Transcript No Slide Title

LECTURES 3/4. CONSTRUCTING and
SCREENING cDNA LIBRARIES to
ISOLATE NEW GENES
ORIGINAL ARTICLES:
CLONING BY COMPLEMENTATION:
Lew, D, Dulic, V, and Reed SI. 1991. Isolation of three
novel human cyclins by rescue of G1 Cyclin (Cln) in yeast.
Cell 66:1127-1206.
DIFFERENTIAL HYBRIDIZATION:
**Davis, RL, Weintraub, H, and Lassar, A. 1987. Expression
of a single transfected cDNA converts fibroblasts to
myoblasts. Cell 51:987-1000.
The Retrovirus Life Cycle
Nobel Laureates 1975
David Baltimore
Renalto Dulbecco
Howard Temin
Reverse Transcriptase: RNA-dependent DNA polymerase
-Requires a primer for polymerization activity (RNA or
DNA)
-Also Converts ssRNA to double stranded DNA
Therefore has DNA-dependent DNA polymerase activity
as well
-No 3' exo-activity (no proofreading); Has RNaseH activity
(destroys RNA in a DNA/RNA hybrid).
How is mRNA primed for RT?
A) mRNA has polyA tails...thus can use oligo dT as a
primer
B) Random primers: synthesize primers (usually
around 10-15mers) of RANDOM sequence (put in all
four base pairs for each cycle of automated DNA
synthesis). This will result in internal, random
priming of mRNA molecules.
Double-Stranded cDNA Enzyme List:
First strand:
-Reverse transcriptase
Second strand:
-RNAse H
-DNA polymerase I
-T4 DNA ligase
First Strand cDNA Synthesis
Primed Synthesis of Second cDNA
Strand
After second strand synthesis, double
stranded, blunt end DNA is produced:
Need to provide complementary ends to
clone into vectors.
1) Homopolymer tailing: Terminal transferase
Terminal transferase is a polymerase that add will add
homopolymer tails to free 3' ends of DNA or RNA
2) addition of adapters (linkers) by ligation
**3) Tailing and primers containing restriction sites
for asymmetric cloning
Synthesis of cDNA: Generation of Asymmetric Ends
Major problem with cDNA
libraries:
Full length cDNAs may not be
present
This depends on SIZE and
SECONDARY STRUCTURE of
the mRNA
Many solutions including RT
PCR RACE.
Rapid Amplification of cDNA ENDS (RACE)
Problem: cDNAs isolated from libraries are
often not full length, often lacking 5' end.
5'
AAAAAAA
3'
primer 1
TTTTT 5'
Solution: 5' RACE
1. Use primer 1 for RT of mRNA
5'
AAAAAAA
+RT
2. Remove RNA (OH-) and Tail the new cDNA
5'
GGGGG
+ Terminal
transferase
3. Use poly C primer (with restriction site at 5' end) a
primer 2 to copy the new cDNA to ds DNA
5'
GGGGG
*CCCC
+ Taq or Klenow
4. PCR using primer 1 and primer 2.
End up with:
5'
AAAAAAA
5'
GGGGG
orginal cDNA
3'
+
TTTTT
Potential Problems with RACE
for 5' ends of cDNA:
1) Secondary structure of RNA
may make it difficult to obtain
5' cDNA (this was probably the
problem in the first place).
2) RNA is very long: may need
to do more than 1 round of 5'
RACE.
Expressed Sequence Tag (EST) Projects:
Random sequencing of cDNA libraries
Make libraries from various tissues, tumors, cell lines, etc.
Randomly sequence library members: usually from the ENDS
Typical mRNA
Incomplete mRNA
Sequence Tag
How to link ESTs from the same gene together?
RIKEN FANTOM PROJECT:
http://genome.gsc.riken.go.jp/home.html
1) Develop technologies to reproducibly produce
full-length cDNA libraries from many tissues and cells
2) Sequence from the 3’ end to find unique ESTs
~1 million cDNAs were sequenced; represent ~128K gene clusters
3) Fully sequence the unique clusters (~82K)
4) Fully annotate the cDNAs sequenced
Shinagawa et al. Functional annotation of a full-length mouse cDNA collection.
Nature. 2001 409(6821):685-90.
-First 20,000 sequences annotated.
How are libraries screened to clone new
1) Cloning by Complementation: rescue o
mutant phenotype by a member of a libra
2) Differential or "Subtraction" Hybridizat
3) Cloning from the protein: either from pr
sequence, or using antibodies, or some
biochemical property of the protein (e.g.,
or DNA binding)
4) By homology (low stringency hybridiza
5) POSITIONAL CLONING
Cloning by Complementation: rescuing yeast cell
cycle mutants with mammalian cDNAs
Cyclin/cdc28 complexes regulate the cell cycle in
yeast
Cells with targeted mutations of cln1, 2, & 3 arrest at "Sta
Hypothesis: mammalian genes ought to recue these muta
yeast cells.
Strategy:
1.Place a cln2 gene under control of a gal inducible
promoter into a yeast strain harboring mutations in all
three Cln genes:
In galactose, cln2 will be made and cells survive
In glucose, promoter is shut off and cells do not grow.
2. Create a human cDNA library in a yeast expression
vector (regulatory sequences that allow for expression in
yeast).
3. Select for growth in glucose (i.e., when cln2 is shut of
41 clones that were not revertants were selected when
two different libraries were screened:
15 cylin B1. 18 cyclin B2, 4 cyclin A, 2 cyclin C, 1 cyclin
D, 1 cyclin E.
Cyclin A and B rescue: cDNAs are truncated
LIMITED REGIONS OF HOMOLOGY
between cyclin family members
cyclin D and cyclin E regulate "Start" in mammalian cells