Uploaded by sarah20042

chapter 3 overview of DNA structure, function and engineering

advertisement
Chapter 3 overview of DNA
structure, function, and engineering
Nucleic Acids
• Nucleic acids represent the fourth major class
of biomolecules (other major classes of
biomolecules are proteins, carbohydrates, fats)
– 1869 Friedrich Miescher
– 1944 Avery, MacLeod, McCarty
– 1953 Watson, Crick
• Genome - the genetic information of an
organism
Biofunctions of nucleotide
• Nucleotides are involved in nearly every facet of
cellular life, participate in
–
–
–
–
Oxidation-reduction reactions
Energy transfer
Intracellular signaling
Biosynthetic reactions
• Their polymers (DNA and RNA) are the primary
player in the storage and decoding of genetic
information
• Nucleotide and nucleic acid also perform
structural and catalytic roles in cells
Biofunctions of nucleotide
• In the view of evolution
the appearance of nucleotides permitted the
evolution of organisms that could harvest and
store energy from their surroundings and
could make copies of themselves
• RNA world is controversial, however, it is
incontrovertible that life as we know it is
inextricably linked to the chemistry of nucleotides
and nucleic acid
Nucleotides Are the Building Blocks of
Nucleic Acids
• Nucleic acids are polynucleotides
• Nucleotides have three components:
(1) A five-carbon sugar
(2) A weakly basic nitrogen base
(3) Phosphate
• Nucleotides are phosphate esters of
nucleosides
Nucleoside
N-9 of purine
N-1 of pyrimidine
nucleotide
Nucleotides
• Nucleotides are phosphorylated derivatives of
nucleosides
• Ribonucleosides contain three potential
hydroxyl groups (2’, 3’ and 5’)
• Deoxyribonucleosides can be phosphorylated
at the 3’ and 5’ positions
• A nucleotide is assumed to be 5’-phosphate
unless specified otherwise
Participate starch synthesis
NUCLEIC ACID STRUCTURE
Nucleotides joined by 3’-5’
phosphodiester linkages to
form nucleic acid
Monomer , dimer, trimer, tetramer …..
to oligomer
• As the size of the polymer
increases, the physical properties
(charge and solubility )may change
A polymer of nonidentical residues
has a property that its component
monomers lack--• it contains information in the form
of its sequence of residues
Base pair rules
1940s Chargaff
DNA has equal numbers of adenine and thymine residues (A=T)
and equal number of guanine and cytosine residues (G=C)
1. DNA’s base composition varies widely among different organisms
It ranges from ~25 to 75 mol % G+C in different species of
bacteria
2. It is more or less constant among related species
in mammals G+C ranges from 39 to 46%
Tautomeric forms of purine and pyrimidine
Major features of Watson-Crick model
of DNA
• Tow polynucleotide chains wind around a
common axis to form a double helix
• The two strands of DNA are antiparallel, but
each forms a right-handed helix
• The bases occupy the core of the helix and
sugar-phosphate chains run along the
periphery. The surface of the double helix
contains two grooves of unequal width: the
major and minor grooves
Major features of Watson-Crick model
of DNA
• Each base is hydrogen bonded to a base in the
opposite strand to form a planar base pair–
complementary base pairing
– Accounts for Chargaff’s rule
– It suggests that each DNA strand can act as a
template for the synthesis of its complementary
strand
– hence that hereditary information is encoded in
the sequence of bases on either strand
Complementary base
pairing in DNA
RNA is a single-stranded nucleic acid
RNA world
• The intricate structures of RNA molecules
provide additional evidence that RNA can do
more than just store and transmit genetic
information
• Certain RNA ca specifically bind small organic
molecules and can catalyze reactions
• Many of the processes essential for life began
through the chemical versatility of small
polynucleotides
OVERVIEW OF NUCLEIC ACID
FUNCTION
DNA carries genetic information
replication
Information specifying protein structure
• Information
• Transcriptionflow:
- copying of the DNA sequence
information intoRNA
RNA
DNA
PROTEIN
• Messenger RNA or mRNA
• Translation - Information in RNA molecules is
translated during polypeptide chain synthesis
• Ribosomal RNA or rRNA
• Transfer RNA or tRNA
• Genomics
the study of the genome’s size, organization and
gene content
• Transcriptomics
the study of gene expression, which focuses on
the set of mRNA molecules (or transcriptome)
that is transcribed from DNA under any
particular set of circumstances
• Proteomics
the study of the proteins (or proteome) produced
as a result of transcription and translation
NUCLEIC ACID SEQUENCING
Restriction enzymes
• Restriction endonucleases
• Discovered by
– Werner Arber and Hamilton Smith
– 1960s Daniel Nathans pioneered their use
• Are found in a wide variety of prokaryotes
– Biological role is to cleave the foreign DNA
molecules but cell’s own DNA is intact
• Recognize specific base sequences in dsDNA
– Cleave at specific places of both strands
restriction enzymes
• Restriction endonuclease
– Type I
– Type II
– Type III
• Type II enzymes
–
–
–
–
Nomenclature
Characteristics of type II RE
Recognition sequence
Type of fragment end produced
Electrophoresis
The chain –terminator method of
sequencing
• Sanger method uses 2’,3’-dideoxynucleoside
triphosphates (ddNTPs) which are incorporated at
the 3’ end of a growing chain in place of a dNTP
• Since ddNTPs lack a 3’-hydroxyl group,
subsequent nucleotide addition cannot take place
• Small amounts of ddNTP’s terminate replication
of some chains at each step, leaving a set of
fragments of different lengths
Newer sequencing technologies use
light or voltage changes
• Pyrosequencing
PPDK(Pyruvate orthophosphate
dikinase)or sulfurylase
http://www.intechopen.com/source/html/18657/media/image8.jpeg
Fiber-optic slide contains
numerous wells
~400,000 templates
Read 300-500 nts
Other detector: Proton
generated on
pyrophosphate release
http://openi.nlm.nih.gov/imgs/512/358/3266102/3266102_JOM-4-10743-g001.png
Data bases
And entire Genome
Metagenomic sequencing
• The DNA sequences of multiple organisms are
analyzed as a single data set
• Used to characterize complex microbial
communities
– Marine environments, or human gut, where individual
species cannot be cultured and sequenced one by one
– Reveal the overall gene number and an estimate of
the collective metabolic capabilities of the community
• In human gut: 3 million genes have been identified and
representing some 1000 bacterial species
Human genome
• 2004
• About half the human genome consists of
repeating sequences of various types
• Up to 80% of the genome may be transcribed to
RNA
• Only 1.2% of the genome encodes protein
• The human genome appears to contain only
~23,000 protein-encoding genes (or ORFs)
– ~6000 ORFs in yeast, ~13,000 ORFs in Drosophila,
~19,000 ORFs in C. elegans, 26,000 ORFs in
Arabidopsis
Human genome
• Only a small fraction of human proteins are
unique to vertebrates; most occur in other if
not all life-forms
• Two randomly selected human genomes differ,
on average, by only 1 nt per 1000;
• any two people are likely to be 〉99.9% genetically
identical
Vertebrate and invertebrate
• It is unlikely to be due to the not-much-larger
numbers of ORFs that vertebrates encode
• Rather, vertebrate proteins themselves are
more complex that those of invertebrates
– Vertebrate proteins tend to have more
domains(modules) than invertebrate proteins
– These modules are more often selectively
expressed through alternative gene splicing
Evolution results from sequence
mutations
• DNA is a dynamic molecule, subject to changes
that alter genetic information
• Point mutations
– DNA misreplication
– DNA damage
• Extensive alteration of genetic information
– Faulty recombination
– transposition (within an organism or between
organisms)
• These changes are the raw material for natural
selection
• A mutation in a gene segment that does not
encode protein might interfere with the binding
of cellular factors that influence the timing of
transcription
• A mutation in a gene encoding an RNA might
interfere with the binding of factors that affect
the efficiency of translation
• A minor rearrangement of genes could disrupt an
entire developmental process resulting in the
appearance of a novel species
Sequence variations can be linked to
human diseases
Single-nucleotide polymorphisms (SNPs, instances where
the DNA sequence differs among individuals at one
nucleotide)
Recombinant DNA technology, molecular cloning, genetic engineering
Cloned DNA is an amplified copy
MANIPULATING DNA
Cloning vectors
• Plasmids
• Viruses:
– Bacteriophage λ
– Baculoviruses -- insect cells
• Artificial chromosomes
– A. bacterial artificial chromosomes (BAC)
• Capacity of this vectors :300 kb
– B. Yeast artificial chromosomes (YAC)
• Capacity of this vectors :1000 kb
DNA libraries
• Are collections of cloned DNA, or the cloned
set of all DNA fragments from a particular
organism
• Genomic library
– Shotgun cloning
– Chromosomal DNA cleaved to fragments of
cloneable size (partial digestion or mechanically
fragmented)
inserted into a cloning vector
• Contains intact representatives of all the organism’s
genes, including those that contain restriction sites
The size of the genomic library
P = 1 – (1 – f )N
The probability P that a set of N clones contains
a fragment that constitutes a fraction f, in bp , of
the organism’s genome
N = log (1 – P)/log(1 - f)
p. 66 sample calculation 3-1
An exercise
• P = 0.99 for a fragments averaging 10 kb in
length for the 4600-kb E. coli chromosome,
N=?
N = log (1 – P)/log(1 - f)
= log (1-0.99)/log(1-10/4600)
N ~2000
• Size of library will decline as the cloning
capacity of cloning vector increases
cDNA library
• Isolating all the cell’s mRNAs
copying them
to cDNA (complementary DNA by using
mRNAs as the template for enzyme reverse
transcriptase)
insert cDNA into cloning
vectors
• A collection of the expressed sequences from
a particular cell type
• DNA microarray (DNA chip)
Screening
library
Colony or in situ
hybridization
1. Genomic lib
probe
2. cDNA lib
Protein products
Probes:
Antibody specific to the
protein product
Polymerase chain reaction
• 1983: PCR Invented
– Kary B. Mullis
– PCR- the polymerase chain reaction
– PCR can make billions of copies of a specific
segment of DNA
– The 1993 Nobel Prize in Chemistry was given for
the invention of PCR.
74
movie
DNA fingerprinting
DNA sequence variations or genetic polymorphism occur among
individuals
Many repetitions do not encode genes
Short tandem repeats (STRs): contain variable numbers of repeating
segments of two to seven base pairs
The number of repeats at any one site on the DNA varies between
individuals, even within family
allele
Practical applications of recombinant
DNA technology
• Cloned gene can be expressed
– Expression vector
• Two problems of expression of eukaryotic genes in
bacteria
– Intron splicing
– Posttranslational modification
Practical applications of recombinant
DNA technology
• Cloned gene can be expressed
– Expression vector
• Two problems of expression of eukaryotic genes in
bacteria
– Intron splicing
– Posttranslational modification
• Site-directed mutagenesis
– Allows predictions about the structural and
functional roles of particular amino acids in a
protein
Oligonucleotidedirected
mutagenesis
Practical applications of recombinant
DNA technology
•
•
Cloned gene can be expressed
– Expression vector
• Two problems of expression of eukaryotic genes in bacteria
– Intron splicing
– Posttranslational modification
Site-directed mutagenesis
– Allows predictions about the structural and functional roles of particular amino acids in
a protein
• Transgenic organisms
– Multicellular organisms expressing a gene from
another organism are said to be
– Transgene
Practical applications of recombinant
DNA technology
•
•
•
Cloned gene can be expressed
– Expression vector
• Two problems of expression of eukaryotic genes in bacteria
– Intron splicing
– Posttranslational modification
Site-directed mutagenesis
– Allows predictions about the structural and functional roles of particular amino acids in a
protein
Transgenic organisms
– Multicellular organisms expressing a gene from another organism are said to be
– Transgene
• Genetic defects can be corrected
– Gene therapy
• Sever combined immunodeficiency disease (SCID)
• Leber’s congenital amaurosis
• X-linked adrenoleukodystrophy