Gene Expression new frontiers

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Gene Expressionnew 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 are learning -
- processing and translation of mRNA is further
controlled (through RNA/Protein complexes), via
ancient, conserved processes.
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Central Dogma addendum
DNA
RNA
Transcription Factors
Protein
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Transcriptional Network(cell cycle)
(example)
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Central Dogma addenda
DNA
TGS Transcriptional Gene Silencing
RNA
Transcription Factors
Protein
PTGS Post Transcriptional Gene
Silencing
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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.

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Ancient History (1)
Cell 75, 843 (1993)
Some development timing genes code for short
anti-sense molecules, appeared to be unique to
C. elegans.
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miRNAmicro-RNA
How would a cell express this?

• Post-transcriptional regulatory genes,
• contain 22 nucleotides (processed),
• are cleaved from somewhat larger double stranded
  RNA (dsRNA) precursors - by a protein called
  Dicer
• are expressed in certain cell types and at
  certain times during differentiation (also
  called short temporal (stRNA).

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Anti-Sense Blocking of Translation
miRNA
Why use RNA to block mRNA function?
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miRNAs

• Conserved amongst eukaryotic cells,
• Often associated with hetrochronic genes,
• difficult to identify in genomic sequences
  because they dont have long ORFs,

How might you locate them?
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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.

• Expect,
• Higher levels of protein,
• Gene-dosage phenotypes,
• Glorious publication.

Frequent Results no protein produced, scorn from
senior scientists.
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Anti-Sense Studies

• Another good idea use a transgene with the
  coding sequence reversed...

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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.

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Ancient History (II)(co-suppression)

• Transgene expression often decreases as the copy
  number of transgenes increased.

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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.

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RNAiRNA 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.

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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,
• regulate gene expression?

• useful for doing Reverse Genetic studies,
• dsRNA triggers sequence specific degradation of
  complementary mRNAs.

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Today
Nature 408 331 - 336
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Ce III 2315 Genes
http//www.wormbase.org
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Functional GenomicsThe 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?

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Differential Interference Contrast
Microscopy Nomarski Optics
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DIC Microscopy Nomarski Optics
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Reverse GeneticsKnockomics, Knockology...

• Sequence to Phenotype to Function

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Forward vs. Reverse Genetics

• Treat thousands of organisms with a mutagen,
• random mutagenesis,
• Identify an individual with a phenotype of
  interest,
• Identify the gene.

• 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.

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Reverse GeneticsFunctional Genomics
Function
Gene DNA Sequence
Phenotype Analysis
Gene Disruption
Development Physiology Cell Biology
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New Data, New Technologynew 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.
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Reverse GeneticsDiscovery 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.

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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 gt 500 bp, or gt 90 of ORF.

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dsRNAs (II)PCR Primers

• T3 or T7 promoter sequences were included in the
  PCR primers...

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T3 and T7 RNA Polymerase

• Bacteriophage T3 and T7 RNA polymerases are
  DNA-dependent 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.

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dsRNAs (III)in vitro transcription

• T3 and T7 polymerases were used to make single
  stranded RNA,
• sRNA (sense) and asRNA (antisense)

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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...

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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)

ssRNA and ds RNA of the same length migrate
differently under electrophoresis.
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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...

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Then What?

• dsRNAs (was) injected ... into the gonads of
  adult wild-type 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 four-cell stage.

320c6
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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 (2040x magnification) for the
  presence of eggs, F1 larvae and their
  developmental stage (normally L2L4).
• Two days after that, the plate was inspected for
  the presence of F1 adults (normally gt100), their
  overall body morphology and the presence of F2
  progeny.
• Partially penetrant embryonic lethality and
  subtle developmental defects were not scored in
  this analysis.
• Moreover, dsRNAs that gave rise to defects in
  less than 5 of the adult progeny were not
  considered as being associated with a phenotype.

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But?

• Its 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.

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So, Firstestablish 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.
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High Throughput Protocols 1st establish
acceptable failure rates...

• Tried mixing (multiplexing) dsRNA from 2 or more
  genes...

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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.

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Then checked the results again...
Conclusion As a result, the DIC phenotypes
reported here almost certainly result from
inactivation of the expected genes.
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For Example...

• Makes sense.

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For Example (II)...

• Surprisingso many translation and ribosomal
  proteins involved in meiosis.

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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!
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Cousins and Orthologs!Everyone and Metazoans
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Successful?

• High throughput Yes,
• Fidelity Yes, 7/7,
• Discovery Yes, gt 100 new genes involved in early
  embryo development, especially cell division,
• Helpful to Metazoan biologists?

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Monday
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Homologous Recombination Range

• Yes...
• mice, many well characterized mammalian cells,
• bacteria,
• yeast, (remember the bar code deletion project),
• No (maybe)...
• C. elegans (no),
• Arabidopsis (done once, not repeated),
• Drosophila (shown in principle, not repeated),
• the rest?

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Huh?
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Other Remodeling RNAs?

• ESTs (Expressed Sequence Tags) cDNA libraries
  are end sequenced,
• lots of non-protein-coding transcripts are
  found,
• Upwards of 60,000 RNA of these transcripts have
  been identified in the human genome,
• ignored until recently
• one active hypothesis is that they are involved
  in chromosome remodeling.

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