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Watson and Crick

Discovered the double helix structure of DNA (1953)

Rosalind Franklin

Rosalind Franklin's research (King's College) produced x-ray diffraction images of DNA
Helped Watson and Crick build their DNA model, for which they received the Nobel Prize

DNA Structure

DNA is a chain of nucleotides

Nucleotides of DNA

Each nucleotide is a complex of three subunits
Phosphoric acid (phosphate)
A pentose sugar (deoxyribose)
A nitrogen-containing base

Four Possible Bases

Adenine (A) - a purine
Guanine (G) - a purine
Thymine (T) - a pyrimidine
Cytosine (C) - a pyrimidine

Complimentary Base Pairing for Adenine (A)

Thymine (T)

Complimentary Base Pairing for Guanine (G)

Cytosine (C)

Complimentary Base Pairing for Thymine (T)

Adenine (T)

Complimentary Base Pairing for Cytosine (C)

Guanine.

Semi-conservative replication (DNA)

Each daughter DNA molecule consists of one new chain of nucleotides and one from the parent DNA molecule

The two daughter DNA molecules will be identical to the parent molecule

Steps of DNA Replication

Before replication begins, the two strands of the parent molecule are hydrogen-bonded together

Enzyme DNA helicase unwinds and "unzips" the double-stranded DNA

New complementary DNA nucleotides fit into place along separated strands by complementary base pairing. These are positioned and joined by DNA polymerase.

DNA ligase seals any breaks in the sugar-phosphate backbone

The two double helix molecules identical to each other and to the original DNA molecule

Ladder Configuration and DNA Replication

Parental DNA molecule contains
so-called old strands
hydrogen-bonded by complementary
base pairing.

Region of replication. Parental DNA
is unwound and unzipped. New
nucleotides are pairing with those
in old strands.

Replication is complete. Each
double helix is composed of an old
(parental) strand and a new
(daughter) strand

How do we get from DNA to proteins (Gene Expression)?

Transcription
Translation

Transcription (DNA)

Enzymes use the base sequence of a gene as a template to make a strand of RNA
This is the first step in protein synthesis (mRNA)
Forms a messager for RNA (mRNA)

Translation

Information in the RNA strand is decoded (translated) into a sequence of amino acids

DNA is a polymer of ______.

nucleotides

A nucleotide is built from how many subunits?

3

The structure of DNA was determined by ______.

Watson and Crick

A purine is always bonded to a pyrimadine. This is called ______.

complimentary base pairing

The two DNA strands are antiparallel. This means that ______.

they run in opposite directions

The process of copying one DNA double helix into two identical double helices is called ______.

DNA replication

Which of the following enzymes "unzips" DNA during DNA replication?

DNA helicase

To complete replication, the enzyme _______ seals any breaks in the sugar-phosphate backbone.

DNA ligase

The nitrogen base __________ occurs in RNA in place of thymine.

uracil

RNA, unlike DNA, ______________.

is single stranded

tRNA transfers __________ to the ribosomes.

amino acids

Once formed, mRNA carries genetic information from DNA to the _____________.

ribosomes

During transcription, a segment of the DNA called a _____ serves as a template for the production of an RNA molecule.

gene

A promoter is ________________.

a region of DNA that contains a special sequence of nucleotides that begins transcription.

Which of the following are removed during the processing of primary mRNA?

introns

What are the three steps required in translation?

Initiation, elongation, and termination

A cluster of genes usually coding for proteins related to a particular metabolic pathway is called a (an) ______.

operon

Active genes in eukaryotic cells are associated with more loosely packed chromatin, called ___________

euchromatin

Environmental influences called _________ cause mutations in humans.

mutagens

Which of the following is (are) primary characteristics of cancer cells?

Cancer cells are genetically unstable
Cancer cells do not correctly regulate the cell cycle
Cancer cells escape the signals for cell death

The hereditary material found in all cells is

DNA.

In the Watson-Crick model of DNA, the "sides" of the ladder are composed of

sugar-phosphate molecules.

A nucleotide contains

a sugar, a phosphate, and a nitrogen base.

Because one original strand of the double-stranded helix is found in each daughter cell, the replication process is called

semi-conservative.

The _____ enzyme is responsible for unwinding the double-helix structure of DNA during replication.

helicase

Information from X-ray crystallographic data collected by _______ was used by Watson and Crick in their development of the model of DNA.

Franklin

Which of the following have nitrogenous bases correctly paired in DNA?

adenine—thymine; guanine—cytosine

The fact that a DNA strand contains equal amounts of the bases A and T allows one to realize that

these molecules bond together.

In the spiraling helix of a DNA molecule, each cross-member (or step) consists of two

nitrogenous bases.

DNA is considered to be the master molecule in cells because it

ultimately controls all cell activity.

Replication MOST accurately refers to the synthesis of

complementary strands of DNA for mitosis

The two molecules resulting from the replication of DNA

are the same in every regard unless a mutation has occurred.

Which of the following is mass-produced directly by cells altered by recombinant DNA techniques?

Insulin.

Which of the following is a result of recombinant DNA

Developing hybrid species of food crops.
Mass-producing molecules for medical treatments.
Producing laboratory clones of organisms for scientific investigations.

Prior to protein synthesis, the DNA

serves as a template for the production of mRNA.

Transcription of part of a DNA molecule with a nucleotide sequence of AAACAACTT results in a mRNA molecule with the complementary sequence of

UUUGUUGAA.

The type of gene mutation in which a nucleotide replaces another nucleotide is termed a(n) ________ mutation

substitution

The function of transfer RNA is to

carry amino acids to ribosomes.

If the normal nucleotide sequence was TACGGCATG, what type of gene mutation is present if the resulting sequence becomes TAGGCATG?

deletion mutation

Which of the following nucleotide bases is found only in DNA, not in RNA?

thymine

Gene Mutations

Permanent change in the sequence of bases in DNA

Effect can range from none to complete inactivity of the protein

Germ-line mutations can be passed to subsequent generations

Some mutations can lead to cancer

Three causes of mutations

Error in replication
Mutagens
Transposons - jumping genes

Error in replication

Proofreading by DNA polymerase usually takes care of this

Mutagens

Radiation and certain organic chemicals
DNA repair enzymes constantly monitor and repair any irregularities

Transposons - jumping genes

Specific DNA sequences that move within and between chromosomes
Sometimes alters neighboring gene expression
Likely all organisms (including humans) have them

Point Mutations - Base pair substitution

Change in a single DNA nucleotide
Possible change in a specific amino acid
May have no effect (silent mutation)
May produce an abnormal protein
Sickle cell
May produce an incomplete protein
New codon now codes for a stop

Frameshift Mutations

One or more nucleotides are either inserted or deleted from DNA

Result can be a completely new sequence of codons and a nonfunctional protein

THE CAT ATE THE RAT
THE ATA TET HER AT

Frameshift Mutations

Nonfunctional Proteins

A single nonfunctioning protein can have a dramatic effect on the phenotype, because enzymes are often a part of metabolic pathways

PKU (phenylketonuria)
Phenylalanine can't be broken down and builds up causing mental impairment

Androgen insensitivity
Faulty receptor makes cells unable to respond
Genetic male looks like a normal female

Mutations and Cancer

Development of cancer involves a series of accumulating mutations that can be different for each type of cancer

Carcinogenesis begins with the loss of tumor suppressor gene activity and/or the gain of oncogene activity

In the United States, the three deadliest forms of cancer are

lung cancer, colon and rectal cancer, and breast cancer

Cancers

Altered cells grow and divide abnormally
Malignant cells may metastasize (break loose and colonize distant tissues)

distant tumor

New metastatic
tumors are found
some distance
from the tumor.

one mutations

Cell (dark pink)
acquires a
mutation for
repeated cell
division.

two mutations

New mutations
arise, and one
cell (green) has
the ability to start
a tumor.

tumor-three mutations

The tumor is at its
place of origin.
One cell (purple)
mutates further.

malignant
tumor

Cancer cells now
have the ability to
invade lymphatic
and blood vessels.

invasive tumor

Cells have gained
the ability to invade
underlying tissues
by producing
a proteinase
enzyme.

Characteristics of Cancer Cells

Genetically unstable
Do not correctly regulate the cell cycle
Escape the signals for cell death
Can survive and proliferate elsewhere in the body

Epigenetics

Changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence, hence the name epi- (Greek: over; above) -genetics.

______________ is the application of computer technologies, specially developed software, and statistical techniques to the study of biological information.

Bioinformatics

Recent research defines a gene as ______.

a genomic sequence directly encoding functional products

The study of the complete genetic sequence of humans and other organisms is ______.

genomics

In which procedure is the gene injected directly into a particular region of the body?

C) in vivo therapy

_____________ is the insertion of genetic material into human cells for the treatment of genetic disorders.

D) Gene therapy

Which of the following procedures is the most common type of DNA microarray used to generate a person's genetic profile?

mutation microarray

What are the two types of DNA testing for mutated genes?

testing for a genetic marker and using a DNA probe

__________ is the use of transgenic farm animals to produce pharmaceuticals.

Gene pharming

Which of the following human proteins are being engineered by transgenic plants?

hormones
clotting factors
antibodies

Which of the following is a result of a transgenic plant that produces potatoes below ground and tomatoes above ground?

pomato

Which of the following is an example of how bacteria can be selected to degrade a specific substance?

Oil-eating bacteria can be genetically engineered to clean up beaches after an oil spill

Which of the following is (are) a biotechnology product produced by bacteria?

insulin
human growth hormone
tissue plasminogen activator

During a process called ___________ an electrical current is used to force DNA through a porous gel material and thus separate them according to their size

gel electrophoresis

Why is short tandem repeat (STR) profiling advantageous as a method of identifying DNA segments?

Short tandem repeat profiling is advantageous because it doesn't require the use of restriction enzymes.

_____________ are short sequences of DNA bases that recur several times.

Short tandem repeats

Which of the following describes the correct order of steps involved in polymerase chain reaction?

denaturation, annealing, extension

Which procedure can create billions of copies of a segment of DNA in a test tube in a matter of hours?

polymerase chain reaction

___________ contains DNA from two or more different sources.

Recombinant DNA

What two enzymes are needed to introduce foreign DNA into vector DNA?

DNA polymerase and DNA ligase

The use of a DNA chip to discover all the particular mutations of an individual is called ______.

genetic profiling

Cloning

Production of identical copies of an organism through asexual means

Gene cloning

Production of many identical copies of a single gene

Cloning Technologies

Somatic cell nuclear transfer
Reproductive cloning
Therapeutic cloning

Somatic cell nuclear transfer

(SCNT) fuses an adult cell with an enucleated egg

Reproductive cloning

technologies produce genetically identical individuals (clones)

Therapeutic cloning

produces embryos that are used for stem cell research

SCNT

To clone an adult, scientists first transform one of its differentiated cells into an undifferentiated cell by turning its unused DNA back on

somatic cell nuclear transfer (SCNT)
Method of reproductive cloning in which a researcher removes the nucleus from an unfertilized egg, then inserts into the egg a nucleus from an adult body cell

Therapeutic Cloning

Researchers are already using SCNT to produce human embryos for research, a practice called therapeutic cloning

The researchers harvest undifferentiated (stem) cells from the cloned human embryos

therapeutic cloning
Use of SCNT to produce human embryos for research purposes
Growing organs for patients
How fatal diseases progress

Cloning Humans

Ethical problems
Still long way to go
Cloned animals tend to have problems:
The first cloned mammal, Dolly the sheep, had old-age problems when she was only two
Clones may be overweight or have enlarged organs
Cloned mice develop lung and liver problems, and almost all die prematurely
Cloned pigs tend to limp and have heart problems: One never did develop a tail or, even worse, an anus

DNA Cloning

Uses of gene cloning

Produce large quantities of the gene's protein product

Learn how a cloned gene codes for a particular protein

Use the genes to alter the phenotypes of other organisms in a beneficial way
Produces transgenic organism
Gene therapy - cloned genes are used to modify a human

Recombinant DNA technology

Recombinant DNA (rDNA)
Contains DNA from two or more different sources
Vector - piece of DNA that foreign DNA can be added to
Plasmids are accessory rings of DNA in bacteria
2 enzymes used to introduce foreign DNA
Restriction enzyme - molecular scissors
DNA ligase

Transgenic organisms are called GMOs

Genetically modified organisms
Products they produce are biotechnology products

Transgenic bacteria

Grown in bioreactors
Bacteria express cloned gene
Gene product collected from the media
Products include insulin, human growth hormone, tPA, and hepatitis B vaccine
Can be selected for their ability to degrade a particular substance

Ability can be enhanced by bioengineering

Eat oil, remove sulfur from coal

Transgenic Plants

Foreign genes introduced into:
Immature plant embryos
Protoplasts - plant cells with cell wall removed
Go on to develop into mature plants
Pomato
Produces potatoes below ground and tomatoes above ground
Pest resistance in cotton, corn, and potato strains
Soybeans resistant to herbicide
Can also be engineered to produce human proteins

Designer Plants

Why do it?
Produce more food at lower cost
Use smaller amounts of toxic pesticides
Resistant to environmental conditions (droughts)
Most widely planted GMO crops:
Corn, sorghum, cotton, soy, canola, alfalfa

"Frankenfood"?

Transgenic Animals

Insert genes into eggs
By hand or by vortex mixing with silicon-carbide eggs

Insert gene for bovine growth hormone (bGH) to produce larger fishes, cows, pigs, rabbits, and sheep

Gene pharming
Use of transgenic farm animals to produce pharmaceuticals
Proteins harvested from animals milk

Modified Humans?

Gene therapy
Transfer of one or more normal or modified genes into an individual's body cells to fix a genetic defect of boost disease resistance

Deliver cells carrying recombinant DNA into a patient's tissues or use viruses as vectors that inject genes into cells

SCID-X1 - severe immune deficiency, bone marrow therapy with the normal gene

Risks?

Eugenic engineering

Selecting the most desired human traits

People with enhance memory, bigger muscles, improved learning abilities..

The Structure of DNA

DNA is composed of four nucleotides, each containing: adenine, cytosine, thymine, or guanine.

The amounts of A = T, G = C, and purines = pyrimidines [Chargaff's Rule].

DNA is a double-stranded helix with antiparallel strands [Watson and Crick].

Nucleotides in each strand are linked by 5'-3' phosphodiester bonds

Bases on opposite strands are linked by hydrogen bonding: A with T, and G with C.

The Basic Principle: Base Pairing to a Template Strand

The relationship between structure and function is manifest in the double helix
Since the two strands of DNA are complementary each strand acts as a template for building a new strand in replication

DNA replication

The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules

Steps in DNA replication

The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C

The first step in replication is separation of the two DNA strands.
Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand

The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each "daughter" DNA molecule consists of one parental strand and one new strand

Semi-conservative

Each 2-stranded daughter molecule is only half new
One original strand was used as a template to make the new strand

DNA Replication

The copying of DNA is remarkable in its speed and accuracy
Involves unwinding the double helix and synthesizing two new strands.
More than a dozen enzymes and other proteins participate in DNA replication
The replication of a DNA molecule begins at special sites called origins of replication, where the two strands are separated

Mechanism of DNA Replication

DNA replication is catalyzed by DNA polymerase which needs an RNA primer
RNA primase synthesizes primer on DNA strand
DNA polymerase adds nucleotides to the 3' end of the growing strand
Nucleotides are added by complementary base pairing with the template strand
The substrates, deoxyribonucleoside triphosphates, are hydrolyzed as added, releasing energy for DNA synthesis.
DNA synthesis on the leading strand is continuous
The lagging strand grows the same general direction as the leading strand (in the same direction as the Replication Fork). However, DNA is made in the 5'-to-3' direction
Therefore, DNA synthesis on the lagging strand is discontinuous
DNA is added as short fragments (Okasaki fragments) that are subsequently ligated together

Replication

Many proteins assist in DNA replication

DNA helicases unwind the double helix, the template strands are stabilized by other proteins

Single-stranded DNA binding proteins make the template available

RNA primase catalyzes the synthesis of short RNA primers, to which nucleotides are added.

DNA polymerase III extends the strand in the 5'-to-3' direction

DNA polymerase I degrades the RNA primer and replaces it with DNA

DNA ligase joins the DNA fragments into a continuous daughter strand

Enzymes in DNA replication

Helicase unwinds
parental double helix
Binding proteins
stabilize separate
strands
Primase adds
short primer
to template strand
DNA polymerase III
binds nucleotides
to form new strands
DNA polymerase I (Exonuclease) removes RNA primer and inserts the correct bases
Ligase joins Okazaki
fragments and seals
other nicks in sugar-phosphate backbone

Proofreading

DNA must be faithfully replicated...but mistakes occur
DNA polymerase (DNA pol) inserts the wrong nucleotide base in 1/10,000 bases
DNA pol has a proofreading capability and can correct errors
Mismatch repair: 'wrong' inserted base can be removed
Excision repair: DNA may be damaged by chemicals, radiation, etc. Mechanism to cut out and replace with correct bases

Mutations

A mismatching of base pairs, can occur at a rate of 1 per 10,000 bases.
DNA polymerase proofreads and repairs accidental mismatched pairs.
Chances of a mutation occurring at any one gene is over 1 in 100,000
Because the human genome is so large, even at this rate, mutations add up. Each of us probably inherited 3-4 mutations!

Proofreading and Repairing DNA

DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides
In mismatch repair of DNA, repair enzymes correct errors in base pairing
In nucleotide excision DNA repair nucleases cut out and replace damaged stretches of DNA

Protein Synthesis

The information content of DNA is in the form of specific sequences of nucleotides along the DNA strands
The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins
The process by which DNA directs protein synthesis, gene expression includes two stages, called transcription and translation

Transcription and Translation

In a eukaryotic cell the nuclear envelope separates transcription from translation
Extensive RNA processing occurs in the nucleus

Transcription

Transcription is the DNA-directed synthesis of RNA
RNA synthesis
Is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides
Follows the same base-pairing rules as DNA, except that in RNA, uracil substitutes for thymine

What is the difference between RNA and DNA?

RNA is single stranded, not double stranded like DNA
RNA is short, only 1 gene long, where DNA is very long and contains many genes
RNA uses the sugar ribose instead of deoxyribose in DNA
RNA uses the base uracil (U) instead of thymine (T) in DNA.

Initiation.

After RNA polymerase binds to
the promoter, the DNA strands unwind, and
the polymerase initiates RNA synthesis at the
start point on the template strand


transcription, the DNA strands re-form a double helix

Elongation.

The polymerase moves downstream, unwinding the
DNA and elongating the RNA transcript 5  3 . In the wake of transcription, the DNA strands re-form a double helix
RNA polymerase synthesizes a single strand of RNA against the DNA template strand (anti-sense strand), adding nucleotides to the 3' end of the RNA chain

As RNA polymerase moves along the DNA it continues to untwist the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides

Termination.

Eventually, the RNA
transcript is released, and the
polymerase detaches from the DNA
Specific sequences in the DNA signal termination of transcription
When one of these is encountered by the polymerase, the RNA transcript is released from the DNA and the double helix can zip up again.

Promoters

signal the initiation of RNA synthesis
Transcription factors help eukaryotic RNA polymerase recognize promoter sequences

Post Termination RNA Processing

Most eukaryotic mRNAs aren't ready to be translated into protein directly after being transcribed from DNA. mRNA requires processing.
Transcription of RNA processing occur in the nucleus. After this, the messenger RNA moves to the cytoplasm for translation.

The cell adds a protective cap to one end, and a tail of A's to the other end. These both function to protect the RNA from enzymes that would degrade

Most of the genome consists of non-coding regions called introns

Non-coding regions may have specific chromosomal functions or have regulatory purposes

Introns also allow for alternative RNA splicing

Thus, an RNA copy of a gene is converted into messenger RNA by doing 2 things:

Add protective bases to the ends

Cut out the introns

Alteration of mRNA Ends

Each end of a pre-mRNA molecule is modified in a particular way
The 5 end receives a modified nucleotide cap
The 3 end gets a poly-A tail

RNA Processing - Splicing

The original transcript from the DNA is called pre-mRNA.

It contains transcripts of both introns and exons.

The introns are removed by a process called splicing to produce messenger RNA (mRNA)

RNA Processing

Proteins often have a modular architecture consisting of discrete structural and functional regions called domains
In many cases different exons code for the different domains in a protein

The Genetic Code

Codons: 3 base code for the production of a specific amino acid, sequence of three of the four different nucleotides

Since there are 4 bases and 3 positions in each codon, there are 4 x 4 x 4 = 64 possible codons

64 codons but only 20 amino acids, therefore most have more than 1 codon

3 of the 64 codons are used as STOP signals; they are found at the end of every gene and mark the end of the protein

One codon is used as a START signal: it is at the start of every protein

Universal: in all living organisms

Start Codon

AUG

Stop Codons

UAA, UGA, UAG

The "anticodon"

is the 3 RNA bases that matches the 3 bases of the codon on the mRNA molecule
The four base-paired regions and three loops are characteristic of all tRNAs, as is the base sequence of the amino acid attachment site at the 3 end. The anticodon triplet is unique to each tRNA type.

Transfer RNA

Consists of a single RNA strand that is only about 80 nucleotides long
Each carries a specific amino acid on one end and has an anticodon on the other end
A special group of enzymes pairs up the proper tRNA molecules with their corresponding amino acids.
tRNA brings the amino acids to the ribosomes,

Ribosomes

Ribosomes facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis
The 2 ribosomal subunits are constructed of proteins and RNA molecules named ribosomal RNA or rRNA

Building a Polypeptide

We can divide translation into three stages
Initiation
Elongation
Termination
The AUG start codon is recognized by methionyl-tRNA or Met
Once the start codon has been identified, the ribosome incorporates amino acids into a polypeptide chain
RNA is decoded by tRNA (transfer RNA) molecules, which each transport specific amino acids to the growing chain
Translation ends when a stop codon (UAA, UAG, UGA) is reached

A summary of transcription and translation in a eukaryotic cell

1. TRANSCRIPTION--RNA is transcribed from a DNA template.
2.RNA PROCESSING--In eukaryotes, the RNA transcript (pre-mRNA) is spliced and modified to produce
mRNA, which moves from the nucleus to the
cytoplasm.
3.FORMATION OF INITIATION COMPLEX--After leaving the nucleus, mRNA attaches to the ribosome.
4.AMINO ACID ACTIVATION--Each amino acid attaches to its proper tRNA with the help of a specific
enzyme and ATP.
5.TRANSLATION--A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved
through the ribosome one codon at a time.(When completed, the polypeptide is released
from the ribosome.)

Post-translation

The new polypeptide is now floating loose in the cytoplasm if translated by a free ribosme.
It might also be inserted into a membrane, if translated by a ribosome bound to the endoplasmic reticulum.
Polypeptides fold spontaneously into their active configuration, and they spontaneously join with other polypeptides to form the final proteins.
Sometimes other molecules are also attached to the polypeptides: sugars, lipids, phosphates, etc. All of these have special purposes for protein function

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