DNA Synthesis•Eukaryotes

•DNA unwinds at a replication origin •Two replication forks form •Bidirectional synthesis of DNA

•Much more DNA; complexed with proteins

•Eukaryotes have much more DNA

•Yeast 250-400 replicons •Mammalian cells up to 25,000

•Solution:

Multiple replication origins

•Yeast 4X that of E. coli •Drosophila 100X that of E. coli

DNA Synthesis in Eukaryotes

Pre-initiation replication complex:

ORC

Cdt1

Cdc6

MCM

Enzyme Function

Prokaryotes Eukaryotes

Helicase

Primase

Polymerase

Proofreading

Sliding clamp

Prevent ss closure

DnaB

DnaG

Pol III

Pol III

b sliding clamp

SSBPs

MCM

Primase s.u. of a-polymerase

Pol d and e

Pol d and e

PCNA (Proliferating Cell Nuclear Antigen)

RPA (Replication Protein A)

Comparison of some replication components

A Problem and a Solution

Telomere Formation

A model for t loop formation

Reproductive cloning

Biotechnology/Cloning

Reproductive cloning

Therapeutic cloning

http://www.rndsystems.com/dam_public/6515.jpg

Therapeutic cloning

First Cloning Experiment

Cohen et al., 1973

“Recombinant DNA”

Cloning Vectors: pBR322 plasmid

Cloning into pBR322

RT-PCR and Cloning