The Lysogenic Cycle the Lytic Cycle

CAMPBELL

TENTH BIOLOGY EDITION Reece • Urry • Cain • Wasserman • Minorsky • Jackson 19 Viruses

Lecture Presentation by Nicole Tunbridge and Kathleen Fitzpatrick

• 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 Figure 19.1 Figure 19.1a 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 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) Experiment Figure 19.2

1 Extracted sap 2 Passed sap 3 Rubbed filtered from tobacco through a sap on healthy plant with porcelain filter tobacco plants tobacco mosaic known to trap disease bacteria

4 Healthy plants became infected Figure 19.2a Figure 19.2b Figure 19.2c 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 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 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 Membranous RNA RNA Head Capsomere DNA envelope Figure 19.3 Capsid DNA Capsomere Tail of capsid sheath Tail fiber Glycoprotein Glycoproteins 18  250 nm 70–90 nm (diameter) 80–200 nm (diameter) 80  225 nm

20 nm 50 nm 50 nm 50 nm (a) Tobacco mosaic (b) Adenoviruses (c) Influenza viruses (d) Bacteriophage T4 virus RNA Capsomere DNA

Figure 19.3a Capsomere of capsid

Glycoprotein 18  250 nm 70–90 nm (diameter)

20 nm 50 nm (a) Tobacco mosaic (b) Adenoviruses virus Figure 19.3aa

20 nm

(a) Tobacco mosaic virus Figure 19.3ab

50 nm

(b) Adenoviruses Membranous RNA envelope Head Capsid DNA Figure 19.3b Tail sheath

Tail fiber Glycoproteins

80–200 nm (diameter) 80  225 nm

50 nm 50 nm (c) Influenza viruses (d) Bacteriophage T4 Figure 19.3ba

50 nm

50 nm (d) Bacteriophage T4 • 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 • 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 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 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 self- assemble into new viruses VIRUS DNA Transcription and Capsid 3 1 Entry and manufacture of Figure 19.4uncoating capsid proteins

HOST CELL 2 Replication Viral DNA

mRNA

Viral DNA

Capsid proteins

4 Self-assembly of new virus particles and their exit from the cell Animation: Simplified Viral Reproductive Cycle 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 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 1 Attachment Figure 19.5-1 1 Attachment

Figure 19.5-2 2 Entry of phage DNA and degradation of host DNA 1 Attachment

Figure 19.5-3 2 Entry of phage DNA and degradation of host DNA

3 Synthesis of viral genomes and proteins 1 Attachment

Figure 19.5-4 2 Entry of phage DNA and degradation of host DNA Phage assembly

3 Synthesis of viral 4 Head Tail Tail Self-assembly genomes and fibers proteins 1 Attachment

Figure 19.5-5 2 Entry of phage DNA and degradation 5 Release of host DNA Phage assembly

3 Synthesis of viral 4 Head Tail Tail Self-assembly genomes and fibers proteins Animation: Phage T4 Lytic Cycle 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 Phage Daughter cell DNA The phage injects its DNA. with prophage Figure 19.6 Many cell divisions Phage DNA create many Tail fiber circularizes. infected Phage bacteria. Bacterial Prophage exits chromosome chromosome.

Lytic Lysogenic cycle cycle Prophage is copied The cell lyses, with bacterial releasing Prophage chromosome. phages.

Phage DNA and proteins Phage DNA integrates are synthesized and into bacterial assembled. chromosome. Phage DNA The phage injects its DNA. Figure 19.6a Phage DNA Tail fiber circularizes. Phage Bacterial chromosome

Lytic cycle

The cell lyses, releasing phages.

Phage DNA and proteins are synthesized and assembled. Daughter cell with prophage Many cell Figure 19.6b divisions create many infected bacteria. Prophage exits chromosome.

Lysogenic cycle Prophage is copied with bacterial Prophage chromosome.

Phage DNA integrates into bacterial chromosome. Animation: Phage Lambda Lysogenic and Lytic Cycles • 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 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 Table 19.1 Table 19.1a Table 19.1b 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 • Other viral membranes form from the host’s nuclear envelope and are then replaced by an envelope made from Golgi apparatus membrane Capsid

RNA Figure 19.7 HOST CELL

Envelope (with glycoproteins) Viral genome Template (RNA) mRNA

Capsid proteins Copy of genome ER (RNA)

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

New virus New HIV leaving a cell Figure 19.8a Viral envelope Glycoprotein Capsid RNA (two identical HOST strands) CELL HIV Reverse Viral RNA Reverse transcriptase transcriptase RNA-DNA hybrid DNA Figure 19.8b NUCLEUS Provirus Chromosomal DNA RNA genome for the progeny viruses mRNA

New virus Figure 19.8c Membrane of HIV white blood cell

0.25 µm HIV entering a cell New HIV leaving a cell Figure 19.8ca

Membrane of HIV white blood cell

0.25 µm HIV entering a cell Figure 19.8cb

0.25 µm HIV entering a cell Figure 19.8cc

0.25 µm New HIV leaving a cell Figure 19.8cd

0.25 µm New HIV leaving a cell Figure 19.8ce

0.25 µm New HIV leaving a cell Animation: HIV Reproductive Cycle 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 • 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 CAMPBELL

TENTH BIOLOGY EDITION Reece • Urry • Cain • Wasserman • Minorsky • Jackson 20 DNA Tools and Biotechnology

Lecture Presentation by Nicole Tunbridge and Kathleen Fitzpatrick

• 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 Figure 20.1 Figure 20.1a • 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 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 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 Figure 20.2

(a) Standard sequencing machine

(b) Next-generation sequencing machines Figure 20.2a

(a) Standard sequencing machine Figure 20.2b

(b) Next-generation sequencing machines Figure 20.3 Technique DNA Primer Deoxyribo- Dideoxyribonucleotides (template strand) 3′ nucleotides (fluorescently tagged) T 5′ C G dATP T T ddATP G T dCTP ddCTP A 5′ C DNA dTTP ddTTP T T polymerase dGTP ddGTP C G A P P P P P P C G G A 3′ A DNA (template Labeled strands 5′ 3′ C strand) dd G T dd A A G dd C C C A dd T T T T C dd G G G G G T dd A A A A A A T dd A A A A A A A C 3′dd G G G G G G G G G dd C C C C C C C C C A T T T T T T T T T C G G G G G G G G G A T T T T T T T T T 3′ A T 5′ T T T T T T T T 5′ Shortest Longest Direction of movement Longest labeled strand of strands

Detector

Laser Shortest labeled strand Results

Last nucleotide G of longest A labeled strand C T G Last nucleotide A A of shortest G labeled strand C Figure 20.3a

Technique DNA Primer Deoxyribo- Dideoxyribonucleotides (template strand) 3′ nucleotides (fluorescently tagged) T 5′ C G dATP T T ddATP G T dCTP ddCTP A 5′ C DNA dTTP ddTTP T T polymerase dGTP ddGTP C G A P P P P P P C G G A 3′ A Figure 20.3b

Technique

DNA (template Labeled strands 5′ 3′ C strand) dd G T dd A A G dd C C C A dd T T T T C dd G G G G G T dd A A A A A A T dd A A A A A A A C dd G G G G G G G G G dd C 3′ C C C C C C C C A T T T T T T T T T C G G G G G G G G G A T T T T T T T T T 3′ A T 5′ T T T T T T T T 5′ Shortest Longest Figure 20.3c

Technique Direction of movement Longest labeled strand of strands

Detector

Laser Shortest labeled strand Results Last nucleotide G of longest A C labeled strand T G A Last nucleotide A of shortest G labeled strand C • “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 Figure 20.4 Technique

1 Genomic DNA is fragmented. Results

4-mer A 2 Each fragment is isolated with T 3-mer G a bead. C

2-mer 3 Using PCR, 106 copies of each fragment are made, each attached 1-mer to the bead by 5′ end.

4 The bead is placed into a well with DNA polymerases and primers. Template strand of DNA 3′ 5′ 3′ 5′ 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

Template C C C strand C C C C C of DNA A A dTTP A dGTP A dCTP A dATP A A A T T T T G G G GC PP TA TA TA T A i PPi DNA GC GC GC GC polymerase GC GC GC GC AG Primer AG AG AG TA TA TA TA

6 If a nucleotide is joined to 7 If a nucleotide is not 8 The process is repeated until every

a growing strand, PPi is complementary to the fragment has a complete complementary released, causing a flash next template base, strand. The pattern of flashes reveals the of light that is recorded. no PPi is released, and sequence. no flash of light is recorded. Figure 20.4a Technique

1 Genomic DNA is fragmented.

2 Each fragment is isolated with a bead.

3 Using PCR, 106 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 3′ 5′ 3′ 5′ 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. Figure 20.4b

Technique

A T G C A T G C

Template C strand C C C of DNA A A dTTP A dATP A T T G G T A T A PPi DNA GC GC polymerase GC GC AG Primer AG TA TA

6 If a nucleotide is joined to 7 If a nucleotide is not a growing strand, PPi is complementary to the released, causing a flash next template base, of light that is recorded. no PPi is released, and no flash of light is recorded. Figure 20.4c

Technique

A T G C A T G C

C C C C A dGTP A dCTP A A T T G GC PP T A T A i GC GC GC GC AG AG TA TA

8 The process is repeated until every fragment has a complete complementary strand. The pattern of flashes reveals the sequence. Figure 20.4d

Results

4-mer A T 3-mer G C

2-mer

1-mer • In “third-generation sequencing,” the techniques used are even faster and less expensive than the previous 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 • 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 Figure 20.5 Bacterium Cell containing gene of interest 1 Gene inserted into plasmid Bacterial Plasmid chromosome DNA of chromosome Recombinant Gene of (“foreign” DNA) DNA (plasmid) interest 2 Plasmid put into bacterial cell

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

4 Basic research Human growth hormone Gene for pest resistance and various treats stunted growth inserted into plants applications

Gene used to alter bacteria Protein dissolves blood clots for cleaning up toxic waste in heart attack therapy Figure 20.5a Bacterium Cell containing gene of interest

1 Gene inserted into plasmid Bacterial Plasmid chromosome DNA of chromosome Recombinant Gene of (“foreign” DNA) DNA (plasmid) interest 2 Plasmid put into bacterial cell

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 Figure 20.5b

Gene of interest Protein expressed from gene of interest

Copies of gene Protein harvested

4 Basic research and various applications

Gene for pest resistance Human growth hormone inserted into plants treats stunted growth

Gene used to alter Protein dissolves bacteria for cleaning blood clots in heart up toxic waste attack therapy • 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 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” • Sticky ends can bond with complementary sticky ends of other fragments • DNA ligase is an enzyme that seals the bonds between restriction fragments Figure 20.6 Bacterial plasmid

Restriction site

5′ 3′ DNA GAATTC CTTAAG 3′ 5′ 1 Restriction enzyme cuts the sugar-phosphate backbones at each arrow. 3′ 5′ 5′ 3′

5′ 3′ 3′ Sticky end 5′ 5′ 3′ 2 Base pairing of sticky ends produces various 3′ combinations. 5′ Fragment from different DNA molecule cut by the same restriction enzyme

5′ 3′ 5′ 3′ 5′ 3′ G AAT T C G AATT C C TTA A G C TTAA G 3′ 5′ 3′ 5′ 3′ 5′ One possible combination 3 DNA ligase seals the strands.

5′ 3′

3′ Recombinant DNA molecule 5′

Recombinant plasmid Figure 20.6a

Bacterial plasmid

Restriction site 5′ 3′ DNA GAATTC CTTAAG 3′ 5′ 1 Restriction enzyme cuts the sugar-phosphate backbones at each arrow. 3′ 5′ 5′ 3′

5′ 3′ 3′ Sticky end 5′ Figure 20.6b

3′ 5′ 5′ 3′

5′ 3′ 3′ Sticky end 5′ 5′ 3′ 2 Base pairing of sticky ends produces various 3′ combinations. 5′ Fragment from different DNA molecule cut by the same restriction enzyme 5′ 3′ 5′ 3′ 5′ 3′ G AATT C G AATT C C TTAA G C TTAA G 3′ 5′ 3′ 5′ 3′ 5′ One possible combination Figure 20.6c 5′ 3′ 5′ 3′ 5′ 3′ G AATT C G AATT C C TTAA G C TTAA G 3′ 5′ 3′ 5′ 3′ 5′ One possible combination 3 DNA ligase seals the strands 5′ 3′

3′ Recombinant DNA molecule 5′

Recombinant plasmid Animation: Restriction Enzymes • 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 Figure 20.7 Mixture of Power DNA mol- source ecules of Cathode Anode different sizes Wells 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. Figure 20.7a

Mixture of Power DNA mol- source ecules of Cathode Anode different sizes Wells

Gel

(a) Negatively charged DNA molecules move toward the positive electrode. Figure 20.7b

Restriction fragments (size standards) (b) Shorter molecules are slowed down less than longer ones, so they move faster through the gel. Video: Biotechnology Lab 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 • 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 Figure 20.8 Technique 5′ 3′ Target sequence Genomic DNA 3′ 5′

1 Denaturation 5′ 3′

3′ 5′ 2 Annealing Cycle 1 yields 2 Primers molecules 3 Extension

New nucleotides

Cycle 2 yields 4 molecules

Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Figure 20.8a

Technique 5′ 3′ Target sequence

Genomic DNA 3′ 5′ Figure 20.8b-1 Technique

1 Denaturation 5′ 3′

3′ 5′

Cycle 1 yields 2 molecules Figure 20.8b-2 Technique

1 Denaturation 5′ 3′

3′ 5′ 2 Annealing Cycle 1 yields 2 Primers molecules Figure 20.8b-3 Technique

1 Denaturation 5′ 3′

3′ 5′ 2 Annealing Cycle 1 yields 2 Primers molecules

3 Extension

New nucleotides 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 (109) molecules match the target sequence. • 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 Figure 20.9 DNA fragments obtained by PCR with restriction sites matching those in the cloning vector

Cut with same restriction enzyme used on cloning A gene that makes bacterial vector cells resistant to an antibiotic is present on the plasmid.

Cloning vector (bacterial plasmid) Mix and ligate

Recombinant DNA plasmid

Only cells that take up a plasmid will survive Animation: Cloning a Gene 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 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 • 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 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 • 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 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 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 Analyzing Gene Expression

• The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made 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 • In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism Figure 20.10 3′ 5′ 3′ 5′ TAACGGTTCCAGC CTCAAGTTGCTCT ATTGCCAAGGTCG GAGTTCAACGAGA 5′ 3′ 5′ 3′ wg mRNA en mRNA Cells Cells expressing expressing the wg gene the en gene Head Thorax Abdomen

50 µm T1 T2 T3 A1 A2 A3 A4 A5 Segment boundary

Head Thorax Abdomen Figure 20.10a 3′ 5′ 3′ 5′ TAACGGTTCCAGC CTCAAGTTGCTCT ATTGCCAAGGTCG GAGTTCAACGAGA 5′ 3′ 5′ 3′ wg mRNA en mRNA Cells Cells expressing expressing the wg gene the en gene Head Thorax Abdomen

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

50 µm T1 T2 T3 A1 A2 A3 A4 A5 Segment boundary

Head Thorax Abdomen Figure 20.10c

Head Thorax Abdomen

50 µm T1 T2 T3 A1 A2 A3 A4 A5 • 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 Figure 20.11-1 DNA in nucleus mRNAs in cytoplasm Figure 20.11-2 DNA in nucleus mRNAs in cytoplasm

Reverse transcriptase mRNA Poly-A tail 5′ AAAAAA 3′ 3′ TTTTT 5′ DNA Primer strand (poly-dT) Figure 20.11-3 DNA in nucleus mRNAs in cytoplasm

Reverse transcriptase mRNA Poly-A tail 5′ AAAAAA 3′ 3′ TTTTT 5′ DNA Primer strand (poly-dT)

5′ AAA AAA 3′ 3′ TTTTT 5′ Figure 20.11-4 DNA in nucleus mRNAs in cytoplasm

Reverse transcriptase mRNA Poly-A tail 5′ AAAAAA 3′ 3′ TTTTT 5′ DNA Primer strand (poly-dT)

5′ AAA AAA 3′ 3′ TTTTT 5′

5′ 3′ 3′ 5′ DNA polymerase Figure 20.11-5 DNA in nucleus mRNAs in cytoplasm

Reverse transcriptase mRNA Poly-A tail 5′ AAAAAA 3′ 3′ TTTTT 5′ DNA Primer strand (poly-dT)

5′ AAA AAA 3′ 3′ TTTTT 5′

5′ 3′ 3′ 5′ DNA polymerase

5′ 3′ 3′ 5′ cDNA Figure 20.12 Technique 1 cDNA synthesis mRNAs

cDNAs Primers 2 PCR amplification

Specific gene 3 Gel electrophoresis

Results Embryonic stages 1 2 3 4 5 6 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 Figure 20.13

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.

Genes expressed ► in neither tissue. DNA microarray (actual size) Figure 20.13a

Each dot is a well containing identical copies of DNA fragments that carry a specific gene. • 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 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 • 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 • 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 • 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 Figure 20.14

A DNA T Normal allele SNP

C G Disease-causing allele 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 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 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 Figure 21.11 Human Chimpanzee chromosome chromosomes Telomere sequences

Centromere sequences

Telomere-like sequences 12

Centromere-like sequences

2 13 Figure 21.12

Human chromosome Mouse chromosomes

16 7 8 16 17 • 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 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 Figure 21.13 Nonsister Gene Transposable chromatids element

Incorrect pairing Crossover of two homologs point during meiosis

and 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 450–500 million years ago • After the duplication events, differences between the genes in the globin family arose from the accumulation of mutations Figure 21.14

Ancestral globin gene

Duplication of ancestral gene Mutation in both copies α β Transposition to different chromosomes α β Further duplications and mutations  Evolutionary time Evolutionary ζ α ϵ β

ζ ζ α α α2 α1 yθ ϵ G A β  β 2 1 α-Globin gene family β-Globin gene family on chromosome 16 on chromosome 11 • 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 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 Figure 21.15

(a) Lysozyme (b) α–lactalbumin

Lysozyme 1 α–lactalbumin 1

Lysozyme 51 α–lactalbumin 51

Lysozyme 101 α–lactalbumin 101 (c) Amino acid sequence alignments of lysozyme and α–lactalbumin 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 Figure 21.16

EGF EGF EGF EGF Epidermal growth factor gene with multiple EGF exons Exon Exon shuffling duplication F F F F Fibronectin gene with multiple “finger” exons F EGF K K

K Plasminogen gene with a Exon “kringle” exon shuffling

Portions of ancestral genes TPA gene as it exists today 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 • 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