Genetics
Ch.19 to 21
AP Biology
CH 19 Microbial Genetics (Bacteria and Viruses)
Why study bacterial genetics?
Its an easy place to start
history
we know more about it
systems better understood
simpler genome
good model for control of genes
build concepts from there to eukaryotes
bacterial genetic systems are exploited in biotechnology
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Bacteria
Bacteria review
one-celled organisms
prokaryotes
reproduce by mitosis
binary fission
rapid growth
generation every ~20 minutes
108 (100 million) colony overnight!
dominant form of life on Earth
incredibly diverse
Bacterial diversity
rods and spheres and spirals… Oh My!
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Bacterial diversity
Borrelia burgdorferi Lyme disease
Treponema pallidum Syphillis
Escherichia coli O157:H7 Hemorrhagic E. coli
Enterococcus faecium skin infections
Bacterial genome
Single circular chromosome
haploid
naked DNA
no histone proteins
~4 million base pairs
~4300 genes
1/1000 DNA in eukaryote
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No nucleus!
No nuclear membrane
chromosome in cytoplasm
transcription & translation are coupled together
no processing of mRNA
no introns
but Central Dogma still applies
use same genetic code
Binary fission
Replication of bacterial chromosome
Asexual reproduction
offspring genetically identical to parent
where does variation come from?
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Variation in bacteria
Sources of variation
spontaneous mutation
transformation
plasmids
DNA fragments
transduction
conjugation
transposons
bacteria shedding DNA
Spontaneous mutation
Spontaneous mutation is a
significant source of variation in rapidly reproducing species
Example: E. coli
human colon (large intestines)
2 x 1010 (billion) new E. coli each day!
spontaneous mutations
for 1 gene, only ~1 mutation in 10 million replications
each day, ~2,000 bacteria develop mutation in that gene
but consider all 4300 genes, then:
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Transformation
Bacteria are opportunists
pick up naked foreign DNA wherever it may be hanging out
have surface transport proteins that are specialized for the uptake of naked DNA
import bits of chromosomes from other bacteria
incorporate the DNA bits into their own chromosome
express new gene
form of recombination
Swapping DNA
Genetic recombination by trading DNA
1 3 2
arg+
trp-
arg- trp+
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Plasmids
Plasmids
small supplemental circles of DNA
5000 - 20,000 base pairs
self-replicating
carry extra genes
2-30 genes
can be exchanged between bacteria
bacterial sex!!
rapid evolution
antibiotic resistance
can be imported from environment
Plasmids This will be important!
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Plasmids & antibiotic resistance
Resistance is futile?
1st recognized in 1950s in Japan
bacterial dysentery not responding to antibiotics
worldwide problem now
resistant genes are on plasmids that are swapped between bacteria
Resistance in Bacteria video
Biotechnology
Used to insert new genes into bacteria
example: pUC18
engineered plasmid used in biotech
antibiotic resistance
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Transduction
Phage viruses carry
bacterial genes from one host to another
Conjugation
Direct transfer of DNA between 2 bacterial cells that are temporarily joined
results from presence of F plasmid with F factor
F for “fertility” DNA
E. coli “male” extends sex pilli, attaches to female bacterium
cytoplasmic bridge allows transfer of DNA
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CONJUGATION IN BACTERIA
CONJUGATION IN BACTERIA
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Viral Genetics
Ebola
Viral diseases
Measles Polio
Hepatitis
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Emerging viruses
Viruses that “jump” host
switch species
Ebola, SARS, bird flu,
hantavirus SARS
Ebola hantavirus
A sense of size
Comparing size
eukaryotic cell
bacterium
virus
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What is a virus? Is it alive?
DNA or RNA enclosed in a protein coat
Viruses are not cells
Extremely tiny
electron microscope size
smaller than ribosomes
~20–50 nm
1st discovered in plants (1800s)
tobacco mosaic virus
couldn’t filter out
couldn’t reproduce on media like bacteria
Variation in viruses
plant virus pink eye
Parasites
lack enzymes for metabolism
lack ribosomes for protein synthesis
need host
“machinery”
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Variation in viruses
bacteriophage influenza
A package of
genes in transit from one host cell to another
“A piece of bad news wrapped in protein”
– Peter Medawar
Viral genomes
Viral nucleic acids
DNA
double-stranded
single-stranded
RNA
double-stranded
single-stranded
Linear or circular
smallest viruses have only 4 genes, while largest have several hundred
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Viral protein coat
Capsid
crystal-like protein shell
1-2 types of proteins
many copies of same protein
Viral envelope
Lipid bilayer membranes cloaking viral capsid
envelopes are derived from host cell membrane
glycoproteins on surface
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Entry
virus DNA/RNA enters host cell
Assimilation
viral DNA/RNA takes over host
reprograms host cell to copy viral nucleic acid & build viral proteins
Self assembly
nucleic acid molecules &
capsomeres then self-
assemble into viral particles
exit cell
Generalized viral lifecycle
Symptoms of viral infection
Link between infection & symptoms varies
kills cells by lysis
cause infected cell to produce toxins
fever, aches, bleeding…
viral components may be toxic
envelope proteins
Damage?
depends…
lung epithelium after the flu is repaired
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Viral hosts
Host range
most types of virus can infect & parasitize only a limited range of host cells
identify host cells via “lock & key” fit
between proteins on viral coat &
receptors on host cell surface
broad host range
rabies = can infect all mammals
narrow host range
human cold virus = only cells lining upper respiratory tract of humans
HIV = binds only to specific white blood cells
Bacteriophages
Viruses that infect bacteria
ex. phages that infect E. coli
lambda phage
20-sided capsid head encloses DNA
protein tail attaches phage to host & injects phage DNA inside
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Bacteriophage lifecycles
Lytic
reproduce virus in bacteria
release virus by
rupturing bacterial host
Lysogenic
integrate viral DNA into bacterial DNA
reproduce with bacteria
Lytic lifecycle of phages
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Lysogenic lifecycle of phages
Defense against viruses
Bacteria have defenses against phages
bacterial mutants with receptors that are no longer recognized by a phage
natural selection favors these mutants
bacteria produce restriction enzymes
recognize & cut up foreign DNA
It’s an escalating war!
natural selection favors phage mutants resistant to bacterial defenses
This will be important!
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RNA viruses
Retroviruses
have to copy viral RNA into host DNA
enzyme = reverse transcriptase
RNA DNA mRNA
host’s RNA polymerase now transcribes viral DNA into viral mRNA
mRNA codes for viral components
host’s ribosomes produce new viral proteins
Why is this significant?
protein RNA
DNA
transcription translation
replication
Retroviruses
HIV
Human ImmunoDeficiency Virus
causes AIDS
Acquired ImmunoDeficiency Syndrome
opportunistic diseases
envelope with glycoproteins for binding to specific WBC
capsid containing 2 RNA strands & 2 copies of
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HIV infection
HIV enters host cell
macrophage & CD4 WBCs
cell-surface receptor
reverse transcriptase
synthesizes double stranded DNA from viral RNA
high mutation rate
Transcription produces more copies of viral RNA
translated into viral proteins
proteins & vRNA self-assemble into virus particles
released from cell by “budding”
or by lysis
HIV treatments
inhibit vRNA replication
AZT
thymine mimic
protease inhibitors
stops cleavage of polyprotein into capsid & enzyme proteins
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Potential HIV treatments
Block receptors
chemokines
bind to & block cell-surface receptors
11% of Caucasians have mutant receptor allele
Block vRNA replication
CAF replication factor
Cancer viruses
Viruses appear to cause certain human cancers
hepatitis B virus
linked to liver cancer
Epstein-Barr virus = infectious mono
linked to lymphoma
papilloma viruses
linked with cervical cancers
HTLV-1 retrovirus
linked to adult leukemia
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Cancer viruses
Transform cells into cancer cells after integration of viral DNA into host DNA
carry oncogenes that trigger cancerous characteristics in cells
version of human gene that normally controls cell cycle or cell growth
Most tumor viruses probably cause
cancer only in combination with other mutagenic events
Prions
Misfolded proteins
infectious
make plaques (clumps) &
holes in brain as neurons die
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Protein as information molecule?!
Prions challenge Central Dogma
transmit information to other proteins
Stanley Prusiner UC School of Medicine
1982 | 1997
proteinaceous infectious molecule
Pn
Pd
Figure 20.2a
Bacterium
Bacterial
chromosome
Plasmid
2
1 Gene inserted into
plasmid Cell containing
gene of interest
Recombinant DNA (plasmid)
Gene of interest
Plasmid put into bacterial cell
DNA of
chromosome (“foreign” DNA) Recombinant
bacterium
Gene Cloning in Bacteria
Figure 20.2b
Host cell grown in
culture to form a clone of cells containing the
“cloned” gene of interest Gene of
interest
Protein expressed from gene of interest
Protein harvested Copies of gene
Basic research and various applications
3
4
Basic research on protein Basic
research on gene
Gene for pest
resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hormone treats stunted growth
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 nucleic acids or proteins by size,
electrical charge, and other properties
• A current is applied that causes charged molecules to move through the gel
• Molecules are sorted into “bands” by their size
Figure 20.9
Mixture of DNA mol- ecules of different sizes
Power source
Power source Longer
molecules
Cathode Anode
Wells
Gel
Shorter molecules TECHNIQUE
RESULTS 1
2
Figure 20.9a
Mixture of DNA mol- ecules of different sizes
Power source
Power source Longer
molecules
Cathode Anode
Wells
Gel TECHNIQUE
2
1
Figure 20.9b
RESULTS
• In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis
• Restriction fragment analysis can be used to compare two different DNA molecules, such as two alleles for a gene, if the nucleotide difference alters a restriction site
Restriction Enzymes OR
Restriction Endonucleases
They are proteins produced by bacteria to
prevent or restrict invasion by foreign DNA by cutting the foreign DNA into pieces so that it cannot function.
Each different restriction enzyme(and there are hundreds, made by many different bacteria)
has its own type of site.
In general, a restriction site is a 4 or 6 base pair sequence that is a palindrome.
Restriction Enzymes OR
Restriction Endonucleases
A DNA palindrome is a sequence in which the “top” strand read from 5’to3’ is the
same as the “bottom” strand read from 5’to3’ For example:
5’ GAATTC 3’
3’ CTTAAG 5’
Palindromes!
RACECAR
Using Restriction Enzymes to Make Recombinant DNA
• Bacterial restriction enzymes cut DNA
molecules at specific DNA sequences called restriction sites
• A restriction enzyme usually makes many cuts, yielding restriction fragments
• The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends.”
Figure 20.3-3
Recombinant DNA molecule One possible combination DNA ligase
seals strands
DNA fragment added from another molecule cut by same enzyme.
Base pairing occurs.
Restriction enzyme cuts sugar-phosphate backbones.
Restriction site DNA
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
3 1
Sticky end
GAATTC CTTAAG
G
G
G
G AATT C AATT C
C TTAA C TTAA
Figure 20.10
Normal -globin allele
Sickle-cell mutant -globin allele
Large fragment
Normal allele
Sickle-cell allele
201 bp 175 bp
376 bp
(a) DdeI restriction sites in normal and sickle-cell alleles of the -globin gene
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles
201 bp 175 bp
376 bp
Large fragment
Large fragment
DdeI DdeI DdeI DdeI
DdeI DdeI DdeI
Figure 20.10a
Normal -globin allele
Sickle-cell mutant -globin allele
(a) DdeI restriction sites in normal and sickle-cell alleles of the -globin gene
201 bp 175 bp
376 bp
Large fragment
Large fragment
DdeI DdeI DdeI DdeI
DdeI DdeI DdeI
Figure 20.10b
Large
fragment
Normal allele
Sickle-cell allele
201 bp 175 bp
376 bp
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles
• A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization
• Specific DNA fragments can be identified by Southern blotting, using labeled probes that
hybridize to the DNA immobilized on a “blot” of gel
Figure 20.11
DNA restriction enzyme
2 3 1
4
TECHNIQUE
I Normal
-globin allele
II Sickle-cell allele
III Heterozygote Restriction
fragments Nitrocellulose
membrane (blot)
Heavy weight
Gel
Sponge Alkaline
solution Paper towels I II III
I II III I II III
Preparation of
restriction fragments
Gel electrophoresis DNA transfer (blotting)
Radioactively labeled probe for -globin gene
Nitrocellulose blot
Probe base-pairs with fragments
Fragment from sickle-cell
-globin allele Fragment from normal - globin allele
Film over blot
Hybridization with labeled probe 5 Probe detection
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
• The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase.
Figure 20.8
Genomic DNA
Target sequence
Denaturation
Annealing
Extension
Primers
New
nucleotides Cycle 1
yields 2 molecules
Cycle 2 yields
4 molecules
Cycle 3 yields 8 molecules;
2 molecules (in white boxes)
match target sequence
5
5
5
5
3
3
3
3
2
3 1 TECHNIQUE
Figure 20.8a
Genomic DNA
Target
sequence 5
5
3
3
TECHNIQUE
Denaturation
Annealing
Extension
Primers
New nucleo- tides Cycle 1
yields 2
molecules
5
5
3
3
2
3 1
Figure 20.8b
Figure 20.8c
Cycle 2 yields
4
molecules
Figure 20.8d
Cycle 3 yields 8 molecules;
2 molecules (in white boxes)
match target sequence
Forensic Evidence and Genetic Profiles
• An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids
• DNA testing can identify individuals with a high degree of certainty
• Genetic profiles can be analyzed using RFLP analysis by Southern blotting
• Variations in DNA sequence are called polymorphisms
• Sequence changes that alter restriction sites are called RFLPs (restriction fragment length
polymorphisms)
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• Even more sensitive is the use of genetic markers called short tandem repeats (STRs), which are variations in the number of repeats of specific
DNA sequences
• PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
• The probability that two people who are not identical twins have the same STR markers is exceptionally small
Figure 20.25
This photo shows
Washington just before his release in 2001,
after 17 years in prison.
(a)
Semen on victim Earl Washington
17,19 16,18
13,16 14,15
12,12 11,12 Source of
sample
STR marker 1
STR marker 2
STR marker 3
• A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse
transcription of all the mRNA produced by a particular cell
• A cDNA library represents only part of the
genome—only the subset of genes transcribed into mRNA in the original cells
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Figure 20.6-5
DNA in nucleus mRNAs in cytoplasm
mRNA
Reverse
transcriptase Poly-A tail
DNA strand
Primer
DNA
polymerase
5 5
5 5
5 5
3 3
3 3
3 3
A A A A A A
A A A A A A T T T T T
T T T T T
Cloning Animals: Nuclear Transplantation
• In nuclear transplantation, the nucleus of an
unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
• Experiments with frog embryos have shown that a transplanted nucleus can often support normal
development of the egg
• However, the older the donor nucleus, the lower the percentage of normally developing tadpoles
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Frog embryo Frog egg cell Frog tadpole UV
Less differ- entiated cell
Donor nucleus trans- planted
Enucleated egg cell
Fully differ- entiated
(intestinal) cell Donor
nucleus trans- planted Egg with donor nucleus
activated to begin development EXPERIMENT
RESULTS
Figure 20.18
Reproductive Cloning of Mammals
• In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by
nuclear transplantation from a differentiated mammary cell
• Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the
original transplanted nucleus
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Figure 20.19a
Mammary cell donor
1 2
3 TECHNIQUE
Cultured mammary cells
Egg
cell from ovary
Egg cell donor
Nucleus removed Cells fused
4
5
6
RESULTS
Grown in culture
Implanted in uterus of a third sheep
Embryonic development
Nucleus from mammary cell
Early embryo
Surrogate mother
Lamb (“Dolly”) genetically
identical to mammary cell donor
Figure 20.19b
Problems Associated with Animal Cloning
• In most nuclear transplantation studies, only a small percentage of cloned embryos have
developed normally to birth, and many cloned animals exhibit defects
• Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for
genes to be expressed or repressed appropriately for early stages of development
Stem Cells of Animals
• A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types
• Stem cells isolated from early embryos at the
blastocyst stage are called embryonic stem (ES) cells; these are able to differentiate into all cell types
• The adult body also has stem cells, which replace nonreproducing specialized cells
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Figure 20.21
Cultured stem cells
Different culture conditions
Different types of
Embryonic stem cells
Adult stem cells
Cells generating all embryonic cell types
Cells generating some cell types
Liver
cells Nerve cells
Blood cells
Genomes vary in size, no. of genes, & gene density
• Free-living bacteria and archaea have 1,500 to
7,500 genes and multicellular eukaryotes have up to at least 40,000 genes
• Number of genes is not correlated to genome size
• For example, it is estimated that the bacteria Escherichia Coli has 4.6 Mb genome size and 44,000 genes, while humans have 3000 Mb genome size and <21000 genes
• Vertebrate genomes can produce more than one
polypeptide per gene because of alternative splicing of RNA transcripts
Gene Density and Noncoding DNA
• Humans and other mammals have the lowest gene density, or number of genes, in a given length of DNA and prokaryotes have highest gene density
• Multicellular eukaryotes have many introns within genes and noncoding DNA between genes
• Sequencing of the human genome reveals that 98.5% does not code for proteins, rRNAs, or tRNAs
Archaea
Most are 16 Mb
Eukarya
Genome size
Number of genes
Gene density
Introns
Other
noncoding
DNA Very little
None in
protein-coding genes
Present in some genes Higher than in eukaryotes
1,5007,500 5,00040,000
Most are 104,000 Mb, but a few are much larger
Lower than in prokaryotes (Within eukaryotes, lower
density is correlated with larger genomes.)
Unicellular eukaryotes:
present, but prevalent only in some species
Multicellular eukaryotes:
present in most genes
Can be large amounts;
generally more repetitive noncoding DNA in
multicellular eukaryotes Bacteria
Figure 21.UN01
Table 21.1
Evo-devo is field of biology that compares developmental process to understand how they may have evolved and
how changes can modify existing organismal features or lead to new ones.
• Duplication, rearrangement, and mutation of DNA contribute to genome evolution
• The size of genomes has increased over
evolutionary time, with the extra genetic material providing raw material for gene diversification
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Alterations of Chromosome Structure
• Humans have 23 pairs of chromosomes, while chimpanzees have 24 pairs
• Following the divergence of humans and
chimpanzees from a common ancestor, two
ancestral chromosomes fused in the human line
• Duplications and inversions result from mistakes during meiotic recombination
• Comparative analysis between chromosomes of humans and seven mammalian species paints a hypothetical chromosomal evolutionary history
Figure 21.12a
Human
chromosome 2 Telomere
sequences
Centromere sequences
Chimpanzee chromosomes
12 Telomere-like
sequences
Centromere-like sequences
13 (a) Human and chimpanzee chromosomes
Interspersed repetitive DNA
Repetitive DNA is spread throughout genome
in primates, at least 5% of genome is made of a family of similar sequences called, Alu elements
300 bases long
Alu is an example of a "jumping gene" –
a transposon DNA sequence that "reproduces" by copying itself & inserting into new chromosome locations
Transposable Elements and Related Sequences
• The first evidence for mobile 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
• These transposable elements move from one site to another in a cell’s DNA; they are present in both prokaryotes and eukaryotes
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Movement of Transposons and Retrotransposons
• Eukaryotic transposable elements are of two types
– Transposons, which move by means of a DNA intermediate
– Retrotransposons, which move by means of an RNA intermediate
Figure 21.9
Transposon
Transposon is copied DNA of
genome
Mobile transposon
Insertion New copy of
transposon
Figure 21.10
Retrotransposon
New copy of retrotransposon
Insertion Reverse
transcriptase
RNA Formation of a single-stranded RNA intermediate
How Transposable Elements Contribute to Genome Evolution
• Multiple copies of similar transposable elements may facilitate recombination, or crossing over, between different chromosomes
• Insertion of transposable elements within a protein-coding sequence may block protein production
• Insertion of transposable elements within a
regulatory sequence may increase or decrease protein production
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• Transposable elements may carry a gene or groups of genes to a new position
• Transposable elements may also create new
sites for alternative splicing in an RNA transcript
• In all cases, changes are usually detrimental but may on occasion prove advantageous to an
organism
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Genetic disorders of repeats
Fragile X syndrome
most common form of
inherited mental retardation
defect in X chromosome
mutation of FMR1 gene causing many
repeats of CGG triplet in promoter region
200+ copies
The more repeats the worse the effects
normal = 6-40 CGG repeats
FMR1 gene not expressed &
protein (FMRP) not produced
function of FMR1 protein unknown
binds RNA