Molecular Genetics
Searching for Genetic Material, I
• Mendel: modes of heredity in pea plants
• Morgan: genes located on chromosomes
• Griffith: bacterial work;
– transformation: change in genotype and phenotype due to
assimilation (uptake) of external substance (DNA) by a cell
• Avery: transformation agent was DNA (stores and transmits
genetic information)
Searching for Genetic
Material, II
• Hershey and Chase
√ bacteriophages (phages) - viruses that attack
bacteria
√ Used radioactive markers to show DNA, not
protein, is the hereditary material
√ sulfur (S) is in protein, phosphorus (P) is
in DNA; only P was found in host cell
DNA Structure
•
Watson & Crick
The Double Helix
nucleotides:
• nitrogenous base
• sugar deoxyribose
• phosphate group
DNA Bonding
• DNA Nitrogen Bases:
– Purines:
• Adenine
• Guanine
– Pyrimidines:
• Cytosine
• Thymine
• Chargaff rules
A-T
C-G
DNA Replication
• Watson & Crick
strands are complementary; nucleotides
line up on template according to base pair rules (Watson)
DNA Replication (Synthesis)
• 2 strands of double helix unwind
– each is template for complementary strand
• Replication is initiated
– RNA primer is synthesized
• DNA strand grows in two directions
3’
5’
Leading strand
3’
RNA
primers
3’
5’
5’
5’3’
3’
5’
Two Okazaki
fragments
Fig. 12-12b, p. 273
Bidirectional Replication, I
• Starting at origin of replication
– proceeding in both directions
• Eukaryotic chromosome
– may have multiple origins of replication
– may replicate at many points at same time
Bidirectional Replication, II
•
•
•
•
Initiation:
Primer (short RNA
sequence~w/primase enzyme),
begins the replication process
Leading strand:
synthesis toward the
replication fork (only in a 5’ to 3’
direction from the 3’ to 5’ master
strand)
Lagging strand:
synthesis away from the
replication fork (Okazaki
fragments); joined by DNA
ligase (must wait for 3’ end to
open; again in a 5’ to 3’
direction)
Bidirectional Replication, III
• Always proceeds in 5′ → 3′ direction
• Leading strand
– synthesized continuously
• Lagging strand
– synthesized discontinuously
– forms short Okazaki fragments
• Key enzymes:
– DNA polymerase adds nucleotide subunits
– DNA primase synthesizes RNA primers
– DNA ligase links Okazaki fragments
3’
5’
Leading strand
DNA helix
RNA primer
DNA polymerase
3’
5’
3’
5’
3’
5’
Replication fork
Lagging strand
(first Okazaki fragment)
Direction of
replication
Fig. 12-12a, p. 273
Protein Synthesis: overview
•
•
•
•
One gene-one enzyme hypothesis
(Beadle and Tatum)
One gene-one polypeptide
(protein) hypothesis
Transcription: synthesis of
Messenger RNA (mRNA) using
DNA code
Translation: actual synthesis of a
polypeptide (protein) using mRNA
code
Protein Synthesis
RNA
• Three types of RNA
– mRNA (messenger RNA) – ling strands of
RNA that are complementary to DNA to one
strand of DNA; travel from nucleus to
ribosome
– tRNA – (transfer RNA) – small segments of
RNA that transport amino acids to the
ribosome
– rRNA – (ribosomal RNA) – component of
ribosomes
The Triplet Code
• The genetic instructions
for a polypeptide chain
are ‘written’ in the DNA as
a series of
3nucleotide ‘words’ called
Codons
• RNA Code uses A, C, G,
but ‘U’ (uracil) replaces ‘T’
Eukaryotic RNA Processing
• Exons – DNA coding that remain in the
final mRNA for protein synthesis; genes
that are expressed
• Introns – DNA coding that does not remain
in mRNA
Transcription
• 2 strands of double helix unwind
– One strand is template for making mRNA
• Key enzyme:
– RNA polymerase – regulates synthesis of
mRNA
Transcription
Translation
• mRNA from nucleus is
‘read’ along its codons by
tRNA’s anticodons at the
ribosome
– Each tRNA anticodon
(nucleotide triplet) is
specific for one amino acid
• Thus, amino acids are
bonded together in the
order translated from
mRNA
Translation
Applying Genetics
• Selective Breeding – allowing only desired
traits to pass onto next generation
• Hybridization – crossing dissimilar
organisms to get the best of both, e.g., crop
plants
• Inbreeding – continued breeding of
organisms with similar characteristics, e.g.,
dog breeds
Applying Genetics
• Increasing Variation
• Producing new kinds of bacteria via
radiation or chemicals to develop oil eating
bacteria
• Producing new kinds of plants, e.g.,
polyploidy plants such as daylilies
Genetic Engineering
Genetic engineering – altering DNA
o DNA extraction – removing DNA from cells
o Cutting and pasting DNA – using restriction enzymes
to cut DNA into pieces that can be analyzed
o Separating DNA – fragments separated via gel
electrophoresis
What can you do with it?
o Read the sequence
o Cut and paste
o Make copies
Genetic Engineering Vocabulary
• Plasmid – bacterial DNA that can be used to replicate foreign DNA
• Genetic marker – a gene that makes it possible to distinguish the
bacteria that carry the plasmid and foreign DNA from those that
don’t
• Transformation – process that results in genetic alteration of a
(bacterial) cell resulting from the uptake DNA from another
(bacterial) cell
• Recombinant DNA – Recombinant DNA is DNA that has been
created artificially. DNA from two or more sources is incorporated
into a single recombinant molecule.
• Gel Electrophoresis – electric current is used to separate
fragments of DNA; DNA fragments travel toward a negative charge
Applications of Genetic
Engineering
Clone – identical cells, DNA
Who is Dolly?
Transgenic organisms – contain genes from other
organisms
Examples:
• Transgenic microorganisms – to produce insulin and
other compounds for health
• Transgenic animals – mice that act physiologically like
humans
• Transgenic plants – plants with natural insecticides
Mutations
• Kinds of Mutations
– Gene Mutations
• Point mutations – change in one or a few nucleotides, i.e.,
substitutions, additions, deletions
• Frameshift mutations – additions (insertions) or deletions can
shift the reading of the strand resulting in more dramatic
changes
– Chromosomal mutations – change in number or
structure of chromosomes
• Deletions – loss of all or part of a chromosome
• Duplications – extra copies of a chromosome
• Inversion – reverse the direction of parts of a chromosome
Mutations
• Most mutations are neutral, but some are
harmful and some are root of genetic
variability. Examples: resistance to
disease such as HIV
• Mutation causes:
Mutagens – chemicals, X rays, gamma
rays
Gene Regulation
• Gene Regulation – turning genes on and
off
• Prokaryotes (bacterium)
• Operon – group of genes that operate
together
– ex: lac genes to use lactose for food.
– Contains an operator, regulatory gene,
promotor and genes that code for proteins.
Gene Regulation
• Operon
– Promotor – Where RNA polymerase first binds. It is the region
where transcription can occur so that proteins can be produced
that allow the transport and break down of lactose. But these
proteins are not needed unless lactose is present.
– Operator – Like a light switch that turns transcription on and off.
In lac operon, O region where repressor proteins are present.
These are present, transcription cannot occur. These proteins fall
off O region when lactose is present.
– Regulatory gene - Mechanism for turning transcription on and
off. In lac operon, makes a repressor protein that binds to the
operator in the promotor sequence and prevents transcription of
the digestive enzyme genes.
Eukaryote Gene Regulation
• Eukaryotes – no operons, but regulation is more
complex due to cell specialization
o Transcription factors – proteins that control
gene expression
o Hox genes – control differentiation in
embryonic development to determine the body
plan of an organism
o RNA Interference – small pieces of doublestranded RNA in the cytoplasm that bind with
mRNA and prevent translation