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Aguilera Lecture 2010Aguilera Lecture 2010
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Introduction to Molecular and Cell Biology
• What is the molecular basis of disease?
• What is the molecular basis of evolution ?
• How did life arise on earth?
• What is the molecular basis of memory?
• How are different cell types produced from a single embryo?
Molecular biology seeks to understand the physical and chemical basis of life.and helps us answer the following????
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Other Important Questions?Other Important Questions?
• What are genes?• How do genes store information?• How is genetic information expressed?• How is this process regulated?• How are genes duplicated during cell
division?• What are mutations?
To answer these questions it is necessary to To answer these questions it is necessary to
understand the nature of genes and proteinsunderstand the nature of genes and proteins
Remembering what you already knowRemembering what you already know
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DNADNA
Cell WallCell Wall
Outer MembraneOuter Membrane
Inner MembraneInner Membrane
Prokaryotic CellProkaryotic Cell
Plasma MembranePlasma MembraneDNADNA
Eukaryotic CellEukaryotic Cell
NucleusNucleus
Nuclear MembraneNuclear Membrane
organellesorganelles
Prokaryotic Cells:Prokaryotic Cells:
No nuclear membraneNo nuclear membrane
No internal compartmentsNo internal compartments
Eukaryotic Cells:Eukaryotic Cells:
NucleusNucleus
Specialized OrganellesSpecialized Organelles
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Permeable to Permeable to
gases and watergases and water
Active transportActive transport
requires energyrequires energy
Eukaryotic Membranes
E. coliE. coli 4 x 104 x 1066 base pairs of DNA base pairs of DNA (1 chromosome)(1 chromosome)
max. potential proteins 3 x 10max. potential proteins 3 x 1033
actually 1actually 1--2 x 102 x 1033
circularcircular
Genetic Information:Genetic Information:
23 pairs23 pairs
HumanHuman 2.9 x 102.9 x 1099 base pairs of DNA base pairs of DNA (46 chromosomes)(46 chromosomes)
cellcell max. potential proteins 2.4 x 10max. potential proteins 2.4 x 1066
actually much less (~30actually much less (~30--40,000) 40,000)
100100--1000 times the total dry weight of bacteria1000 times the total dry weight of bacteria
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Human Chromosome pairsHuman Chromosome pairs
Although human beings appear to be much more Although human beings appear to be much more
complex and sophisticated than other complex and sophisticated than other
organisms, we contain and express very similar organisms, we contain and express very similar
genes that are highly evolutionarily conservedgenes that are highly evolutionarily conserved
The function of some genes is so important, that The function of some genes is so important, that
they are highly conserved in sequence and they are highly conserved in sequence and
functionfunction
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Expression of Genes follows an ordered developmental planExpression of Genes follows an ordered developmental plan
HOXHOX
Humans and Flies express similar genes but obviously have different pattern of expression (function?)
During evolution, it is easier to adapt an existing gene than to create it from scratch
Biological molecules are interdependentBiological molecules are interdependent
DNADNA
You need protein to make DNA/RNAYou need protein to make DNA/RNA
and you need nucleic acids to make proteinsand you need nucleic acids to make proteins
Central DogmaCentral Dogma
RNARNA ProteinProtein
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•• Prevalent view in early 1900Prevalent view in early 1900’’s was that genetic s was that genetic
information was contained within proteinsinformation was contained within proteins
Why?
Proteins are more complex than nucleic acids (20 amino acids vs 4 different nucleotides)
Nucleic acids, DNA, was believed to play structural role in cell
nnnnnnnn Early 1940Early 1940’’s s DNADNA was finally implicated as thewas finally implicated as the
genetic material genetic material
DNA is the Genetic MaterialDNA is the Genetic Material
nnnnnnnn Pure DNA extracted from one bacterial strain Pure DNA extracted from one bacterial strain
could provide genetic information to another could provide genetic information to another
bacteria by a process known as transformation bacteria by a process known as transformation
nnnnnnnn Makes sense since DNA is very stable, it isMakes sense since DNA is very stable, it is
present in two copies in eukaryotes (diploid), present in two copies in eukaryotes (diploid),
exist as doubleexist as double--stranded molecule, andstranded molecule, and
most importantly can be faithfully copiedmost importantly can be faithfully copied
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GenesGenes
��Defined as a unit of DNA that encodes the Defined as a unit of DNA that encodes the
information for the synthesis of a proteininformation for the synthesis of a protein
��Prokaryotes contain less genetic informationProkaryotes contain less genetic information
(genes) than eukaryotes(genes) than eukaryotes
��Genes are copied (transcribed) into messenger Genes are copied (transcribed) into messenger
RNA and it is this message that is translated RNA and it is this message that is translated
into proteininto protein
�� In prokaryotes, genes involved in the sameIn prokaryotes, genes involved in the same
pathway are commonly linked closed togetherpathway are commonly linked closed together
In most eukaryotes, each gene is generally In most eukaryotes, each gene is generally
independently copied or independently copied or transcribed transcribed into a single into a single
RNA that is then RNA that is then translatedtranslated into a single proteininto a single protein
Three genes in two chromosomesThree genes in two chromosomes
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Most genes in mammals and plants contain Most genes in mammals and plants contain intronsintrons that are that are
removed during RNA processingremoved during RNA processing
ExonsExons encode the information for protein synthesis encode the information for protein synthesis
mature messagemature message
IntronsIntrons spliced out (deleted)spliced out (deleted)
Mutations within genes can cause disease or deathMutations within genes can cause disease or death
� Mutation in an exon can cause the production of an
abnormal or truncated (shorter) protein
� Mutation in an intron can cause no effect or can
alter or destroy the normal processing of the mRNA
�Mutation in regulatory regions can cause the gene to
not be expressed at all or over-expressed
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AAlternative Splicing can produce different or altered proteins from the same gene
Even though humans encode for ~30,000 proteins Even though humans encode for ~30,000 proteins
(based on gene count), the # of different proteins is (based on gene count), the # of different proteins is
higher due to added complexityhigher due to added complexity
Fig.9-2
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��In eukaryotes, each gene is independently In eukaryotes, each gene is independently
copied and generally encodes information for copied and generally encodes information for
a specific product (protein)a specific product (protein)
��Eukaryotic mRNA is a product of several Eukaryotic mRNA is a product of several
modifications which include removal of modifications which include removal of intronsintrons
��All DNA is arranged in a structure called a All DNA is arranged in a structure called a
doubledouble--helix that is composed of two identical helix that is composed of two identical
strands which adds to the chemical stability of strands which adds to the chemical stability of
this moleculethis molecule
DNA Double HelixDNA Double HelixWatson & CrickWatson & Crick
(1953)(1953)
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HO
O
P
O-O Sugar
Base 2
OHSugar
Base 1
O
O
P
O-O Sugar
Base 2
Sugar
Base 1
5’ 3’
3’5’
Linkage of Nucleic AcidsLinkage of Nucleic Acids
--HH22OO
O
HH
H
O
HH
CH2
O- OP
O-
O
O
H
H
H
OH
HH
CH2
O- OP
O
Base 2
Base 1
33’’
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POH
A AC T G
5’ ACTAG 3’UGAUC
T only in DNAT only in DNA
And U only in RNAAnd U only in RNA
Held by manyHeld by many
H bonds and H bonds and
HydrophobicHydrophobic
InteractionsInteractions
Sugar PhosphateSugar Phosphate
backbone is on backbone is on
the outsidethe outsideBases stackon inside
DNA HelixDNA Helix
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To maintain To maintain
the geometry the geometry
of this structureof this structure
small bases,small bases,
pyrimidinespyrimidines,,
(C or T) must (C or T) must
pair with largerpair with larger
bases, bases, purinespurines,,
(A or G)(A or G)
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CHN
NNC
C
N
NC
HC
N
H
C
(Deoxyribose)
HC
C
NN
NC
C O
CH3
O H H
C
(Deoxyribose)
ThymidineThymidine (T)(T) Adenine (A)Adenine (A)
AA--T Base PairT Base Pair
Base pair complementarity due to size, shape,
and chemical composition of bases
CHN
NNC
C
N
H NC
CH N
O
Guanine (G)Guanine (G)
HC
HC
NN
N
C
C O
N H
Cytosine (C)Cytosine (C)
H
HC
(Deoxyribose)
C
(Deoxyribose)
CC--G Base PairG Base Pair
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Genes are arranged in ChromosomesGenes are arranged in Chromosomes
in specialized structuresin specialized structures
Humans and other higher organisms have more junk DNA
Widely separated from each otherWidely separated from each other
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��The E. coli bacterial genome is approximately The E. coli bacterial genome is approximately
1mm long which is about 1000 x the size (1mm long which is about 1000 x the size (volvol) of ) of
a single bacteriaa single bacteria
��DNA needs to be highly compactedDNA needs to be highly compacted
DNA packaging within cellsDNA packaging within cells
Eukaryotic DNAEukaryotic DNA
nnnnnnnn Can be up to hundred thousand timesCan be up to hundred thousand times
longer than the cell that contains itlonger than the cell that contains it
nnnnnnnn Found in highly compacted units calledFound in highly compacted units called
chromosomeschromosomes
nnnnnnnn DNA is wound around special proteinsDNA is wound around special proteins
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Eukaryotic genes are arranged in chromosomesEukaryotic genes are arranged in chromosomes
Longest human chromosomesLongest human chromosomes
22--3 x 103 x 1088 base pairs ~10 cm longbase pairs ~10 cm long
gene 1 gene 1
gene 2 gene 2
gene 3gene 3
gene 4gene 4
Contents of ChromosomesContents of Chromosomes
Chromosomes contain different types of sequences Chromosomes contain different types of sequences
such as:such as:
nnnnnnnn Single copy genes, gene families, defective genes
nnnnnnnn Non-coding DNA and repetitive sequences(can compose a significant part of genome)
nnnnnnnn Viral DNA and other transposable elements (few)
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DNA cloningDNA cloning
Cloning comes from the Cloning comes from the greekgreek word word klonklon which means twigwhich means twig
Plants can be cloned by taking a cutting and replanting themPlants can be cloned by taking a cutting and replanting them--
they should turn out identical to original plant.they should turn out identical to original plant.
The first cloning experiment was performed in 1973 (only 37 The first cloning experiment was performed in 1973 (only 37
yrs ago) by Cohen and Boyeryrs ago) by Cohen and Boyer
They used They used bacterial plasmidsbacterial plasmids which are small circular replicating which are small circular replicating
fragments of DNAfragments of DNA
They also used enzymes that cut DNA into specific fragments. They also used enzymes that cut DNA into specific fragments.
These enzymes are called These enzymes are called restriction restriction endonucleasesendonucleases (enzymes (enzymes
that cleave nucleic acids)that cleave nucleic acids)
Plasmids are Plasmids are ““selfishselfish”” pieces of DNA that are circular and replicate pieces of DNA that are circular and replicate
inside of bacterial hostinside of bacterial host
AmpicillinAmpicillin resistance generesistance gene
Origin of replication
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When plasmids replicate they make two identical copiesWhen plasmids replicate they make two identical copies
Fig. 7.2
Restriction Enzyme Cleaves DNA at specific sitesRestriction Enzyme Cleaves DNA at specific sites
They are called sticky ends because they have a tendency of pairThey are called sticky ends because they have a tendency of pairing ing
or or ““rere--pairingpairing”” with each other (or other foreign fragments)with each other (or other foreign fragments)
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Fragment of foreign DNA can be readily introduced into plasmidsFragment of foreign DNA can be readily introduced into plasmids
Mix plasmid with Mix plasmid with E. coliE. coli bacteria and perform transformationbacteria and perform transformation
Heat Shock
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Restriction EnzymesRestriction Enzymes
Restriction enzymes Restriction enzymes (RE)(RE) are the bacteriaare the bacteria’’s defense against virusess defense against viruses
These enzymes These enzymes restrict restrict the host range of the virusesthe host range of the viruses
There are more than 100 different restriction enzymes from a There are more than 100 different restriction enzymes from a
large number of different bacterialarge number of different bacteria
Biotech companies have been making millions of dollars selling Biotech companies have been making millions of dollars selling
specific enzymes to researchers specific enzymes to researchers