Download Lectures prepared by Christine L. Case Chapter 8 Microbial Genetics

Chapter 8
Microbial Genetics
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Lectures prepared by Christine L. Case
Lectures prepared by Christine L. Case
Structure and Function of the Genetic Material
 Genetics (遺傳學): The study of
 what genes are
 how they carry information
 how information is expressed
 how genes are replicated
 Gene: A segment of DNA that encodes a functional
product, usually a protein.
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 Genome (基因組):
 The genetic information in a cell
 A cell’s genome includes its chromosome and plasmid
 Chromosome (染色體):
 Structures containing DNA that physically carry
hereditary information
 The chromosomes contain the genes
 Gene (基因): The segment of DNA that code for
functional product
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Genotype and phenotype
 Genomics (基因體學): The molecular study of
genomes
 Genotype (基因型): The genes of an organism
 Phenotype (外表型): Expression of the genes
 In microbes, most proteins are either enzymatic or
structural.
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DNA and Chromosomes
 DNA in cells exists
 as a double-stranded helix
 associated with various proteins that regulate genetic
activity
 the two strands are held together by hydrogen bonds
between specific nitrogenous base pairs (complementary) :
A=T
C = G.
 bacterial DNA is circular
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 Chromosome of E. coli
 contains about 4.6 million base
pairs
 1 mm
 takes up only about 10% of the
cell’s volume because the DNA
is supercoiled
Disrupted E. coli cell
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Figure 8.1a
 The location of genes on
a bacterial chromosome
was determined by
experiments on the
transfer of genes from
one cell to another.
 The bacterial map is
marked in minutes.
KEY
Amino acid metabolism
Carbohydrate metabolism
DNA replication and repair
Membrane synthesis
Lipid metabolism
Genetic map of the chromosome of E. coli
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Figure 8.1b
 The entire genome does not consist of back-to-back
genes.
 Noncoding regions called short tandem repeats
(STRs).
 Open-reading frame (ORF, 開放譯讀區): region of
DNA that are likely to encode a protein.
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Flow of Genetic Information
Parent cell
DNA
重組
基因表現
expression
recombination
Genetic information is used within a cell to
produce the proteins needed for the cell to
function.
Genetic information can be
Insert
Fig 8.2
transferred between cells
of the same generation.
複製
replication
Genetic information can
be transferred between
generations of cells.
New combinations
of genes
Transcription
轉錄
Translation
轉譯
Cell metabolizes and grows
Recombinant cell
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Daughter cells
Figure 8.2
Transcription
 genetic information in DNA is
copied into a complementary
base sequence of RNA
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Figure 8.2
Translation
 mRNA is translated in
codons (three nucleotides)
 Translation of mRNA begins
at the start codon: AUG
 Translation ends at a stop
codon: UAA, UAG, UGA
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Figure 8.2
DNA replication
DNA
 Polymer of nucleotides: Adenine
(腺嘌呤), thymine (胸腺嘧啶),
cytosine (胞嘧啶), and guanine
(鳥糞嘌呤)
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Figure 8.3b
 Double helix associated with
proteins
 "Backbone" is deoxyribosephosphate
 Strands are held together by
hydrogen bonds between
AT and CG.
 Strands are antiparallel.
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Figure 8.3a
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Figure 8.4
 DNA is copied by DNA polymerase
 In the 5'  3' direction
 Initiated by an RNA primer
 Leading strand is synthesized continuously
 Lagging strand is synthesized discontinuously
 Okazaki fragments
 RNA primers are removed and Okazaki fragments
joined by a DNA polymerase and DNA ligase
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REPLICATION
Proteins stabilize the
unwound parental DNA.
The leading strand is
synthesized continuously
by DNA polymerase.
3'
DNA polymerase
5'
Enzymes
unwind the
1
parental double
helix.
Replication
fork
RNA primer
Insert Fig 8.5
Primase
5'
DNA polymerase
3'
Parental
strand
Okazaki fragment
DNA
polymerase
The lagging strand is
synthesized discontinuously.
Primase, an RNA polymerase,
synthesizes a short RNA
primer, which is then extended by
DNA polymerase.
DNA polymerase
digests RNA primer
and replaces it with DNA.
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DNA ligase
3'
5'
DNA ligase joins
the discontinuous
fragments of the
lagging strand.
Figure 8.5
Important Enzymes
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 DNA replication is semiconservative (半保留).
Replication forks
REPLICATION
An E. coli1chromosome in the process of replicating
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Figure 8.6a
An E. coli 1chromosome in the process of replicating
Origin of replication
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Figure 8.6b
The Meselson and
Stahl experiment:
evidence
demonstrating
semiconservative
replication
Semiconservative replication
 Because each double-stranded DNA molecule contains
one original and one new strand, the replication process
is called semiconservative replication
 DNA polymerase proofreads new molecules of DNA and
removes mismatched bases before continuing DNA
synthesis.
 Errors only occur ~1 time for every 1010 bases added.
 Each daughter bacterium receives a chromosome
identical to the parent's.
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RNA and protein synthesis
Transcription
 DNA is transcribed to make RNA (mRNA, tRNA, and rRNA).
 Transcription begins when RNA polymerase binds to the
promoter (啟動子) sequence
 Transcription proceeds in the 5'  3' direction
 Transcription stops when it reaches the terminator
sequence
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Figure 8.7 (1 of 2)
啟動子
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Figure 8.7 (2 of 2)
Translation
 Degeneracy (退化性)
 Sense codons and
nonsense codons
 Anticodon
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Figure 8.8
Translation steps:
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Figure 8.9, step 1-2
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Figure 8.9, step 3-4
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Figure 8.9, step 5-6
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Figure 8.9, step 7-8
RNA polymerase
Met
 In prokaryotic cells, the translation of mRNA into
protein can begin even before transcription is complete.
Met
RNA
Ribosome
Peptide
1
Insert Fig 8.10
Met
Met
Met
Met
Direction of transcription
RNA
polymerase
DNA
5′
Peptide
Polyribosome
Ribosome
Direction of translation
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mRNA
Figure 8.10
RNA processing in Eukaryotes
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Figure 8.11
The Regulation of Bacterial Gene Expression
 Constitutive enzymes, perhaps 60-80%, are expressed
at a fixed rate.
 Other enzymes are expressed only as needed.
Repressible enzymes
Inducible enzymes
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Pre-transcriptional control
Two genetic control mechanisms regulate the transcription
of mRNA:
 Repression (抑制作用)
 To inhibit gene expression and decrease the
synthesis of enzymes
抑制物
 To be mediated by regulatory proteins, repressors
 To block the ability of RNA polymerase to initiate
transcription
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 Induction (誘導作用)
 To turn on the transcription of gene or genes
 Inducer, to act to induce transcription
 Inducible enzyme: enzymes that are synthesized in
the presence of inducers (enzyme induction, βgalactosidase)
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The operon model of gene expression
Operon: a unit of prokarytoic gene expression and
regulation which typically includes:
1.
Structural genes for enzymes in a specific biosynthetic
pathway whose expression is coordinately controlled.
2.
Control elements, such as operator and promoter.
3.
Regulator gene(s) whose products recognize the control
elements. Regulator gene(s) sometimes are encoded by
the gene under the control of a different promoter.
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Operon
lac operon
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trp operon
Figure 8.12, 8.13
An inducible operon
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Figure 8.12, step 2-3
A repressible operon
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Figure 8.13, step 2-3
Positive regulation
Bacteria growing on glucose as the
sole carbon source grow faster
than on lactose.
Figure 8.14
Bacteria growing in a medium containing glucose
and lactose first consume the glucose and then,
after a short lag time, the lactose. During the lag
time, intracellular cAMP increases, the lac operon
is transcribed, more lactose is transported into the
cell, and β-galactosidase is synthesized to break
down lactose.
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CAP: catabolic activator protein
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cyclic AMP (cAMP)
Figure 8.15
Epigenetic control (表觀調控)
 Eukaryotic and bacterial cells can turn genes
off by methylating certain nucleotides.
 can pass to offspring
 isn’t permanent
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Post-transcriptional control
 Some regulatory mechanisms stop protein synthesis
after transcription has
occurred:
DNA
Transcription of
miRNA occurs.
miRNA
miRNA binds to target
mRNA that has at least six
complementary bases.
 Ex: microRNAs (miRNAs)
mRNA
mRNA is degraded.
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Figure 8.16
Mutation: Change in the Genetic material
 A change in the genetic material
 Mutations may be neutral, beneficial, or harmful.
 Mutagen (致突變原): Agent that causes mutations
 Spontaneous mutations (自發性突變): Occur in the
absence of a mutagen
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Types of mutations
 Base substitution
(point mutation)
 Silent mutation
 Missense
mutation
 Nonsense
mutation
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Figure 8.17
 Base substitution
Silent mutation
 Result in no change in amino acid
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Figure 8.8
 Base substitution
Missense mutation
 Result in change in amino acid
Figure 8.18a–b
 Ex: Sickle-cell disease – A single missense mutation, a
change from an A to a T at a specific site, results in the
change from glutamic acid to valine in the protein of
hemoglobin.
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 Base substitution
Nonsense mutation
 Results in a nonsense codon
Figure 8.18a, c
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 Frameshift mutation
 Insertion or deletion of one or more nucleotide pairs
Figure 8.18a, d
 Huntington’s disease – A progressive neurological
disorder caused by extra bases inserted into a particular
gene.
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 Spontaneous mutations occur without the presence
of a mutagen. The spontaneous mutation rate
varies from genome to genome.
 Copying errors in the genetic material during cell
division
 Induced mutations occur as a result of applying
mutagens.
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Mutagens
 Mutagens (致突變原) are agent in environment that
brings about DNA mutation. Usually chemically or
physically interact with DNA to cause change. Once
mistake is fixed into the DNA the change is
permanent.
 Chemical mutagens
 Radiation
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Chemical mutagens
 Nitrous acid: converts A so it pairs with C instead of T
亞硝酸
Adenosine
Figure 8.19
thymine or uracil
Adenosine
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cytosine
Figure 8.19
 Nucleoside analogs: have chemical structure similar to
a base but do not base pair correctly
 2-aminopurine and 5-bromouracil incorporated into
DNA in place of A and T but base pairs with C
 Advantage: antiviral and antitumor drugs. Ex: AZT for
HIV
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Figure 8.20
苯芘
 Benzpyrene (cigarette smoke): causes frameshift
mutations
 binds between bases and offsets the double helix
strands
 Another ex: aflatoxin, produced by Aspergillus flavus
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Radiation
 x-rays and -rays: create ions and free radicals that
break molecular bonds
 UV causes crosslinking of T bases (Thymine dimer)
which can prevent unwinding for replication or
transcription. Cells have light repair enzymes called
photolyases which cut out damaged Ts and replace
them.
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Nucleotide excision repair
= enzymes that can cut out
and replace DNA damage
Figure 8.21
 Nucleotide excision repairs mutations
 damaged parts are removed leaving gap in strand
 gap is filled by complementary base pairing from other
strand
 often repair restores correct sequence
 sometimes errors are made during repair: once
nucleotide excision repair mechanisms seal the DNA,
mutation is permanent
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Damage on one strand
Damage on both strands
ATGCTAGGCTATTATCG
TACGATCCGATAATAGC
ATGCTAGGCTATTATCG
TACGATCCGATAATAGC
ATGCT GCTATTATCG
TACGAT GATAATAGC
ATGCTA?GCTATTATCG
TACGAT?CGATAATAGC
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The frequency of mutation
 Mutation rate: probability that gene will mutate when
cell divides
 Spontaneous mutation rate = 1 in 109 replicated base
pairs or 1 in 106 replicated genes
 If harmful, organism dies. If beneficial, organism
thrives and passes mutation to offspring (drives
adaptation and evolution).
 Mutagens increase to 10–5 or 10–3 per replicated gene.
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Identifying mutation
 Mutants can be detected by selecting or testing for an
altered phenotype
 Positive (direct) selection detects mutant cells because
they grow or appear different. Ex: penicillin resistant
 Negative (indirect) selection detects mutant cells
because they do not grow.
 Technique: replica plating
 very effective
 the mutant is known as an auxotroph (營養需求突變株)
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Replica Plating (複製平板法)
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Figure 8.22
Identifying chemical carcinogens
 Carcinogen (致癌物): substances that cause cancer in
animal.
 The Ames test (安氏試驗) is a relatively inexpensive and
rapid test for identifying possible chemical carcinogens.
 The test assumes that a mutant cell can revert to a normal cell
in the presence of mutagen, and that many mutagens are
carcinogens.
 Histidine auxotrophs (組蛋白營養需求突變株) of Samonella
are exposed to an enzymatically treated potential carcinogen,
and reversions to the nonmutant state are selected.
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The Ames Test for Chemical Carcinogens
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Figure 8.22
Genetic Transfer and Recombination
 Genetic recombination
 refer to the exchange of
genes between two
DNA molecules to form
new combinations of
genes on chromosome
 involves crossing over
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Figure 8.24
 Genetic recombination contributes to population
diversity: recombination more likely than mutation to
provide beneficial change since it tends not to destroy
gene function.
 Eukaryotes: recombination during meiosis for sexual
reproduction.
 creates diversity in offspring but parent remains
unchanged
 vertical gene transfer = genes passed from organism to
offspring
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 Prokaryotes: recombination via gene transfer between
cells or within cell by transformation (轉型), conjugation
(接合), or transduction (轉導)
 original cell is altered
 horizontal gene transfer = genes passed to neighboring
microbes of same generation
 transfer involves donor cell that gives portion of DNA to
recipient cell
 when donor DNA incorporated into recipient, recipient now
called recombinant cell
 if recombinant cell acquired new function/characteristic as
result of new DNA, cell has been transformed
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 Generation of recombinant cells is very low frequency
event (less than 1%): very few cells in population are
capable of exchanging and incorporating DNA.
 Three methods of prokaryotic gene transfer:
 Transformation (轉型)
 Conjugation (接合)
 Transduction (轉導)
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Transformation in bacteria
 Genes are transferred as “naked” DNA in solution.
 It can occur between unrelated genus/species.
 It was discovered by F. Griffith 1928 who studied
Streptococcus pneumoniae
 virulent strain had capsule
 non-virulent stain did not
 in mouse, dead virulent strain could pass “virulence
factor” to live nonvirulent strain
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Griffith’s experiment
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Figure 8.25
 Competent cells (勝任細胞) can pick up DNA from dead
cells and incorporate it into genome by recombination
(e.g. antibiotic resistance).
 Transformed cell than passes genetic recombination to
progeny.
 Transformation works best when donor and recipient
are related but they do not have to be.
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 competent =
permeable to DNA:
alterations in cell wall
that allow large
molecule like DNA to
get through (in lab we
use chemical agents
to poke holes)
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Figure 8.26
Conjugation in bacteria
 Genes are transferred between two live cells via sex
pilus (Gram -) or surface adhesion molecules (Gram +).
 Transfer is mediated by a plasmid: small circle of DNA
separate from genome that is self replicating but
contains no essential genes.
 Plasmid has genes for its own transfer.
 Gram negative plasmids have genes for pilus. Gram
positive plasmids have genes for surface adhesion
molecules.
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 Conjugation requires cell to cell contact between two
cells of opposite mating type, usually the same
species, must be same genus.
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Figure 8.27
 During conjugation plasmid is replicated and single
stranded copy is transferred to recipient. Recipient
synthesizes complementary strand to complete plasmid.
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Figure 8.28a
 Plasmid can remain as separate circle or plasmid can
be integrated into host cell genome resulting in
permanent chromosomal changes.
高頻重
組細胞
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Figure 8.28b
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Figure 8.28c
Transduction in bacteria
 DNA from a donor is carried by a virus to a recipient
cell.
 Bacteriophage (Phage) is a kind of virus that infects
bacterial cells
 Each phage is species specific (donor and recipient
are the same species).
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 Transduction mechanism:
1. Phage attaches to donor cell and injects phage DNA.
2. Phage DNA directs donor cell to synthesize phage
proteins and DNA, phage enzymes digest the bacterial
chromosome.
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Figure 8.29, step 1-2
3. New phages are assembled: phage DNA is
packaged into capsids. Occasionally bacterial DNA
is packaged by mistake.
4. Capsid containing bacterial DNA “infects” new host
recipient cell by injecting the DNA.
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Figure 8.29, step 3-4
5. Donor DNA does not direct viral replication (not
viral DNA): instead integrates into recipient
genome.
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Figure 8.29, step 5
Plasmids and Transposons
Plasmids
 Plasmids are self-replicating circle of DNA containing
“extra” genes.
1. Conjugative plasmid: used in bacterial conjugation.
Carries genes for sex pili and transfer of the plasmid.
2. Dissimilation plasmids (異化質體): Encode enzymes for
catabolism of unusual compounds.
3. Pathogenicity plasmids: carry genes that code for
virulence traits. Ex: capsules, toxins, adhesion
molecules, bacteriocins.
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4. Resistance factor (R factor): carry genes for resistance
to antibiotic and toxins. R factor has two parts:
 Resistance transfer factor (RTF)
 R-determinant
抗汞
抗磺胺劑
抗鏈黴素
抗
性
移
轉
因
子
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r決
定
體
四環素
抗氯黴素
Figure 8.30
 Plasmids can be transferred between species:
 allows spread of antibiotic resistance between different
pathogens
 wide use of antibiotics has put selective pressure on
microbes to “develop” and “share” resistance genes
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Transposons (轉位子)
 Transposons are small segments of DNA that can
move independently from one region of DNA to another.
 discovered 1950s by McClintock: mosaic pattern in
indian corn (Nobel Prize 1983)
 transposons pop out and randomly insert at rate of 10-5
to 10-7 per generation
 integration is random: can disrupt genes
 at minimum transposons carry genetic info to carry out
own transposition, may also carry other genes
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 Simplest transposon = insertion sequence (IS)
 gene for transposase (轉位酶) (enzyme that cuts DNA
at recognition sites and religates it elsewhere in
genome)
 two recognition sites called inverted repeats, mark
ends of transposon, recognized by transposase
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Figure 8.31a
 Complex transposons have inverted repeats outside
other genes.
 Genes will get carried with transposon when it moves.
 Depending on where it inserts and what genes it
carries, it can mediate good or bad genetic changes.
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Figure 8.31b
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Figure 8.31c