Download Lecture 9

Whole Genome of Ancient Human is Decoded
Gene Expression
new frontiers
…the processes by which information contained in genes and
genomes is decoded by cells, in order to produce
molecules that determine the phenotypes observed in
organisms,
– transcription is controlled so that the correct DNA sequences are
expressed as mRNA in the right cells, at the right time, and in the
right amount.
- and, now we’re learning - processing and translation of mRNA is further controlled (through
RNA/Protein complexes), via ancient, conserved processes.
Central Dogma
addendum
DNA
transcription
RNA
Alt. Splicing
Alt. Poly-A,
Alt. Translatin Start
translation
Protein
Transcription
Factors
Transcriptional Network
(cell cycle)
(example)
Science. 2002 Oct 25;298(5594):799-804.
Central Dogma
addenda
DNA
TGS: Transcriptional
Gene Silencing
TGS
transcription
RNA
PTGS
Transcription
Factors
translation
Protein
PTGS: Post Transcriptional
Gene Silencing
tiny RNAs
(20-to-25 nt RNAs)
• in eukaryotic cells, tiny RNAs function as transcriptional
regulators of gene expression in (at least) three distinct
pathways,
– small interfering RNAs (siRNAs) direct RNA
destruction via the RNA interference (RNAi) pathway,
• and transcriptional regulation via epigenetic processes,
– micro (miRNAs) regulate RNA translation.
Ancient History (1)
Cell 75, 843 (1993)
Some development timing genes code for short anti-sense
molecules, …appeared to be unique to C. elegans.
How would a cell express this?
miRNA
micro-RNA
• Post-transcriptional regulatory
“genes”,
– contain ~22 nucleotides (processed),
– are cleaved from somewhat larger
double stranded RNA (dsRNA)
precursors - by a protein complex
called Dicer;
– are expressed in certain cell types
and at certain times during
differentiation (also called short
temporal (stRNA).
miRNA
Anti-Sense Blocking of Translation
“Anti-Sense”
“Sense Strand”
Why use RNA to block mRNA function?
miRNAs
• Conserved amongst
eukaryotic cells,
– Often associated with
hetrochronic genes,
– difficult to identify in
genomic sequences because
they don’t have long ORFs,
How might you locate them?
Ancient History (II)
(co-suppression)
Transgene expression often decreases as the copy number of transgenes
increased.
Over Expression Studies
• Make a gene construct with,
– Structural Gene,
– Active promoter (often from a virus promoter),
– Marker gene to be able to determine transformation.
Marker Gene (w/P)
Active promoter
Gene of Interest
• Expect,
– Higher levels of protein,
– Gene-dosage phenotypes,
– Glorious publication.
Frequent Results: no protein produced, scorn from senior scientists.
Anti-Sense Studies
• Another good idea: use a transgene with the coding
sequence reversed...
Native promoter
5’
Gene of Interest
mRNA
Marker Gene
3’
Active promoter
5’
tseretnI fo eneG
anti-sense RNA
Duplex RNA formation.
3’
Expected Results
• Low, to no detectable single stranded transcript,
• Low, to no protein products,
• Glorious publication detailing gene function.
Actual Results (Wacky)
• Phenotypes ranged from death to over-expression,
• Transcript levels were also extremely variable,
• Scorn from senior scientists.
Co-suppression Modes
...Transcriptional Gene Silencing
(TGS),
– RNA functions in the
methylation of promoters and
structural elements of genes,
...Post-Transcritional Gene
Silencing (PTGS),
– involves the specific
degradation of mRNA via a
double-stranded RNA
intermediate, dsRNA.
RNAi
RNA interference
...while attempting to do anti-sense KO of gene expression
in C. elegans, Guo and Kemphues, Cell 81, 611 (1995)
observed that sense and anti-sense strands worked equally,
– in an anti-sense experiment, a gene is constructed so that it
produces a complementary strand to an expressed transcript,
• the goal is to complement, thus inactivate the mRNA.
...following up, other researchers found that dsRNA worked
at least an order of magnitude better that either sense or
anti-sense strands.
RNAi
...siRNA control of gene expression by RNA processing is
now considered a common element in eukaryotic cells,
– defense against viruses,
– control of transposable elements,
– adapted to regulate gene expression?
…stolen for doing Reverse Genetic studies,
– dsRNA triggers sequence specific degradation of complementary
mRNAs.
delivery
amplification
Today
Nature 408: 331 - 336
Ce III
2315 Genes
http://www.wormbase.org
Functional Genomics
The Question(s)
Can we establish a high throughput system to assign
cellular function to genes identified in metazoans?
- using cell division and associated processes as the
scorable phenotype,
In the process, can we learn about…
–
–
–
–
cell division genes,
embryology,
general development,
anything else?
Differential Interference Contrast Microscopy
Nomarski Optics
DIC Microscopy
Nomarski Optics
Reverse Genetics
Knockomics, Knockology...
Sequence to Phenotype to Function
Forward vs. Reverse Genetics
• Treat thousands of organisms with a mutagen,
– random mutagenesis,
• Identify an individual with a phenotype of interest,
• Identify the gene.
Forward
• Treat thousands of organisms with a mutagen (usually),
– random mutagenesis, or other gene expression block,
• Identify individual(s) with a genotype of interest,
• Identify the phenotype.
Reverse
Reverse Genetics
Functional Genomics
Gene DNA
Sequence
Gene Disruption
Genetically Link
Phenotype
Analysis
Function
Development
Physiology
Cell Biology
New Data, New Technology
new paradigms
• The C. elegans genome is sequenced, and we can identify
2315 candidate sequences on Chromosome III.
• We can see cell division through a microscope, and further,
we are able to identify many abnormalities.
• We have RNAi technology at hand to selectively “knock
down” any gene we are interested in.
Further, RNAi can be added to cells prior to fertilization,
mitosis commences after fertilization.
Reverse Genetics
Discovery Research (High Throughput)
• Few, if any, hypothesis going in,
• High throughput, (2232 genes),
• Lots of “negative” results, (87.1% of the genes
tested),
• Value is in (12.9%)…
– the analysis of the data in concert with annotations in the
data sets and references in the literature,
– the generation of materials for further “hypothesis” - or “discovery” driven research.
dsRNAs (I)
Where do they come from?
• PCR primer pairs were designed for each of the genes
discovered via bioinformatic analysis of the sequenced
chromosome,
– and confirmed through EST sequences, or experimental
expression studies,
– shortest region > 500 bp, or > 90% of ORF.
gene
dsDNA
dsRNAs (II)
PCR Primers +
• T3 or T7 promoter sequences were included in the PCR
primers...
T3 sequence tacked onto the reverse primer.
gene
5’ -
GTAATACGACTCACTATAGGG GCTAAGCTATTCGATGCTA
T7 promoter sequence
gene specific sequence
- 3’
T3 and T7 RNA Polymerase
• Bacteriophage T3 and T7 RNA polymerases are DNAdependent RNA polymerases with high sequence
specificity for T3 or T7 promoters.
• T3 and T7 RNA polymerases synthesize RNA 5' to 3'.
• These enzymes are isolated from an overproducing
recombinant E. coli clone, and are available
commercially.
dsRNAs (III)
in vitro transcription
• T3 and T7 polymerases were used to make single stranded
RNA,
– sRNA (sense) and asRNA (antisense)…
dsDNA
- two reactions -
T7 polymerase
sRNA
T3 polymerase
asRNA
dsRNAs (IV)
Where do they come from?
• sRNA and asRNA are then mixed, and form dsRNA,
• Done for 2232 genes, all in 96 well plates...
dsDNA
- two reactions -
T7 polymerase
T3 polymerase
sRNA
asRNA
dsRNA
dsRNAs (VI) x 2232
• Quality control…
– Each dsRNA reaction product was run out on a gel,
assayed to see if it migrated as a ssRNA or dsRNA
based on the estimated size of the product(s)…
dsRNA
ssRNA
…ssRNA and ds RNA of the same length
migrate differently under electrophoresis.
dsRNAs (IV)
Where do they come from?
• sRNA and asRNA are then mixed, and form dsRNA,
• Done for 2232 genes, all in 96 well plates...
dsDNA
- two reactions -
T7 polymerase
T3 polymerase
sRNA
asRNA
dsRNA
Then What?
• dsRNAs (was) injected ... into the gonads of adult wildtype hermaphrodites, which were left at 20 °C for 24 h,
• Embryos were then removed and analyzed for potential
defects in cell-division processes, capturing 1 image every
5 s using time-lapse Nomarski Differential Interference
Contrast (DIC) microscopy,
•
A minimum of three embryos from three different worms
were filmed from shortly after fertilization until the fourcell stage.
http://fire.biol.wwu.edu/young/470/rnai_movies.html
C. elegans
Life Cycles
And More…
Progeny Tests
• Three animals were transferred to a fresh plate 24 h after
injection, and left at 20 °C.
– Two days later, the plate was inspected with a stereomicroscope
(20–40x magnification) for the presence of eggs, F1 larvae and
their developmental stage (normally L2–L4).
– Two days after that, the plate was inspected for the presence of F1
adults (normally >100), their overall body morphology and the
presence of F2 progeny.
• Partially penetrant embryonic lethality and subtle developmental
defects were not scored in this analysis.
• dsRNAs that gave rise to defects in less than 5% of the adult progeny
were not considered as being associated with a phenotype.
But?
• It’s supposed to be high throughput, so experiments were
designed to minimize the time required,
– in part to make the acquisition of so much “meaningless” data
palatable (89.1%),
– in part because it is a whole lot of work no matter how you
approach it,
• Remember, along with discovery, this experiment was
designed to establish a workable paradigm for future large
scale analysis of metazoan (and other complex) organisms.
So, First
establish reliability
• Injected 13 dsRNAs targeted to known components of the
cell division process,
– all 13 known mutations were observable using DIC photography,
• This control tested RNAi efficiency, and the efficacy of
DIC phenotype scoring...
13 of 13 genes were disrupted,
based on clear DIC image
acquisition.
High Throughput Protocols
1st establish acceptable failure rates...
• Tried mixing (multiplexing) dsRNA from 2
or more genes...
92% Rate
deemed
acceptable.
1. Then did it,
2. Then checked the results...
• When a phenotype was observed…
– to see which of the two dsRNAs caused the phenotype,
fresh worms were injected with the dsRNA (one at a
time),
– genomic sequence was examined to make sure that only
the dsRNA targeted gene was responsible,
• Gene families,
• Miscalled ORFs.
Then checked the results again...
Conclusion: “As a result, the DIC phenotypes reported here
almost certainly result from inactivation of the expected genes”.
For Example...
• Makes sense….
For Example (II)...
• Surprising…so many translation and ribosomal
proteins involved meiosis.
Forward vs. Reverse Scorecard
• 7 of 7 known chromosome III DIC observable,
early embryo phenotypes observed,
• 9 of 14 late embryo phenotypes observed,
• 9 of 31 larvae/adult phenotypes observed.
7 of 7 known, plus 126 new genes!
Cousins and Orthologs!
Everyone and Metazoans
Successful?
• High throughput: Yes,
• Fidelity: Yes, 7/7,
• Discovery: Yes, > 100 new genes involved in
early embryo development, especially cell
division,
• Helpful to Metazoan biologists?
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