions create many infected bacteria. The cell lyses, releasing phages. Lytic cycle Lysogenic cycle Prophage Prophage is copied with bacterial chromosome. Phage DNA and proteins are synthesized and assembled. Phage DNA integrates into bacterial chromosome.

38 Figure 19.6a Phage Phage DNA Tail fiber The phage injects its DNA. Bacterial chromosome Phage DNA circularizes. Lytic cycle The cell lyses, releasing phages. Phage DNA and proteins are synthesized and assembled.

39 Figure 19.6b Daughter cell with prophage Prophage exits chromosome. Many cell divisions create many infected bacteria. Lysogenic cycle Prophage Prophage is copied with bacterial chromosome. Phage DNA integrates into bacterial chromosome.

40 Animation: Phage Lambda Lysogenic and Lytic Cycles

41 An environmental signal can trigger the virus genome to exit the bacterial chromosome and switch to the lytic mode Phages that use both the lytic and lysogenic cycles are called temperate phages

42 Replicative Cycles of Animal Viruses There are two key variables used to classify viruses that infect animals An RNA or DNA genome A single-stranded or double-stranded genome Whereas few bacteriophages have an envelope or an RNA genome, many animal viruses have both

43 Table 19.1

44 Table 19.1a

45 Table 19.1b

46 Viral Envelopes Many viruses that infect animals have a membranous envelope Viral glycoproteins on the envelope bind to specific receptor molecules on the surface of a host cell Some viral envelopes are derived from the host cell s plasma membrane as the viral capsids exit

47 Other viral membranes form from the host s nuclear envelope and are then replaced by an envelope made from Golgi apparatus membrane

48 Capsid Figure RNA19.7 HOST CELL Envelope (with glycoproteins) mrna Template Viral genome (RNA) Glycoproteins ER Capsid proteins Copy of genome (RNA) New virus

49 RNA as Viral Genetic Material The broadest variety of RNA genomes is found in viruses that infect animals Retroviruses use reverse transcriptase to copy their RNA genome into DNA HIV (human immunodeficiency virus) is the retrovirus that causes AIDS (acquired immunodeficiency syndrome)

50 The viral DNA that is integrated into the host genome is called a provirus Unlike a prophage, a provirus remains a permanent resident of the host cell RNA polymerase transcribes the proviral DNA into RNA molecules The RNA molecules function both as mrna for synthesis of viral proteins and as genomes for new virus particles released from the cell

51 Glycoprotein Figure 19.8 HIV Reverse transcriptase Viral envelope Capsid RNA (two identical strands) Viral RNA RNA-DNA hybrid DNA Chromosomal DNA RNA genome for the progeny viruses mrna HOST CELL Reverse transcriptase NUCLEUS Provirus HIV Membrane of white blood cell 0.25 µm HIV entering a cell New virus New HIV leaving a cell

52 Figure 19.8a Glycoprotein HIV Reverse transcriptase Viral envelope Capsid RNA (two identical strands) Viral RNA RNA-DNA hybrid DNA HOST CELL Reverse transcriptase

53 Figure 19.8b Chromosomal DNA RNA genome for the progeny viruses mrna NUCLEUS Provirus New virus

54 Figure 19.8c HIV Membrane of white blood cell 0.25 µm HIV entering a cell New HIV leaving a cell

55 Figure 19.8ca HIV Membrane of white blood cell 0.25 µm HIV entering a cell

56 Figure 19.8cb 0.25 µm HIV entering a cell

57 Figure 19.8cc New HIV leaving a cell 0.25 µm

58 Figure 19.8cd New HIV leaving a cell 0.25 µm

59 Figure 19.8ce New HIV leaving a cell 0.25 µm

60 Animation: HIV Reproductive Cycle

61 Evolution of Viruses Viruses do not fit our definition of living organisms Since viruses can replicate only within cells, they probably evolved as bits of cellular nucleic acid Candidates for the source of viral genomes include plasmids and transposons Plasmids, transposons, and viruses are all mobile genetic elements

62 The largest virus yet discovered is the size of a small bacterium, and its genome encodes proteins involved in translation, DNA repair, protein folding, and polysaccharide synthesis There is controversy about whether this virus evolved before or after cells

63 CAMPBELL BIOLOGY TENTH EDITION Reece Urry Cain Wasserman Minorsky Jackson 20 DNA Tools and Biotechnology Lecture Presentation by Nicole Tunbridge and Kathleen Fitzpatrick 2014 Pearson Education, Inc.

64 The DNA Toolbox Recently the genome sequences of two extinct species Neanderthals and wooly mammoths have been completed Advances in sequencing techniques make genome sequencing increasingly faster and less expensive

65 Figure 20.1

66 Figure 20.1a

67 Biotechnology is the manipulation of organisms or their components to make useful products The applications of DNA technology affect everything from agriculture, to criminal law, to medical research

68 Concept 20.1: DNA sequencing and DNA cloning are valuable tools for genetic engineering and biological inquiry The complementarity of the two DNA strands is the basis for nucleic acid hybridization, the base pairing of one strand of nucleic acid to the complementary sequence on another strand Genetic engineering is the direct manipulation of genes for practical purposes

69 DNA Sequencing Researchers can exploit the principle of complementary base pairing to determine a gene s complete nucleotide sequence, called DNA sequencing The first automated procedure was based on a technique called dideoxy or chain termination sequencing, developed by Sanger

70 Figure 20.2 (a) Standard sequencing machine (b) Next-generation sequencing machines

71 Figure 20.2a (a) Standard sequencing machine

72 Figure 20.2b (b) Next-generation sequencing machines

73 Figure 20.3 Technique DNA (template strand) 5 C T G A C TT A 3 A 5 C G AC C T G A C TT C G AC DNA (template strand) dd Primer 3 T G T T 5 DNA polymerase G 3 dd CT dd A G dd Deoxyribonucleotides datp Labeled strands A A G dctp dttp dgtp dd G AA G C T G dd T G AA G Dideoxyribonucleotides (fluorescently tagged) C T G A A G ddatp ddctp ddttp ddgtp P P P P P P G dd dd A C T G A A G dd G 3 AC T G AA G CT G A 3 A C T G T T 5 Shortest Direction of movement of strands C C C C C G TT T TGTT TGTT T TGTT TGTT TGTT Longest labeled strand Detector G TT 5 Longest Results Laser Last nucleotide of longest labeled strand Last nucleotide of shortest labeled strand G A C T G A A G C Shortest labeled strand

74 Figure 20.3a Technique DNA (template strand) 5 3 C T G A C T T C G A C A A Primer 3 T G T T 5 DNA polymerase Deoxyribonucleotides datp dctp dttp dgtp Dideoxyribonucleotides (fluorescently tagged) ddatp ddctp ddttp ddgtp P P P P P P G G

75 Figure 20.3b Technique 5 3 C T G A C TT C G AC A A DNA (template strand) dd C T G T T dd 3 G C T G T dd A G C T G T dd Labeled strands A A G C T G T dd G A A G C T G T 5 T T T T T T T 5 Shortest Longest dd T G A A G C T G T dd C T G AA G C T G TT dd A C T G AA G C T G T dd G A C T G AA G C T G T 3

76 Figure 20.3c Technique Direction of movement of strands Longest labeled strand Detector Results Laser Last nucleotide of longest labeled strand Last nucleotide of shortest labeled strand G A C T G A A G C Shortest labeled strand

77 Next-generation sequencing techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments Thousands or hundreds of thousands of fragments (400 1,000 nucleotides long) are sequenced in parallel This is a type of high-throughput technology

78 Figure 20.4 Technique 1 Genomic DNA is fragmented. Results 2 Each fragment is isolated with a bead. 4-mer 3-mer A T G C 3 Using PCR, 10 6 copies of each fragment are made, each attached to the bead by 5 end. 2-mer 1-mer 4 The bead is placed into a well with DNA polymerases and primers. Template strand of DNA Primer A T G C 5 A solution of each of the four nucleotides is added to all wells and then washed off. The entire process is then repeated. A T G C A T G C A T G C A T G C DNA polymerase Template C C strand C C C C C C A of DNA A dttp A dgtp A A datp A A A T T T T G G G GC TA PP TA i TA T A GC GC GC GC GC GC GC GC AG Primer AG AG AG TA TA TA TA dctp PP i 6 If a nucleotide is joined to a growing strand, PP i is released, causing a flash of light that is recorded. 7 If a nucleotide is not complementary to the next template base, no PP i is released, and no flash of light is recorded. 8 The process is repeated until every fragment has a complete complementary strand. The pattern of flashes reveals the sequence.

79 Figure 20.4a Technique 1 Genomic DNA is fragmented. 2 3 Each fragment is isolated with a bead. Using PCR, 10 6 copies of each fragment are made, each attached to the bead by 5 end. 4 The bead is placed into a well with DNA polymerases and primers. Template strand of DNA Primer A T G C 5 A solution of each of the four nucleotides is added to all wells and then washed off. The entire process is then repeated.

80 Figure 20.4b Technique A T G C A T G C DNA polymerase C C A A T G T A GC GC AG TA Template strand of DNA datp PP i Primer C C A A T G T A GC GC AG TA dttp 6 If a nucleotide is joined to 7 a growing strand, PP i is released, causing a flash of light that is recorded. If a nucleotide is not complementary to the next template base, no PP i is released, and no flash of light is recorded.

81 Figure 20.4c Technique A T G C A T G C C C A A T G T A GC GC AG TA dgtp C C A A T GC T A GC GC AG TA dctp PP i 8 The process is repeated until every fragment has a complete complementary strand. The pattern of flashes reveals the sequence.

82 Figure 20.4d Results 4-mer 3-mer A T G C 2-mer 1-mer

83 In third-generation sequencing, the techniques used are even faster and less expensive than the previous

84 Making Multiple Copies of a Gene or Other DNA Segment To work directly with specific genes, scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome Researchers can insert DNA into plasmids to produce recombinant DNA, a molecule with DNA from two different sources

85 Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA This results in the production of multiple copies of a single gene The production of multiple copies of a single gene is a type of DNA cloning called gene cloning

86 Figure 20.5 Bacterial chromosome Bacterium Plasmid 1 Recombinant DNA (plasmid) Gene inserted into plasmid 2 Cell containing gene of interest Gene of interest Plasmid put into bacterial cell DNA of chromosome ( foreign DNA) Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the cloned gene of interest Gene of interest Protein expressed from gene of interest Copies of gene Protein harvested Gene for pest resistance inserted into plants 4 Basic research and various applications Human growth hormone treats stunted growth Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy

87 Figure 20.5a Bacterium Cell containing gene of interest Bacterial chromosome Plasmid 1 Recombinant DNA (plasmid) Gene inserted into plasmid Gene of interest DNA of chromosome ( foreign DNA) 2 Plasmid put into bacterial cell Recombinant bacterium Gene of interest 3 Host cell grown in culture to form a clone of cells containing the cloned gene of interest Protein expressed from gene of interest

88 Figure 20.5b Gene of interest Copies of gene Protein expressed from gene of interest Protein harvested 4 Basic research and various applications Gene for pest resistance inserted into plants Human growth hormone treats stunted growth Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy

89 A plasmid used to clone a foreign gene is called a cloning vector Bacterial plasmids are widely used as cloning vectors because they are readily obtained, easily manipulated, easily introduced into bacterial cells, and once in the bacteria they multiply rapidly Gene cloning is useful for amplifying genes to produce a protein product for research, medical, or other purposes

90 Using Restriction Enzymes to Make a Recombinant DNA Plasmid Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites A restriction enzyme usually makes many cuts, yielding restriction fragments The most useful restriction enzymes cut DNA in a staggered way, producing fragments with sticky ends

91 Sticky ends can bond with complementary sticky ends of other fragments DNA ligase is an enzyme that seals the bonds between restriction fragments

92 Figure 20.6 Bacterial plasmid Restriction site 1 5 GAATTC DNA CTTAAG 3 Restriction enzyme cuts the sugar-phosphate backbones at each arrow Base pairing of sticky ends produces various combinations. 5 3 Sticky end Fragment from different DNA molecule cut by the same restriction enzyme G AAT T C G AATT C C TTA A G C TTAA G DNA ligase One possible combination seals the strands Recombinant DNA molecule 5 Recombinant plasmid

93 Figure 20.6a Bacterial plasmid Restriction site 1 DNA 5 Restriction enzyme cuts the sugar-phosphate backbones at each arrow G A AT T C C T TA A G Sticky end 5

94 Figure 20.6b Base pairing of sticky ends produces various combinations. 5 3 Sticky end G AATT C G AATT C C TTAA G C TTAA G One possible combination Fragment from different DNA molecule cut by the same restriction enzyme

95 Figure 20.6c 3 DNA ligase seals the strands G AATT C G AATT C C TTAA G C TTAA G One possible combination Recombinant DNA molecule 5 Recombinant plasmid

96 Animation: Restriction Enzymes

97 To check the recombinant plasmid, researchers might cut the products again using the same restriction enzyme To separate and visualize the fragments produced, gel electrophoresis would be carried out This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size, charge, or other physical properties

98 Figure 20.7 Mixture of DNA molecules of different sizes Power source Cathode Wells Anode Gel (a) Negatively charged DNA molecules move toward the positive electrode. Restriction fragments (size standards) (b) Shorter molecules are slowed down less than longer ones, so they move faster through the gel.

99 Figure 20.7a Mixture of DNA molecules of different sizes Power source Cathode Wells Anode Gel (a) Negatively charged DNA molecules move toward the positive electrode.

100 Figure 20.7b Restriction fragments (size standards) (b) Shorter molecules are slowed down less than longer ones, so they move faster through the gel.

101 Video: Biotechnology Lab

102 Amplifying DNA: The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning 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

103 The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase PCR uses a pair of primers specific for the sequence to be amplified PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells

104 Figure 20.8 Technique 5 3 Target sequence Genomic DNA Denaturation 5 3 Cycle 1 yields 2 molecules 2 Annealing 3 5 Primers 3 Extension New nucleotides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

105 Figure 20.8a Technique Genomic DNA Target sequence

106 Figure 20.8b-1 Technique 1 Denaturation Cycle 1 yields 2 molecules

107 Figure 20.8b-2 Technique 1 Denaturation Cycle 1 yields 2 molecules 2 Annealing Primers

108 Figure 20.8b-3 Technique 1 Denaturation Cycle 1 yields 2 molecules 2 Annealing Primers 3 Extension New nucleotides

109 Figure 20.8c Technique Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Results After 30 more cycles, over 1 billion (10 9 ) molecules match the target sequence.

110 PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors The resulting clones are sequenced and error-free inserts selected

111 Figure 20.9 DNA fragments obtained by PCR with restriction sites matching those in the cloning vector A gene that makes bacterial cells resistant to an antibiotic is present on the plasmid. Cut with same restriction enzyme used on cloning vector Cloning vector (bacterial plasmid) Mix and ligate Only cells that take up a plasmid will survive Recombinant DNA plasmid

112 Animation: Cloning a Gene

113 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

114 Bacterial Expression Systems Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active bacterial promoter

115 Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes Researchers can avoid this problem by using cdna, complementary to the mrna, which contains only exons

116 Eukaryotic DNA Cloning and Expression Systems Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells, such as yeasts, as hosts for cloning and expressing genes Even yeasts may not possess the proteins required to modify expressed mammalian proteins properly In such cases, cultured mammalian or insect cells may be used to express and study proteins

117 One method of introducing recombinant DNA into eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes Alternatively, scientists can inject DNA into cells using microscopically thin needles Once inside the cell, the DNA is incorporated into the cell s DNA by natural genetic recombination

118 Cross-Species Gene Expression and Evolutionary Ancestry The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species For example, Pax-6 is a gene that directs formation of a vertebrate eye; the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye) The Pax-6 genes in flies and vertebrates can substitute for each other

119 Concept 20.2: Biologists use DNA technology to study gene expression and function Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function

120 Analyzing Gene Expression The most straightforward way to discover which genes are expressed in certain cells is to identify the mrnas being made

121 Studying the Expression of Single Genes mrna can be detected by nucleic acid hybridization with complementary molecules These complementary molecules, of either DNA or RNA, are nucleic acid probes

122 In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mrnas in place in the intact organism

123 Figure TAACGGTTCCAGC CTCAAGTTGCTCT ATTGCCAAGGTCG GAGTTCAACGAGA wg mrna en mrna Cells expressing the wg gene Head Thorax Abdomen Cells expressing the en gene 50 µm Segment boundary T1 T2 T3 A1 A2 A3 A4 A5 Head Thorax Abdomen

124 Figure 20.10a 3 TAACGGTTCCAGC ATTGCCAAGGTCG wg mrna Cells expressing the wg gene CTCAAGTTGCTCT GAGTTCAACGAGA en mrna 5 3 Cells expressing the en gene Head Thorax Abdomen 50 µm T1 T2 T3 A1 A2 A3 A4 A5

125 Figure 20.10b Head Thorax Abdomen 50 µm Segment boundary T1 T2 T3 A1 A2 A3 A4 A5 Head Thorax Abdomen

126 Figure 20.10c Head Thorax Abdomen 50 µm T1 T2 T3 A1 A2 A3 A4 A5

127 Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mrnas in several samples at the same time Reverse transcriptase is added to mrna to make complementary DNA (cdna), which serves as a template for PCR amplification of the gene of interest The products are run on a gel and the mrna of interest is identified

128 Figure DNA in nucleus mrnas in cytoplasm

129 Figure DNA in nucleus mrnas in cytoplasm 5 Reverse transcriptase mrna Poly-A tail 3 DNA strand A A A A A A TTTTT 3 5 Primer (poly-dt)

130 Figure DNA in nucleus mrnas in cytoplasm 5 Reverse transcriptase mrna Poly-A tail 3 DNA strand A A A A A A TTTTT 3 5 Primer (poly-dt) 5 3 A A A A A A TTTTT 3 5

131 Figure DNA in nucleus mrnas in cytoplasm 5 Reverse transcriptase mrna Poly-A tail 3 DNA strand A A A A A A TTTTT 3 5 Primer (poly-dt) 5 3 A A A A A A TTTTT DNA polymerase

132 Figure DNA in nucleus mrnas in cytoplasm 5 Reverse transcriptase mrna Poly-A tail 3 DNA strand A A A A A A TTTTT 3 5 Primer (poly-dt) 5 3 A A A A A A TTTTT DNA polymerase cdna

133 Figure Technique 1 cdna synthesis mrnas 2 PCR amplification cdnas Primers 3 Gel electrophoresis Specific gene Results Embryonic stages

134 Studying the Expression of Interacting Groups of Genes Automation has allowed scientists to measure the 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

135 Figure Each dot is a well containing identical copies of DNA fragments that carry a specific gene. Genes expressed in first tissue. Genes expressed in second tissue. Genes expressed in both tissues. DNA microarray (actual size) Genes expressed in neither tissue.

136 Figure 20.13a Each dot is a well containing identical copies of DNA fragments that carry a specific gene.

137 With rapid and inexpensive sequencing methods available, researchers can also just sequence cdna samples from different tissues or embryonic stages to determine the gene expression differences between them By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets

138 Determining Gene Function 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

139 Gene expression can also be silenced using RNA interference (RNAi) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the gene s mrna

140 In humans, researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition These genome-wide association studies test for genetic markers, sequences that vary among individuals SNPs (single nucleotide polymorphisms), single nucleotide variants, are among the most useful genetic markers

141 SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene SNPs are rarely directly involved in the disease; they are most often in noncoding regions of the genome

142 Figure DNA SNP A T Normal allele C G Disease-causing allele

143 Concept 21.5: Duplication, rearrangement, and mutation of DNA contribute to genome evolution The basis of change at the genomic level is mutation, which underlies much of genome evolution The earliest forms of life likely had only those genes necessary for survival and reproduction The size of genomes has increased over evolutionary time, with the extra genetic material providing raw material for gene diversification

144 Duplication of Entire Chromosome Sets Accidents in meiosis can lead to one or more extra sets of chromosomes, a condition known as polyploidy The genes in one or more of the extra sets can diverge by accumulating mutations; these variations may persist if the organism carrying them survives and reproduces In this way genes with novel functions can evolve

145 Alterations of Chromosome Structure Humans have 23 pairs of chromosomes, while chimpanzees have 24 pairs Following the divergence of humans and chimpanzees from a common ancestor, two ancestral chromosomes fused in the human line Duplications and inversions result from mistakes during meiotic recombination Comparative analysis between chromosomes of humans and seven mammalian species paints a hypothetical chromosomal evolutionary history

146 Figure Telomere sequences Human chromosome Chimpanzee chromosomes Centromere sequences Telomere-like sequences 12 Centromere-like sequences 2 13

147 Figure Human chromosome Mouse chromosomes

148 The rate of duplications and inversions seems to have accelerated about 100 million years ago This coincides with when large dinosaurs went extinct and mammals diversified Chromosomal rearrangements are thought to contribute to the generation of new species

149 Duplication and Divergence of Gene-Sized Regions of DNA Unequal crossing over during prophase I of meiosis can result in one chromosome with a deletion and another with a duplication of a particular region Transposable elements can provide sites for crossover between nonsister chromatids

150 Figure Nonsister chromatids Gene Transposable element Incorrect pairing of two homologs during meiosis Crossover point and

151 Evolution of Genes with Related Functions: The Human Globin Genes The genes encoding the various globin proteins evolved from one common ancestral globin gene, which duplicated and diverged about million years ago After the duplication events, differences between the genes in the globin family arose from the accumulation of mutations

152 Figure Evolutionary time Ancestral globin gene Duplication of ancestral gene Mutation in both copies α β Transposition to different chromosomes Further duplications and mutations ζ α α ϵ β β ζ ζ α α 2 α 1 β 2 α 1 y θ ϵ G β A α-globin gene family on chromosome 16 β-globin gene family on chromosome 11

153 Subsequent duplications of these genes and random mutations gave rise to the present globin genes, which code for oxygen-binding proteins The similarity in the amino acid sequences of the various globin proteins supports this model of gene duplication and mutation

154 Evolution of Genes with Novel Functions The copies of some duplicated genes have diverged so much in evolution that the functions of their encoded proteins are now very different For example the lysozyme gene was duplicated and evolved into the gene that encodes -lactalbumin in mammals Lysozyme is an enzyme that helps protect animals against bacterial infection -lactalbumin is a nonenzymatic protein that plays a role in milk production in mammals

155 Figure (a) Lysozyme (b) α lactalbumin Lysozyme α lactalbumin Lysozyme α lactalbumin Lysozyme α lactalbumin (c) Amino acid sequence alignments of lysozyme and α lactalbumin

156 Rearrangements of Parts of Genes: Exon Duplication and Exon Shuffling The duplication or repositioning of exons has contributed to genome evolution Errors in meiosis can result in an exon being duplicated on one chromosome and deleted from the homologous chromosome In exon shuffling, errors in meiotic recombination lead to some mixing and matching of exons, either within a gene or between two nonallelic genes

157 Figure EGF EGF EGF EGF Epidermal growth factor gene with multiple EGF exons F F F F Fibronectin gene with multiple finger exons Exon shuffling F EGF Exon duplication K K K Plasminogen gene with a kringle exon Exon shuffling Portions of ancestral genes TPA gene as it exists today

158 How Transposable Elements Contribute to Genome Evolution Multiple copies of similar transposable elements may facilitate recombination, or crossing over, between different chromosomes Insertion of transposable elements within a protein-coding sequence may block protein production Insertion of transposable elements within a regulatory sequence may increase or decrease protein production

159 Transposable elements may carry a gene or groups of genes to a new position Transposable elements may also create new sites for alternative splicing in an RNA transcript In all cases, changes are usually detrimental but may on occasion prove advantageous to an organism