Download File - adv biology aims

yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Document related concepts

DNA repair wikipedia, lookup

DNA profiling wikipedia, lookup

Homologous recombination wikipedia, lookup

Helicase wikipedia, lookup

DNA virus wikipedia, lookup

DNA nanotechnology wikipedia, lookup

DNA replication wikipedia, lookup

United Kingdom National DNA Database wikipedia, lookup

DNA polymerase wikipedia, lookup

Microsatellite wikipedia, lookup

Replisome wikipedia, lookup

Helitron (biology) wikipedia, lookup

Studies of bacteria and viruses ushered in the
field of molecular biology, the study of heredity
at the molecular level, and revealed the role of
DNA in heredity.
At first, people believed that proteins served
as the genetic material, even though DNA
had already been discovered (1869). It
wasn't until the 1940s that this theory was
People believed that proteins were
responsible for storing genetic information
because of its complexity. They are made of
20 different amino acids, but DNA has only
4 bases. It was concluded that more
complexity would account for diversity in
But as more research was done, it was
discovered that DNA was actually the
genetic material.
The Griffith
Frederick Griffith, established that there was a
transforming principle in bacterial genetics in a
ground-breaking experiment, performed in
Frederick Griffith
He postulated that information could somehow
be transferred between different strains of
bacteria. This was long before the discovery
of DNA and was an inspired piece of
scientific detective work.
The Experiment
Griffith used two strains of Pneumococcus bacteria, type III-S and type II-R. The III-S strain has a smooth polysaccharide
coat which makes it resistant to the immune system of mice, whereas the II-R strain lacks this coat and so will be
destroyed by the immune system of the host.
1. For the first stage, Griffith showed that mice injected with III-S died but when injected with II-R lived and showed few
2. The next stage showed that if the mice were injected with type III-S that had been killed by heat, the mice all lived,
indicating that the bacteria had been rendered ineffective.
3. The interesting results came with the third part of the experiment, where mice were injected with a mixture of heat
killed III-S and live II-R. The mice all died, indicating that some sort on information had been passed from the dead
type III-S to the live type II-R. Blood sampling showed that the blood of the dead mice contained both live type III-S
and live type II-R bacteria.
Somehow the type III-S had been transformed into the type III-R strain, a process he named the transforming principle.
Griffith concluded that the type II-R had been
"transformed" into the lethal III-S strain by a
"transforming principle" that was somehow part of
the dead III-S strain bacteria.
Today, we know that the "transforming principle"
Griffith observed was the DNA of the III-S strain
bacteria. While the bacteria had been killed, the
DNA had survived the heating process and was
taken up by the II-R strain bacteria. The III-S strain
DNA contains the genes that form the protective
polysaccharide capsule. Equipped with this gene,
the former II-R strain bacteria were now protected
from the host's immune system and could kill the
host. The exact nature of the transforming principle
(DNA) was verified in the experiments done by
Avery, McLeod and McCarty and by Hershey and
The Avery-McLeodMcCarty Experiment
Three scientists, Oswald Avery, Colin MacLeod,
and Maclyn McCarty, managed to show that
Frederick Griffith’s transforming factor was in
fact DNA, i.e. DNA is the heritable substance.
In 1944, Oswald Avery and colleagues expanded
upon the findings of Frederick Griffith to
demonstrate that DNA is the genetic material.
The Experiment
They prepared cultures containing the
heat-killed III-S strain and then
removed lipids and carbohydrates from
the solution.
Next they treated the solutions with
different digestive enzymes (DNase,
RNase or protease) to destroy the
targeted compound.
Finally, they introduced living II-R cells
to the culture to see which cultures
would develop transformed III-S
Only in the culture treated with DNase did the
III-S strain bacteria fail to grow (i.e. no DNA
= no transformation)
The results indicated that DNA was the genetic
component that was being transferred between
cells. It was the first experimental evidence that
showed that DNA was the genetic material.
Despite this finding, the scientific community was
reluctant to accept the role of DNA as a genetic
material. It was only 8 years later, when Hershey
and Chase conducted their experiment, that the
concept was accepted.
The Hershey-Chase
Alfred Hershey and Martha Chase did their
famous experiment in 1952 which helped
confirm that DNA is the genetic material.
The Experiment
Viruses (T2 bacteriophage) were grown in one of
two isotopic mediums in order to radioactively
label a specific viral component .
Viruses grown in radioactive sulfur (35S)
had radiolabelled proteins (sulfur is
present in proteins but not DNA).
Viruses grown in radioactive phosphorus
(32P) had radiolabeled DNA (phosphorus
is present in DNA but not proteins).
1. The viruses were then allowed to infect a
bacterium (E. coli) and then the virus and
bacteria were separated via centrifugation.
The larger bacteria formed a solid pellet
while the smaller viruses remained in the
The bacterial pellet was found to be radioactive when
infected by the 32P–viruses (DNA) but not the 35S–
viruses (protein).
DNA and RNA are nucleic acids. They
are polymers of nucleotides
One of the two strands of DNA is a DNA
polynucleotide, a nucleotide polymer
A nucleotide is composed of a
a. nitrogenous base,
b. five-carbon sugar, and
c. phosphate group.
• It was known that DNA is a polymer of nucleotides, each
consisting of a nitrogenous base, a sugar, and a phosphate group.
• A nucleotide without a phosphate is called nucleoside
• Each type of DNA nucleotide has a different nitrogen-containing
1. adenine (A) ,
2. cytosine (C)
3. thymine (T)
4. and guanine (G)
• RNA (ribonucleic acid) is unlike DNA in that it uses the sugar ribose
(instead of deoxyribose in DNA) and RNA has the nitrogenous base uracil
(U) instead of thymine
• The nucleotides are joined to one another by a sugar-phosphate
• In 1950, Erwin Chargaff reported that DNA composition varies from one
species to the next. Chargaff’s rules state that in any species there is an
equal number of A and T bases, and an equal number of G and C bases
Scientific Discovery: DNA
• DNA is a double-stranded helix
• In 1952, after the Hershey-Chase experiment demonstrated that the
genetic material was most likely DNA, a race was on to describe the
structure of DNA and b. explain how the structure and properties of
DNA can account for its role in heredity.
• In 1953, James D. Watson and Francis Crick deduced the secondary
structure of DNA, using X-ray crystallography data of DNA from the
work of Rosalind Franklin and Maurice Wilkins and Chargaff’s
observation that in DNA,
• the amount of adenine was equal to the amount of thymine and ii.
the amount of guanine was equal to that of cytosine
• Watson and Crick reported that DNA consisted of two polynucleotide
strands wrapped into a double helix.
• The sugar-phosphate backbone is on the outside. b. The strands are antiparallel to each other
• The nitrogenous bases are perpendicular to the backbone in the interior.
• Specific pairs of bases give the helix a uniform shape.
• A pairs with T, forming two hydrogen bonds, and G pairs with C forming
three hydrogen bonds.
• In 1962, the Nobel Prize was awarded to James D. Watson, Francis Crick, and
Maurice Wilkins. Rosalind Franklin probably would have received the prize as
well but for her death from cancer in 1958. Nobel Prizes are never awarded
• The Watson-Crick model gave new meaning to the words genes and
chromosomes. The genetic information in a chromosome is encoded in the
nucleotide sequence of DNA
The bacterial chromosome is a double-stranded, circular DNA molecule associated with a small
amount of protein
Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein
In a bacterium, the DNA is “supercoiled” and found in a region of the cell called the nucleoid
Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells
Histones are proteins that are responsible for the first level of DNA packing in chromatin.
Histones have large amounts of positively charged amino acids (lysine and arginine) thus they can
bond with negatively charged phosphate groups
Chromatin is organized into fibers 10-nm fiber. DNA winds around histones to form nucleosome
“beads”. Nucleosomes are strung together like beads on a string by linker DNA 30-nm fiber.
Interactions between nucleosomes cause the thin fiber to coil or fold into this thicker fiber 300nm fiber. The 30-nm fiber forms looped domains that attach to proteins
Most chromatin is loosely packed in the nucleus during interphase and condenses prior to
mitosis. Loosely packed chromatin is called eu-chromatin, and is the one transcribe during
transcription. During interphase a few regions of chromatin (centromeres and telomeres) are
highly condensed into heterochromatin. Dense packing of the heterochromatin makes it difficult
for the cell to express genetic information coded in these regions.
• Histones can undergo chemical modifications that result in changes in
chromatin organization.
• Acetylation : Adding an acetyl group neutralizes the positive charge and
activates genes by opening up the chromatin structure. In formation of the
Barr body the H4 histone is greatly under acetylated
• Methylation : Addition of methyl groups to arginine and lysine correlates with
inactivation of genes
• Phosphorylation
• Phosphate groups can be added to hydroxyl groups of amin acids,
serine and histidine. This introduces a negative charge to the protein.
Increased phosphorylation is associated with specific times during the
cell cycle and has been linked to gene activation
• DNA replication depends on specific base pairing
• In their description of the structure of DNA, Watson and Crick noted that
the structure of DNA suggests a possible copying mechanism.
• DNA replication follows a semiconservative model.
• The two DNA strands separate.
• Each strand is used as a pattern to produce a complementary strand,
using specific base pairing.
• Each new DNA helix has one old strand with one new strand.
• Experiments by Matthew Meselson and Franklin Stahl supported the
semiconservative model
• They labeled the nucleotides of the old strands with a heavy isotope of
nitrogen, while any new nucleotides were labeled with a lighter isotope.
• The first replication produced a band of hybrid DNA, eliminating the
conservative model
• A second replication produced both light and hybrid DNA, eliminating the
dispersive model and supporting the semiconservative model (see figure
on the next page).
DNA Replication: A Closer Look
• DNA replication proceeds in two directions at many sites simultaneously
• DNA replication begins at special sites called the “origins of replication”
a. DNA unwinds at the origin to produce a “bubble,”
b. Replication proceeds in both directions from the origin, and
• replication ends when products from the bubbles merge with each other.
• At the end of each replication bubble is a replication fork, a Y-shaped
region where new DNA strands are elongating
• DNA replication occurs in the 5´ to 3´ direction.
• a. Replication is continuous on the 3´ to 5´ template.
• b. Replication is discontinuous on the 5´ to 3´ template, forming short
• Two key proteins are involved in DNA replication.
a. DNA ligase joins small fragments into a continuous chain.
b. DNA polymerase adds nucleotides to a growing chain and proofreads and
corrects improper base pairings.
• Helicases are enzymes that untwist the double helix at the replication
• Single-strand binding protein binds to and stabilizes single-stranded DNA
until it can be used as a template
• Topoisomerase corrects “over winding” ahead of replication forks by
breaking, swiveling, and rejoining DNA strands. It cuts and rejoins the helix
The sequence of DNA replication is as follows
• Enzyme DNA Helicase unwinds our double helix into two strands. Singlestrand binding proteins bind to the unwound DNA strands to keep them
• An enzyme called primase start an RNA chain from scratch and adds RNA
nucleotides one at a time using the parental DNA as a template to form an
initial nucleotide strand that is a short RNA primer.
• A primer is needed because DNA polymerases do not intitiate synthesis of
a polynucleotide. They only add nucleotides to the 3` end. The primer is
short (5–10 nucleotides long), and the 3 end on the template serves as
the starting point for the new DNA strand.
• Enzymes DNA polymerases catalyze the elongation of new DNA at a
replication fork. The rate of elongation is about 500 nucleotides per
second in bacteria and 50 per second in human cells. The incoporation of
nucleotides by DNA polymerase is shown in the figure on the right
• DNA polymerases add nucleotides only to the free 3 end of a growing
strand; therefore, a new DNA strand can elongate only in the 5 to 3
• Along one template strand of DNA, the DNA polymerase synthesizes a
leading strand continuously, moving toward the replication fork
• To elongate the other new strand, called the lagging strand, DNA
polymerase must work in the direction away from the replication
fork. The lagging strand is synthesized as a series of segments called
Okazaki fragments, which are joined together by DNA ligase (see
figure on left). The proteins that participate in DNA replication form
a large complex, a “DNA replication machine
Proofreading and Repairing DNA
• DNA polymerases proofread newly made DNA, replacing any incorrect
nucleotides. DNA polymerases and DNA ligase also repair DNA damaged by
harmful radiation and toxic chemicals.
• In mismatch repair of DNA, repair enzymes correct errors in base pairing.
• DNA can be damaged by chemicals, radioactive emissions, X-rays, UV
light, and certain molecules (in cigarette smoke for example)
• In nucleotide excision repair, a nuclease cuts out and replaces damaged
stretches of DNA
• Limitations of DNA polymerase create problems for the linear DNA of
eukaryotic chromosomes
• The usual replication machinery provides no way to complete the 5
• ends, so repeated rounds of replication produce shorter DNA molecules
• Eukaryotic chromosomal DNA molecules have at their ends nucleotide
sequences called telomeres
• Telomeres do not prevent the shortening of DNA molecules, but they do
postpone the erosion of genes near the ends of DNA molecules
• It has been proposed that the shortening of telomeres is connected to
• DNA replication ensures that all the somatic cells in a multicellular
organism carry the same genetic information.
• The Flow of Genetic Information from DNA to RNA to Protein.
DNA Genotype Expression The DNA genotype is expressed as proteins, which provide the
molecular basis for phenotypic traits.
Gene expression, the process by which DNA directs protein synthesis, includes two stages:
transcription and translation
DNA specifies traits by dictating protein synthesis.
The molecular chain of command is from DNA in the nucleus to RNA and RNA in the cytoplasm
to protein.
Transcription is the synthesis of RNA under the direction of DNA.
Translation is the synthesis of proteins under the direction of RNA.
The connections between genes and proteins
The initial one gene–one enzyme hypothesis was based on studies of inherited metabolic
The one gene–one enzyme hypothesis was expanded to include all proteins.
Most recently, the one gene–one polypeptide hypothesis recognizes that some proteins are
composed of multiple polypeptides (see figure below).
Translation of Codons into Amino Acid Sequence
• Genetic information written in codons is translated into amino acid
• Genetic information written in codons is translated into amino acid
• Protein construction requires a conversion of a nucleotide sequence
to an amino acid sequence
• Transcription rewrites the DNA code into RNA, using the same
nucleotide “language
• The flow of information from gene to protein is based on a triplet
code: The genetic instructions for the amino acid sequence of a
polypeptide chain are written in DNA and RNA as a series of nonoverlapping three-base “words” called codons.
• Translation involves switching from the nucleotide “language” to
the amino acid “language.”
• Each amino acid is specified by a codon
i. 64 codons are possible.
ii. Some amino acids have more than one possible codon.
The Genetic Code
• The genetic code dictates how codons are translated into amino
 Characteristics of the genetic code
o Three nucleotides specify one amino acid.
i. 61 codons correspond to amino acids.
ii. AUG codes for methionine and signals the start of transcription.
iii. 3 “stop” codons signal the end of translation (See the dictionary
of the genetic code).
The genetic code is
a. redundant, with more than one codon for some amino acids,
b. unambiguous in that any codon for one amino acid does not code
for any other amino acid,
c. nearly universal—the genetic code is shared by organisms from the
simplest bacteria to the most complex plants and animals, and
d. without punctuation in that codons are adjacent to each other with
no gaps in between
DNA Transcription
• Transcription produces genetic messages in the form of RNA
• An RNA molecule is transcribed from a DNA template by process that
resembles the synthesis of a DNA strand during DNA replication.
• RNA nucleotides are linked by the transcription enzyme RNA
• Specific sequences of nucleotides along the DNA mark where
transcription begins and ends
• The “start transcribe” signal is a nucleotide sequence called a promoter.
• Transcription begins with initiation, as the RNA polymerase attaches to the
• During the second phase, elongation, the RNA grows longer. As the RNA
peels away, the DNA strands rejoin.
• Finally, in the third phase, termination, the RNA polymerase reaches a
sequence of bases in the DNA template called a terminator, which signals
the end of the gene.
• The polymerase molecule now detaches from the RNA molecule and the
RNA Processing
• Eukaryotic RNA is processed before leaving the nucleus as
• Messenger RNA (mRNA) encodes amino acid sequences and
conveys genetic messages from DNA to the translation
machinery of the cell, which in
i. prokaryotes occurs in the same place that mRNA is made
ii. but in eukaryote, mRNA must exit the nucleus via nuclear
pores to enter the cytoplasm
• Eukaryotic mRNA has introns, interrupting sequences that
separate exons, the coding regions.
• Eukaryotic mRNA undergoes processing before leaving the
a. RNA splicing removes introns and joins exons to produce a
continuous coding sequence.
b. A cap and tail of extra nucleotides are added to the ends
of the mRNA to
i. facilitate the export of the mRNA from the nucleus,
ii. protect the mRNA from attack by cellular enzymes, and
iii. help ribosomes bind to the mRNA.
• tRNA Molecules Transfer RNA molecules serve as interpreters during
• A tRNA molecule consists of a single RNA strand that is only about 80
nucleotides long
• Flattened into one plane to reveal its base pairing, a tRNA molecule
looks like a cloverleaf. Because of hydrogen bonds, tRNA actually
twists and folds into a three-dimensional molecule tRNA is roughly Lshaped as shown in the figure below
• Transfer RNA (tRNA) molecules function as a language interpreter,
converting the genetic message of mRNA into the language of
• Transfer RNA molecules perform this interpreter task by picking up
the appropriate amino acid and using a special triplet of bases, called
an anticodon, to recognize the appropriate codons in the mRNA.
• Accurate translation requires two steps: – First: a correct match
between a tRNA and an amino acid, done by the enzyme aminoacyltRNA synthetase – Second: a correct match between the tRNA
anticodon and an mRNA codon
• The viral genome is the complete
genetic complement contained in a
DNA or RNA molecule in a virus.
• Viral DNA may become part of the
host chromosome
• A virus is essentially “genes in a box
,” an infectious particle consisting of
a bit of nucleic acid, wrapped in a
protein coat called a capsid and in
some cases, a membrane envelop.
Reproductive cycle of viruses
• Viruses have two types of reproductive cycles.
a. In the lytic cycle,
i. viral particles are produced using host cell
ii. the host cell lyses, and
iii. viruses are released
•Lysogenic cycle
i. Viral DNA is inserted into the host
chromosome by recombination.
ii. Viral DNA is duplicated along with the host
chromosome during each cell division.
iii. The inserted phage DNA is called a
iv. Most prophage genes are inactive.
• Environmental signals can cause a switch to the lytic
cycle, causing the viral DNA to be excised from the
bacterial chromosome and leading to the death of the
host cell
• A phage that reproduces only by the lytic cycle is called
a virulent phage
• Bacteria have defenses against phages, including
restriction enzymes that recognize and cut up certain
phage DNA
• Many viruses cause disease in animals and plants
 Viruses can cause disease in animals and plants.
DNA viruses and RNA viruses cause disease in animals.
 A typical animal virus has a membranous outer envelope and projecting
spikes of glycoprotein.
The envelope helps the virus enter and leave the host cell.
 Many animal viruses have RNA rather than DNA as their genetic material.
These include viruses that cause the common cold, measles, mumps,
polio, and AIDS.
 The reproductive cycle of the mumps virus, a typical enveloped RNA virus,
has seven major steps:
Seven major steps
Entry of the protein-coated RNA into the cell,
Uncoating—the removal of the protein coat,
RNA synthesis—mRNA synthesis using a viral enzyme,
Protein Synthesis—mrna Is Used To Make Viral Proteins,
New viral genome production—mRNA is used as a template to
synthesize new viral genomes,
6) Assembly—the new coat proteins assemble around the new viral RNA,
7) Exit—the viruses leave the cell by cloaking themselves in the host cell’s
plasma membrane.
• Some animal viruses, such as herpesviruses, reproduce in the cell nucleus. Most
plant viruses are RNA viruses.
To infect a plant, they must get past the outer protective layer of the plant.
Viruses spread from cell to cell through plasmodesmata.
Infection can spread to other plants by insects, herbivores, humans, or farming
There are no cures for most viral diseases of plants or
Human Health & Viruses
• Emerging viruses threaten human health
• Viruses that appear suddenly or are new to medical scientists are
called emerging viruses. These include the AIDS virus, Ebola virus,
West Nile virus, and SARS virus.
• Three processes contribute to the emergence of viral diseases:
a. mutation—RNA viruses mutate rapidly.
b. contact between species—viruses from other animals spread to
c. spread from isolated human populations to larger human
populations, often over great distances.
HIV Virus & AIDS
• The AIDS virus makes DNA on an RNA template
• AIDS (acquired immunodeficiency syndrome) is
caused by HIV (human immunodeficiency virus).
• HIV is an RNA virus, has two copies of its RNA
genome, and carries molecules of reverse
transcriptase, which causes reverse transcription,
producing DNA from an RNA template.
• After HIV RNA is uncoated in the cytoplasm of the host cell,
Reverse transcriptase makes one DNA strand from RNA,
Reverse transcriptase adds a complementary DNA strand,
Double-stranded viral DNA enters the nucleus and integrates into the chromosome,
becoming a provirus,
The provirus DNA is used to produce mRNA,
The viral mRNA is translated to produce viral proteins, and
New viral particles are assembled, leave the host cell, and can then infect other cells.
Viroids & Prions
• Viroids and prions are formidable pathogens in plants and
• Some infectious agents are made only of RNA or protein.
 Viroids are small, circular RNA molecules that infect plants.
replicate within host cells without producing
proteins and interfere with plant growth.
 Prions are infectious proteins that cause
degenerative brain diseases in animals.
Prions appear to be misfolded forms of normal
brain proteins, which convert normal protein to
misfolded form.
Bacterial Genetics
• Bacteria can transfer DNA in three ways
• Viral reproduction allows researchers to learn more about the
mechanisms that regulate DNA replication and gene expression in
living cells.
• Bacteria are also valuable but for different reasons.
Bacterial DNA is found in a single, closed loop chromosome.
 Bacterial cells divide by replication of the bacterial chromosome and
then by binary fission.
Because binary fission is an asexual process, bacteria in a colony are
genetically identical to the parent cell.
• Bacteria use three mechanisms to move genes from
cell to cell These include:
• Transformation is the uptake of DNA from the
surrounding environment.
• Transduction is gene transfer by phages.
• Conjugation is the transfer of DNA from a donor to a
recipient bacterial cell through a cytoplasmic (mating)
• Once new DNA gets into a bacterial cell, part of it may
then integrate into the recipient’s chromosome.
• Bacterial plasmids can serve as
carriers for gene transfer
• The ability of a donor E. coli
cell to carry out conjugation is
usually due to a specific piece
of DNA called the F factor.
• During conjugation, the F
factor is integrated into the
bacterium’s chromosome.
• The donor chromosome starts replicating at
the F factor’s origin of replication.
• The growing copy of the DNA peels off and
heads into the recipient cell.
• The F factor serves as the leading end of the
transferred DNA.
• An F factor can also exist as a plasmid, a
small circular DNA molecule separate from
the bacterial chromosome
• The F factor serves as the leading end of the transferred
• An F factor can also exist as a plasmid, a small circular DNA
molecule separate from the bacterial chromosome.
a. Some plasmids, including the F factor, can bring about
conjugation and move to another cell in linear form.
b. The transferred plasmid re-forms a circle in the recipient
• R plasmids pose serious problems for human medicine by
carrying genes for enzymes that destroy antibiotics.