Download Genome Control - University of California, Los Angeles

Genome Control
Bacterial chromosome
•
•
•
•
•
•
1 circular chromosome
Virus < bacteria < eukaryotes (# of bases)
Nucleiod region
Replication proceeds in both directions
Rapid reproduction
Single cell to colony (10^7 to 10^8 bacteria) within 12
hours
• Plasmids
• Variation in bacteria?
Transduction: Phage carries genes from one bacterium to another
Defective phage
Conjugation: And you thought
bacteria couldn’t have sex?
F factor
Exist as part of chromosome (episome) or as a plasmid (contains mostly
genes for making sex pili
F+  F+ and F+
F-  F- and FDuring conjugation
F+  F+
F-  F+
Hfr – F factor on
chromosome
Genes transferred
depends on initiation
and termination
R plasmids
• Function of natural
antibiotics?
• Advantage of
antibiotic resistant
genes
• Consequence of
prescribing
antibiotics?
Transposable elements
Recombination within a genome
(between chromosome and plasmid
or between plasmids
• Insertion sequences
– Only in bacteria
– Transposase recognizes
the inverted repeats
– May disrupt coding
sequence or regulatory
genes
• Transposons
– More complex (extra genes)
– Can add antibiotic resistance
genes to plasmi already carrying
genes. How would this spread?
– Found in eukaryotic genomes as
well
Transformation
• Uptake of exogenous DNA, resulting in newly
acquired traits
• Must be in competency (state able to take in DNA
facilitated by membrane bound proteins
• Competency is induced in E. Coli with CaCl2, MgCl2,
or RbCl, and sudden changes of heat and cold
(makes cell membrane permeable)
• How do the Griffith and Avery experiments
relate to transformation?
Transformation
efficiency
• Amount of cells transformed/μg DNA
• Through selection, colonies are
counted
• Each colony grew from a transformed
cell
pUC8 Plasmid
• Present in E. Coli
• Replicates independent of bacterial
chromosome
• Genetically engineered
– Lac Z gene (β galactosidase)
– MCR (can facilitate insertion of DNA
(not required for replication)
pUC8 plasmid
• With Inducers (IPTG and X-Gal)
colonies of bacteria appear blue
– Lac Z  β galactosidase  X-Gal
cleaved  blue product
• With inserted gene, lac Z is
interrupted and colonies appear white
– No Lac Z  No β galactosidase  X-Gal
not cleaved  white product
pUC8 plasmid
• Engineered E. Coli cells only
synthesizes carboyxl terminal of β
galactosidase protein
• pUC8 plasmid contains gene for amino
terminal
• If pUC8 transforms cells, gene is
fully functional
Further selection
• E. Coli NOT resistant to antibiotic
ampicillin
• pUC8 contains ampicillin resistant gene
• The enzyme B-lactamase exits cell and
inactivates ampicillin
• Satellite colonies can appear around blue
colonies (which color would they be , why?)
Overview of
Genome Control
• Gene expression must
respond to external cues
• Differentiation allows
cells to become
specialized
• Expression usually
regulated at transcription
level by DNA-binding
proteins
Genome packing (1st level)
• Prokaryotic
– Coiled and looped
with protein
(chromatin)
• Eukaryotic
– More complex
– Highly condensed
and coiled during
metaphase around
histones (4 + H1)
– Histones leave only
during DNA
replication (what
about
transcription?)
– Heterochromatin
vs. euchromatin
- Charged
phosphates
+ charged a.a.
Non histones
Chromatin modification
• Chromatin function
– Compact DNA
– Control transcription
• Euchromatin vs. heterochromatin
• Gene’s location relative to nucleosome and scaffold
• DNA methylation – inactivates genes
• Histone acetylation – increase gene
expression by changing conformation of
histones
Distal
Control elements
(noncoding DNA)
Proximal
Recognizes TATA box
-Transcription will
occur, but
inefficiently without
activators
-Repressors can bind
to silencers to
repress transcription
(methylation)
Recognizes
proteins,
including
RNA
Polymerase
Transcription factors
• DNA-binding domain (helix-turnhelix, zinc finger, leucine zipper)
• Protein binding domain
Coordinately controlled
genes
• Similar control elements before each
gene allows simultaneous gene
expression (analogous to operons)
• steroid hormones and growth factors
can act as signals to control
expression
Posttranscriptional control
• Alternative splicing
• mRNA degradation (hemoglobin example)
– Poly(A) tail shortened  5’ cap removed  mRNA
degraded (enzymatic action)
– mRNA stability controlled by part of sequence near 3’
end
• Translational control - Preventing mRNA attachment to
ribosome
(could help
store mRNA for
later use)
Post translational control
• Protein modification
– Cleavage
– Chemical modification
– Transportation
• Posttranslational degradation
Relevant tutorials
• Campbell site – 19D, 19E, 19F
• http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapt
er18/animations.html#
– Transcription Complex and Enhancers (586.0K)
– Control of Gene Expression in Eukaryotes
(959.0K)
Cancer caused by genetic
changes
All result in oncogenes
“cancer genes”
Agents of cancer – Spontaneous mutation,
chemical carcinogens, radiation, viruses
Signaling pathway that
regulates cell growth
oncogene
Mutated
tumor
suppressor
Multiple mutations
leading to cancer
Monday – drip Oxaliplatin
Tuesday - 1/2 hour drip 5-fluorouracil
Take home and use leucovorin for 22 hours
Organization at the DNA
level
• Prokaryotic
– DNA codes for
protein, tRNA, or
rRNA
– Noncoding regions:
Regulatory
sequences
– Coding sequence
along a gene is
continuous
• Eukaryotic
– Only 3% coding
region
– Noncoding regions:
Regulatory regions,
repeated
sequences, introns
– Multigene families
Repetitive DNA
- Different nucleotide
composition, making the
density distinct (appears
as a satellite band)
- Fragile X, Huntington’s
- Near centromeres,
telomeres
Alu elements
-5% of genome
-300 pairs long
-Transcribed into RNA
- Unknown function
Gene Families
-Almost like
extensive
repetitive DNA
-Some consist
of identical
genes, tandemly
clustered
-Most code for
RNA
-lack
regulatory
sequences
-Higher affinity for oxygen
-Transcription separated temporally
Gene amplification
• Higher rate of
transcription
during early
development
Genome rearrangement
•
•
•
•
•
Transposons - Stretches
of DNA that can move
within a genome
Can interrupt gene
expression
Can carry a gene that
becomes expressed
when inserted next to a
promoter
Retrotransposon – needs
RNA intermediate (Alu
elements)
50% of maize, 10% of
humans
Relevant tutorials
• Campbell site – 19A, 19B
Problems cloning DNA
• Getting cloned
eukaryotic gene
into a prokaryotic
setting
• Introns
• Eukaryotic versus
prokaryotic
genome control
• Engineer cloning
vector to include
prokaryotic
promoters
• Extract mature
mRNA and make
cDNA
• Use yeast cells,
artificial
chromosomes
VNTRs (type of DNA
polymorphism)
http://www.lsic.ucla.edu/ls3/tutorials/gene_cloning.html
PCR of VNTRs
• Extract DNA from sample
• Lyse cell membranes to release
DNA
• Add nucleotides, primers, Taq
polymerase to DNA sample
• Thermocycler amplifies DNA
• Gel electrophoresis of amplified
DNA
Sample results
ladder
More repeats
homozygous
heterozygous
• Will this be enough evidence to convict
someone matching the DNA sample?
Mapping a genome
• Linkage mapping
– Based on
recombinant
frequencies
• Physical mapping
(chromosome
walking)
• DNA sequencing
Comparison of methods
Relevant tutorials
• Campbell site – 19G, 20F
• http://highered.mcgrawhill.com/sites/0072437316/student_view0
/chapter20/animations.html
– How Tumor Suppressor Genes Block Cell
Division (694.0K)
Group presentations
• 1 – Genomic and cDNA libraries (367368)
• 2 – Gel Electrophoresis and RFLPs
(372-374)
• 3 – Southern Blotting (372, 373, 375)
• 4 – Sanger Method (378)
• 5 – DNA microarray assays and in vitro
mutagenesis (379)
Relevant tutorials
• Campbell site: 20B, 20D, 20F
• http://highered.mcgrawhill.com/sites/0072437316/student_
view0/chapter16/animations.html
Genomic and cDNA
libraries
Gel Electrophoresis and RFLPs
Southern Blottting
Sanger Method
DNA microarray assays
and in vitro mutagenises