Download PPT - Yavapai College

Molecular Genetics
•
•
•
•
•
•
DNA
Chromosomes
Genes and genome
Protein synthesis
DNA replication
Changes in DNA sequence: phages
and transposons
• Genetic engineering
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
DNA and GENETICS
• Cells divide and pass on
instructions coded in DNA of
chromosomes
• Each chromosome is a huge
DNA molecule with coded
information
• DNA has dual role:
– DNA replicates to pass on
information
– DNA is transcribed to make
proteins that run cell
metabolism
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Prokaryote DNA/chromosome
• Single (usually)
chromosome is one long
DNA molecule
• Small secondary DNA
molecules are called
plasmids
• Localized in region of cell
called "nucleoid" but not
bound by membrane as is
nucleus of eukaryotes
• DNA molecule is
packaged in loose loops
within cell
• DNA of prokaryotes is
invisible in light
microscope
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
DNA and chromosomes
• Long DNA molecules
(millions of base pairs long)
in nucleus are called
chromosomes
• Each chromosome is
organized and packaged or
wrapped up with proteins
giving it a certain shape
• In humans, 23 pairs of
chromosomes
– 1 of each pair from mother
– 1 of each pair from father
• Total view of all 23 pairs is
called karyotype
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Chromosomes
• Each chromosome is a single
DNA molecule wrapped up within
a special group of proteins giving
it a particular shape
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Genes and Genome
• Genome:
– entire DNA of cell (all DNA molecules)
– also includes DNA of mitochondria, chloroplasts in
eukaryotes
– Thought question: Are viruses, phages, transposons part
of genome?
• Gene:
– Region along DNA molecule that codes for 1 protein
– usually 1000's of base pairs long
– E. coli lac operon is first gene whose regulation and
function was understood at molecular level in coding of
DNA (see more below)
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
DNA is transcribed to make proteins
that run cell metabolism
•
•
•
•
•
DNA is transcribed to mRNA
mRNA is translated to amino acid sequence
Amino acid sequence folds up into protein
Proteins catalyze reactions of cell metabolism
This process is called “gene expression”—the
information in one region of the DNA—a
“gene”—is being expressed so that the cell’s
metabolism can function
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
2 steps of gene expression
1. Transcription – DNA
is read to make a
mRNA in the
nucleus of our cells
2. Translation –
Reading the mRNA
to make a protein in
the cytoplasm
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Transcription
• Happens in nucleus
• DNA double helix
“opens up”
• mRNA transcript is
made from DNA
template
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Translation
• Happens outside
nucleus
• Ribosomes (special
RNA particles or
organelles) do the
translation
• They glom onto
mRNA and line up
amino acids
according to mRNA
sequence (see next
slide for “code”
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
RNA-protein
translation code
• Every three RNA
bases codes for one
amino acid
• This code is very
evolutionary
conservative—works
almost the same in all
forms of life
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Overview of transcription and translation
REMEMBER: A particular region of DNA that has the code to make a particular protein is called a “gene.”
Details in web link video animations
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
How does cell decide when to activate
which genes to produce what proteins?
• DNA must be unpackaged
and uncoiled in order to be
transcribed to mRNA
• Lampbrush chromosome
shows loops of DNA that
are being transcribed
• What determines which
regions or genes are going
to be transcribed and
translated?
• This is called regulation of
gene expression
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Regulation of gene expression
•
•
•
Gene expression is regulated—not all genes are
constantly active and having their protein produced
The regulation or feedback on gene expression is
how the cell’s metabolism is controlled.
This regulation can happen in different ways:
1. Transcriptional control (in nucleus):
•
e.g. chromatin density and transcription factors
2. Posttranscriptional control (nucleus)
•
e.g. mRNA processing
3. Translational control (cytoplasm)
•
e.g. Differential ability of mRNA to bind ribosomes
4. Posttranslational control (cytoplasm)
•
•
e.g. changes to the protein to make it functional
When regulation of gene expression goes wrong—
cancer!
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
The lac operon: model for gene expression
•
•
•
•
•
•
Region along DNA molecule
Includes three protein
synthesis coding region-sometimes called "genes"
as well as region of
chromosome that controls
transcription of genes
Genes for proteins involved
in the catabolism or
breakdown of lactose
Thus, when lactose is
absent, no transcription of
gene since no need for
these proteins (a in figure
below)
When lactose is present,
transcription of genes takes
place so proteins are
available to catalyze
breakdown of lactose (b in
figure below)
See animation on course
website
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
DNA is structured to replicate
• DNA is “double helix”—
two complementary
strands wound in a spiral
• Strands separate and
DNA replicates by filling
in other half of each
separated strand
• Famous Watson-Crick
model (Nobel prize)
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
DNA replicates to pass on information
(to daughter cells in mitosis)
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Changes in DNA sequence
• Mutation—change
in DNA sequence.
• Example:
Changes in DNA
allow bacteria to
resist effects of
antibiotics
• Mutation changes
DNA sequence,
which in turn
changes protein
sequence that
codes for a
specific protein in
one of the many
cell metabolic
pathways
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Mutations can spread through a population
• Mutation allows cell to survive in a new
environment--in this case, in the
presence of penicillin. This type of
beneficial mutations can result in more
individual cells that are resistant to
penicillin and can thereby grow colonies
• The offspring or daughter
cells then continue to
spread throughout the
population, especially
where penicillin is
present. This is natural
selection leading to
evolutionary change!! We
have seen it happen...in the
laboratory...in the "wild."
• Fifty years, ago, almost no
bacteria were resistant to
penicillin. Today, some
penicillin resistant cells are
found in virtually all
bacterial populations.
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Horizontal gene transfer
• Antibiotic resistant genes can also spread by "horizontal
gene transfer," even between different species of
bacteria.
• Three ways that genes can transfer between adult cells:
• transformation--cell takes up DNA from environment,
presumably released by another dead cell...or provided,
in a laboratory, by a researcher.
• Transduction--phage delivers DNA to cell
• Conjugation--"bacterial sex." Cells form connection to
transfer genes.
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Transposons
•
•
•
•
•
•
"Jumping genes" can move from one location to another within
the cell's DNA. For example, in bacteria, can move from main
chromosome to plasmid.
Always have palindrome to remove and insert into DNa
Complex transposons carry genes for particular traits
Example: "R factor" is transposon that carries gene that confers
resistance to antibiotics (in the case of penicillin, presumably by
changing the cell wall synthesis process and avoiding the killing
effect of penicillin)
Transposons can be carried by phage (transduction) or passed
cell to cell (conjugation), even among different species of
bacteria.
Here is replication and transfer by conjugation of a plasmid
carrying "F factor" which gives "fertility" or the ability to conjugate
to a cell. This plasmid also contains a transposon or jumping
gene that allows it to reintegrate into the cell's main chromosome
Larry M. Frolich, Ph.D.
or DNA molecule:
Biology Department, Yavapai College
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Recombinant DNA tools: Restriction Enzymes
• Cut DNA at particular, usually
palindromic sequence:
• Cells make restriction enzymes
normally to allow for plasmids,
transposons, even viruses to insert
into genome
• Molecular biologists use restriction
enzymes to cut DNA at the same
"restriction site" in order to create
specific fragments of DNA
• These fragments can then be run out
on a gel, multiplied using PCR, and
even "recombined" or inserted into a
new organisms, usually E. coli.
• If the restriction fragment contains a
protein coding sequence, then that E.
coli cell will produce that protein:
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Use of restriction enzyme to insert HGH into bacteria
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Common used restriction enzymes
Blunt or
Sticky
End
Enzyme
Organism
Recognition
Sequence
EcoRI
Escherichia Coli
GAATTC
Sticky
BamHI
Bacillus amyloliquefaciens
GFATCC
Sticky
BglII
Bacillus globigii
AGATCT
Sticky
PvuI
Proteus vulgaris
CGATCG
Sticky
PvuII
Proteus vulgaris
CAGCTG
Blunt
HindIII
Haemophilus influenzae Rd
AAGCTT
Sticky
Hinfl
Haemophilus influenzae Rf
GANTC
Sticky
Sau3A
Staphylococcus aureus
GATC
Sticky
AluI
Arthrobacter luteus
AGCT
Blunt
TaqI
Thermus aquaticus
TCGA
Sticky
HaeIII
Haemophilus aegyptius
GGCC
Blunt
NotI
Nocardia otitidis-caviarum
GCGGCCGC
Sticky
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Recombinant DNA tools: Reverse transcriptase
• Reverse transcriptase
• Used by RNA viruses to transcribe
RNA into DNA (note reverse of
central dogma)
• DNA can then insert into cell's
genome
• Cell then makes RNA transcripts of
that DNA and new RNA viruses are
manufactured and released from
cell
• Molecular biologists use reverse
transcript to make cDNA--DNA
made from RNA transcripts. This
cDNA is the DNA that codes for
proteins (not the entire genome
codes for proteins
• It is useful to have only the proteincoding regions in order to figure out
what a particular gene does or to
be able to force a bacterial cell to
make that protein.
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Recombinant DNA tools: Gel electrophoresis
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Recombinant DNA tools: Gel electrophoresis
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Recombinant DNA tools: PCR—polymerase chain reaction
•
•
•
Basically is the process of replicating DNA,
but done in a test tube for a particular DNA
sequence (usually a restriction fragment)
Allows for making many copies of a
particular gene
In PCR, a heat-stable DNA polymerase is
used, most commonly Taq Polymerase from
the thermophilic microbe Thermus
aquaticus.
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College
Recombinant DNA tools: Genetic maps, Genetic
sequences, and Gene libraries
• Using combination of above techniques to fully
characterize the genome of a particular cell
• A genetic map puts the restriction fragments and the
genes they might contain in order along the chromosome
• A gene library is a bank of cells, each of which contains
a cloned gene, often on a plasmid that can be easily
manipulated, perhaps because it has an antibiotic
resistance gene attached as well
• A genetic sequence is the complete DNA code of a
particular region of a chromosome or the entire
genome. It is obtained by knocking the last nucleotide
off of a restriction fragment, one at a time, and then
identifying the tailing nucleotide using specific molecular
markers.
Larry M. Frolich, Ph.D.
Biology Department, Yavapai College