molecular genetics dna. what is the significance of dna? why is it so important? because it is...

MOLECULAR GENETICSDNA

What is the significance of DNA? Why is it so important?

• Because it is universal. It is the genetic material for all forms of life.

• It points to a common evolutionary origin of all living things.

• Why is it relevant in your life today?The knowledge of DNA have changed our

lives drastically. From forensics to the biotechnology industry, in agriculture and in medicine to name just a few areas.

Where is DNA and what does it do?• DNA is packed in the chromosomes

and the chromosomes are in the nucleus of all eukaryotic cells.

What is DNA’s function?

DNA’s function is to store and transfer genetic information from one generation to the next.

To do this we need mitosis and meiosis so we can copy our own DNA and pass it on.

What kind of information is encoded in DNA?

• Instructions of how to make proteins.

What does DNA look like?• DNA is a nucleic acid.

• Nucleic acids are made up of nucleotides.

( remember that a nucleotide consists of three parts: a 5 carbon sugar, a phosphate group and a nitrogen base)

DNA looks like a twisted staircase. The handrails or sides are also called its backbone and are made up of sugars and phosphate groups connected to each other. The paired bases made up the rungs of the ladder connecting the two strands.

– The structure of DNA• Consists of two nucleotide strands wrapped around

each other in a double helix

Figure 10.3C Twist

DNA polynucleotide

A

C

T

G

T

Sugar-phosphate backbone

Phosphate group

Nitrogenous base

SugarA

C

T

G

T

Phosphategroup

O

O–

OO P CH2

H3C C

C

C

CN

C

N

H

H

O

O

C

O

O

H

C H H

H

C

H

Nitrogenous base(A, G, C, or T)

Thymine (T)

Sugar(deoxyribose)

DNA nucleotide

DNA nucleotide

•DNA and RNA are polymers of nucleotides– DNA is a nucleic acid

• Made of long chains of nucleotide monomers

Figure 10.2A

Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Fig. 16.5

– DNA has four kinds of nitrogenous bases• A, T, C, and G

A pairs with T and G pairs with C

CC

C

CC

C

O

N

C

H

H

ONH

H3C

H H

H

H

N

N

N

H

OC

H HN

H C

N

N N

N

C

CC

C

H

H

N

N

H

C

CN

C HN

CN

H C

O

H

H

Thymine (T) Cytosine (C) Adenine (A) Guanine (G)

PurinesPyrimidines

Figure 10.2B

– RNA is also a nucleic acid• But has a slightly different sugar• And has U instead of T

Nitrogenous base (A, G, C, or U)

Phosphategroup

O

O–

OO P CH2

HC

C

C

CN

C

N

H

H

O

O

C

O

O

H

C H H

OH

C

H

Uracil (U)

Sugar(ribose)

KeyHydrogen atomCarbon atom

Nitrogen atom

Oxygen atom

Phosphorus atom

Figure 10.2C, D

• In April 1953, James Watson and Francis Crick – published a model for the structure of

deoxyribonucleic acid or DNA.– won the Nobel prize for it

• Your genetic material is the DNA you inherited from your parents.

• Nucleic acids are unique – direct their own replication.

• The resemblance of offspring to their parents – depends on the precise replication of DNA– transmission from one generation to the next.

A little history…


• Once Thomas Morgan’s group showed that genes are located on chromosomes, the two constituents of chromosomes - proteins and DNA - were the candidates for the genetic material.

• Until the 1940s, abundance and variety of proteins seemed to indicate that proteins were the genetic material.

The Search for Genetic Material


Early 1900”s everyone “knew” that the genetic material was protein. In 1928 Fred Griffith trying to produce a

vaccine against pneumonia made scientist doubt .

• Griffith said that protein was not the genetic material because heat denatures proteins and in his experiments heat did not denatured the genetic material

• Still nobody knew what was the genetic material

• Griffith called this phenomenon transformation, a change in genotype and phenotype due to the assimilation of a foreign substance (now known to be DNA) by a cell.


Fig. 16.1

1944, Oswald Avery, McCarty and MacLeod announced that the transforming substance was DNA.

• Still, many biologists were skeptical.– this reflected a belief that the genes of bacteria

could not be similar in composition and function to those humans.

– Avery confirmed Griffith discovery and that the genetic material was a nucleotide and not a protein

THE STRUCTURE OF THE GENETIC MATERIAL• Experiments showed that DNA is the genetic material– The Hershey-Chase experiment showed that

certain viruses reprogram host cells• To produce more viruses by injecting their DNA

Figure 10.1A

Head

Tail

Tail fiber

DNA

300

,00

0

What did Hershey and Chase do?

• They showed that the genetic material of a virus is DNA.

• They separated the protein coat and the DNA of the virus (a phage known as T2) and tagged it with a radioactive isotope and then traced it. The protein coat remained outside and only the DNA when inside the bacteria.

• In 1952, Alfred Hershey and Martha Chase showed that DNA was the genetic material of the phage T2.( a phage is a virus that attacks bacteria)

• The T2 phage (the virus), consisting almost entirely of DNA and protein, attacks Escherichia coli (E. coli), a common intestinal bacteria of mammals.

• This phage can quickly turn an E. coli cell into a T2-producing factory that releases phages when the cell ruptures.


Fig. 16.2a

– The Hershey-Chase experiment

Phage

Bacterium

Radioactiveprotein

DNA

Phage DNA

Empty protein shell Radioactivity

in liquid

PelletCentrifuge

Batch 1Radioactiveprotein

Batch 2RadioactiveDNA

RadioactiveDNA

Centrifuge

Pellet

Radioactivityin pellet

Figure 10.1B

Mix radioactively labeled phages with bacteria. The phages infect the bacterial cells.

1 Agitate in a blender to separate phages outside the bacteria from the cells and their contents.

2 Centrifuge the mixture so bacteria form a pelletat the bottom of the testtube.

3 Measure the radioactivity in the pellet and the liquid.

4

Chargaff’s Rules• By 1947, Erwin Chargaff already knew that DNA was a

polymer of nucleotides consisting of a nitrogenous base, deoxyribose ( a sugar), and a phosphate group.

By 1947 Chargaff had developed rules saying that

The bases could be adenine (A), thymine (T), guanine (G), or cytosine (C).

Chargaff rule:

A always pairs with T and

C always pairs with G

• In any one species, the four bases are found in characteristic, but not necessarily equal, ratios.


• He found a regular ratio of nucleotide bases which are known as Chargaff’s rules.

• The number of adenines was approximately equal to the number of thymines (%T = %A).

• The number of guanines was approximately equal to the number of cytosines (%G = %C).– Human DNA is 30.9% adenine, 29.4% thymine,

19.9% guanine and 19.8% cytosine.


By 1950 DNA was accepted by scientists as the genetic material but nobody knew its

structure • Many scientists wanted to be the first to

discover it

• By the beginnings of the 1950’s, the race was on to move from the structure of a single DNA strand to the three-dimensional structure of DNA.– Among the scientists working on the problem

were Linus Pauling, in California, and Maurice Wilkins and Rosalind Franklin, in London.

Race for the Prize


The story of DNA :The social nature of science

• In 1951 James Watson went to Europe (after completing his PhD) and met Crick. Francis Crick was a graduate student working in hemoglobin in Cambridge, England. They talked about DNA

• Crick and Watson teamed up to work on the structure of DNA.

• Watson went to London to hear a lecture by Rosalind Franklin on DNA. She and Maurice Wilkins were authorities on X-ray diffraction and had been working on the DNA structure on the same lab. R.Franklin made observations about her X ray diffraction of DNA.

• Watson didn’t understand about X-ray crystallography so he didn’t get much out of Franklin’s lecture.

• Rosalind Franklin was a well know physical chemist because of her previous work on carbon fiber technology. She was invited to King’s College in London to set up an X-ray diffraction lab. She was not told of Wilkins interest in her DNA work.

• Wilkins did not know about Franklin. He treated her as a lab technician or a secretary, attacked her behind her back.

• Watson and Crick made a model of DNA that was all wrong because they did not have Franklin’s information. Watson believed that the sugar-phosphate backbone was on the inside and the bases sticking out. Franklin knew it was on the inside.

The story continues….• In December 1952 Watson and Crick learned that Linus Pauling was

working on DNA structure. They asked Pauling for a copy of his paper and Pauling sent one to Watson. They also got the information from Chargaff about the bases A-t ans C-G.

• Wilkins allowed Crick to get Franklin’s information from her lab’s annual report without asking Franklin. He gave Franklin’s X ray pictures to Crick.

• They learned all the positions and measurements from Franklin’s picture and build a model in which everything fit except the bases.

• Examining Chargaff rules they build an accurate model of DNA in March 1953.

• When Watson and Crick wrote their results in a scientific journal Franklin was given no credit

• Franklin died in 1958 (breast cancer) without ever knowing her data was used to figure the structure of DNA.

• Franklin’s failure was due to her social isolation because Watson and Crick had many contacts in her lab, lots of friends and people supporting and consulting with them.

•DNA is a double-stranded helix– James Watson and Francis Crick

• Worked out the three-dimensional structure of DNA, based on work by Rosalind Franklin

Figure 10.3A, B

• Maurice Wilkins and Rosalind Franklin used X-ray crystallography to study the structure of DNA.– In this technique, X-rays are diffracted as they passed

through aligned fibers of purified DNA.– The diffraction pattern can be used to deduce the three-

dimensional shape of molecules.

• James Watson learned from their research that DNA was helical in shape and he deducedthe width of the helixand the spacing of bases.

Fig. 16.4

• James Watson and his colleague Francis Crick began to work on a model of DNA with two strands, the double helix. They decided on a helix shape after seeing Franklin’s X ray.

• They build their model to scale to conform to the

X ray data.

Using molecular models made of wire, they first tried to place the sugar-phosphate chains on the inside..

However, this did not fit the X-ray measurements and other information on the chemistry of DNA. In 1953 Watson and Crick won the Nobel prize for discovering the structure of DNA

Watson and Crick

• The key breakthrough came when Watson put the sugar-phosphate chain on the outside and the nitrogen bases on the inside of the double helix.

– The sugar-phosphate chains of each strand are like the side ropes of a rope ladder.

– Pairs of nitrogen bases, one from each strand, form rungs.

– The ladder forms a twist every ten bases.


The Molecule Comes Together

• The nitrogenous bases are paired in specific combinations: adenine with thymine and guanine with cytosine.

• Pairing like nucleotides did not fit the uniform diameter indicated by the X-ray data.– A purine-purine pair would be too wide and a pyrimidine-

pyrimidine pairing would be too short.– Only a pyrimidine-

purine pairing would produce the 2-nm diameter indicated by the X-ray data.


• In addition, Watson and Crick determined that chemical side groups off the nitrogen bases would form hydrogen bonds, connecting the two strands.– Based on details of their

structure, adenine would form two hydrogen bonds only with thymine and guanine would form three hydrogen bonds only with cytosine.

– This finding explained Chargaff’s rules.


Fig. 16.6

– The structure of DNA• Consists of two nucleotide strands wrapped around

each other in a double helix

Figure 10.3C Twist


Fig. 16.5

Protein synthesis

DNA RNA protein

What type of chemical bonds hold the DNA molecule together?

• The sugar-phosphates are joined by covalent bonds which are strong

• The base pairs that form the rungs of the ladder are hydrogen bonds.

• Hydrogen bonds are easily broken which is good when the DNA molecule is going to replicate.

Replication• When? Replication occurs during the “S”

phase of the cell cycle

• It requires lots of enzymes

• Ex: enzymes unwinds the double helix breaks the hydrogen bonds between the paired bases and other enzymes separate the two strands and add new nucleotides.

• The main team of enzymes are the

DNA polymerases

• The specific pairing of nitrogenous bases in DNA was the flash of inspiration that led Watson and Crick to the correct double helix.

• The possible mechanism for the next step, the accurate replication of DNA, was clear to Watson and Crick from their double helix model.

• In a second paper Watson and Crick published their hypothesis for how DNA replicates.– Essentially, because each strand is complementary to each other,

each can form a template when separated.– The order of bases on one strand can be used to add in

complementary bases and therefore duplicate the pairs of bases exactly.

Form Predicts Function


– DNA replication is a complex process• Due in part to the fact that some of the helical DNA

molecule must untwist

Figure 10.4B

G C

A T

G C

A T

C G

AGA

C

GC

GC

G

T

AG

C

T

AT

A

A

TT

A

CG

C

G

CG

T

A

G

C

T

A

T

A

A

T

T

A

T

C

T

• It takes E. coli less than an hour to copy 5 million base pairs in its chromosome and divide to form two identical daughter cells.

• A human cell can copy its 6 billion base pairs and divide into daughter cells in only a few hours.

• This process is remarkably accurate, with only one error per billion nucleotides.

• More than a dozen enzymes and other proteins participate in DNA replication.

Replication is Relatively Fast and Very Accurate

Enzymes: Helicase, DNA polymerase and ligase

• Helicase: Is the enzyme that untwists the double helix at the replication forks

• DNA polymerase: is the enzyme that adds nucleotides to the existing chain.

It catalizes the elongation of the new DNA strand. It also does proof-reading to remove mistakes.

• Ligase: a linking enzyme. It bonds fragments, like glue

DNA REPLICATION• DNA replication depends on base pairing– DNA replication starts with the separation of DNA strands (done by

an enzyme)– Then enzymes use each strand as a template

to assemble new nucleotides into complementary strands.( DNA polymerase and ligase)

A with T C with G

Figure 10.4A

A T

C G

G C

A T

T A

A T

C G

G C

A T

T A

A T

C G

G C

A T

T A

A T

C G

G C

A

T

A T

C G

AC

T

A

Parental moleculeof DNA

Both parental strands serve as templates

Two identical daughtermolecules of DNA

Nucleotides

• When a cell copies a DNA molecule, each strand serves as a template for ordering nucleotides into a new complimentary strand.– One at a time, nucleotides line up along the template

strand according to the base-pairing rules.– The nucleotides are linked to form new strands.

Each strand serves as a template (blueprint) for the synthesis of a new strand

• DNA polymerase can only work in one direction so each new molecule is made up of an old and a new strand.

• Since DNA is a very long molecule replication occurs simultaneously at many sites called replication forks the enzyme ligase joins them

Errors• Errors are made frequently during

replication. DNA polymerase may skip a nucleotide or add an extra one or put one in the wrong place.

• Very few errors remain because a system of enzymes ( repair nuclease) that detect and repair the errors.

• Those errors that remain are called MUTATIONS

• Watson and Crick’s model, semiconservative replication, predicts that when a double helix replicates each of the daughter molecules will have one old strand and one newly made strand.

• Other competing models, the conservative model and the dispersive model, were also proposed.


Fig. 16.8


Fig. 16.9

•The first replication in the 14N medium produced a band of hybrid (15N-14N) DNA, eliminating the conservative model.

•A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model.

– Each strand of the double helix• Is oriented in the opposite direction

Figure 10.5B

P

P

P

P

P

P

P

P

HO

OH

A

C

G

T

T

C

G

A

2 134

5

15 4

32

5 end 3 end

3 end 5 end

– Using the enzyme DNA polymerase• The cell synthesizes one daughter strand as a continuous

piece

– The other strand is synthesized as a series of short pieces

• Which are then connected by the enzyme DNA ligase

Figure 10.5C

3

53

53

5

53

Daughter strandsynthesizedcontinuously

Daughter strandsynthesizedin pieces

Parental DNA

DNA ligase

DNA polymerasemolecule

Overall direction of replication

Protein synthesis

DNA RNA protein

THE FLOW OF GENETIC INFORMATION FROM DNA TO RNA TO PROTEIN

• The DNA genotype is expressed as proteins, which provide the molecular basis for phenotypic traits

• The information constituting an organism’s genotype– Is carried in its sequence of its DNA bases

• A particular gene, a linear sequence of many nucleotides– Specifies a polypeptide

• Genetic information written in codons is translated into amino acid sequences– The “words” of the DNA “language”

• Are triplets of bases called codons

– The codons in a gene • Specify the amino acid sequence of a polypeptide

DNA strand

Transcription

Translation

Polypeptide

RNA

Amino acid

Codon

A A A C C G G C A A A A

U U U G G C C G U U U U

Gene 1

Gene 2

Gene 3

DNA molecule

Figure 10.7

• The genetic code is the Rosetta stone of life– Nearly all organisms

• Use exactly the same genetic code

Figure 10.8A

UUC

UGUUGC

UGA Stop

Met or start

Phe

Leu

Leu

Ile

Val Ala

Thr

Pro

Ser

Asn

Lys

His

Gln

Asp

Glu

Ser

Arg

Arg

Gly

CysTyr

G

A

C

U

U C A G

Th

ird

bas

e

Second base

Fir

st b

ase

UUA

UUU

CUC

CUU

CUG

CUA

AUC

AUU

AUG

AUA

GUC

GUU

GUG

GUA

UCC

UCU

UCG

UCA

CCC

CCU

CCG

CCA

ACC

ACU

ACC

ACA

GCC

GCU

GCG

GCA

UAC

UAU

UAG Stop

UAA Stop

CAC

CAU

CAGCAA

AAC

AAU

AAG

AAA

GAC

GAU

GAG

GAA

UGG Trp

CGC

CGU

CGGCGA

AGCAGU

AGG

AGA

GGC

GGU

GGG

GGA

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

UUG

– An exercise in translating the genetic code

Figure 10.8B

T A C T T C A A A A T C

A T G A A G T T T T A G

A U G A A G U U U U A G

Transcription

Translation

RNA

DNA

Met Lys PhePolypeptide

Startcondon

Stopcondon

Strand to be transcribed

• Transcription produces genetic messages in the form of RNA– A close-up view of transcription

RNApolymerase

RNA nucleotides

Direction of transcription

Template Strand of DNA

Newly made RNA

TC

AT C C A A T

T

GG

CC

AATTGGAT

G

U

C A U C C AA

U

Figure 10.9A

– In the nucleus, the DNA helix unzips• And RNA nucleotides line up along one strand of the

DNA, following the base pairing rules

– As the single-stranded messenger RNA (mRNA) peels away from the gene

• The DNA strands rejoin

– Transcription of a geneRNA polymerase

DNA of gene

PromoterDNA Terminator

DNA

Area shownIn Figure 10.9A

GrowingRNA

Completed RNARNApolymerase

Figure 10.9B

1 Initiation

2 Elongation

3 Termination

• Eukaryotic RNA is processed before leaving the nucleus– Noncoding segments called introns are spliced

out• And a cap and a tail are added to the ends

Exon Intron Exon Intron Exon

DNA

Cap TranscriptionAddition of cap and tail

RNAtranscript with capand tail

Introns removedTail

Exons spliced together

mRNA

Coding sequence Nucleus

Cytoplasm

Figure 10.10

• Transfer RNA molecules serve as interpreters during translation– Translation

• Takes place in the cytoplasm

– A ribosome attaches to the mRNA • And translates its message into a specific

polypeptide aided by transfer RNAs (tRNAs)

Amino acid attachment site

Hydrogen bond

RNA polynucleotide chain

AnticodonFigure 10.11A

• Ribosomes build polypeptides– A ribosome consists of two subunits

• Each made up of proteins and a kind of RNA called ribosomal RNA

tRNAmolecules

mRNA Small subunit

Growingpolypeptide

Largesubunit

Figure 10.12A

– The subunits of a ribosome• Hold the tRNA and mRNA close together during

translation

Largesubunit

mRNA-binding site

Smallsubunit

tRNA-binding sites

Growing polypeptide

Next amino acid to be added to polypeptide

mRNA

tRNA

Codons

Figure 10.12B, C

• An initiation codon marks the start of an mRNA message

Start of genetic message

End

Figure 10.13A

– mRNA, a specific tRNA, and the ribosome subunits

• Assemble during initiation

Met Met

Initiator tRNA

1 2mRNA Small ribosomal

subunit

Startcodon

Large ribosomalsubunit

A siteU A CAU C

A U G A U G

P site

Figure 10.13B

• Elongation adds amino acids to the polypeptide chain until a stop codon terminates translation– Once initiation is complete

• Amino acids are added one by one to the first amino acid

– Each addition of an amino acid• Occurs in a three-step elongation process

Polypeptide

P site

mRNA Codons

mRNAmovement

Stopcodon

NewPeptidebond

Anticodon

Aminoacid

A site

Figure 10.14

1 Codon recognition

2 Peptide bondformation

3 Translocation

– The mRNA moves a codon at a time• And a tRNA with a complementary anticodon pairs

with each codon, adding its amino acid to the peptide chain

– Elongation continues• Until a stop codon reaches the ribosome’s A site,

terminating translation

• Review: The flow of genetic information in the cell is DNARNAprotein– The sequence of codons in DNA, via the

sequence of codons • Spells out the primary structure of a polypeptide

Polypeptide

TranscriptionDNA

mRNA

RNApolymerase

Amino acid Translation

tRNA

Enzyme

Anticodon

ATP

InitiatortRNA

Largeribosomalsubunit

Start Codon

Codons

mRNA

Stop codon

Smallribosomalsubunit

Growingpolypeptide

New peptidebond forming

mRNA

Figure 10.15

– Summary of transcription and translation

mRNA is transcribed from a DNA template.1

Each amino acidattaches to its propertRNA with the help of aspecific enzyme and ATP.

2

Initiation ofpolypeptide synthesis

The mRNA, the first tRNA,and the ribosomal subunits come together.

3

Elongation4

A succession of tRNAsadd their amino acids to the polypeptide chain as the mRNA is moved through the ribosome, one codon at a time.

5

The ribosome recognizes a stop codon. The poly-peptide is terminated and released.

Termination

• Mutations can change the meaning of genes– Mutations are changes in the DNA base

sequence caused by errors in DNA replication or recombination, or by mutagens

C T T C A T

Normal hemoglobin

Mutant hemoglobin DNA

G A A G U A

Sickle-cell hemoglobin

Normal hemoglobin DNA

Glu Val

mRNA mRNA

Figure 10.16A

– Substituting, inserting, or deleting nucleotides alters a gene

• With varying effects on the organism

Normal gene

mRNA

Base substitution

Base deletion Missing

Met Lys Phe Gly Ala

Met Lys Phe Ser Ala

Met Lys Leu Ala His

A U G A A G U U U G G C G C A

A U G A A G U U U A G C G C A

A U G A A G U U G G C G C A U

U

Protein

Figure 10.16B

MICROBIAL GENETICS

• Viral DNA may become part of the host chromosome– Viruses

• Can be regarded as genes packaged in protein

– When phage DNA enters a lytic cycle inside a bacterium

• It is replicated, transcribed, and translated

– The new viral DNA and protein molecules• Then assemble into new phages, which burst from

the host cell

– In the lysogenic cycle• Phage DNA inserts into the host chromosome and is

passed on to generations of daughter cells

– Much later• It may initiate phage production

The AIDS virus makes DNA on an RNA template– HIV, the AIDS virus

• Is a retrovirus

Envelope

Glycoprotein

Protein coat

RNA (two identical strands)

Reverse transcriptase

Figure 10.21A

– Inside a cell, HIV uses its RNA as a template for making DNA

• To insert into a host chromosome

Viral RNA

RNAstrand

Double-strandedDNA

Viral RNAand proteins

CYTOPLASM

NUCLEUSChromosomal DNA

Provirus DNA

RNA

Figure 10.21B

1

2

3

45

6

• 10.22 Bacteria can transfer DNA in three ways– Bacteria can transfer genes from cell to cell

by one of three processes• Transformation, transduction, or conjugation

DNA enterscell

Fragment of DNAfrom anotherbacterial cell

Bacterial chromosome

(DNA)

Phage

Fragment of DNA fromanotherbacterial cell(former phagehost)

Phage

Sex pili

Mating bridge

Donor cell(“male”)

Recipient cell(“female”)

Figure 10.22A–C

– Once new DNA gets into a bacterial cell• Part of it may then integrate into the recipient’s

chromosome

Recipient cell’schromosome

Recombinantchromosome

Donated DNACrossovers Degraded DNA

Figure 10.22D

• Bacterial plasmids can serve as carriers for gene transfer– Plasmids

• Are small circular DNA molecules separate from the bacterial chromosome

– Plasmids can serve as carriers• For the transfer of genes

Plasmids

Co

loriz

ed

TE

M 2

,00

0

Cell now male

Plasmid completes transferand circularizes

F factor starts replication and transfer

Male (donor) cell

Bacterial chromosome

F factor (plasmid)

Recombination can occur

Only part of the chromosome transfers

F factor starts replication and transfer of chromosome

Origin of F replicationBacterial chromosome

Male (donor) cellF factor (integrated)

Recipient cell

Figure 10.23A–C

molecular genetics dna. what is the significance of dna? why is it so important? because it is...

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