Download Molecular Basis for Relationship between Genotype and Phenotype

Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
transcription
DNA sequence
replication
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
Refer to Figure 7-24 from Introduction to Genetic Analysis, Griffiths et al., 2012.
Initiation at Origin of Replication
Prokaryotes:
Fixed origin
DnaA proteins
DnaB (helicase)
Eukaryotes:
Multiple origins
ORC protein complex
Cdc6 and Cdt1
MCM complex (helicase)
Overview of DNA Synthesis
DNA polymerases synthesize
new strands in 5’ to 3’
direction.
Primase makes RNA primer.
Lagging strand DNA consists of
Okazaki fragments.
In E. coli, pol I fills in gaps in
the lagging strand and removes
RNA primer.
Fragments are joined by DNA
ligase.
The Problem of Replicating Chromosome Ends
telomere
3’ 5’
A
B
3’ 5’
A
+
DNA
Replication
5’ 3’
3’ 5’
B
5’ 3’
3’ 5’
A
RNA primer
removal and
DNA ligation
5’ 3’
3’ 5’
B
+
5’ 3’
5’ 3’
Last section of lagging strand cannot be primed. Result is
a shorter chromosome after each round of replication.
The Problem of Replicating Chromosome Ends
Here is another angle. Note again that the last section
of lagging strand cannot be primed. A shorter
chromosome is produced after each round of replication.
It is theorized that as cells age (generations), telomeres
shorten, eventually leading to their death.
Is there a mechanism to maintain chromsome length?
Telomere Lengthening
Telomerase (a reverse
transcriptase) adds
repeats to telomeric
DNA.
It carries an
RNA molecule
that serves as
template for
DNA synthesis.
Telomere Lengthening
The Telomeric Cap Structure
Telomeric end is protected by a “cap”. It consists of
TRF1 and TRF2 (that bind to telomeric repeats) and
proteins such as WRN that bind to TRF1 and TRF2.
Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
Making Recombinant DNA: Donor DNA
Genomic DNA:
DNA obtained from chromosomes of an organism
Complementary DNA (cDNA):
double-stranded DNA version of mRNA obtained
by reverse transcription
Chemically Synthesized DNA:
DNA sequence obtained by automated chemical
reactions
Cutting DNA: Restriction Endonucleases
Formation of a recombinant DNA molecule
Circular ds DNA
is cut with one
restriction
enzyme.
Linear ds DNA is
cut with the same
restriction
enzyme.
Both restriction
fragments are
linear and have
sticky ends (in
this case).
By complementary base
pairing, the sticky ends
can hybridize.
The result is a
recombinant DNA
molecule.
Inserting a gene into a recombinant DNA plasmid
Vector is a
cloning vehicle.
Both vector
and donor DNA
are cut with the
same
restriction
enzyme.
Restriction
fragments are
mixed; sticky
ends hybridize.
Recombinant
vector is the
result.
DNA ligase seals
gaps by forming
phophodiester
linkages.
How amplification works
Recombinant vectors
are introduced into
bacterial host cells.
Replication and cell
division produce
many copies of the
recombinant vector.
Clones of donor DNA
fragments result.