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114 Cards in this Set

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allows us to isolate specific genes from any genome so that we can study their function.
recombinant DNA technology
(Figure 11-3)
it can be made from any organism by inserting DNA fragments into a cloning
Recombinant DNA molecules
Plasmid or Virus
Contains an o of replication for gene amplification.
Contains an antibiotic resistance gene 4 selection
Makes specific cuts in DNA by cleaving phosphodiester bonds of each strand of the DNA duplex "digestion".
Restriction Enzymes
cohesive "sticky" ends
blunt ends
A. 180° axis of symmetry.
B. Usually a 4 (1/256) or 6 nt sequence (1/4096).
Characteristics of Restriction Sites
1.Digest 2.mix with sticky or blunt 3. DNA Ligase for desired gene 5.purify rocombinant gene 6. determine DNA sequence and OFRs 7. study gene ans protien function
gene cloning (Figure 11-5,6)
Small, circular, origin of replication, antibiotic resistance gene. Can clone several kb.
Plasmids (Figure 11-7)
Special plasmids that contain t'scription and t'lation signals to allow overproduction of the
protein encoded by the gene.
Express euk genes in bacteria (e.g., human insulin).
Expression Vectors
Contain origins of replication for two organisms.
Clone in E. coli, purify DNA, transfect mammalian cell line.
Shuttle Vectors
Based on the F plasmid. ~150 kb can be cloned.

Contain an o of rep, telomeres, and a centromere.
~1000 kb can be cloned.
Bacterial Artificial Chromosomes (BACs)

Yeast Artificial Chromosomes (YACs)
DNA generated from mRNA and reverse transcriptase, thus no introns.
cDNA (complementary DNA)
Random c'somal or cDNA frags cloned into any vectors.
A random pop of clones should contain every gene.
Isolate a specific gene by selection (cloning by complementation) or screening.
DNA Library
1. Isolate a mutant strain giving the desired phenotype.
2. Transform the mutant strain with a DNA library and directly select for the positive clone by its
ability to complement the mutant defect.
Cloning by complementation
A radioactive DNA fragment that is complementary to the gene you want to clone.
Can be the homologous gene from a related organism.
(e.g., clone a human gene using the cloned mouse gene as a probe)
DNA Probe
Used to fractionate DNA, RNA or proteins based on their size.
(Digest DNA and run on a gel)
Electrophoresis (Figure 11-13)
1)Probing for a DNA fragment using a DNA probe.
2)Probing for an RNA fragment using a DNA probe.
3)Probe for a protein using antibodies.
1)Southern Blot (Figure 11-14)
2)Northern Blot
3)Western Blot
Restriction sites in a DNA fragment can be used to subclone fragments within the fragment.
1. Digest DNA with enzymes.
2. Run digested DNA on an agarose gel to sep frags.
3. Stain DNA with bromide which between bases.
4. View under UV light
5. Single, double or partial digests.
Restriction Mapping
(Figure 11-16)
Used to determine the nt seq of any gene. Can resolve DNA fragments differing by 1 nt.
DNA Sequencing
(Gilbert and Sanger shared the Nobel Prize)
nuc lacking a 3’ OH group can’t be extended by DNA pol once incorporated. Random incorporation because of a mixture of dNTPs and one ddNTP. (ddATP, ddCTP, ddGTP, ddTTP)
Dideoxy Sequencing
(Figures 11-17 and 11-18)
Uses fluorescent dyes
Automated Sequencing
(Figure 11-19)
Used to amplify specific regions of DNA. Uses a thermostable DNA polymerase. (Taq) Can amplify DNA from a single cell.
Polymerase Chain Reaction (PCR) (Mullis Nobel Prize)
(Figure 11-21)
Directing point mutations, insertions or deletions into cloned DNA fragments by PCR.
Site-Directed Mutagenesis
1. Clone selectable marker in the middle of a gene.
2. Linearize with restriction enzyme.
3. Transform organism.
4. Double X-over results in replace of WT gene with disrupted gene.
5. Study the effect of the mutation.
Gene Inactivation (Suicide vector)
1. Clone the regulatory region (promoter) adjacent to a
reporter gene .Expression of reporter gene depends on cloned regulatory elements.
2. Study regulation.
3. Repeat with deletions or point mutations in regulatory region.
Studying Gene Regulation
Recessive disorders cause over 500 genetic diseases.
perhaps determine if individual carries mutant gene(s).
Human Genetic Disorders
Sickle cell anemia affects 0.25% of U.S. Afri-Americans.
mutation eliminates an MstII restriction site.
detected by Southern blotting. Change in banding pattern diagnostic for sickle allele.
Restriction Fragment Length Polymorphism (RFLP)
(Figure 11-24)
Humans1-5 kb sequences consisting of repeats 15-100 nt long.
1. Digest DNA with restriction enzyme that does not cut within VNTRs.
2. Run DNA on gel.
3. Southern blot with VNTR probe.
4. Pattern on autoradiograph is highly individualistic.
DNA samples can be amplified by PCR using trace amounts of blood, semen, hair.
DNA Fingerprinting.Used in forensic medicine.
Variable Number Tandem Repeats (VNTRs)
E.coli 4.6 million bp
Human 3 billion bp
Plants even larger
Specialized techniques were developed to handle large genomes (e.g., YACs).
Eukaryotic Transgenic Technology
1)Methods used to transfect eukaryotic cells.
2)Organism that develops from the transfected cell.
3)Ti (Tumor Inducing) Plasmidfrom tumefaciens Causes crown gall (plant tumors).
1)Transgenic Technology
2)Transgenic Organism
3)Transgenic Plants
(Figure 11-28)
Applying similar techniques to study the function of animal genes.
Can be used for gene therapy in humans.
Transgenic Animals
Correct genetic defects by transferring WT genes into the germ line (gametes)
or other actively dividing tissue (e.g., stem cells).
Human Gene Therapy
"Boy in the Bubble Disease".
No functional immune system.
Has been cured in 9 of 11 individuals in a clinical trial with gene therapy (France).
One individual developed leukemia due to the point of insertion in the genome.
SCID (Severe Combined Immunodeficiency Disease)
The study of entire genomes.
> 250 completed bacterial genomes.
Theoretically possible to complete a bacterial genome in a single day using automated technology.
Genomics (Figure 12-3)
Sequence random clones and then assemble the sequence into a complete chromosome by looking
for overlaps within the sequenced clones.
Requires more sequencing (10 genome equivalents) but no need to order clones.
Sequence the entire genome
A. Shotgun sequencing. (Figure 12-2)
Less sequencing and no need to assemble the genome but it takes time to order the clones.
seguence entire genome
B. Sequence ordered clones.
Annotate the genome
A. Identify all of the Open Reading Frames (ORFs) encoded in the genome.
1. Computationally remove introns in higher eukaryotes.
2. Compare to full-length cDNA sequences.
3. Predict regulatory regions "docking sites for regulators."
(comparative genomics)
A hit suggests that the gene is real.
5. Codon bias.
2. Bioinformatics
Figures 12-25 and 12-26)(Figure 12-22)
~25,000 genes
There are about 3 alternative splicing pathways per gene.
Thus, the proteome is thought to be about 3 times the size of the genome.
(i.e., ~75,000 proteins)
Human Genome
Analyze the expression patterns of all genes simultaneously under various growth conditions, times
during development, when a regulatory protein is mutated, etc.
Microarray studies (i.e., gene chips)
A. Transcriptome analysis
(Figure 12-27)
Using 2-D gels and mass spectroscopy to identify changes in protein levels under various growth
conditions, times during development, when a regulatory protein is mutated, etc.
Complementary to transcriptome analysis but also identified translationally controlled genes.
Proteome analysis
Identification of all phenotypes associated with the inactivation of each gene.
D. Phenome analysis
"Jumping Genes"
Genetic elements than can move or "transpose" from one position to another.
Transposable Genetic Elements
who won the Nobel Prize (1983) for Ac/Ds elements.
Transposable elements are present in essentially all organisms.
Barbara McClintock
mobile piece of bacteria that can inactovate a gene
insertion sequences
1)mobile DNA flanked by terminal repeats bears genes coding for transposition.
2)genetic elements move within genome
3) any gene unit that can move
1)Transposons (Tn)
3)transposable elements
F (fertility factor) integration into the chromosome to generate Hfr strains occurs via X-overs between IS
elements in the F plasmid and chromosomal IS elements.
F (fertility factor) integration
(Figure 13-8)
Composite elements that contain tnp and additional genes (typically a drug resistance gene).
a type of bacterial TE containing a variety of genes that reside b/w 2 identical IS elements
Transposase comes from one IS elements and both IS elements function as the IR
A. Composite Transposon
(Figure 13-9a)
Composed of a transposase gene and typically a drug resistance gene.
IRs similer to those for IS elements.
B. Simple Transposon
(Figure 13-9b)
Transposition involves cleavage of the DNA target followed by insertion of the transposable element.
Subsequent filling in of the resulting single-stranded gaps generates target site duplications.
(Characteristic of transposable elements)
Transposase is responsible for target site selection and DNA cleavage.
Mechanism of Transposition
One copy of the transposable element remains in the original site and a second copy inserts into a new site.
Requires DNA replication.
A. Replicative
No DNA replication.
The transposable element is excised and moved to a new site.
Disrupted genes at the original site revert to wild-type.
B. Conservative (non-replicative)(Figure 13-11)
Transposons can jump from a naturally occurring plasmid to a chromosome or from plasmid to plasmid.
The plasmids can move from bacteria to bacteria via transformation and/or conjugation.
Can lead to multiple antibiotic resistant strains of bacteria.
Multiple Antibiotic Resistance
(Figure 13-10)
(Huge problem in a clinical setting)
Can inactivate a gene, cause chromosome breaks, and transpose to new locations (replicative or nonreplicative).
Eukaryotic Transposable Elements
Must be transcribed into RNA, then reverse transcribed to DNA, then inserted.
Class I elements can't excise and restore function to an interrupted gene.
(e.g., yeast Ty, Drosophila copia, human Alu elements)
A. Class I > Retro-transposons (RNA elements)
(both have gag and pol genes).
gag>involved in RNA maturation
pol>reverse transcriptase

The absence of the env (envelope) gene prevents packaging of the genome into a vial particle.
Instead, the DNA is inserted into a new place in the genome.
retroviruses simialar to retro-transposons
(Figure 13-15)
1. Eject RNA genome.
2. Reverse transcriptase synthesizes dsDNA from the ssRNA.
A. First DNA strand synthesized using RNA as template.
B. Second DNA strand synthesized using the first DNA strand as template while simultaneously
degrading the RNA strand.
3. Integration into the mammalian genome.
RetrovirusesssRNA animal viruses. (e.g., HIV)
found in both prok and euk participates directly with transposition

Excision from a gene can lead to reversion to wild-type.
(Figures 13-5 and 13-6)
B. Class II> DNA transposons
1)Fully functional (can transpose by themselves).
(e.g., Ac elements of Maize)
2)Requires an autonomous element to supply transposase for transposition.
(e.g., Ds elements of Maize)
1)Autonomous Elements
2)Non-autonomous elements
Transposable elements can be VERY abundant in genomes.
~ 50% of the human genome is derived from transposable elements.
>1 million Alu sequences alone.
There is 20 times more human DNA corresponding to transposable elements than protein coding DNA.
LINEs (autonomous) and SINEs (nonautonomous) are retro-transposons.
Alu is a SINE.
The majority of transposable elements are inserted in introns or between genes.
Natural selection reduces exon insertions being fixed in the population.
Transposable elements in Humans
1)A mutation in a specific gene resulting in a new allele.
Wild type (WT) compared to a mutant or variant.
2) An individual or strain carrying a mutation.
3)Agents that increase the rate of mutations.
4)Any change from the WT allele.
5)Change to the WT allele (true reversion).
1)Gene Mutation
4)Forward Mutation
5)Reverse Mutation
A change in the same gene or a second gene resulting in a complete or partial phenotypic reversion to WT
(second site reversion).
Second Site Suppressor
1)Results in the loss of activity.
2)Results in a new or altered activity.
1)Loss of Function Mutation
2)Gain of Function Mutation
1)The mutation changes one codon for an amino acid into another codon for the same amino acid.
2)The codon for one amino acid is replaced by a codon for another amino acid.
3)The codon for an amino acid AA is replaced by a stop codon.
A. Silent (Synonymous) Substitution
B. Missense Mutation
C. Nonsense Mutation
1)Removal of one nucleotide.

2)Addition of one nucleotide.

Both results in frameshift
D. Deletion
E. Insertion
Produced when a cell or organism is exposed to a mutagen. (e.g., chemicals or UV light)
Molecules that are similar in structure to bases (base analogs) but have different pairing properties
can replace the normal base in the DNA during replication.
A. Base Replacement
Mutagens that alter the structure of a base lead to mispairing.
(e.g. alkylating agents)
B. Base Alteration (Figure 14-9)
Damage so severe that pairing can't take place.
Will lead to a replication block (lethal if not repaired).
(e.g., UV-induced cyclobutane pyrimidine dimmers OR 6-4 photoproduct)
C. Base Damage (Figure 14-12)
Occur in all cells without a mutagen.
When the N-glycosidic bond between the base and the sugar is broken. loss of a purine bases

The resulting apurinic site (AP site) can’t specify a complementary base during replication.
(Put in part of Figure 14-14)
Loss of an amino group from the base. Deamination of dCytosol yields dUricil.

dU pairs with dA.
(DNA repair required)
(Figure 14-18)
Normally A, C, G, and T are in the keto form.
Errors occur during DNA replication when rare imino forms of A and C or rare enol forms of T and
G are incorporated by DNA polymerase. These tautomers pair with the wrong base.

The DNA pol III editing function removes mismatches when the rare forms change back unless
polymerization has already moved past the mismatch. (DNA Repair required)
Errors in DNA Replication
1. Keto-Enol Shift
(Figure 14-5)(Figure 14-6)
Results in deletions or insertions that can cause frameshifts.

Triplet repeat expansion diseases in humans is thought to occur via replication slippage.
(e.g., Fragile X syndrome, Huntington;s disease, myotonic dystrophy)
Errors in DNA Replication
2. Replication Slippage
(Figure 14-21)
Byproducts of aerobic metabolism produce compounds that cause damage.
spontanious DNA damage
D. Oxidative Damage
Some enzymes neutralize damaging compounds.
(e.g., Detoxification of molecules that cause oxidative damage).
Superoxide dismutase converts oxygen radicals to hydrogen peroxide.

Then catalase converts hydrogen peroxide to water.
Classes of Repair Pathways
I. Prevention of Errors Before they Happen
these dimers are repaired by photolyase.

Requires visible light for the enzyme to work.
Classes of Repair Pathways
II. Direct Reversal

A. Cyclobutane pyrimidine
(Figure 14-26)
Alkyltransferases responsible for direct reversal.

(e.g.,. Methyltransferase of E. coli)
Classes of Repair Pathways
II. Direct Reversal

B. Removal of alkyl groups added to bases
Removal of damaged bases, along with several neighboring bases, and then repairing the gap by DNA synthesis.

E. coli>An excinuclease (excision nuclease)cuts on both sides of the damaged base removing
ssDNA containing the damaged base(s).
Gap filled in by DNA pol I.
DNA ligase seals the nick.
Nucleotide Excision Repair (General Excision Repair)
(Figure 14-28)
AP endonuclease removes AP site by breaking a phosphodiester bonds at the AP site.
Then the general excision repair pathway takes over.
B. AP Endonuclease Repair Pathway
(Figure 14-27)
DNA glycosylases recognize certain damaged bases and cleave the N-glycosidic bond between the base and the sugar leaving an AP site.

The resulting AP site is cleaved by AP endonuclease.
Then the general excision repair pathway takes over.
DNA Glycosylase Repair Pathway
(Figure 14-27)
DNA editing by DNA pol III did not occur.
1) Recognition of themismatch.
2) Determine which mismatched base is incorrect. (Crucial step)
3) Excise the incorrect base.
4) General excision repair takes over.
Adenine methylase methylates A residues following replication in the sequence: 5' GATC 3'
Since it takes a few minutes for the newly synthesized strand to be methylated, the old methylated
strand is distinguished from the newly synthesized unmethylated strand.
An enzyme introduces a cleavage in the backbone of the unmethylated strand near the mismatch and adjacent to the nearest GATC sequence
ssDNA gap is filled in by DNA pol I.
Ligase seals the nick.
Postreplication Repair
A. Mismatch Repair (Figure 14-30)
1)Cells with one chromosome set (e.g., gametes).
2)Cells with two chromosome set (e.g., somatic cells).
1)Haploid Cells (n)
2)Diploid Cells (2n)
Number of chromosomes in the basic set of an organism.

Monoploid = 1n; Diploid = 2n; Triploid = 3n; etc...
Monoploid Number (n)
1)Organisms with multiples of the monoploid number.
2)Euploid with more than two sets of chromosomes.
male bees, wasps, ants.
Males develop parthogenetically from unfertilized eggs.
These organisms produce gametes via mitosis.
Monoploid organisms
Generate a monoploid plant from a diploid.
Then generate a drug resistant diploid from the monoploid plant.
Colchicine inhibits mitotic spindle formation.
After mutant selection and growth, use colchicine for one cell division.
Plant Engineering (Figure 15-11)
1)Multiple chromosome sets from within one species.
Arise spontaneously from accidental doubling (2X to 4X) or use colchicine.
Advantages: larger plant and fruit

2)Multiple chromosome sets from closely related species.
(e.g., cotton, wheat)
1)Autopolyploids(Figure 15-6)
Problems during meiotic segregation (sterile).
(4x) Tetraploid X (2x) Diploid
= (3x) Triploid
(e.g., Seedless watermelons and bananas.)
Triploids (3x) (3 c'somes)
Leeches, brine shrimp, flatworms
Common in amphibians and reptiles.
Salmon and trout originated through polyploidy.
Oysters (3n)>No spawning>palatable all year.
Most human triploids die in utero. If born, none survive.
Polyploidy in animals
An individual whose chromosome number differs from WT by part of a chromosome set.
(Usually one chromosome)
Caused by nondisjunction during meiosis
If an (n-1) gamete is involved in fertilization,
the resulting zygote will be monosomic for that particular chromosome (2n-1).
If an (n+1) gamete is involved in fertilization,
the resulting zygote will be trisomic for that particular chromosome (2n+1).
Aneuploidy(Figure 15-13).
Missing chromosome disturbs homeostasis.
The individual is hemizygous for that chromosome.
Deleterious because recessive alleles are expressed phenotypically.
Monosomics (2n-1) (Deleterious)
~10% of all human conceptions have a major chromosome abnormality.
Most are spontaneously aborted.
Human Aneuploidy
(1/5000 females)
44 autosomes with 1X chromosome.
Sterile, normal intelligence.
1. Turner Syndrome

1)(1/1000 males)
Lanky builds, retarded, sterile
Fertile. Aggressive behavior
3.Trisomy 13
Severe physical and mental abnormalities (~ 3 month survival).
5.Trisomy 18
Severe physical and mental abnormalities (survive a few weeks).
1. Klinefelter Syndrome
2. Mean Man Syndrome? (1/1000 m)
3. Patau Syndrome
5. Edward Syndrome
Trisomy 21
Most common human aneuploid.
This more common than the translocation form.
No family history.
Older mothers at greater risk. (Figure 15-18)
Mental retardation.
Males infertile.
Females may be fertile producing normal and trisomic children
Down Syndrome (1.5/1000 births)
47/58 patients were aneuploid for either chromosome 8, 9, or 21.
Acute Myeloid Leukemia
Loss of a chromosomal region.
Usually fatal if homozygous.
Often fatal if heterozygous.
Some small deletions are viable as a heterozygote.
Deletions can never revert to WT.
Chromosomal Rearrangements
A. Deletions
Visualized as a deletion loop during meiosis.
(Figure 15-28)
Deletion will “uncover” recessive alleles, thus the recessive phenotype is expressed.

Small deletions can be mapped due to pseudodominance.
Usually caused by a new germinal mutation in one parent.
(e.g., cri du chat syndrome. Tip of chromosome 5 deleted).
deletions on humans
(Figure 15-30)
Gain of a chromosomal region.
Can be adjacent to each other or the second copy may be in a novel location in the genome.
3 copies/cell in a diploid.
Usually difficult to detect phenotypically.
A loop structure may be detected during meiosis.
B. Duplications
Tandem Duplication A B C B C D
Reverse Duplication A B C C B D
Human homozygous duplications have never been detected (probably lethal).
Homologous Chromosomes for duplications
Chromosomal region rotated 180°.
~ 2% of humans carry inversions.
No net change of genetic material.
Viable without phenotypic abnormalities unless breakage occurs in an essential gene.
Then they are lethal if homozygous.
Paired homologs form an inversion loop during meiosis if heterozygous .
Inversions (Figure 15-21b)
Centromere outside of the inversion.
A B●C D E F--->A B●C E D F
X-overs between a paracentric inversion and a WT chromosome result in a dicentric and an acentric
Acentric fragment is lost.
Dicentric breaks randomly in bridge
Paracentric Inversion
(Figure 15-22)
Inversion spans around the centromere
X-overs between a pericentric inversion and a WT chromosome result in products with a deletion and a
duplication of different parts of the chromosome.
Inviable deletion products and inhibition of pairing in the inverted region reduces the number of X-over
recombinants among the progeny of inversion heterozygotes.
Pericentric Inversion
(Figure 15-23)
1. Decreased recombinant freq.
2. Inversion loops.
3. Inverted arrangements of chromosomal landmarks.
(e.g.) Centromere position
Diagnostic features of inversions
Exchange of parts of non-homologous chromosomes.
Viable unless the breakpoint is in an essential gene.
A region from one chromosome is exchanged with a region from another nonhomologous chromosome so
that two translocation products are generated simultaneously (most common).
Reciprocal Translocation
(Figure 17-23)
1. Establishes new linkage groups.
(i.e.) gene is now on a different chromosome
2. May alter the size of chromosomes and centromere position.
Diagnostic features of translocations
Always in heterozygous state.
Human Translocations
Missing the tip of chromosome 5 due to translocation.
Cri du chat syndrome
Two normal chromosomes 21s, and an additional region of 21 due to translocation.
Results in high occurrence in family tree.
Down syndrome
Part of chromosome 22 is translocated to chromosome 9.
Often found in individuals with chronic myeloid leukemia.
Philadelphia Chromosome
When a gene is translocated to a region near heterochromatin of another chromosome.

In some cells the heterochromatin will engulf the gene shutting off expression leading to mutant phenotype.
Position Effect Variegation
(Figure 15-27)