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Viruses called bacteriophages can infect and set in motion a genetic takeover of bacteria, such as Escherichia coli Bacteria are prokaryotes w ith cells.

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Presentation on theme: "Viruses called bacteriophages can infect and set in motion a genetic takeover of bacteria, such as Escherichia coli Bacteria are prokaryotes w ith cells."— Presentation transcript:

1 Viruses called bacteriophages can infect and set in motion a genetic takeover of bacteria, such as Escherichia coli Bacteria are prokaryotes w ith cells much smaller and more simply organized than those of eukaryotes Viruses a re smaller and simpler than bacteria

2 Tobacco mosaic disease stunts growth of tobacco plants and gives their leaves a mosaic coloration In the late 1800s, 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)

3 Viruses are not cells Viruses are very small infectious particles consisting of nucleic acid enclosed in a protein coat and, in some cases, a membranous envelope Viral genomes may consist of – Double- or single-stranded DNA – Double- or single-stranded RNA Depending on its type of nucleic acid, a virus is called a DNA virus or an RNA virus

4 Capsomere of capsid RNA 18  250 mm Tobacco mosaic virus 20 nm A capsid is the protein shell that encloses the viral genome and can have various structures

5 Capsomere Glycoprotein 70–90 nm (diameter) DNA Adenoviruses 50 nm

6 Some viruses have structures that have membranous envelopes that help them infect hosts These viral envelopes surround the capsids of influenza viruses and many other viruses found in animals Viral envelopes, which are derived from the host cell’s membrane, contain a combination of viral and host cell molecules

7 Glycoprotein 80–200 nm (diameter) RNA Capsid Influenza viruses 50 nm Membranous envelope

8 Bacteriophages, also called phages, are viruses that infect bacteria Phages have an elongated capsid head that encloses their DNA A protein tailpiece attaches the phage to the host and injects the phage DNA inside Viruses use enzymes, ribosomes, and small host molecules to synthesize progeny viruses

9 80  225 nm DNA Head Tail sheath Tail fiber Bacteriophage T4 50 nm

10 DNA VIRUS Capsid HOST CELL Viral DNA Replication Entry into cell and uncoating of DNA Transcription Viral DNA mRNA Capsid proteins Self-assembly of new virus particles and their exit from cell

11 The Lytic Cycle The lytic cycle is a phage reproductive cycle that culminates in the death of the host cell The lytic cycle produces new phages and digests 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

12 Attachment Entry of phage DNA and degradation of host DNA Synthesis of viral genomes and proteins Assembly Release Phage assembly Head Tails Tail fibers

13 The Lysogenic Cycle The lysogenic cycle replicates the phage genome without destroying the host The viral DNA molecule is incorporated by genetic recombination 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 Phages that use both the lytic and lysogenic cycles are called temperate phages

14 Phage DNA The phage attaches to a host cell and injects its DNA. Phage DNA circularizes Bacterial chromosome Lytic cycle The cell lyses, releasing phages. Lytic cycle is induced or Lysogenic cycle is entered Certain factors determine whether Lysogenic cycle Occasionally, a prophage exits the bacterial chromosome, initiating a lytic cycle. The bacterium reproduces normally, copying the prophage and transmitting it to daughter cells. Prophage Many cell divisions produce a large population of bacteria infected with the prophage. Daughter cell with prophage Phage DNA integrates into the bacterial chromosomes, becoming a prophage. New phage DNA and proteins are synthesized and assembled into phages.

15 Reproductive Cycles of Animal Viruses Two key variables in classifying viruses that infect animals: – DNA or RNA? – Single-stranded or double-stranded? 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 Viral Envelopes

16 RNA ER Capsid HOST CELL Viral genome (RNA) mRNA Capsid proteins Envelope (with glycoproteins) Glyco- proteins Copy of genome (RNA) Capsid and viral genome enter cell New virus Template

17 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 is the retrovirus that causes AIDS

18 Capsid Viral envelope Glycoprotein Reverse transcriptase RNA (two identical strands)

19 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 The host’s 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

20 HOST CELL Reverse transcription Viral RNA RNA-DNA hybrid DNA NUCLEUS Chromosomal DNA Provirus RNA genome for the next viral generation mRNA New HIV leaving a cell HIV entering a cell 0.25 µm HIV Membrane of white blood cell

21 Viroids and Prions: The Simplest Infectious Agents Viroids are circular RNA molecules that infect plants and disrupt their growth Prions are slow-acting, virtually indestructible infectious proteins that cause brain diseases in mammals Prions propagate by converting normal proteins into the prion version

22 Normal protein New prion Prion Original prion Many prions

23 The Bacterial Genome and Its Replication The bacterial chromosome is usually a circular DNA molecule with few associated proteins Many bacteria also have plasmids, smaller circular DNA molecules that can replicate independently of the chromosome Bacterial cells divide by binary fission, which is preceded by replication of the chromosome

24 Mutation and Genetic Recombination as Sources of Genetic Variation Since bacteria can reproduce rapidly, new mutations quickly increase genetic diversity More genetic diversity arises by recombination of DNA from two different bacterial cells Three processes bring bacterial DNA from different individuals together: – Transformation – Transduction – Conjugation

25 Transformation and Transduction Transformation is the alteration of a bacterial cell’s genotype and phenotype by the uptake of naked, foreign DNA from the surrounding environment For example, harmless Streptococcus pneumoniae bacteria can be transformed to pneumonia-causing cells In the process known as transduction, phages carry bacterial genes from one host cell to another

26 A+A+ Phage DNA A+A+ Donor cell B+B+ A+A+ B+B+ Crossing over A+A+ A–A– B–B– Recipient cell A+A+ B–B– Recombinant cell

27 Conjugation and Plasmids Conjugation is the direct transfer of genetic material between bacterial cells that are temporarily joined The transfer is one-way: One cell (“male”) donates DNA, and its “mate” (“female”) receives the genes

28 Sex pilus 5 µm

29 Mutant strain arg + trp – Mutant strain arg + trp – Mixture No colonies (control) No colonies (control) Colonies grew Mutant strain arg – trp + Mutant strain arg – trp +

30 The F Plasmid and Conjugation Cells containing the F plasmid, designated F + cells (fertile), function as DNA donors during conjugation F + cells transfer DNA to an F  recipient cell Chromosomal genes can be transferred during conjugation when the donor cell’s F factor is integrated into the chromosome A cell with a built-in F factor is called an Hfr cell The F factor of an Hfr (high frequency of recombination) cell brings some chromosomal DNA along when transferred to an F – cell

31 F plasmid Bacterial chromosome F + cell Mating bridge F + cell Bacterial chromosome F – cell Conjunction and transfer of an F plasmid from and F + donor to an F – recipient F + cell Hfr cell F factor Hfr cell F – cell Temporary partial diploid Recombinant F – bacterium Conjugation and transfer of part of the bacterial chromosome from an Hfr donor to an F – recipient, resulting in recombination

32 R plasmids and Antibiotic Resistance R plasmids confer resistance to various antibiotics When a bacterial population is exposed to an antibiotic, individuals with the R plasmid will survive and increase in the overall population The DNA of a cell can also undergo recombination due to movement of transposable elements within the cell’s genome Transposable elements, often called “jumping genes,” contribute to genetic shuffling in bacteria Transposition of Genetic Elements

33 Insertion Sequences The simplest transposable elements, called insertion sequences, exist only in bacteria An insertion sequence has a single gene for transposase, an enzyme catalyzing movement of the insertion sequence from one site to another within the genome

34 Insertion sequence Transposase gene 5 3 Inverted repeat 3 5 Inverted repeat

35 Transposons Transposable elements called transposons are longer and more complex than insertion sequences In addition to DNA required for transposition, transposons have extra genes that “go along for the ride,” such as genes for antibiotic resistance

36 Transposon Insertion sequence Insertion sequence Antibiotic resistance gene Transposase gene Inverted repeat

37 Operons: The Basic Concept In bacteria, genes are often clustered into operons, composed of – An operator, an “on-off” switch – A promoter – Genes for metabolic enzymes An operon can be switched off by a protein called a repressor A corepressor is a small molecule that cooperates with a repressor to switch an operon off

38 Promoter DNA trpR Regulatory gene RNA polymerase mRNA 3 5 Protein Inactive repressor Tryptophan absent, repressor inactive, operon on mRNA 5 trpE trpD trpC trpBtrpA Operator Start codon Stop codon trp operon Genes of operon E Polypeptides that make up enzymes for tryptophan synthesis D C B A

39 DNA Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off mRNA Active repressor

40 DNA Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off mRNA Active repressor No RNA made

41 Repressible and Inducible Operons: Two Types of Negative Gene Regulation A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription The trp operon is a repressible operon An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription The classic example of an inducible operon is the lac operon, which contains genes coding for enzymes in hydrolysis and metabolism of lactose

42 DNA lacl Regulatory gene mRNA 5 3 RNA polymerase Protein Active repressor No RNA made lacZ Promoter Operator Lactose absent, repressor active, operon off

43 DNAlacl mRNA 5 3 lac operon Lactose present, repressor inactive, operon on lacZ lacYlacA RNA polymerase mRNA 5 Protein Allolactose (inducer) Inactive repressor  -Galactosidase Permease Transacetylase

44 Inducible enzymes usually function in catabolic pathways Repressible enzymes usually function in anabolic pathways Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor

45 Positive Gene Regulation Some operons are also subject to positive control through a stimulatory activator protein, such as catabolite activator protein (CAP) When glucose (a preferred food source of E. coli ) is scarce, the lac operon is activated by the binding of CAP When glucose levels increase, CAP detaches from the lac operon, turning it off

46 DNA cAMP lacl CAP-binding site Promoter Active CAP Inactive CAP RNA polymerase can bind and transcribe Operator lacZ Inactive lac repressor Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized

47 DNA lacl CAP-binding site Promoter RNA polymerase can’t bind Operator lacZ Inactive lac repressor Inactive CAP Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

48 Two features of eukaryotic genomes are a major information-processing challenge: – First, the typical eukaryotic genome is much larger than that of a prokaryotic cell – Second, cell specialization limits the expression of many genes to specific cells The DNA-protein complex, called chromatin, is ordered into higher structural levels than the DNA-protein complex in prokaryotes

49 Nucleosomes, or “Beads on a String” Proteins called histones are responsible for the first level of DNA packing in chromatin The association of DNA and histones seems to remain intact throughout the cell cycle In electron micrographs, unfolded chromatin has the appearance of beads on a string Each “bead” is a nucleosome, the basic unit of DNA packing

50 DNA double helix Histone tails His- tones Linker DNA (“string”) Nucleosome (“bead”) 10 nm 2 nm Histone H1 Nucleosomes (10-nm fiber)

51 30 nm Nucleosome 30-nm fiber

52 300 nm Loops Scaffold Protein scaffold Looped domains (300-nm fiber)

53 Metaphase chromosome 700 nm 1,400 nm

54 Differential Gene Expression Differences between cell types result from differential gene expression, the expression of different genes by cells within the same genome In each type of differentiated cell, a unique subset of genes is expressed Many key stages of gene expression can be regulated in eukaryotic cells

55 Signal NUCLEUS DNA RNA Chromatin Gene available for transcription Gene Exon Intro Transcription Primary transcript RNA processing Cap Tail mRNA in nucleus Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Translation Degradation of mRNA Polypeptide Cleavage Chemical modification Transport to cellular destination Degradation of protein Active protein Degraded protein

56 Regulation of Chromatin Structure Genes within highly packed heterochromatin are usually not expressed Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression

57 Histone Modification In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails This process seems to loosen chromatin structure, thereby promoting the initiation of transcription

58 Histone tails Amino acids available for chemical modification DNA double helix Histone tails protrude outward from a nucleosome Acetylation of histone tails promotes loose chromatin structure that permits transcription Unacetylated histones Acetylated histones

59 DNA Methylation DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species In some species, DNA methylation causes long-term inactivation of genes in cellular differentiation In genomic imprinting, methylation turns off either the maternal or paternal alleles of certain genes at the start of development … lions, tigers, ligers?

60 The Roles of Transcription Factors To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors General transcription factors are essential for the transcription of all protein-coding genes In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors

61 Enhancers and Specific Transcription Factors Proximal control elements are located close to the promoter Distal control elements, groups of which are called enhancers, may be far away from a gene or even in an intron An activator is a protein that binds to an enhancer and stimulates transcription of a gene

62 Enhancer (distal control elements) Proximal control elements Upstream DNA Promoter ExonIntron ExonIntron Exon Downstream Transcription Poly-A signal sequence Termination region Intron ExonIntron Exon RNA processing: Cap and tail added; introns excised and exons spliced together Poly-A signal Cleaved 3 end of primary transcript 3 Poly-A tail 3 UTR (untranslated region) 5 UTR (untranslated region) Start codon Stop codon Coding segment Intron RNA 5 Cap mRNA Primary RNA transcript (pre-mRNA) 5 Exon

63 Distal control element Activators Enhancer DNA DNA-bending protein TATA box Promoter Gene General transcription factors Group of mediator proteins RNA polymerase II RNA polymerase II RNA synthesis Transcription Initiation complex

64 Control elements EnhancerPromoter Albumin gene Crystallin gene Available activators Available activators Albumin gene not expressed Albumin gene expressed Liver cell Lens cell Crystallin gene not expressed Crystallin gene expressed Liver cell nucleus Lens cell nucleus Some transcription factors function as repressors, inhibiting expression of a particular gene Some activators and repressors act indirectly by influencing chromatin structure

65 Primary RNA transcript DNA or Exons RNA splicing mRNA In alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns

66 mRNA Degradation The life span of mRNA molecules in the cytoplasm is a key to determining the protein synthesis The mRNA life span is determined in part by sequences in the leader and trailer regions RNA interference by single-stranded microRNAs (miRNAs) can lead to degradation of an mRNA or block its translation The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi)

67 Dicer Hydrogen bond Protein complex miRNA Target mRNA Degradation of mRNA OR Blockage of translation

68 Protein Processing and Degradation After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control Proteasomes are giant protein complexes that bind protein molecules and degrade them

69 Protein to be degraded Ubiquitinated protein Proteasome Protein entering a proteasome Protein fragments (peptides) Proteasome and ubiquitin to be recycled Ubiquitin

70 Types of Genes Associated with Cancer Genes that normally regulate cell growth and division during the cell cycle include: – Genes for growth factors – Their receptors – Intracellular molecules of signaling pathways Mutations altering any of these genes in somatic cells can lead to cancer

71 Oncogenes and Proto-Oncogenes Oncogenes are cancer-causing genes Proto-oncogenes are normal cellular genes that code for proteins that stimulate normal cell growth and division A DNA change that makes a proto-oncogene excessively active converts it to an oncogene, which may promote excessive cell division and cancer

72 Proto-oncogene DNA Translocation or transposition: gene moved to new locus, under new controls New promoter Gene amplification: multiple copies of the gene Point mutation within a control element Oncogene Point mutation within the gene Normal growth-stimulating protein in excess Normal growth-stimulating protein in excess Normal growth-stimulating protein in excess Hyperactive or degradation- resistant protein

73 Tumor-Suppressor Genes Tumor-suppressor genes encode proteins that inhibit abnormal cell division Any decrease in the normal activity of a tumor- suppressor protein may contribute to cancer

74 Interference with Normal Cell-Signaling Pathways Many proto-oncogenes and tumor suppressor genes encode components of growth- stimulating and growth-inhibiting pathways, respectively The Ras protein, encoded by the ras gene, is a G protein that relays a signal from a growth factor receptor to a cascade of protein kinases Many ras oncogenes have a mutation that leads to a hyperactive Ras protein that issues signals on its own, resulting in excessive cell division

75 The p53 gene,“guardian angel of the genome” encodes a tumor-suppressor protein that is a specific transcription factor that promotes synthesis of cell cycle–inhibiting proteins Mutations that knock out the p53 gene can lead to excessive cell growth and cancer Increased cell division, possibly leading to cancer, can result if the cell cycle is over stimulated or not inhibited when it normally would be

76 Protein overexpressed EFFECTS OF MUTATIONS Protein absent Cell cycle not inhibited Increased cell division Cell cycle overstimulate Effects of mutations Active form of p53 DNA DNA damage in genome UV light Protein kinases MUTATION Defective or missing transcription factor, such as p53, cannot activate transcription Protein kinases (phosphorylation cascade) Cell cycle-inhibiting pathway Cell cycle-stimulating pathway Protein that inhibits the cell cycle NUCLEUS DNA Gene expression Transcription factor (activator) Receptor G protein Growth factor MUTATION Hyperactive Ras protein (product of oncogene) issues signals on its own Protein that stimulates the cell cycle

77 Colon Colon wall Loss of tumor- suppressor gene APC (or other) Normal colon epithelial cells Small benign growth (polyp) Larger benign growth (adenoma) Activation of ras oncogene Loss of tumor- suppressor gene DCC Loss of tumor- suppressor gene p53 Additional mutations Malignant tumor (carcinoma) Individuals who inherit a mutant oncogene or tumor- suppressor allele have an increased risk of developing certain types of cancer (Inherited Predisposition to Cancer)

78 Transposable Elements and Related Sequences The first evidence for wandering DNA segments came from geneticist Barbara McClintock’s breeding experiments with Indian corn McClintock identified changes in the color of corn kernels that made sense only by postulating that some genetic elements move from other genome locations into the genes for kernel color

79

80 Movement of Transposons and Retrotransposons Eukaryotic transposable elements are of two types: – Transposons, which move within a genome by means of a DNA intermediate – Retrotransposons, which move by means of an RNA intermediate

81 DNA of genome Transposon is copied Mobile transposon Transposon Insertion New copy of transposon Transposon movement (“copy-and-paste” mechanism) Retrotransposon movement DNA of genome Insertion RNA Reverse transcriptase Retrotransposon New copy of retrotransposon

82 Genes and Multigene Families Most eukaryotic genes are present in one copy per haploid set of chromosomes The rest of the genome occurs in multigene families, collections of identical or very similar genes Globin gene family clusters also include pseudogenes, nonfunctional nucleotide sequences that are similar to the functional genes

83 DNA Non-transcribed spacer RNA transcripts Transcription unit DNA 18S 5.8S 28S rRNA 18S 5.8S 28S Part of the ribosomal RNA gene family

84 Heme Hemoglobin  -Globin  -Globin  -Globin gene family  -Globin gene family Chromosome 11 Chromosome 16    11 11 22      AA Embryo Fetus Adult Fetus and adult The human  -globin and  -globin gene families

85 DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes

86 DNA Cloning and Its Applications To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called gene cloning Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids Cloned genes are useful for making copies of a particular gene and producing a gene product

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

88 Using Restriction Enzymes to Make Recombinant DNA Bacterial restriction enzymes cut DNA molecules at DNA sequences called restriction sites usually makes many cuts, yielding restriction fragments The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary “sticky ends” of other fragments DNA ligase is an enzyme that seals the bonds between the “sticky ends” restriction fragments

89 Restriction site DNA Restriction enzyme cuts the sugar-phosphate backbones at each arrow. One possible combination DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. Fragment from different DNA molecule cut by the same restriction enzyme DNA ligase seals the strands. Recombinant DNA molecule Sticky end

90 Isolate plasmid DNA and human DNA. Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Bacterial cell lacZ gene (lactose breakdown) Human cell Restriction site amp R gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow. Colony carrying non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene Bacterial clone

91 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

92 Genomic DNA Target sequence Primers Denaturation: Heat briefly to separate DNA strands Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Extension: DNA polymerase adds nucleotides to the 3 end of each primer Cycle 1 yields 2 molecules New nucleo- tides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

93 Gel Electrophoresis One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis This technique uses a gel as a molecular sieve to separate nuclei acids or proteins by size In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene

94 Cathode Power source Anode Mixture of DNA molecules of differ- ent sizes Gel Glass plates Longer molecules Shorter molecules

95 Normal  -globin allele 175 bp201 bpLarge fragment Sickle-cell mutant  -globin allele 376 bpLarge fragment Ddel Ddel restriction sites in normal and sickle-cell alleles of  -globin gene Normal allele Sickle-cell allele Large fragment 376 bp 201 bp 175 bp Electrophoresis of restriction fragments from normal and sickle-cell alleles

96 Medical Applications One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases Scientists can diagnose many human genetic disorders by using PCR and primers corresponding to cloned disease genes, then sequencing the amplified product to look for the disease-causing mutation Even when a disease gene has not been cloned, presence of an abnormal allele can be diagnosed if a closely linked RFLP marker has been found

97 DNA RFLP marker Disease-causing allele Normal allele Restriction sites

98 Human Gene Therapy Gene therapy is the alteration of an afflicted individual’s genes Gene therapy holds great potential for treating disorders traceable to a single defective gene Vectors are used for delivery of genes into cells Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

99 Cloned gene Retrovirus capsid Bone marrow cell from patient Inject engineered cells into patient. Insert RNA version of normal allele into retrovirus. Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Bone marrow

100 Pharmaceutical Products Some pharmaceutical applications of DNA technology: – Large-scale production of human hormones and other proteins with therapeutic uses – Production of safer vaccines

101 Forensic Evidence DNA “fingerprints” obtained by analysis of tissue or body fluids can provide evidence in criminal and paternity cases A DNA fingerprint is a specific pattern of bands of RFLP markers on a gel The probability that two people who are not identical twins have the same DNA fingerprint is very small Exact probability depends on the number of markers and their frequency in the population

102 Defendant’s blood (D) Blood from defendant’s clothes Victim’s blood (V)

103 Animations and Videos Processing of Gene Information Control of Gene Expression in Eukaryotes RNA Interface Regulatory Proteins Regulation by Repression Tryptophan Repressor Lac Operon Bozeman - Lac Operon The Lac Operon in E. coli Bozeman - Gene Regulation

104 Animations and Videos Bozeman - Restriction Enzyme DNA Transformation – 1 DNA Transformation – 2 Conjugation: Transfer of Chromosomal DNA Conjugation - Transfer of F Plasmid Integration and Excision of a Plasmid Transduction (Generalized) Bacterial Transformation Lamda Phage Replication Cycle

105 Animations and Videos Early Genetic Engineering Experiment Genetic Engineering Cloning Steps in Cloning a Gene Tutorial - Human Cloning Human Cloning - Reproductive Cloning Human Cloning - Therapeutic Cloning Genetic Engineering to Produce Insulin Polymerase Chain Reaction

106 Animations and Videos PCR – 1 PCR – 2 Gel Electrophoresis – 1 Gel Electrophoresis – 2 DNA Fingerprinting Construction of a DNA Library DNA Restriction Enzymes Restriction Enzyme Digestion of DNA Restriction Endonucleases

107 Animations and Videos Principles of Biotechnology Applications of Biotechnology Constructing Vaccines DNA Probe (DNA Hybridization) Restriction Length Ploymorphisms cDNA Southern Blot Entry of Virus into Host Cell Replication Cycle of a Retrovirus

108 Animations and Videos HIV Replication Mechanism for Releasing Enveloped Viruses Treatment of HIV Prions Disease How Prions Arise Stem Cells Embryonic Stem Cells Human Embryonic Stem Cells Human Stem Cells

109 Animations and Videos Microarray Sanger Sequencing DNA Fingerprinting RNA Interface miRNA Dicer DNA Microarrays Bozeman - DNA Fingerprinting Bozeman - Viral Replication

110 Animations and Videos Genes into Plants Using the Ti-plasmid Highput Through Sequencing Sequencing the Genome Cycle Sequencing Bozeman - Effects of Change in Pathways Chapter Quiz Questions – 1 Chapter Quiz Questions – 2 Chapter Quiz Questions – 3 Chapter Quiz Questions – 4

111 What does the operon model attempt to explain? the coordinated control of gene expression in bacteria bacterial resistance to antibiotics how genes move between homologous regions of DNA the mechanism of viral attachment to a host cell horizontal transmission of plant viruses

112 What does the operon model attempt to explain? the coordinated control of gene expression in bacteria bacterial resistance to antibiotics how genes move between homologous regions of DNA the mechanism of viral attachment to a host cell horizontal transmission of plant viruses

113 When tryptophan (an amino acid) is present in the external medium, the bacterium brings in the tryptophan and does not need to make this amino acid. Which of the following is true when there is no tryptophan in the medium? The repressor is active and binds to the operator. The repressor is inactive, and RNA polymerase moves through the operator. The operator is bound, and mRNA is made. Genes are inactive. The corepressor binds to the repressor.

114 When tryptophan (an amino acid) is present in the external medium, the bacterium brings in the tryptophan and does not need to make this amino acid. Which of the following is true when there is no tryptophan in the medium? The repressor is active and binds to the operator. The repressor is inactive, and RNA polymerase moves through the operator. The operator is bound, and mRNA is made. Genes are inactive. The corepressor binds to the repressor.

115 Each of a group of bacterial cells has a mutation in its lac operon. Which of the following will make it impossible for the cell to metabolize lactose? mutation in lac  (  -galactosidase gene) mutation in lac  (cannot bind to operator) mutation in operator (cannot bind to repressor) mutation in lac  (cannot bind to inducer)

116 Each of a group of bacterial cells has a mutation in its lac operon. Which of the following will make it impossible for the cell to metabolize lactose? mutation in lac  (  -galactosidase gene) mutation in lac  (cannot bind to operator) mutation in operator (cannot bind to repressor) mutation in lac  (cannot bind to inducer)

117 Which element(s) from the following list constitute(s) a bacterial operon? repressor gene promoter inducer repressor protein all of the above

118 Which element(s) from the following list constitute(s) a bacterial operon? repressor gene promoter inducer repressor protein all of the above

119 Which of the following statements about specific transcription factors is false? The binding of specific transcription factors to the control elements of enhancers influences the rate of gene expression. Specific transcription factors include activators and repressors. MyoD is one. Some act indirectly by affecting chromatin structure. Interaction of specific transcription factors and RNA polymerase II with a promoter leads to a low rate of initiation and production of a few RNA transcripts.

120 Which of the following statements about specific transcription factors is false? The binding of specific transcription factors to the control elements of enhancers influences the rate of gene expression. Specific transcription factors include activators and repressors. MyoD is one. Some act indirectly by affecting chromatin structure. Interaction of specific transcription factors and RNA polymerase II with a promoter leads to a low rate of initiation and production of a few RNA transcripts.

121 Approximately what proportion of the DNA in the human genome codes for proteins or functional RNA? 83% 46% 32% 13% 1.5%

122 Approximately what proportion of the DNA in the human genome codes for proteins or functional RNA? 83% 46% 32% 13% 1.5%

123 A specific gene is known to code for three different but related proteins. This could be due to which of the following? premature mRNA degradation alternative RNA splicing use of different enhancers protein degradation differential transport

124 A specific gene is known to code for three different but related proteins. This could be due to which of the following? premature mRNA degradation alternative RNA splicing use of different enhancers protein degradation differential transport

125 RNA is cut up into small 22-nucleotide fragments to regulate another “target” mRNA. Which of the following is/are true? The target mRNA is degraded, and its protein is not made. The RNA fragments enhance protein synthesis by the mRNA. The RNA fragments bind the ribosome to enhance use of the mRNA and protein synthesis. The target mRNA is blocked from being used in translation. The RNA fragments act on the ribosome to shut down translation of all mRNAs.

126 RNA is cut up into small 22-nucleotide fragments to regulate another “target” mRNA. Which of the following is/are true? The target mRNA is degraded, and its protein is not made. The RNA fragments enhance protein synthesis by the mRNA. The RNA fragments bind the ribosome to enhance use of the mRNA and protein synthesis. The target mRNA is blocked from being used in translation. The RNA fragments act on the ribosome to shut down translation of all mRNAs.

127 Even though the two cells have numerous transcription factors and many are present in both cells, the lens cell makes the crystallin protein (not albumin), whereas the liver cell makes albumin (not crystallin). Which of the following explains this cell specificity? Specific transcription factors made in the cell determine which genes are expressed. At fertilization, specific cells are destined for certain functions. The activators needed for expression of the crystallin gene are present in all cells. The promoters are different for the different genes.

128 Even though the two cells have numerous transcription factors and many are present in both cells, the lens cell makes the crystallin protein (not albumin), whereas the liver cell makes albumin (not crystallin). Which of the following explains this cell specificity? Specific transcription factors made in the cell determine which genes are expressed. At fertilization, specific cells are destined for certain functions. The activators needed for expression of the crystallin gene are present in all cells. The promoters are different for the different genes.

129 Differential gene expression (different genes turned on in different cells) leads to different tissues developing in the embryo. Which of the following is not a cause of differential gene expression? cytoplasmic determinants induction the environment around a particular cell corepressor proteins

130 Differential gene expression (different genes turned on in different cells) leads to different tissues developing in the embryo. Which of the following is not a cause of differential gene expression? cytoplasmic determinants induction the environment around a particular cell corepressor proteins

131 Initially, cytoplasmic determinants are localized in one part of a zygote and could be which of the following? (Choose more than one answer.) gene mRNA transcription factor ribosome myoblast

132 Initially, cytoplasmic determinants are localized in one part of a zygote and could be which of the following? (Choose more than one answer.) gene mRNA transcription factor ribosome myoblast

133 Scientists showed that bicoid mRNA, and then its Bicoid protein, is normally found in highest concentrations in the fly’s anterior. What would happen if Bicoid were injected at the posterior end? Anterior structures would form at both ends. Posterior structures would form at both ends. The embryo would have no dorsal-ventral axis. Bicoid mRNA wouldn’t be translated into protein.

134 Scientists showed that bicoid mRNA, and then its Bicoid protein, is normally found in highest concentrations in the fly’s anterior. What would happen if Bicoid were injected at the posterior end? Anterior structures would form at both ends. Posterior structures would form at both ends. The embryo would have no dorsal-ventral axis. Bicoid mRNA wouldn’t be translated into protein.

135 Mutations in _______ genes caused the development of legs in the place of antennae. homeotic embryonic lethal myoD Ras wild-type Wild type Eye Mutant

136 Mutations in _______ genes caused the development of legs in the place of antennae. homeotic embryonic lethal myoD Ras wild-type Wild type Eye Mutant

137 The shape of an organ, the number of brain cells in an embryonic brain, the removal of mutated cells, and the webbing cells between the toes of a human embryo are all regulated by which of the following? certain cells becoming much larger certain cells shrinking certain cells dying formation of embryonic cells concentration of Bicoid protein

138 The shape of an organ, the number of brain cells in an embryonic brain, the removal of mutated cells, and the webbing cells between the toes of a human embryo are all regulated by which of the following? certain cells becoming much larger certain cells shrinking certain cells dying formation of embryonic cells concentration of Bicoid protein

139 Which of the following would not typically cause a proto-oncogene to become an oncogene? gene suppression translocation amplification point mutation retroviral activation

140 Which of the following would not typically cause a proto-oncogene to become an oncogene? gene suppression translocation amplification point mutation retroviral activation

141 Which of the following statements about the APC gene is false? It is a tumor-suppressor gene. It is mutated in 60% of colorectal cancers. It regulates cell migration and adhesion. It may be deleted in colon cancer. Mutations in one allele are enough to lose the gene’s function.

142 Which of the following statements about the APC gene is false? It is a tumor-suppressor gene. It is mutated in 60% of colorectal cancers. It regulates cell migration and adhesion. It may be deleted in colon cancer. Mutations in one allele are enough to lose the gene’s function.

143 The diagrams on the next slide show an intact DNA sequence (top) and three experimental DNA sequences. A red X indicates the possible control element (1, 2, or 3) that was deleted in each experimental DNA sequence. The area between the slashes represents the approximately 8 kilobases of DNA located between the promoter and the enhancer region. The horizontal bar graph shows the amount of reporter gene mRNA that was present in each cell culture after 48 hours relative to the amount that was in the culture containing the intact enhancer region (top bar = 100%). Scientific Skills Exercise

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145 What was the independent variable in this experiment? the length of time that the cells were incubated the relative level of reporter gene mRNA the distance between the promoter and the enhancer the possible control element that was deleted

146 What was the independent variable in this experiment? the length of time that the cells were incubated the relative level of reporter gene mRNA the distance between the promoter and the enhancer the possible control element that was deleted

147 What was the dependent variable in this experiment? the length of time that the cells were incubated how many of the artificial DNA molecules were taken up by the cells the relative level of reporter gene mRNA the distance between the promoter and the enhancer

148 What was the dependent variable in this experiment? the length of time that the cells were incubated how many of the artificial DNA molecules were taken up by the cells the relative level of reporter gene mRNA the distance between the promoter and the enhancer

149 What was the control treatment in this experiment? the reporter gene the construct that had no DNA deleted from the enhancer the temperature, pH, and salt concentration of the incubation medium the construct that resulted in the lowest amount of reporter mRNA

150 What was the control treatment in this experiment? the reporter gene the construct that had no DNA deleted from the enhancer the temperature, pH, and salt concentration of the incubation medium the construct that resulted in the lowest amount of reporter mRNA

151 Do the data suggest that any of these possible control elements are actual control elements? Only control elements 1 and 2 appear to be control elements. Only control element 3 appears to be a control element. All three appear to be control elements. None of the possible control elements appear to be actual control elements.

152 Do the data suggest that any of these possible control elements are actual control elements? Only control elements 1 and 2 appear to be control elements. Only control element 3 appears to be a control element. All three appear to be control elements. None of the possible control elements appear to be actual control elements.

153 Did deletion of any of the possible control elements cause a reduction in reporter gene expression? How can you tell? Deletion of element 3 caused a reduction in reporter gene expression; that construct resulted in less than 50% of the control level of mRNA. Deletion of elements 2 and 3 caused a reduction in reporter gene expression; those constructs resulted in less than the highest level of mRNA. None of the deletions caused a reduction in reporter gene expression; all of them still resulted in reporter mRNA being made.

154 Did deletion of any of the possible control elements cause a reduction in reporter gene expression? How can you tell? Deletion of element 3 caused a reduction in reporter gene expression; that construct resulted in less than 50% of the control level of mRNA. Deletion of elements 2 and 3 caused a reduction in reporter gene expression; those constructs resulted in less than the highest level of mRNA. None of the deletions caused a reduction in reporter gene expression; all of them still resulted in reporter mRNA being made.

155 If deletion of a control element causes a reduction in gene expression, what must be the normal role of that control element? To repress gene expression; without the control element, repressors are not able to bind to the enhancer, and the level of gene expression decreases. To activate gene expression; without the control element, activators are not able to bind to the enhancer, and the level of gene expression decreases. To repress gene expression; without the control element, repressors are not able to bind to the enhancer, and the level of gene expression increases. To activate gene expression; without the control element, repressors are not able to bind to the enhancer, and the level of gene expression increases.

156 If deletion of a control element causes a reduction in gene expression, what must be the normal role of that control element? To repress gene expression; without the control element, repressors are not able to bind to the enhancer, and the level of gene expression decreases. To activate gene expression; without the control element, activators are not able to bind to the enhancer, and the level of gene expression decreases. To repress gene expression; without the control element, repressors are not able to bind to the enhancer, and the level of gene expression increases. To activate gene expression; without the control element, repressors are not able to bind to the enhancer, and the level of gene expression increases.

157 Did deletion of any of the possible control elements cause an increase in reporter gene expression? How can you tell? Deletion of control element 1 or 2 caused an increase in reporter gene expression; both constructs resulted in over 100% of the control level of mRNA. Deletion of control element 1 caused an increase in reporter gene expression; that construct resulted in the highest level of mRNA. Deletion of control element 3 caused an increase in reporter gene expression; that construct resulted in less reporter mRNA than the control. All of the deletions caused an increase in reporter gene expression; all of them still resulted in reporter mRNA being made.

158 Did deletion of any of the possible control elements cause an increase in reporter gene expression? How can you tell? Deletion of control element 1 or 2 caused an increase in reporter gene expression; both constructs resulted in over 100% of the control level of mRNA. Deletion of control element 1 caused an increase in reporter gene expression; that construct resulted in the highest level of mRNA. Deletion of control element 3 caused an increase in reporter gene expression; that construct resulted in less reporter mRNA than the control. All of the deletions caused an increase in reporter gene expression; all of them still resulted in reporter mRNA being made.

159 If deletion of a control element causes an increase in gene expression, what must be the normal role of that control element? To activate gene expression; without the control element, repressors are not able to bind to the enhancer, and the level of gene expression increases. To repress gene expression; without the control element, activators are not able to bind to the enhancer, and the level of gene expression decreases. To repress gene expression; without the control element, repressors are not able to bind to the enhancer, and the level of gene expression increases. To activate gene expression; without the control element, activators are not able to bind to the enhancer, and the level of gene expression decreases.

160 If deletion of a control element causes an increase in gene expression, what must be the normal role of that control element? To activate gene expression; without the control element, repressors are not able to bind to the enhancer, and the level of gene expression increases. To repress gene expression; without the control element, activators are not able to bind to the enhancer, and the level of gene expression decreases. To repress gene expression; without the control element, repressors are not able to bind to the enhancer, and the level of gene expression increases. To activate gene expression; without the control element, activators are not able to bind to the enhancer, and the level of gene expression decreases.

161 Which of the following is a property of life shared by prokaryotic cells and eukaryotic cells, but not viruses? nucleic acids used to store hereditary information order and complexity in arrangement of biological molecules the ability to process energy through metabolic reactions the capacity to evolve

162 Which of the following is a property of life shared by prokaryotic cells and eukaryotic cells, but not viruses? a)nucleic acids used to store hereditary information b)order and complexity in arrangement of biological molecules c)the ability to process energy through metabolic reactions d)the capacity to evolve

163 Which of the following is characteristic of the lytic cycle? Viral DNA is incorporated into the host genome. The virus-host relationship usually lasts for generations. A large number of phages are released at a time. Many bacterial cells containing viral DNA are produced. The viral genome replicates without destroying the host.

164 Which of the following is characteristic of the lytic cycle? Viral DNA is incorporated into the host genome. The virus-host relationship usually lasts for generations. A large number of phages are released at a time. Many bacterial cells containing viral DNA are produced. The viral genome replicates without destroying the host.

165 What is the function of reverse transcriptase in retroviruses? It converts host cell RNA into viral DNA. It hydrolyzes the host cell's DNA. It uses viral RNA as a template for making complementary RNA strands. It translates viral RNA into proteins. It uses viral RNA as a template for DNA synthesis.

166 What is the function of reverse transcriptase in retroviruses? It converts host cell RNA into viral DNA. It hydrolyzes the host cell's DNA. It uses viral RNA as a template for making complementary RNA strands. It translates viral RNA into proteins. It uses viral RNA as a template for DNA synthesis.

167 Why are viruses referred to as obligate parasites? They use the host cell to reproduce. Viral DNA always inserts itself into host DNA. They invariably kill any cell they infect. They can incorporate nucleic acids from other viruses. They must use enzymes encoded by the virus itself.

168 Why are viruses referred to as obligate parasites? They use the host cell to reproduce. Viral DNA always inserts itself into host DNA. They invariably kill any cell they infect. They can incorporate nucleic acids from other viruses. They must use enzymes encoded by the virus itself.

169 Which of the following molecules make up the viral envelope? viral glycoproteins capsid phospholipids from human host cell membrane membrane proteins from human host cell viral DNA

170 Which of the following molecules make up the viral envelope? viral glycoproteins capsid phospholipids from human host cell membrane membrane proteins from human host cell viral DNA

171 You have isolated viral particles from a patient, but you are not sure whether they are adenoviruses or influenza viruses. The presence of which class of biological molecules would allow you to distinguish between the two types of virus? RNA phospholipids proteins glycoproteins DNA

172 You have isolated viral particles from a patient, but you are not sure whether they are adenoviruses or influenza viruses. The presence of which class of biological molecules would allow you to distinguish between the two types of virus? RNA phospholipids proteins glycoproteins DNA

173 The HIV virus attacks only a certain type of white blood cells, and not other cell types. Why? HIV receptors are not found on the other cell types. Reverse transcriptase cannot transcribe RNA to DNA. Viral mRNA cannot be transcribed from the integrated provirus. Viruses cannot bud from the host cell.

174 The HIV virus attacks only a certain type of white blood cells, and not other cell types. Why? HIV receptors are not found on the other cell types. Reverse transcriptase cannot transcribe RNA to DNA. Viral mRNA cannot be transcribed from the integrated provirus. Viruses cannot bud from the host cell.

175 Which is not an accepted theory about the evolution of viruses: a)Viruses originated from naked bits of cellular nucleic acids. b)Genes coding for capsid proteins allowed viruses to bind cell membranes. c)Plasmids and transposons may have been the original sources of viral genomes. d)Viruses are the descendents of precellular life forms.

176 Which is not an accepted theory about the evolution of viruses: a)Viruses originated from naked bits of cellular nucleic acids. b)Genes coding for capsid proteins allowed viruses to bind cell membranes. c)Plasmids and transposons may have been the original sources of viral genomes. d)Viruses are the descendents of precellular life forms.

177 AZT is a nucleoside analog used to treat HIV infections. It is a modified nucleoside. Which step does AZT hamper in the reproductive cycle of the HIV virus? entry into the cell synthesis of DNA from RNA catalyzed by reverse transcription transcription of RNA from proviral DNA viral assembly within the cell

178 AZT is a nucleoside analog used to treat HIV infections. It is a modified nucleoside. Which step does AZT hamper in the reproductive cycle of the HIV virus? entry into the cell synthesis of DNA from RNA catalyzed by reverse transcription transcription of RNA from proviral DNA viral assembly within the cell

179 Which of the following most likely describes the vertical transmission of a plant virus? The plant shows symptoms of disease after being grazed on by herbivores. Sap from one plant is rubbed on the leaves of a second plant; both plants eventually show disease symptoms. Seeds are planted and reared under protected conditions, but mature plants show disease symptoms. After a gardener prunes several plants with the same shears, they all show disease symptoms.

180 Which of the following most likely describes the vertical transmission of a plant virus? The plant shows symptoms of disease after being grazed on by herbivores. Sap from one plant is rubbed on the leaves of a second plant; both plants eventually show disease symptoms. Seeds are planted and reared under protected conditions, but mature plants show disease symptoms. After a gardener prunes several plants with the same shears, they all show disease symptoms.

181 The photograph shows Rainbow and CC (CC is Rainbow’s clone). Why is CC’s coat pattern different from Rainbow’s given that CC and Rainbow are genetically identical? random X chromosome inactivation heterozygous at coat color gene locus environmental effects on gene expression all of the above

182 The photograph shows Rainbow and CC (CC is Rainbow’s clone). Why is CC’s coat pattern different from Rainbow’s given that CC and Rainbow are genetically identical? random X chromosome inactivation heterozygous at coat color gene locus environmental effects on gene expression all of the above

183 Which is an incorrect statement about STRs (Short Tandem repeats)? They are tandemly repeated units of 5- to 10 nucleotide sequences The number of repeats is polymorphic from person to person Two alleles of an STR may differ in an individual They occur in specific regions of the genome PCR is used to amplify particular STRs.

184 Which is an incorrect statement about STRs (Short Tandem repeats)? They are tandemly repeated units of 5- to 10 nucleotide sequences The number of repeats is polymorphic from person to person Two alleles of an STR may differ in an individual They occur in specific regions of the genome PCR is used to amplify particular STRs.

185 Which of the following beneficial traits have not resulted from DNA technology and genetic engineering of crop plants? Delayed ripening Resistance to drought Resistance to herbicides Resistance to salinity Superweeds

186 Delayed ripening Resistance to drought Resistance to herbicides Resistance to salinity Superweeds Which of the following beneficial traits have not resulted from DNA technology and genetic engineering of crop plants?

187 Which of the following is not a correct statement about third generation sequencing? A single DNA molecule is sequenced on its own Different bases interrupt an electric current for a particular length of time a compound and an isotope; a molecule DNA moves through a small nanopore a molecule and a compound; a molecule DNA must be cut into fragments or amplified

188 A single DNA molecule is sequenced on its own Different bases interrupt an electric current for a particular length of time a compound and an isotope; a molecule DNA moves through a small nanopore a molecule and a compound; a molecule DNA must be cut into fragments or amplified Which of the following is not a correct statement about third generation sequencing?

189 Place the steps in a cycle of PCR (Polymerase Chain Reaction) in the correct order: Annealing—Cool to allow primers to form hydrogen bonds with ends of target sequence 2.Extension—DNA polymerase adds nucleotides to the 3 end of each primer 3.Denaturation—Heat briefly to separate DNA strands

190 Place the steps in a cycle of PCR (Polymerase Chain Reaction) in the correct order: 1.Annealing—Cool to allow primers to form hydrogen bonds with ends of target sequence 2.Extension—DNA polymerase adds nucleotides to the 3 end of each primer 3.Denaturation—Heat briefly to separate DNA strands

191 Which of the following is an example of “recombinant DNA”? combining alternate alleles of a gene in a single cell manipulating a meiotic crossing-over event cloning genes from homologous pairs of chromosomes introducing a human gene into a bacterial plasmid alternate alleles assorting independently

192 combining alternate alleles of a gene in a single cell manipulating a meiotic crossing-over event cloning genes from homologous pairs of chromosomes introducing a human gene into a bacterial plasmid alternate alleles assorting independently Which of the following is an example of “recombinant DNA”?

193 This segment of DNA is cut at restriction sites 1 and 2, which creates restriction fragments A, B, and C. Which of the following electrophoretic gels represents the separation of these fragments? a) b) c) d)

194 This segment of DNA is cut at restriction sites 1 and 2, which creates restriction fragments A, B, and C. Which of the following electrophoretic gels represents the separation of these fragments? a) b) c) d)

195 1) The top diagram depicts the very large regulatory region upstream of the Hoxd13 gene. The area between the slashes represents the DNA located between the promoter and the regulatory region. 2) The diagrams to the left of the bar graph show, first, the intact DNA and, next, the three altered DNA sequences. A red X indicates the segment (A, B, and/or C) that was deleted in each line of transgenic mice. 3) The horizontal bar graph shows the amount of Hoxd13 mRNA that was present in the digit-formation zone of each transgenic 12.5-day- old embryo paw relative to the amount that was in the digit-formation zone of a wild-type mouse that had the intact regulatory region (top bar = 100%). The paw images have blue stain visible where the Hoxd13 mRNA is located. Scientific Skills Exercise

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197 Which of the four treatments was the control for the experiment? the wild-type mouse C the transgenic mouse with all three segments deleted N the transgenic mouse with segments B and C deleted the transgenic mouse with only segment C deleted

198 the wild-type mouse C the transgenic mouse with all three segments deleted N the transgenic mouse with segments B and C deleted the transgenic mouse with only segment C deleted Which of the four treatments was the control for the experiment?

199 The hypothesis was that all three segments of the regulatory region are required for highest expression of the Hoxd13 gene. Is this hypothesis supported by the results? Yes; when any of the segments were deleted, the expression level dropped to less than 100% of the control. No; they did not delete the promoter, so the gene could still be expressed even without the segments. Yes; when all three segments were present, the expression level was at 100%. No; even when segments were deleted, the Hoxd13 gene was still being expressed.

200 The hypothesis was that all three segments of the regulatory region are required for highest expression of the Hoxd13 gene. Is this hypothesis supported by the results? Yes; when any of the segments were deleted, the expression level dropped to less than 100% of the control. No; they did not delete the promoter, so the gene could still be expressed even without the segments. Yes; when all three segments were present, the expression level was at 100%. No; even when segments were deleted, the Hoxd13 gene was still being expressed.

201 What was the effect on the amount of Hoxd13 mRNA when segments B and C were both deleted? Only about 60% of the control amount of Hoxd13 mRNA was produced. The deletion of segments B and C had no effect on the amount of Hoxd13 mRNA produced. Only about 35% of the control amount of Hoxd13 mRNA was produced. Only about 5% of the control amount of Hoxd13 mRNA was produced.

202 What was the effect on the amount of Hoxd13 mRNA when segments B and C were both deleted? Only about 60% of the control amount of Hoxd13 mRNA was produced. The deletion of segments B and C had no effect on the amount of Hoxd13 mRNA produced. Only about 35% of the control amount of Hoxd13 mRNA was produced. Only about 5% of the control amount of Hoxd13 mRNA was produced.

203 Look at the blue stain in the in situ hybridization for the transgenic mouse lacking segments B and C. How would you describe the spatial pattern of gene expression in the embryo paw as compared to the control? There is very light blue stain in the center of each digit zone as compared to the control. The blue stain is generally lighter than in the control, but all four digit zones are still visible. There is almost no blue stain anywhere in the paw as compared to the control. There is no blue stain at the base of the paw as compared to the control.

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205 Look at the blue stain in the in situ hybridization for the transgenic mouse lacking segments B and C. How would you describe the spatial pattern of gene expression in the embryo paw as compared to the control? There is very light blue stain in the center of each digit zone as compared to the control. The blue stain is generally lighter than in the control, but all four digit zones are still visible. There is almost no blue stain anywhere in the paw as compared to the control. There is no blue stain at the base of the paw as compared to the control.

206 What was the effect on the amount of Hoxd13 mRNA when just segment C was deleted? The deletion of segment C had no effect on the amount of Hoxd13 mRNA produced. Only about 60% of the control amount of Hoxd13 mRNA was produced. Only about 35% of the control amount of Hoxd13 mRNA was produced. Only about 5% of the control amount of Hoxd13 mRNA was produced.

207 What was the effect on the amount of Hoxd13 mRNA when just segment C was deleted? The deletion of segment C had no effect on the amount of Hoxd13 mRNA produced. Only about 60% of the control amount of Hoxd13 mRNA was produced. Only about 35% of the control amount of Hoxd13 mRNA was produced. Only about 5% of the control amount of Hoxd13 mRNA was produced.

208 How would you describe the spatial pattern of gene expression in the embryo paw lacking segment C as compared to the control and to the paw lacking segments B and C? The digit zones are not visibly stained as they are in the control and the paw lacking B and C. The top of the paw is stained darker than both the control and the paw lacking B and C. The base of the paw is stained darker than both the control and the paw lacking B and C. The digit zones are defined with darker stain than both the control and the paw lacking B and C.

209 How would you describe the spatial pattern of gene expression in the embryo paw lacking segment C as compared to the control and to the paw lacking segments B and C? The digit zones are not visibly stained as they are in the control and the paw lacking B and C. The top of the paw is stained darker than both the control and the paw lacking B and C. The base of the paw is stained darker than both the control and the paw lacking B and C. The digit zones are defined with darker stain than both the control and the paw lacking B and C.

210 Suppose the researchers had only measured the amount of Hoxd13 mRNA and not done the in situ hybridizations. What important information about the role of the regulatory segments would have been missed? The interaction of the regulatory region with the promoter would have been missed. The interaction among the different segments of the regulatory region would have been missed. The mRNA would not have been blue; therefore it could not have been measured for the results shown in the bar graph. The spatial patterns of Hoxd13 gene expression in the paws would have been missed.

211 Suppose the researchers had only measured the amount of Hoxd13 mRNA and not done the in situ hybridizations. What important information about the role of the regulatory segments would have been missed? The interaction of the regulatory region with the promoter would have been missed. The interaction among the different segments of the regulatory region would have been missed. The mRNA would not have been blue; therefore it could not have been measured for the results shown in the bar graph. The spatial patterns of Hoxd13 gene expression in the paws would have been missed.

212 Suppose the researchers had only done the in situ hybridizations and not measured the amount of Hoxd13 mRNA. What important information would have been missed? The information about which regulatory segments were deleted would have been missed. The spatial patterns of Hoxd13 gene expression in the paws would have been missed. Qualitative data about Hoxd13 mRNA levels would have been missed. Quantitative data about Hoxd13 mRNA levels would have been missed.

213 Suppose the researchers had only done the in situ hybridizations and not measured the amount of Hoxd13 mRNA. What important information would have been missed? The information about which regulatory segments were deleted would have been missed. The spatial patterns of Hoxd13 gene expression in the paws would have been missed. Qualitative data about Hoxd13 mRNA levels would have been missed. Quantitative data about Hoxd13 mRNA levels would have been missed.


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