• Viruses called bacteriophages can infect and set in motion a genetic takeover of bacteria, such as Escherichia coli
• Bacteria are prokaryotes with cells much smaller and more simply organized than those of eukaryotes
• Viruses are smaller and simpler than bacteria
• 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)
• 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
Capsomereof capsid
RNA
18 250 mm
Tobacco mosaic virus20 nm
A capsid is the protein shell that encloses the viral genome and can have
various structures
• 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
• 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
DNAVIRUS
Capsid
HOST CELL
Viral DNA
Replication
Entry into cell anduncoating of DNA
Transcription
Viral DNA
mRNA
Capsidproteins
Self-assembly ofnew virus particlesand their exit from cell
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
Attachment
Entry of phage DNAand degradation of host DNA
Synthesis of viralgenomes and proteins
Assembly
ReleasePhage assembly
Head Tails Tail fibers
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
Phage
Phage DNA
The phage attaches to ahost cell and injects its DNA.
Phage DNAcircularizes
Bacterial chromosome
Lytic cycle
The cell lyses, releasing phages.Lytic cycleis induced
or Lysogenic cycleis entered
Certain factorsdetermine whether
Lysogenic cycle
Occasionally, a prophageexits the bacterial chromosome,initiating a lytic cycle.
The bacterium reproducesnormally, copying the prophageand transmitting it to daughter cells.
Prophage
Many cell divisionsproduce a large population of bacteria infected withthe prophage.
Daughter cellwith prophage
Phage DNA integrates into thebacterial chromosomes, becoming aprophage.
New phage DNA and proteins aresynthesized and assembled into phages.
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
RNA
ER
Capsid
HOST CELL
Viral genome (RNA)
mRNA
Capsidproteins
Envelope (withglycoproteins)
Glyco-proteins Copy of
genome (RNA)
Capsid and viral genomeenter cell
New virus
Template
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
• 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
HOST CELL
ReversetranscriptionViral RNA
RNA-DNAhybrid
DNA
NUCLEUS
ChromosomalDNA
Provirus
RNA genomefor thenext viralgeneration
mRNA
New HIV leaving a cell
HIV entering a cell
0.25 µm
HIVMembrane ofwhite blood cell
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
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
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
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
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
Mutantstrain
arg+ trp–
Mutantstrain
arg+ trp–
Mixture
Mixture
Nocolonies(control)
Nocolonies(control)
Coloniesgrew
Mutantstrain
arg– trp+
Mutantstrain
arg– trp+
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
F plasmid Bacterial chromosome
F+ cellMatingbridge
F+ cell
F+ cellBacterial 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
Temporarypartialdiploid
Recombinant F–
bacterium
Conjugation and transfer of part of the bacterial chromosome from anHfr donor to an F– recipient, resulting in recombination
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
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
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
53
35
Transposon
Insertion sequence
Insertion sequence
Antibioticresistance gene
Transposase geneInverted repeat
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
Promoter Promoter
DNA trpR
Regulatorygene
RNApolymerase
mRNA
3
5
Protein Inactiverepressor
Tryptophan absent, repressor inactive, operon on
mRNA 5
trpE trpD trpC trpB trpA
OperatorStart codon Stop codon
trp operon
Genes of operon
E
Polypeptides that make upenzymes for tryptophan synthesis
D C B A
DNA
Protein
Tryptophan(corepressor)
Tryptophan present, repressor active, operon off
mRNA
Activerepressor
DNA
Protein
Tryptophan(corepressor)
Tryptophan present, repressor active, operon off
mRNA
Activerepressor
No RNA made
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
DNA lacl
Regulatorygene
mRNA
5
3
RNApolymerase
ProteinActiverepressor
NoRNAmade
lacZ
Promoter
Operator
Lactose absent, repressor active, operon off
DNA lacl
mRNA5
3
lac operon
Lactose present, repressor inactive, operon on
lacZ lacY lacA
RNApolymerase
mRNA 5
Protein
Allolactose(inducer)
Inactiverepressor
-Galactosidase Permease Transacetylase
• 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
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
DNA
cAMP
lacl
CAP-binding site
Promoter
ActiveCAP
InactiveCAP
RNApolymerasecan bindand transcribe
Operator
lacZ
Inactive lacrepressor
Lactose present, glucose scarce (cAMP level high): abundant lacmRNA synthesized
DNA lacl
CAP-binding site
Promoter
RNApolymerasecan’t bind
Operator
lacZ
Inactive lacrepressor
InactiveCAP
Lactose present, glucose present (cAMP level low): little lacmRNA synthesized
• 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
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
DNA double helix
Histonetails
His-tones
Linker DNA(“string”)
Nucleosome(“bead”)
10 nm
2 nm
Histone H1
Nucleosomes (10-nm fiber)
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
Signal
NUCLEUS
DNA
RNA
Chromatin
Gene availablefor transcription
Gene
Exon
Intro
Transcription
Primary transcript
RNA processing
Cap
Tail
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Translation
Degradationof mRNA
Polypeptide
CleavageChemical modificationTransport to cellular
destination
Degradation of protein
Active protein
Degraded protein
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
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
Histonetails
Amino acidsavailablefor chemicalmodification
DNAdouble helix
Histone tails protrude outward from a nucleosome
Acetylation of histone tails promotes loose chromatinstructure that permits transcription
Unacetylated histones Acetylated histones
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?
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
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
Enhancer(distal control elements)
Proximal control elements
Upstream
DNA
Promoter
Exon Intron Exon Intron Exon
DownstreamTranscription
Poly-A signalsequence
Terminationregion
Intron Exon Intron Exon
RNA processing:Cap and tail added;introns excised andexons spliced together
Poly-A signal
Cleaved 3 endof primarytranscript
3
Poly-Atail
3 UTR(untranslated
region)
5 UTR(untranslated
region)
Startcodon
Stopcodon
Coding segment
Intron RNA
5 Cap
mRNA
Primary RNAtranscript(pre-mRNA)
5Exon
Distal controlelement Activators
Enhancer
DNA
DNA-bendingprotein
TATAbox
PromoterGene
Generaltranscriptionfactors
Group ofmediator proteins
RNApolymerase II
RNApolymerase II
RNA synthesisTranscriptionInitiation complex
Controlelements
Enhancer Promoter
Albumingene
Crystallingene
Availableactivators
Availableactivators
Albumingene notexpressed
Albumingeneexpressed
Liver cell Lens cell
Crystallin genenot expressed Crystallin gene
expressed
Liver cellnucleus
Lens cellnucleus
Some transcription factors function as repressors, inhibiting expression of a particular geneSome activators and repressors act indirectly by influencing chromatin structure
PrimaryRNAtranscript
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
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)
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
Protein tobe degraded
Ubiquitinatedprotein
Proteasome
Protein entering aproteasome
Protein fragments(peptides)
Proteasomeand ubiquitinto be recycled
Ubiquitin
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
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
Proto-oncogene
DNA
Translocation or transposition:gene moved to new locus, under new controls
Newpromoter
Gene amplification:multiple copies of the gene
Point mutationwithin a controlelement
Oncogene Oncogene
Point mutationwithin the gene
Normal growth-stimulatingprotein in excess
Normal growth-stimulatingprotein in excess Normal growth-stimulating
protein in excessHyperactive ordegradation-resistant protein
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
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
• 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
Protein overexpressed
EFFECTS OF MUTATIONS
Protein absent
Cell cycle notinhibited
Increased celldivision
Cell cycle overstimulate
Effects ofmutations
Activeformof p53
DNADNA damagein genome
UVlight
Protein kinasesMUTATION
Defective ormissingtranscriptionfactor, such as p53, cannotactivatetranscription
Protein kinases(phosphorylationcascade)
Cell cycle-inhibitingpathway
Cell cycle-stimulatingpathway
Protein thatinhibitsthe cell cycle
NUCLEUS
DNA
Gene expression
Transcriptionfactor (activator)
Receptor
G protein
Growthfactor
MUTATIONHyperactiveRas protein(product ofoncogene)issues signalson its own
Protein thatstimulatesthe cell cycle
Colon
Colon wall
Loss oftumor-suppressorgene APC (orother)
Normal colonepithelial cells
Small benigngrowth (polyp)
Larger benigngrowth (adenoma)
Activation ofras oncogene
Loss oftumor-suppressorgene DCC
Loss oftumor-suppressorgene p53
Additionalmutations
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)
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
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
DNA of genomeTransposon
is copied
Mobile transposon
Transposon
Insertion
New copy oftransposon
Transposon movement (“copy-and-paste” mechanism)
Retrotransposon movement
DNA of genome
Insertion
RNA
Reversetranscriptase
RetrotransposonNew copy of
retrotransposon
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
DNA
Non-transcribedspacer
RNA transcripts
Transcription unit
DNA18S 5.8S 28S
rRNA
18S
5.8S28S
Part of the ribosomal RNA gene family
Heme
Hemoglobin
-Globin
-Globin
-Globin gene family -Globin gene family
Chromosome 11Chromosome 16
1 12 A
Embryo Embryo Fetus AdultFetus
and adult
The human -globin and -globin gene families
• 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
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
Bacterium
Bacterialchromosome
Plasmid
Gene inserted intoplasmid
Cell containing geneof interest
Gene ofinterest DNA of
chromosome
RecombinantDNA (plasmid)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest
Protein expressedby gene of interest
Protein harvested
Gene ofinterest
Copies of gene
Basicresearchon gene
Basicresearchon protein
Basic research andvarious applications
Gene for pestresistance insertedinto plants
Gene used to alterbacteria for cleaningup toxic waste
Protein dissolvesblood clots in heartattack therapy
Human growth hor-mone treats stuntedgrowth
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
Restriction site
DNA 53
35
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow.
One possible combination
DNA fragment from anothersource is added. Base pairingof sticky ends producesvarious combinations.
Fragment from differentDNA molecule cut by thesame restriction enzyme
DNA ligaseseals the strands.
Recombinant DNA molecule
Sticky end
Isolate plasmid DNAand human DNA.
Cut both DNA samples withthe same restriction enzyme.
Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many nonrecombinant plasmids.
Bacterial cell lacZ gene(lactosebreakdown)
Humancell
Restrictionsite
ampR gene(ampicillinresistance)
Bacterialplasmid Gene of
interest
Stickyends
Human DNAfragments
Recombinant DNA plasmids
Introduce the DNA into bacterial cellsthat have a mutation in their own lacZgene.
Recombinantbacteria
Plate the bacteria on agarcontaining ampicillin and X-gal.Incubate until colonies grow.
Colony carrying non-recombinant plasmidwith intact lacZ gene
Colony carryingrecombinantplasmid withdisrupted lacZ gene
Bacterialclone
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
Genomic DNA
Targetsequence
5
3
3
5
5
3
3
5
Primers
Denaturation:Heat brieflyto separate DNAstrands
Annealing:Cool to allowprimers to formhydrogen bondswith ends oftarget sequence
Extension:DNA polymeraseadds nucleotides tothe 3 end of eachprimer
Cycle 1yields
2molecules
Newnucleo-
tides
Cycle 2yields
4molecules
Cycle 3yields 8
molecules;2 molecules
(in white boxes)match target
sequence
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
Cathode
Powersource
Anode
Mixtureof DNAmoleculesof differ-ent sizes
Gel
Glassplates
Longermolecules
Shortermolecules
Normal -globin allele
175 bp 201 bp Large fragment
Sickle-cell mutant -globin allele
376 bp Large fragment
Ddel Ddel Ddel Ddel
Ddel Ddel Ddel
Ddel restriction sites in normal and sickle-cell alleles of-globin gene
Normalallele
Sickle-cellallele
Largefragment
376 bp201 bp175 bp
Electrophoresis of restriction fragments from normaland sickle-cell alleles
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
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
Cloned gene
Retroviruscapsid
Bonemarrowcell frompatient
Inject engineeredcells into patient.
Insert RNA version of normal alleleinto retrovirus.
Viral RNA
Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured.
Viral DNA carrying the normalallele inserts into chromosome.
Bonemarrow
Pharmaceutical Products• Some pharmaceutical applications of DNA
technology:– Large-scale production of human hormones and
other proteins with therapeutic uses– Production of safer vaccines
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
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
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
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
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
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
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
Animations and Videos
• Microarray• Sanger Sequencing• DNA Fingerprinting• RNA Interface• miRNA• Dicer• DNA Microarrays• Bozeman - DNA Fingerprinting• Bozeman - Viral Replication
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
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
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
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.
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.
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)
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)
Which element(s) from the following list constitute(s) a bacterial operon?
• repressor gene• promoter• inducer• repressor protein• all of the above
Which element(s) from the following list constitute(s) a bacterial operon?
• repressor gene• promoter• inducer• repressor protein• all of the above
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.
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.
Approximately what proportion of the DNA in the human genome codes for proteins or functional RNA?
• 83%• 46%• 32%• 13%• 1.5%
Approximately what proportion of the DNA in the human genome codes for proteins or functional RNA?
• 83%• 46%• 32%• 13%• 1.5%
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
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
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.
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.
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.
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.
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
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
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
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
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.
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.
Mutations in _______ genes caused the development of legs in the place of antennae.
• homeotic• embryonic lethal• myoD• Ras• wild-type
Wild type
Eye
Mutant
Mutations in _______ genes caused the development of legs in the place of antennae.
• homeotic• embryonic lethal• myoD• Ras• wild-type
Wild type
Eye
Mutant
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
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
Which of the following would not typically cause a proto-oncogene to become an oncogene?• gene suppression• translocation• amplification• point mutation• retroviral activation
Which of the following would not typically cause a proto-oncogene to become an oncogene?
• gene suppression• translocation• amplification• point mutation• retroviral activation
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.
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.
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
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
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
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
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
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
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
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
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.
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.
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.
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.
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.
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.
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
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
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
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
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.
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.
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.
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.
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
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
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.
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.
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
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
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.
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.
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
• 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?
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
• 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?
Place the steps in a cycle of PCR (Polymerase Chain Reaction) in the correct order:
• 3-1-2• 3-2-1• 1-2-3• 2-3-1• 1-3-2• 2-1-3
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
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
• 3-1-2• 3-2-1• 1-2-3• 2-3-1• 1-3-2• 2-1-3
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
• 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”?
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)
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)
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
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
• 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?
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.