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LECTURES 3/4. CONSTRUCTING and

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.

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LECTURES 3/4. CONSTRUCTING and

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  1. 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.

  2. The Retrovirus Life Cycle

  3. 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.

  4. First Strand cDNA Synthesis

  5. Primed Synthesis of Second cDNA Strand

  6. Synthesis of cDNA: Generation of Asymmetric Ends

  7. 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.

  8. 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.

  9. 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 Sequence Tag Incomplete mRNA How to link ESTs from the same gene together?

  10. RIKEN FANTOM PROJECT: http://genome.gsc.riken.go.jp/home.html • Develop technologies to reproducibly produce • full-length cDNA libraries from many tissues and cells • 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.

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

  12. Cyclin/cdc28 complexes regulate the cell cycle in yeast

  13. Cyclin A and B rescue: cDNAs are truncated

  14. LIMITED REGIONS OF HOMOLOGY between cyclin family members

  15. cyclin D and cyclin E regulate "Start" in mammalian cells

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