DNA: The Molecule of Life

Molecular Genetics

DNA and RNA Genes are segments of DNA on a

chromosome that code for specific traits DNA – nucleic acid called deoxyribonucleic

acid that contains the instructions necessary for a cell to build proteins from amino acids.

RNA – ribonucleic acid plays a role in gene expression and protein synthesis

Isolating the Material of Heredity 1869 Friedrich Miescher isolated a weakly acidic

phosphorus-containing substance from the nuclei of white blood cells– Called it “nucleic acid”

Early 1900’s Pheobus Levene isolated two types of nucleic acid– Called them “ribose nucleic acid” (RNA) and “deoxyribose

nucleic acid” (DNA) Soon after, Thomas Hunt Morgan provided the first

experimental evidence that genes are located on chromosomes– Working with fruit flies

Isolating the Material of Heredity In 1928 Fredrick Griffith designed an

experiment to study the bacteria that were responsible for a pneumonia epidemic in London– He discovered that the dead pathogenic bacteria

passed on their disease-causing properties to live, non-pathogenic bacteria

– He called this the transforming principle Griffith died during world war II but several

scientists built on his work

http://www.juliantrubin.com/bigten/dnaexperiments.html

Isolating the Material of Heredity

In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty discovered:– When they treated heat-killed pathogenic

bacteria with a protein-destroying enzyme, transformation still occurred

– When they treated heat-killed pathogenic bacteria with a DNA-destroying enzyme, transformation did not occur

The results provided evidence that DNA has a role in transformation

http://courses.cm.utexas.edu/emarcotte/ch339k/fall2005/Lecture-Ch8-1.html

Isolating the Material of Heredity

In 1952, Alfred Hershey and Martha Chase used radioactive labeling to show that genes are made of DNA– They used a virus that contains a protein

coat surrounding a length of DNA– This virus attaches to a bacteria cell and

injects genetic information into the cell– The infected cell produces new viruses and

bursts which releases the new viruses to infect other cells

Isolating the Material of Heredity

Hershey and Chase created two batches of the virus– In one they labeled the protein coat with

radioactive sulfur– In the other, they labeled the DNA with

radioactive phosphorus– They found that the radioactive phosphorus

was found in the bacterial cells– They concluded that DNA must direct the cell

to produce new viruses– animation

                                                                   

http://www.accessexcellence.org/RC/VL/GG/hershey.html

Structure of DNA After isolating DNA and RNA, Levene

determined that both molecules are made up of nucleotides (in long chains)

Nucleotide is composed of:– 5-carbon sugar (deoxyribose in DNA, ribose in

RNA)– Phosphate– Nitrogen base (4 different types)

The 4 nitrogen bases belong to two chemical groups called purines and prymidines

Purines = Adenine (A) and Guanine (G) Prymidines = Thymine (T) and Cytosine (C)

Structure of DNA In the late 1940’s Erwin Chargaff found that

nucleotides are not present in equal amounts in DNA and RNA– Nucleotides are present in varying proportions– He found that the number of adenine in DNA is

equal to the number of thymine in a sample– The amount of cytosine is approximately equal to

the amount of guanine

This constant relationship is called Chargaff’s rule

Video on Chargaff’s rule

Structure of DNA In the early 1950’s, a British scientist Rosaslind Franklin used

x-ray photography to analyze the structure of DNA– DNA has a helical structure with two regularly repeating patterns– Nitrogenous bases are located on the inside of the structure, and the

sugar-phosphate backbone is located on the outside (near the watery nucleus)

– Many argue that Franklin should have shared in the Nobel Prize for discovering the structure of DNA, but she died before it was handed out

NOVA program on photo 51.

Structure of DNA In 1953, James Watson and Francis Crick were the first to

produce a structural model for DNA Watson and Crick’s model of DNA closely resembles a twisted

ladder– Deoxyribose sugar and phosphate molecules make up backbone

(handrails of the ladder)– Paired nitrogen bases held together by weak hydrogen bonds make up

the rungs (steps) of the ladder– The ladder twists to form a double helix

From Franklin’s images, Watson and Crick knew the distance between the sugar-phosphate handrails remained constant

Structure of DNA The two strands that make up

DNA are not identical, they are complementary to eachother

– This is due to the complementary base pairs of A-T and C-G

The two strands are also antiparallel

– The phosphate bridges run in opposite directions in the two strands

– Each end of a double stranded DNA molecule contains the 5’ end of one strand and the 3’ end of the complementary strand

http://www.synapses.co.uk/genetics/tsg19.html

In a segment of DNA, the number of purines equals the number of pyrimidines; this is because of the base pairing rule

RULE: nitrogen bases always pair in complementary pairs– Adenine = Thymine– Guanine = Cytosine

Ex) if 15% of the bases were thymine, what percentage would be

a) adenine

b) guanine

c) cytosine

Example

Determine the complementary strand of DNA:A T G C A G C

I I I I I I I

Ribonucleic Acid (RNA) compared to DNA

The sugar component of RNA is ribose RNA does not have the nucleotide

thymine (T), in its place is uracil (U) RNA remains single stranded There are several types of RNA

– mRNA, rRNA, tRNA

DNA, RNA animation

DNA Replication (Synthesis)

Replication – DNA has the ability to replicate (or duplicate) itself.

This is why one cell is able to divide into two cells and each cell has identical genetic information

Replication takes place during S phase of interphase

DNA Replication (Synthesis)

Replication is semi-conservative – Each new molecule of DNA contains one strand of the

original complementary DNA and one new parent strand– Each new strand conserves half of the original molecule– Meselson-Stahl Experiment

Replication takes place at several locations along the DNA molecule simultaneously– The steps are described in sequence to better

understand the concept – BioFlix Replication

1) Replication starts at a specific nucleotide sequence called the replication origin

2) During replication, weak hydrogen bonds that hold complementary nitrogen bases together are broken (This causes the two edges to “unzip”) with a special group of enzymes called helicases (gyrase breaks the hydrogen bonds)

3) This creates two y-shaped areas (replication forks) at the end of the unwound area, the unwound area is called a replication bubble

4) The parent (original) strands are conserved while two new strands created from nucleotides are formed with them (they act as a template)

5) Free floating nucleotides (from diet) are attached to the exposed nitrogen bases according to the base pair rule with an enzyme called DNA polymerase– This process is called elongation– DNA polymerase attaches new nucleotides to the free 3’ end

of a preexisting chain of nucleotides– Elongation can only take place in a 5’ to 3’ direction – This means that replication occurs in opposite directions along

each strand of the parent DNA• One strand is replicated continuously, it is called the leading strand• The other strand is replicated in short segments, it is called the lagging

strand– The short segments are called Okazaki fragments– These fragments are spliced together by an enzyme called DNA ligase

6) Since DNA polymerase cannot synthesize new fragments of DNA, and RNA primer serves as a starting point for the elongation of each new DNA strand An enzyme called primase is required to

construct a primer When finished the strand, DNA polymerase

removes the primer by eliminating the nucleotides in a 5’ to 3’ direction

7) Hydrogen bonds form between the nitrogen bases

8) Special proofreading enzymes (DNA polymerase) check the new strand of DNA for mistakes. Errors are removed by cutting the mistake out and using an endonuclease and replacing it with the correct nitrogen base

As soon as the newly formed strands are complete, they rewind automatically into the helix structure

Replication continues until the new strands are complete and the two DNA molecules separate from eachother– This is called termination

This replication produces two strands of DNA from one where each strand is composed of “half-old and half-new”

http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2005/Durnbaugh/yfp.html

Replication Fork Adding NucleotidesReplication AnimationReplication Review

http://www.biosci.ohio-state.edu/~mgonzalez/Micro521/04.html

http://distancelearning.ksi.edu/demo/bio378/lecture.htm

Genetic Engineering and Recombinant DNA

Human Genome Project - animation Genetic Engineering – refers to the

alteration of an organism’s genome (complete set of genes) by selectively removing, adding, or modifying DNA

Recombinant DNA – the process of cutting out DNA from one genome and placing the DNA into another genome resulting in a transgenic organism

Examples of transgenic organisms:– Genetically modified bacteria for use in

medicine and bioremediation (environmental clean-up)

– Transgenic plants to improve crop yield and resistance to environmental effects

– Cloned animals (livestock) and organs for human use

Recombinant DNA – How do they do it? Use restriction enzymes

(endonucleases) to cut strands of DNA within their interior (at specific sequences)– animation

Then ligase (enzyme that fuses segments of DNA) is used as a biological glue

Production of human insulin

Gene in the human genome that codes for insulin is cut out using restriction enzymes

The plasmid of an E-coli bacteria is cut using restriction enzymes so that the gene for insulin can be inserted using a ligase

Bacteria can read the DNA and produce insulin for us to later harvest and use

Another example is the insertion of the gene that codes for growth hormone into animals so that they grow faster

Note: Biotechnology refers to the use of organisms or biological products for commercial and/or industrial processes

- video

What are the Issues?– Costs/where money is spent– Motivation for the product– Biological characteristics of the product– Heath effects– Environmental effects– Freedom of Information/Privacy– Who Owns the technology/patents– Issues Animation

Gel Electrophoresis Technique used to separate DNA fragments

by size for the purpose of identification in paternal or criminal suits (animation)

1) Sample of DNA is cut using restriction enzymes from hair, blood, skin, etc. This produces a number of DNA segments of different lengths.

2) The different pieces of DNA (referred to as restriction-fragment-length-polymorphisms or RFLP) are tagged with a radioactive isotope

3) Using an agarose gel that contains holes or wells along one side, the samples of DNA are inserted into the wells. A known sample is loaded with it as a comparison

4) Electric current is run through the gel, causing the movement of the negatively charged DNA fragments. The shortest strands move the farthest (lowest weight) and the longer strands (heavier) will not move as far.

5) This causes separation of the DNA into bands. The gel is left to set

6) When combined with staining or X-ray film, the patterns are used to determine the presence or absence of particular DNA or proteins

DNA Fingerprinting

Developed in 1985 – used to identify whether or not a sample of DNA comes from a specific person

People have similar DNA, however every human (with the exception of identical twins, triplets, etc.) have some unique noncoding segments of DNA called introns; exons are segments of DNA that actually code for proteins

Sample of DNA is placed through gel electrophoresis as well as samples from individuals who are suspected as “owners” of the sample

Because of introns, each individual will have a different number of sites where the restriction enzyme will cut

This results in a unique number and length of fragments which produces a unique banding pattern (fingerprint) when x-rayed

Fingerprints are used to identify criminals, paternity or kinship

Animation

Lane A: DNA from crime scene cut with Enzyme 1 Lane B: DNA from crime scene cut with Enzyme 2 Lane C: DNA from Suspect 1 cut with Enzyme 1 Lane D: DNA from Suspect 1 cut with Enzyme 2 Lane E: DNA from Suspect 2 cut with Enzyme 1 Lane F: DNA from Suspect 2 cut with Enzyme 2

Protein Synthesis

Genetic code is determined by the arrangement of nitrogen bases within the strands of DNA

Each gene codes for the production of a specific protein– DNA RNA protein

transcription translation

Proteins

Proteins are composed of 20 different amino acids that are strung together in endless combinations

Compose cell membranes, cell organelles, muscle filaments, hair, hair color, enzymes (regulate speed of chemical reactions in cells), antibodies (disease-control agents), hormones

Genetic Code It takes the code of 3 nucleotides (a codon)

to code for one amino acid Humans can make 12 of the 20 amino acids,

we must consume the other 8 essential amino acids

Simple protein = 8 amino acids Complex protein = 50 000 amino acids Sequencing amino acids is determined by

DNA Replacement of a single amino acid can

change a protein

Genetic Code

The genetic code has three important characteristics

1. The genetic code is redundant (more than one codon can code for the same amino acid)

2. The genetic code is continuous (reads as a series of three letter codons without spaces, punctuation or overlap)

3. The genetic code is nearly universal (almost all organisms use the same code – this is good for genetic engineering and biotechnology)

Role of DNA in protein synthesis DNA is in nucleus, but protein synthesis

occurs on the ribosomes in the cytoplasm

Carrier molecule (mRNA – messenger RNA) is responsible for reading the information from the DNA (transcription) and carry it to the ribosomal RNA (rRNA) in the cytoplasm where it will be translated into an amino acid sequence by transfer RNA (tRNA)

RNA (Ribonucleic Acid)

Different from DNA in that:1) It’s single stranded2) It contains the sugar ribose3) It is located throughout the cell (DNA is only in

the nucleus with some also in the mitochondria)

4) It contains the base uracil (U) instead of thymine (T)

5) There are three types: mRNA, rRNA, tRNA6) It’s shorter (no introns)

Transcription Protein synthesis begins with

transcription (RNA synthesis) of DNA DNA never leaves the nucleus

(protected)

Steps of Transcription1) DNA molecule unzips (like in replication),

however, RNA nucleotides are now added to the necessary areas (exons) by RNA Polymerases For each gene, only one strand of the DNA is

transcribed, this is called the sense strand. The other is called the anti-sense strand.

2) As double helix uncoils, nucleotides from the mRNA find the appropriate pair by using single DNA strand as a template (5’ to 3’ direction)

3) Uracil binds to exposed adenine bases and cytosine binds to exposed guanine bases

4) mRNA joined and fused in a long chain5) mRNA move away from DNA and the DNA

strands rejoin again6) mRNA leave the nucleus in search of the

ribosomes

http://fig.cox.miami.edu/~cmallery/150/gene/mol_gen.htm

_____________________________DNA

I I I I I I I I

A G C T T A T C

U C G A A U A G

I I I I I I I I

_____________________________RNA

mRNA reads code from DNA RNA codes for amino acids Some codes in mRNA are not for amino acids

but are terminators and initiators Terminators – end protein synthesis (stop

codon) Initiators – turning protein synthesis on (start

codon). Also called promoter site, starts RNA transcription

Transcription Animation

http://www.coolschool.ca/lor/BI12/unit6/U06L01.htm

Example Original DNA sequence: AAT GCC AGT GGT TCG CAC AAA

a) Write the complementary DNA sequence

b) Write the mRNA sequence

c) How many amino acids are there?

d) What is the amino acid sequence?

Do the same for the following DNA sequence:

TAC CAC GTG GAC TGA GGA CTC CTC ATC ATA

Translation Translation is the next stage of protein synthesis Involves translating codons found on the mRNA into

a chain of amino acids (to form a protein) Transfer RNA, tRNA is made up of a single strand of

RNA that folds into a clover-leaf shape– One lobe contains the anticodon, three nucleotides

that are complementary to the mRNA codon– At the opposite end is a binding site for the amino acid

that corresponds to the codon Ribosomal RNA, rRNA is a linear strand of RNA that

remains associated with the ribosomes

More on tRNA

The exposed bases are called the anticodon Each kind of tRNA molecule has a specific

anticodon Ex) Glutamate carried by a tRNA molecule

that carries either the CUU or the CUC anticodon (opposite to mRNA codons)

Ex) Valine carried by a tRNA molecule that has the CAA anticodon

Steps of translation1) Translation is activated when an mRNA molecule binds to an

active ribosome complex in such a way that two codons are exposed

2) The first tRNA molecule carrying the amino acid methionine, base pairs with the start codon on the mRNA, (AUG)

3) Another tRNA molecule arrives at the codon next to the first tRNA, and the first tRNA passes its amino acid on to the second tRNA

4) Enzymes catalyze the formation of a peptide bond between the two amino acids

5) The ribosome moves along the mRNA strand one codon at a time6) The first tRNA molecule detaches from the mRNA and picks up

another amino acid as another tRNA attaches to the mRNA.

NOTE: The process begins with the presence of an initiator (start codon) AUG and ends with the presence of a stop (terminator codon) UAA, UGA, or UAG on the mRNA.

Remember that the sequence of amino acids was originally derived from the message carried by mRNA from the nucleus (DNA)

Translation animation 1 Translation animation 2 Translation 3 Protein Synthesis Process

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Translation.html

http://www.scq.ubc.ca/?p=263

Example

Write a tRNA and amino acid sequence for the following DNA sequence:

TAC CAC TGA GGA CTC CTC CAT CAT

Mutations A mutation is a permanent change in

the DNA sequence caused by mutagens (mutagenic agents)

Mutations – are inheritable– Arise from mistakes in DNA replication

when one nitrogen base is substituted for another

– Creates a new genetic code that gives new instructions to make amino acids (causes a different protein to be made)

Mutagenic agents Physical:

– Pushing or tugging chromosomes Chemical:

– Carcinogens, mustard gas, poor nutrition Medications:

– Some antibiotics Radiation:

– X-ray, ultraviolet radiation, cosmic rays Replication mistakes:

– Natural mistakes occur during mitosis or meiosis Nutritional:

– Lacking certain nucleotides in diet means you are unable to provide the proper free nucleotide base and this causes a mismatch

Biological:– Most viruses use genetic material of chromosomes to reproduce.

They join existing DNA to cause permanent damage

Mutation Animation If a mutation is present in the gametes, it will

be passed on to further generations. This is why it is particularly dangerous for pregnant women

Mutations can be grouped under 2 categories:

1) Chromosomal mutations

2) Point mutations

Chromosomal mutations

Large mutations that visibly effect the structure or number of chromosomes– Ex) nondisjunction, fragile-X-syndrome

Point Mutations

Alter a single gene. There are several types:

1) Base substitution – a foreign base replaces the normal base in each strand of DNA. This could result in one amino acid being different animation– ACGCCA becomes CCGCCA– Ex) Sickle cell anemia – substitution of one

nitrogen base causes an inability to carry sufficient oxygen

2) Insertion: A base is added into the normal DNA sequence– ACGCCA becomes AACGCCA

3) Deletion: a base is removed from the normal DNA sequence– ACGCCA becomes CGCCA– Ex) Cystic fibrosis – deletion of 7th, 8th, and

9th nitrogen bases causes an inability to produce protein that regulates chloride channels

– animation

NOTE: both insertion and deletion result in a frame-shift mutation because every amino acid after the point of mutation may be affected

4) Translocation: a sequence of nitrogen bases is removed from one area and placed in another– ABCDEFGHIJ becomes ABFGHIJCDE

5) Inversion: reversal of a sequence of nitrogen bases– ABCDEFGHIJ becomes ABEDCFGHIJ

6) Duplication: duplicating a set or sequence of nitrogen bases twice in one location– ABCDEFGHIJ becomes ABCABCDEFGHIJ

7) Silent mutations: no phenotypic effect because certain amino acids have more than one code– GTA (CAU) and GTG (CAC) both code for

histidine

NOTE: The body can repair some mutations, but not all

Oncogenes and Cancer In normal cells, protein synthesis is

carried out by structural genes only when required

Because protein synthesis is not always required, a regulator gene produces a repressor protein which switches off protein synthesis and reducing the rate of cell division

P53 animation

Uncontrolled cell division is cancer. Cancer is caused by a mutation due to an

environmental factor or carcinogen Mutation could be a base substitution that

prevents the production of the repressor protein, or the movement of a gene from one part of the chromosome to another

If the structural gene is separated from its regulator gene, it cannot be turned off (cancer forms)– These genes that cannot be turned off are called

oncogenes

Most common oncogene = ras Found in 50% of colon cancers and

30% of lung cancers Ras makes a protein that acts as an

“on” switch for cell division. Once a sufficient number of cells are produced, it should shut off

Cancer-causing oncogene produces a protein that blocks the “off” switch (causes cell division to continue at an accelerated rate)

Diagnosing Genetic Disorders

Amniocentesis and Chorionic Villus Sampling can take cell samples from a developing fetus or embryo– This sample can be screened for genetic

markers for certain disorders• Uses a DNA probe which identifies problem

genes

Gene Therapy

Targets genetic causes of diseases rather than their symptoms

A DNA vector carries foreign DNA into target cells of the patient – The vector is usually a virus that has been

genetically altered to carry a desired gene– The virus will eventually transfer the new

gene into the cell’s genome

Gene Therapy

Somatic Gene Therapy – correcting genetic disorders in somatic cells– Mutations can still be passed on to offspring

Germ-line Therapy – correcting the genetic information in sperm and egg – Could have many negative effects on future

generations– Currently banned in Canada

Ames Test Technology to determine quickly,

cheaply, and accurately if a chemical is mutagenic.

Any chemical that is mutagenic has potential to be carcinogenic

We must test products for their cancer-causing agents

Performed on bacteria that have been mutated so they cannot produce histadine on their own (we must supply them with it in order for them to survive)

Plate this bacteria on a petri dish with no histidine and the chemical being tested (expect no growth)

If bacteria are found, conclusion can be made that microbe has been mutated and is now producing its own histidine

» The more colonies that are found, the higher the strength of mutagenic the chemical is which indicates it is highly carcinogenic

Often chemical is added to liver enzymes because chemicals are often harmless to an individual until they are broken down in the liver into toxic metabolites

NOTE: some chemicals can cause cancer in some individuals and not in others (because of different nitrogen base sequences in each individual)

Biological Warfare

Most disease-causing agents can be exploited for biological weaponry

Microbe or toxin produced by microbe may be harmful to livestock, grains, bacteria in soil, or humans

Fortunately, few organisms are suited for mass destruction

Examples

AIDS – not transmitted by air Clostridium botulinum – deadly in water

(not air)…one kg of toxin in water reservoir kills ~50 000 people (60% of population dies in 24 hrs)

Research/Testing Stations

Britain = Porton Down USA = Camp Detrick in Maryland Canada = Suffield in Alberta

Preferred microbe was anthrax bacillus (affects cattle and humans).

Anthrax

Deadly spores (rod-shaped bacterium) live long periods of time, are highly contagious, and resistant to many environmental factors

We currently have the ability to create the “superbug” through merging genes for rapid reproduction and environmental resistance…what do you think would happen then?

Mitochondrial DNA

Mitochondria = responsible for cellular respiration

1960’s – discovered that mitochondria contains its own DNA (mtDNA)

They have an amount of control over what they do (not completely controlled by nuclear DNA)

Endosymbiotic Hypothesis

Mitochondria once were free living bacteria that were engulfed by other cells.

The two cells developed mutualistic relationship (mitochondria had protection and food, engulfing cell had a source of energy and oxygen)

Evidence to support theory1) MtDNA resembles the loops of DNA found

in bacteria and viruses2) The mtDNA is tiny compared to nuclear

DNA3) Mitochondria divide and replicate

independently of the cell itself* the same theory is used to explain how

photosynthetic cells gained chloroplasts

NOTE: The mtDNA in our bodies is maternal because sperm’s mitochodria are lost when their tail falls off.