Chapter 29 Slides

Download Report

Transcript Chapter 29 Slides

Biochemistry 2/e - Garrett & Grisham
CHAPTER 29
DNA: Genetic Information,
Recombination, and Mutation
to accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
All rights reserved. Requests for permission to make copies of any part of the work
should be mailed to: Permissions Department, Harcourt Brace & Company,
6277 Sea Harbor Drive, Orlando, Florida 32887-6777
Biochemistry 2/e - Garrett & Grisham
Outline
• 29.1 DNA Carries Genetic Information
• 29.2 Genetic Information in Bacteria
• 29.3 Molecular Mechanism of
Recombination
• 29.4 The Immunoglobulin Genes
• 29.5 Molecular Nature of Mutation
• 29.6 RNA as Genetic Material
• 29.7 Transgenic Animals
Biochemistry 2/e - Garrett & Grisham
The Transforming Principle is
DNA
• Griffith in 1928 injected bacteria into
mice
• Combination of heat-killed type S and
non-virulent type R killed the mice
• Showed that type S had been
transformed by the type R!
• In 1944, Oswald Avery showed that the
active transforming agent was DNA!
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Further Proof
• In 1952, Hershey and Chase, studying
bacteriophages, labelled DNA with 32P
and protein with 35S
• Bacteriophage progeny produced by
infection of bacteria contained 32P (thus
DNA from the original phage), but not
35S (from the protein)!
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Genetic Information Can Be
Transferred Between Bacteria
• In 1946, Lederberg and Tatum showed
that two different strains of bacteria with
different growth requirements could
exchange genes
• Lederberg and Tatum surmised that the
bacterial cells must interact with each
other - the process is now known as
sexual conjugation
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Chromosome Mapping
by Interrupted Mating
• Passage of the ‘F factor’ from Hfr cells
to F- cells also brings adjacent genes
• Genes from the Hfr chromosome are
transferred in a fixed order
• This order can be mapped by
‘interrupted mating’ methods
• The genetic map obtained reveals a
circular arrangement of genes
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Mechanism of Recombination
• General recombination: any pair of
homologous DNA segments as
substrates
• In 1964, Robin Holliday proposed a
model involving single-stranded nicks at
homologous sites
• Duplex unwinding, strand invasion and
ligation create a Holliday junction
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Enzymology of Recombination
• RecBCD initiates recombination in
E.coli
• RecA forms nucleoprotein filament for
strand invasion and homologous pairing
• RuvA, RuvB, RuvC drive branch
migration and process the Holliday
junction into recombination products
• Eukaryotic systems are probably similar
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
The RecA Protein
• 38 kD enzyme that catalyzes ATPdependent DNA strand exchange,
leading to formation of Holliday junction
• RecA forms a helical filament with a
groove to accommodate DNA
• RecA:ssDNA complex binds dsDNA at
secondary site and searches for regions
homologous with the bound ssDNA,
then forms the desired duplex
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Resolving Holliday Junctions
• Ruv proteins resolve the junction into
recombination products
• RuvA and RuvB act as a helicase that
dissociates the RecA filament and
catalyzes branch migration
• RuvC is an endonuclease that binds at
the junction and cuts pairs of DNA
strands of similar polarity. Splice and
patch recombinants result.
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Transposons
• In 1950, Barbara McClintock showed
that activator genes in corn could move
freely about the genome.
• This was at first viewed as heresy and
McClintock was ostracized
• Molecular biologists in the late 1970s
rediscovered what McClintock knew
• She received a MacArthur Award in
1981 and a Nobel in 1983
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Immunoglobulin Genes
• Immunoglobulin genes maximize
protein diversity with a limited amount of
genetic information
• DNA rearrangement (genetic
recombination) during assembly of IG
genes accounts for this diversity
• Three separate genes are combined to
form the L-chain gene
• Four genes combine to form H-gene
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Molecular Nature of Mutation
• Point mutations - one base for another arise from mispairing, insertion of
analogs or chemical mutagens
• Mutagens include nitrous acid,
hydroxylamine and alkylating agents
• Insertions and deletions result in
frameshift mutations
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Prions
Proteins as Genetic Agents?
• Prions are ‘protein infectious particles’
• Agents composed only of protein
• Responsible for kuru, Creutzfeld-Jacob
disease, mad-cow disease, etc.
• The ‘infection’ appears to involve a
change of secondary structure and
conformation (or conformations!) in the
prion protein
• A Nobel for Stanley Prusiner in 1997
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham
Transgenic Animals
• Genes can be introduced into animals
by transfection - injection of plasmid
DNA into recipient cells
• Plasmids can be injected into fertilized
eggs in mice
• Expression is usually variable, because
the gene is inserted randomly
• Growth hormone transfection produces
mice that are very large!
Biochemistry 2/e - Garrett & Grisham
Biochemistry 2/e - Garrett & Grisham