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

Reverse Transcriptase: RNA-dependent DNA polymerase-Requires a primer for polymerization activity (RNA or DNA)-Also Converts ssRNA to double stranded DNATherefore 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

primerB) 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.

                               

Nobel Laureates 1975

                                                               

David Baltimore

Howard TeminRenalto Dulbecco

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 transferaseTerminal 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.

AAAAAAA

TTTTT

Solution: 5' RACE

5'

5'3'primer 1

1. Use primer 1 for RT of mRNA

AAAAAAA 5'

+RT

2. Remove RNA (OH-) and Tail the new cDNA5'GGGGG

+ Terminal transferase

3. Use poly C primer (with restriction site at 5' end) as primer 2 to copy the new cDNA to ds DNA

5'GGGGG *CCCC

+ Taq or Klenow

4. PCR using primer 1 and primer 2.

AAAAAAA 5'

5'GGGGG

TTTTT3'+

End up with:

orginal cDNA

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?

http://genome.gsc.riken.go.jp/home.html

RIKEN FANTOM PROJECT:

1) Develop technologies to reproducibly produce full-length cDNA libraries from many tissues and cells2) Sequence from the 3’ end to find unique ESTs~1 million cDNAs were sequenced; represent ~128K gene clusters3) 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 genes?

1) Cloning by Complementation: rescue of a mutant phenotype by a member of a library2) Differential or "Subtraction" Hybridization3) Cloning from the protein: either from protein sequence, or using antibodies, or some biochemical property of the protein (e.g., ligand or DNA binding)4) By homology (low stringency hybridization)5) POSITIONAL CLONING

Cloning by Complementation: rescuing yeast cell cycle mutants with mammalian cDNAs

Cyclin/cdc28 complexes regulate the cell cycle in yeast

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 surviveIn 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 off)

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.

Cells with targeted mutations of cln1, 2, & 3 arrest at "Start" Hypothesis: mammalian genes ought to recue these mutant yeast cells.

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