Introduction to Molecular and Cell Biologyutminers.utep.edu/raguilera/10-1301molbio.pdf · 2010-08-30 · Aguilera Lecture 2010 4 Permeable to gases and water Active transport requires

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’’


1313

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

Documents

Introduction to Molecular and Cell Biologyutminers.utep.edu/raguilera/10-1301molbio.pdf · 2010-08-30 · Aguilera Lecture 2010 4 Permeable to gases and water Active transport requires