경희대학교 의과대학·의학전문대학원

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Fundamentals of

Molecular Biology

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4 Fundamentals of Molecular Biology

Chapter Outline



_{Heredity, Genes, and DNA}



Expression of Genetic Information



_{Recombinant DNA}



Detection of Nucleic Acids and

Proteins

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Introduction

The focus of molecular biology concerns the mechanisms responsible for

transmission and expression of genetic information.

Fundamental experiments on model organisms such as bacteria and viruses have provided information on molecular mechanisms that operate in all organisms.

Recombinant DNA technology allowed fundamental principles and experimental approaches to be extended to eukaryotic cells.

Recombinant DNA allowed isolation and characterization of individual genes, and determination of complete genome sequences.

All organisms inherit genetic information specifying their structure and function from their parents.

All cells arise from pre-existing cells, so the genetic material must be

replicated and passed from parent to progeny at each cell division.--- _왜

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Heredity, Genes, and DNA

The classical principles of genetics were deduced by Gregor

Mendel in 1865, from his experiments with pea plants.

He studied well-defined traits such as seed color, and could predict patterns of inheritance by assuming that each trait is determined by a pair of inherited factors, now called genes. One gene copy (allele) specifying each trait is inherited from

each parent.

Example: strains with identical alleles specifying yellow (Y) or green (y) seeds, are crossed.

The progeny (F₁ generation) are hybrids (Yy) and have yellow seeds: yellow is dominant, green is recessive.

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Genotype is the genetic makeup of an individual. Phenotype is the resulting physical appearance.

The genotype of the F₁ generation: Yy The phenotype: yellow.

Mendel’s results were ignored until 1900 when their importance was recognized.

Shortly afterward, chromosomes were identified as the carriers of genes.

Q) Differences between prokaryotic and eukaryotic genes?

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Most cells of plants and animals are diploid: they have two copies of each chromosome.

Formation of germ cells (sperm and egg) involves a type of cell division (meiosis) in which only one

member of each chromosome pair is transmitted to the progeny cell.

The sperm and egg are haploid: they have only one copy of each chromosome.

At fertilization, two haploid cells are combined to make a new diploid cell.

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Fundamentals of mutation, genetic linkage, and relationships between genes and chromosomes were established by

experiments with the fruit fly Drosophila melanogaster. In the early 1900s, mutations (genetic alterations) were

observed in Drosophila that affected characters such as eye color and wing shape.

Genetic linkage

Breeding experiments showed that some of these genes are inherited independently of each other.

This suggested that the genes are on different chromosomes that segregate independently during meiosis.

Genes located on the same chromosome are inherited together and are said to be linked.

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The relationship between genes and proteins began to emerge in 1909:

It was realized that the inherited disease

phenylketonuria results from a genetic defect in the enzyme needed to metabolize the amino acid phenylalanine -- 발달장애 , 경련 : hydroxylase 결핍 In 1941, experiments with the fungus Neurospora

crassa by Beadle and Tatum found more evidence linking genes with the synthesis of enzymes.

Mutant strains of Neurospora required particular amino acids for growth.

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Each mutation resulted in a deficiency in a specific metabolic pathway, which is governed by enzymes. This led to the conclusion that genes specify enzyme

structure. : one gene and one protein

one gene and one enzyme

It is now known that some genes specify RNAs rather than proteins.

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Evidence that DNA is the genetic material first came from experiments with the bacterium that causes pneumonia (Pneumococcus).

A “transforming principle” was responsible for

inducing the genetic transformation of one strain of the bacteria to another.

The transforming principle was later identified as DNA when it was shown that the activity of the transforming principle is abolished by enzymatic digestion of DNA: but not by digestion of proteins.

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Other studies with viruses confirmed that DNA was the genetic material.

It was shown that when a bacterial virus infects a cell, the viral DNA must enter the cell in order for the virus to replicate (not the viral protein).

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The structure of DNA was determined in 1953 by Watson and Crick, using information from X-ray crystallography and ideas on hydrogen bonding in the α helix of proteins.

DNA is a double helix with the sugar- phosphate backbones on the outside of the molecule.

The bases are on the inside; hydrogen bonds are formed between purines and pyrimidines on

opposite chains.----DNA breathing

The base pairing is very specific: A always pairs with T and G with C.

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DNA replication

Complementary base pairing suggested the mechanism for DNA replication.

The DNA molecule separates, and each strand

becomes a template for synthesis of a new strand:

Semiconservative replication: one strand of

parental DNA is conserved in each progeny DNA molecule.

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Experimental support for semi-conservative DNA

replication:

Meselson and Stahl grew E. coli in medium labeled with the heavy isotope 15N.

The heavier DNA could be separated from light DNA (with 14N) by equilibrium ultracentrifugation in a CsCl

solution.

E. coli grown with 15N were transferred to 14N medium

and allowed to replicate once more.

The DNA from these bacteria was intermediate in weight.

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Further evidence: an enzyme purified from E. coli

(DNA polymerase) could catalyze DNA replication

in vitro.

In the presence of DNA to act as a template, DNA

polymerase directed the incorporation of nucleotides into a complementary DNA molecule.

Genes determine the structure of proteins.

The information in DNA is specified by the order of the four bases.

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Expression of Genetic Information

Proteins are made up of 20 different amino acids, the sequence of which determines the protein function.

The first direct link between a genetic mutation and alteration in a protein was made in 1957.

Patients with inherited sickle-cell anemia had

hemoglobin that differed from normal by a single amino acid substitution.

The next step was determining the relationship between DNA and proteins.

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The simplest explanation: the order of nucleotides in DNA specifies the order of amino acids in proteins (colinearity).

Mutations (changes in the nucleotide sequence) should lead to corresponding changes in the protein.

Using E. coli, Yanofsky and colleagues mapped a series of mutations in the gene that encodes an enzyme for tryptophan synthesis.

The relative positions of the amino acid alterations were the same as those of the corresponding

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Although DNA appeared to specify the order of amino acids in proteins, it did not necessarily follow that DNA itself directs protein synthesis. DNA is located in the nucleus of eukaryotic cells,

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RNA was the likely candidate to be an intermediate because of its similar structure.

RNA is single-stranded, its sugar is ribose, and it contains uracil (U) instead of thymine (T), but this does not affect base-pairing.

The pathway for the flow of genetic information: DNA → RNA → Protein

is now known as the central dogma of molecular biology.

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RNA is synthesized from DNA templates (transcription); proteins are synthesized from RNA templates (translation).

RNA polymerase catalyzes synthesis of messenger RNA (mRNA) from a DNA template.

Ribosomal RNA (rRNA) is a component of ribosomes, sites of protein synthesis.

Transfer RNAs (tRNAs) serve as adaptor molecules that align amino acids along the mRNA template.

Each amino acid is attached by a specific enzyme to its appropriate tRNA.

Base pairing between the tRNA and a complementary sequence on the mRNA directs the attached amino acid to its correct position on the mRNA template.

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How can four nucleotide bases specify the sequence of 20 amino acids?

The nucleotides are used as triplets to encode the different amino acids: genetic code.

Evidence for the triplet code came from bacteriophage T4 with mutations in a gene called rII.

Mutants with the addition of one or two bases always exhibited the mutant phenotype.

Changes in three bases frequently led to the wild-type phenotype.

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Figure 4.12 The triplet UUU encodes phenylalanine

Assigning nucleotide triplets to their

corresponding amino acids was done using

in vitro systems that

could carry out protein synthesis (in vitro

translation).

The systems contain ribosomes, amino acids, tRNAs,

enzymes, and synthetic mRNA with known

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All 64 possible triplets (called codons) were assigned in this way.

61 specify an amino acid; three are stop codons

that signal the termination of protein synthesis. The code is degenerate: many amino acids are

specified by more than one codon.

With few exceptions, all organisms utilize the same genetic code—strong support for the conclusion that all present-day cells evolved from a common ancestor.

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Some viruses contain RNA instead of DNA.

The mode of replication of viral RNA was determined by studies of RNA bacteriophages of E. coli.

These viruses encode an enzyme that catalyzes

synthesis of RNA from an RNA template (

RNA-directed RNA synthesis).

Most animal viruses replicate in this way, but one group (RNA tumor viruses) requires DNA synthesis in

infected cells.

These viruses (now called retroviruses) replicate via synthesis of a DNA intermediate, a DNA provirus.

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This hypothesis was initially met with

disbelief because it reverses the

central dogma.

Later, an enzyme that catalyzes

synthesis of DNA from an RNA

template (reverse transcription) was

discovered.

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Key Experiment, Ch. 4, p. 117

Howard M. Temin

Nobel Prize

in Physiology or Medicine 1975

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Reverse transcription has other broad implications. It also occurs in cells and is frequently responsible

for transposition of DNA from one chromosomal location to another.

Reverse transcriptase can be used experimentally to generate DNA copies of any RNA molecule.

This has allowed mRNAs of eukaryotic cells to be studied using the molecular approaches currently applied to the manipulation of DNA.

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Recombinant DNA

Recombinant DNA technology allows scientists to isolate, sequence, and manipulate individual genes from any type of cell.

It has enabled detailed molecular studies of the structure and function of eukaryotic genes and genomes, and revolutionized our understanding of cell biology.

Restriction endonucleases: enzymes that cleave DNA at

specific sequences.

First identified in bacteria, where they provide defense against the entry of foreign DNA.

Bacteria have a variety of restriction endonucleases that cleave DNA at more than 100 distinct recognition sites.

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Recombinant DNA

EcoRI recognizes the sequence GAATTC.

This sequence is present at five sites in DNA of

bacteriophage λ, so it is digested into six fragments ranging from 3.6 to 21.2 kb long.

1 kilobase (kb) = 1000 base pairs

The fragments can be separated by gel

electrophoresis:

A gel of agarose or polyacrylamide is placed between two electrodes and the sample is added to the gel. Nucleic acids are negatively charged so they migrate toward the positive electrode.

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Recombinant DNA

Smaller molecules move through the gel more

rapidly, allowing the fragments to be separated by size.

The order of restriction fragments can also be determined, and maps of restriction sites

generated.

Detailed restriction maps of viral DNA molecules have been produced.---RFLP

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Recombinant DNA

For larger DNA molecules, restriction

endonuclease digestion alone does not provide sufficient resolution.

For example, the human genome would yield more than 500,000 EcoRI fragments.

Purified DNA fragments can be obtained through molecular cloning.

In molecular cloning a DNA fragment is inserted into a DNA molecule (a vector) that can replicate independently in a host cell.

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Recombinant DNA

The result is a recombinant molecule or molecular

clone.

Fragments of human DNA can be cloned in plasmid

vectors: small circular DNA molecules that can replicate independently in bacteria.

Recombinant plasmids with human DNA inserts can be introduced into E. coli, where they replicate along with the bacteria to yield millions of copies of plasmid DNA. The fragment can be isolated from the rest of the plasmid

DNA by restriction endonuclease digestion and gel electrophoresis, allowing a pure fragment of human DNA to be analyzed and further manipulated.

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Recombinant DNA

Restriction endonucleases cleave the

recognition sequences at staggered

sites, leaving overhanging

single-stranded tails that can associate with

each other by complementary base

pairing.

DNA ligase can then seal the ends

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Recombinant DNA

Synthetic DNA “linkers” containing desired restriction endonuclease sites can be added to the ends of any DNA fragment.

This allows virtually any fragment of DNA to be ligated to a vector and isolated as a molecular clone.

RNA can also be cloned.

RNA is copied using reverse transcriptase. The resulting DNA (cDNA) is ligated to a vector DNA.

This allows mRNA to be isolated as a molecular clone, and allows exploration of the noncoding sequences in eukaryotic genes (introns).

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Recombinant DNA

Plasmids are often used for cloning DNA inserts up to a few thousand base pairs long.

Plasmids have an origin of replication—the DNA sequence that signals the host DNA polymerase to start replication.

Plasmid vectors also carry genes that confer resistance to antibiotics, so bacteria carrying the plasmids can be selected for.--- Selection markers

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Recombinant DNA

Bacteriophage λ vectors can accommodate larger

DNA fragments.

Sequences not needed for virus replication are

removed and replaced with unique restriction sites for insertion of cloned DNA.

The recombinant molecules are then put into E. coli, where they replicate to yield millions of progeny

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Recombinant DNA

For even larger fragments of DNA, five major types of vectors are used.

1. Cosmid vectors contain bacteriophage λ

sequences, origins of replication, and genes for antibiotic resistance, so they are able to replicate as plasmids in bacterial cells.

2. Bacteriophage P1 vectors allow recombinant molecules to be packaged in vitro into P1 phage particles and replicated as plasmids in E. coli.

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Recombinant DNA

3. P1 artificial chromosome (PAC) vectors also have bacteriophage P1 sequences, but are

introduced directly as plasmids into E. coli.

4. Bacterial artificial chromosome (BAC) vectors are derived from a naturally occurring plasmid of

E. coli (the F factor).

5. Yeast artificial chromosome (YAC) vectors contain yeast origins of replication and other

sequences that allow them to replicate as linear chromosome-like molecules in yeast

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Recombinant DNA

Nucleotide sequencing aids the study of protein

structure, gene sequences that regulate

expression, and gene function.

DNA sequencing is usually done with automated

systems.

One method is based on premature termination

of DNA synthesis.

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Recombinant DNA

Dideoxynucleotides are included along with the

normal nucleotides. They are labeled with different fluorescent dyes.

The dideoxynucleotides stop DNA synthesis because no 3 OH group is available for addition of the next nucleotide.

The fragments are separated by gel electrophoresis, and a laser beam excites the fluorescent dyes.

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Recombinant DNA

Next-generation sequencing

allows DNA

to be sequenced faster and less

expensively.

These methods simultaneously determine

sequences of millions of templates by

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Recombinant DNA

Molecular cloning is also used to make large amounts of protein for study.

Many proteins in cells are present in small amounts and can’t be purified.

Cloned genes can be used to engineer vectors that lead to high levels of gene expression in bacteria or

eukaryotic cells.

In bacteria, cDNA is cloned into a plasmid or phage vector (an expression vector).

Inserted genes can be expressed at levels as high as 10% of the total bacterial protein.

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Recombinant DNA

Expression in eukaryotic cells instead of bacteria

may be needed, (e.g., if

posttranslational

modification

of the protein is required).

Cloned genes are inserted into virus vectors.

One such system uses infection of insect cells by

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Detection of Nucleic Acids and Proteins

Detection of specific nucleic acids and

proteins is important for a variety of studies:



Mapping of genes to chromosomes

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_{Analysis of gene expression}



Localization of proteins to subcellular

organelles.

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To isolate large amounts of a single DNA molecule, an

alternative to molecular cloning is the polymerase chain

reaction (PCR), developed in 1988.

DNA polymerase is used in vitro for repeated replication of a defined segment of DNA.

A specific region of DNA can be amplified if the nucleotide sequence surrounding the region is known.

Two primers are designed to initiate DNA synthesis in opposite directions at the desired point.

The synthetic primers are 15–20 bases long.

The reaction starts by heating the template DNA to 95°C to separate the strands.

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The DNA polymerase used (Taq polymerase) is a heat-stable enzyme from Thermus aquaticus, a bacterium that lives in hot springs.

The temperature is then lowered to allow primers to pair and DNA synthesis proceeds.

Two DNA molecules are synthesized from one template.

The cycle can be repeated multiple times, with a two-fold increase in DNA for each cycle.

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PCR amplification can be performed rapidly and automatically.

RNA can also be amplified by PCR if reverse

transcriptase is used to synthesize a cDNA copy first.

If enough of a gene sequence is known so that

primers can be made, PCR can selectively amplify it from complex mixtures, such as total cell DNA or

RNA.

This extraordinary sensitivity has made PCR an important method for a variety of applications.

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The key to detection of specific nucleic acid sequences is base pairing.

In nucleic acid hybridization, DNA strands are separated by high temperatures; when cooled, they re-form double-stranded molecules by

complementary base pairing.

Cloned DNA can be labeled with radioactive

nucleotides or nucleotides modified to fluoresce. This labeled DNA is then used as a probe that

hybridizes with complementary DNA or RNA in complex mixtures.

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Southern blotting:

DNA is digested with a restriction

endonuclease, and the fragments separated

by gel electrophoresis.

The gel is then overlaid with a nitrocellulose

or nylon membrane to which the DNA

fragments are transferred (blotted). The

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Northern blotting is used for detection

of RNA instead of DNA.

It is often used in studies of gene

expression, for example, to determine

whether specific mRNAs are present.

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Recombinant DNA libraries are

collections of clones that contain all the

genomic or mRNA sequences of a

particular cell type.

A genomic library of human DNA can be

made by cloning random DNA fragments

of about 15 kb in a bacteriophage λ

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Any gene for which a probe is available

can be isolated from a recombinant

library.

cDNA clones can be used as probes to

isolate corresponding genomic clones.

Or a gene cloned from one species (e.g.,

mouse) can be used to isolate a related

gene from a different species (e.g.,

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Hybridization to DNA microarrays allows tens of

thousands of genes to be analyzed simultaneously. A DNA microarray is a glass slide or membrane filter

onto which oligonucleotides or fragments of cDNAs are printed by a robotic system in small spots at a high density.

One application of DNA microarrays is in studies of gene expression.

Example: a comparison of the genes expressed by two different types of cells.

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In situ hybridization can be used to detect

homologous DNA or RNA sequences in cell extracts, chromosomes, or intact cells.

Hybridization of fluorescent probes to specific cells or subcellular structures is analyzed by microscopic examination.

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Antibodies can be used as protein probes.

Antibodies are proteins produced by immune system cells (B lymphocytes) that react against foreign

molecules (antigens).

Different antibodies recognize unique antigens. Antibodies can be generated by inoculation of an

animal with any foreign protein.

Monoclonal antibodies can be produced by

culturing clonal lines of B lymphocytes from immunized animals (usually mice).

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Two common methods using antibodies: 1. Immunoblotting (Western blotting)

Proteins are separated by size by

SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

The negatively charged detergent sodium dodecyl sulfate (SDS) denatures the protein and gives it an overall negative charge.

The proteins will migrate towards the positive electrode and are then reacted with labeled antibodies.

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2. Immunoprecipitation:

Cells are incubated with radioactive amino acids to label their proteins, then incubated with antibodies. The resulting antigen-antibody complexes are

isolated and subjected to electrophoresis.

Antibodies can be used to visualize proteins in intact cells.

Cells can be stained with antibodies labeled with fluorescent dyes or tags visible by electron

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Gene Function in Eukaryotes

In classical genetics, gene function has been revealed by the altered phenotypes of mutant organisms.

It is now possible to study the function of a cloned

gene directly by reintroducing it into eukaryotic cells. Yeasts (e.g., Saccharomyces cerevisiae) are used in

studies of eukaryotic cells because they are easily grown in culture, reproduce rapidly, and have a

small genome.

Mutants that have specific nutrient requirements can be easily isolated.

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Temperature-sensitive mutants encode

proteins that are functional at one

temperature (permissive temperature) but

not another (nonpermissive temperature).

The ability to isolate temperature-sensitive

mutants has allowed identification of

genes controlling many fundamental cell

processes.

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A gene corresponding to any yeast mutation

can be cloned based on its functional

activity.

Yeast genes encoding a wide variety of

essential proteins have been identified in

this manner.

In many cases, such genes have also been

useful in identifying and cloning related

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Cloned DNA can also be introduced into

plant and animal cells (gene transfer).

Transfection uses infectious viral

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Other methods include:

• _{Direct microinjection}

_{into the nucleus}

• _{Coprecipitation of DNA with calcium}

phosphate to form small particles that are

taken up by the cells

• Incorporation of DNA into

liposomes

that

fuse with the plasma membrane

• Exposure of cells to brief electric pulses

that open pores in the plasma membrane

(

electroporation

)

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In most of the cells, the DNA is transported to the nucleus, and is transcribed for several days:

transient expression.

In about 1% of cells, the foreign DNA is integrated into the genome and transferred to progeny cells at cell division.

If the transfected DNA contains a selectable marker, the stably transformed cells can be isolated and studied.

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Animal viruses, especially retroviruses,

can be used as vectors to introduce

cloned DNAs into cells.

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Cloned genes can also be introduced

into the germ line of multicellular

organisms.

Mice that carry foreign genes

(transgenic mice) are produced by

microinjection of cloned DNA into the

pronucleus of a fertilized egg.

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Embryonic stem (ES) cells are also

used to get cloned genes into mice.

Cloned DNA is introduced into ES cells

in culture, then transformed cells are

introduced back into mouse embryos.

The offspring are chimeric: a mixture of

cells that arise from normal and

transfected embryonic cells.

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Introducing specific mutations into cloned DNAs (in

vitro mutagenesis) is a powerful tool to study

expression and function of eukaryotic genes.

Sometimes called reverse genetics: a mutation is introduced into a gene first, and its functional

consequence is determined second.

In vitro mutagenesis allows detailed characterization of

the functional roles of both regulatory and protein-coding sequences of cloned genes.

The most common method uses synthetic

oligonucleotides to generate changes in a DNA sequence.

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To determine the role of a cloned gene,

activity of the normal gene copy must

be eliminated.

Homologous recombination: the

mutated copy of the cloned gene

replaces the normal gene copy in the

chromosomal DNA.

This occurs frequently in yeast but is

rare in mammalian cells.

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Gene Function in Eukaryotes: transgenic vs knockout mice

Genes can be inactivated in mouse embryonic stem cells, which can grow into transgenic mice.

The mice yield progeny with mutated copies of the gene on both homologous chromosomes.

The effects of inactivation of a gene can then be investigated in the context of the intact animal.

Homologous recombination has been used to systematically inactivate (knockout) every gene in yeast.

A collection of genome-wide yeast mutants is available for scientists to use to study the function of any desired gene. In mice, there is an international effort to knockout all mice

genes to develop a genome-wide collection of mutant mice for research.

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