DNA in Detail

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DNA in Detail

DNA Structure & Replication

How We Discovered Genetic Material: DNA vs. Proteins

1952: Alfred Hershey and Martha Chase set out to determine whether DNA or protein was the genetic

material

Virus Reproduction

Viruses are made of 2 parts: Inner core made of nucleic acid Outer coat, called the capsid, made of protein

T2(type 2) virus reproduces inside bacteria Hershey and Chase reasoned that if they could determine which part of the virus, DNA or protein,

enters bacteria to produce more viruses, they would know if genes are made up of DNA or proteins Set up 2 experiments to test this outHershey-Chase Experiments

Experiment 1: Labeled DNA with radioactive 32P (red) Viruses were allowed to attach to bacteria and inject genetic material Agitated mixture in a kitchen blender to shake off whatever was left on the outside of the bacteria Centrifuged (spin at super high speed) culture so bacteria collected in a pellet at bottom of

centrifuge tube

Tested pellet: found most of32

P-labeled DNA in bacteria and not in the liquid medium Conclusion: DNA entered bacteria

Experiment 2: Labeled protein capsid with radioactive 35S (red) Viruses were allowed to attach to bacteria and inject genetic material Agitated mixture to shake off whatever was on the outside of the bacteria Centrifuged culture to form a pellet on bottom of centrifuge tube No 35S found in pellet; most 35S found in liquid medium Conclusion: Radioactive protein capsids remained on outside of bacteria and were shaken off by

blender into the liquid medium

Hershey & Chase Conclusion

Experiments proved that the DNA of a virus, and not its protein, enters the host, where virusreproduction occurs

Thus, DNA is genetic material: it transmits all genetic information needed to produce new viruses.

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DNA Structure

Review

DNA is a polynucleotide Composed of 3 subunits: nitrogenous base, phosphorus group, and pentose sugar 4 bases: 2 purines (Adenine, Guanine) with double ring; 2 pyrimidines (Cytosine, Thymine) with

single ring

Backbone made of sugar and phosphates Double helix structure of 2 twisting strands held together by hydrogen bonds between bases (AT, C

G)

5 and 3

The 2 DNA strands are antiparallel; they run in opposite directions Sugar molecules are oriented differently (see diagram below) 5thCarbon is uppermost in one strand, 5 (5 prime) 3rdCarbon (attached to phosphate) is uppermost in 2ndstrand, 3 (3 prime)Read the Science Focus on Finding the Structure of DNA(page and chapter varies between textbooks)

Who discovered the structure of DNA? James Watson, an American biologist, and Francis Crick, a British Physicist (early 1950s) Used previous research (including Chargaffs rules and the work of Franklin and Wilkins) to

deduce that DNA has a twisted, ladder-like structure, with sides made of sugar-phosphate

molecules and rungs made of bases

Determined that A is hydrogenbonded with T, and G is hydrogen

bonded with C; this model suggests

that complementary base pairing

plays a role in DNA replication

What is Chargaffs rule? Regardless of the species, DNA

always contains an equal number of

purines and pyrimidines

the frequency of A equals that of T the frequency of G equals that of C

How did Rosalind Franklin and MauriceWilkins contribute to the research? Prepared an X-ray diffraction

photograph of DNA

Showed that DNA is a double helixof constant diameter, and that the

bases are regularly stacked on top of

one another

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DNA Replication

Process of copying one DNA helix into 2 identical helices Each old strand serves as a template for the formation of a complementary new strand Semiconservative: each new helix has one old (conserved) strand and one new (not conserved) strand

Video: DNA Replication Process

Parent-Daughter DNA replication

1. Strands H-bonded to each other2. DNA helicase(an enzyme) unwinds and unzips the double-stranded DNA (breaks up weak H-bonds)3. Complementary DNA nucleotides are joined to old strand byDNA polymerase(an enzyme)4.

DNA ligase(an enzyme) seals any breaks in sugar-phosphate bonds5. The 2 new double helices are identical to the original strand

Cancer Treatment

Some chemotherapeutic drugs are analogs that have similar, but not the same, structure as the 4nucleotides.

When cancer cells mistakenly use these to synthesize DNA, replication stops and cancer cells die off

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Homework

1. What do we mean when we say that 2 strands of DNA are antiparallel?2. Label the 5 Carbons in ribose and attach a phosphate group and a base to the right Carbon; connect

them to another nucleotide that has its Carbons labeled too.

3. Draw 3 sugar-phosphate backbones (using pentagons and circles and sticks); label the 5 Carbon and3 Carbon in each one.

4. Using 2 different colours (1 for old strand, 1 for new strand), draw a simple ladder-like DNA doublehelix. In a cartoon, show how the helix unwinds, how new bases are added, and why the new strands

are semiconservative.

Gene Expression

Gene: segment of DNA thatspecifies a specific amino acid

sequence of a protein (in the 21st

century, we also know that portionsof a gene may help in its

expression/regulation)

RNA

contains the bases A, U, G, and C Single stranded, not double Does not form a double helix3 Major Types:

mRNA: messenger RNA Takes message from DNA

and brings it to ribosomes (DNA stays in nucleus; ribosomes are in cytoplasm)

tRNA: transfer RNA Transfers amino acids to the ribosomes

rRNA: ribosomal RNA Along with proteins makes up the ribosome, where new proteins are made (synthesized)

Transcription and Translation

Gene expression (production of a protein) requires 2 processes: Step 1) transcription Step 2) translation

Transcription (in brief)

Genetic information on DNA in nucleus is transferred (transcribed) to RNA Polynucleotide is transcribed letter by letter from DNA template (master copy) to mRNA copy

(actually a mirror copy, as in AU, TA, CG, and GC)

The mirror copy is later used to retranslate the original message

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Translation (in brief)

The RNA transcript (message fragment produced during Step 1: transcription) directs the sequence ofamino acids that are added to a polypeptide

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Nucleotide vs. Amino Acid Sequence

Nucleotide sequence is very different from amino acid sequence The nucleotide sequence coded by mRNA helps direct the addition of amino acids by the ribosomes

Step 1: Transcription in Detail

RNA polymerase: an enzyme that binds to a promoter region and opens up the double helix (unzipsit) so that complementary base pairing can occur just in this region

Promoter region: region that contains special sequence of DNA that signals, Start here! RNA polymerase also joins the complementary base pairs to form an RNA sequence, which results in

an mRNA molecule

Video: Transcription

mRNA Maturation

Before entering the cytoplasm, the mRNA must be processed. The original DNA and the transcribed mRNA contain intron and exon regions. Intronsare genetic areas that do not code for proteins and must be removed before the mRNA leaves

for the cytoplasm. They are called introns because they are intragene segments.

Exonsare portions of the gene that are ultimately expressed. Only the exons result in a protein product. Modifications to mRNA:1) A cap and tail are added to the primary RNA Cap is composed of an altered guanine nucleotide Tail is composed of many adenosine nucleotides. It is often referred to as a poly-A tail because

poly means many and A stands for Adenosine.2) Introns are removed and exons rejoined together Splicing done by a complex of both RNA and protein (RNA is the enzyme here, not protein, and

we call it a ribozyme).

Capped, tailed, spliced (so there are exons only), mRNA is now a mature mRNA fragment.All or None? Or Some?

Processing normally brings together all the exons of a gene; however, in some cases, some exons arekept while others are removed or not expressed.

This results in a different protein for each different combination.

We suspect white blood cell recognition of specific antibodies for each antigen encountered is a resultof this.

Step 2: Translation in Detail

2ndstep of gene expression that leads to protein synthesis. Requires several enzymes: mRNA, tRNA, rRNAGenetic Code

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Just 4 nucleotides for 20 aminoacids! How does the code work?

If 1 base = 1 amino acid:4 possibilities

If 2 bases = 1 amino acid:16 possibilities

If 3 bases = 1 amino acid:64 possibilities (refer to

table on right)

DNA is based on a triplet code. Codon: a 3 letter (nucleotide)

unit of mRNA that codes for an

amino acid.

61/64 code for amino acids 3/64 code for stop codon:

signals polypeptide termination Methionine codon (AUG) is

also the start codon: signals

polypeptide initiation

Why repetition? Why do some aminoacids have many codons? (ie. several

have 4 codons, and one has 6 codons)

Allows for some protection fromharmful mutations (while also

promoting variation)

tRNA (transfer RNA)

brings amino acids to ribosomes is single-stranded boot-shaped nucleic acid that loops

back on itself and carries a specific amino

acid to the forming polypeptide chain

based on its anticodon, the CBP for a

specific codon of mRNA

Codon-Anticodon

The anticodon of a tRNA-amino acidcomplex pairs with the mRNA codon

The amino acid is added to theforming polypeptide chain

Once the amino acid is taken from thetRNA, that tRNA leaves the area

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The mRNA codon sequence determines the order that tRNA-amino acids arrive at the ribosomeVideo: Protein Synthesis: Translation Process

Ribosomes

Small structural bodies found in cytoplasm and endoplasmic reticulum where translation occurs Composed of many proteins and several ribosomal RNA (rRNA) rRNA produced in nucleolus joins with proteins formed in cytoplasm to form 2 ribosomal subunits

(the large and small subunits), which leave and join together in cytoplasm for protein synthesis

1 binding site for mRNA 2 binding sites for 2 tRNA at a time Ribosome moves down mRNA; new tRNAs then arrive and add amino acids to the growing

polypeptide chain

Ribosomes dissociate once protein is fully formedPolyribosomes

Often, as one ribosome moves along an mRNA molecule, another ribosome will attach to thebeginning of the same mRNA

Allows many proteins to be formed at the same time from one mRNA strand The entire complex (ie. mRNA + ribosomes) is called a polyribosome (much more efficient than

making a single protein from one mRNA strand)

Video: Polyribosome

Translation in 3 Steps:

Translation must be very orderly so that the amino acid sequence in a polypeptide is correct1) Chain Initiation: steps necessary to begin translation2) Chain Elongation: steps necessary to effect polypeptide synthesis3) Chain Termination: steps necessary to end the process of translationVideo: mRNA Splicing

Video: Translation

Chain Initiation

Step 1: small ribosomal subunit attaches to mRNA around start codon (AUG) Step 2: anticodon of the initiator tRNA-methionine complex binds with this codon Step 3: large ribosomal subunit joins with small ribosomal subunit in order to begin elongationChain Elongation

a ribosome has 2 binding sites for tRNAeach newly-arrived tRNA at site 2 receives a peptide froma tRNA at site 1

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tRNA at site 1 leaves, and tRNA at site 2 moves to site 1, making room for another tRNA to nowarrive at site 2

this movement of ribosomes along the mRNA during protein synthesis is called translocation Ribozyme: an RNA enzyme that is part of the larger ribosomal subunit Attaches peptide to newly arrived amino acid (formation of a peptide bond) Occurs over and over as protein is synthesized tRNAs that leave are now free to pick up a new amino acid and return

entire cycle of CBP to new tRNA, transfer of peptide, and translocation is rapid (15 times/s in E. coli)Chain Termination

occurs whenprotein synthesis comes to an end at a stop codon peptide is enzymatically cleaved from the last tRNA by a release factor tRNA and polypeptide leave, and ribosome dissociates into its 2 subunitsIn short,

Step 1: TranscriptionDNA base sequence is copied into mRNA Step 2: TranslationmRNA serves as a template for tRNA to bring amino acids that form a protein

Genes and Gene Mutations

Gene: a sequence of DNA bases that code for a product, which is usually a protein Gene Mutation: a change in sequence of bases within a gene May lead to malfunctioning proteins in cells (or it may not).

Causes of Mutation

3 general types: Errors in replication Mutagens Transposons

If it occurs in gametes, then offspring may be affected in cells (somatic), then cancer may result

Errors in Replication

Mistakes in DNA replication occur, but are rare DNA polymerase proofreads new strand against old strand and corrects mismatched pairs: only 1

mistake out of 1 000 000 000 replicated nucleotides occur

Mutagens

Mutagens: environmental influences that cause mutations in cells eg. radiation (radioactive elements and isotopes, X-rays, UV light, gamma rays), certain organic

chemicals (some chemicals found in pesticides or in cigarette smoke)

Mutation rate ends up being low because of repair rate and efficiency of DNA repair enzymes

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Transposons

Transposons: segments of DNA that have ability to move within and between chromosomes Movement affects neighbouring genes by increasing their expression (up-regulating) or

decreasing it (down-regulating)

Jumping genes: genes that have been up- or down- regulated due to transposons; responsible forcertain genetic conditions (Charcot-Marie-Tooth disease) in humans and plants (white corn

kernels)

There are 2 major types of mutation:

Frameshift Mutations

Frameshift Mutation: one or more nucleotides are inserted or deleted from a sequence, resulting in anincorrect reading of mRNA that leads to an incorrect amino acid sequence in the polypeptide

eg. CCC GGG AAA TTT add a T in the 2ndpositionPRO, GLY, LYS, PHECTC CGG GAA ATT T (frame has shifted 1 over)LEU, ARG, GLU, ILE

Point Mutations: involve the substitution of one nucleotide for another

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Silent Mutation: Consider CTT: what does it code for? Substitute an A in the 3rdposition: what does it code for? Substitute a C in the 3rdposition: what does it code for? Substitute a G in the 3rdposition: what does it code for? Answer: they all code for leucine (LEU)

Nonsense Mutation:Consider TGT (CYS): Substitute an A in the 3rdposition: what does it code for? Stop Substitute a C in the 3rdposition: what does it code for? CYS Substitute a G in the 3rdposition: what does it code for? TRP Which one do you think is a nonsense mutation? Answer: TGA is a nonsense mutation because it results in a premature stop codon.

Missense Mutation: Consider the above cases. Which one(s) do you think are missense mutations?How would you define missense mutation?

Answer: TGG is the missense mutation. Substitution of an incorrect amino acid results in achange in the protein which may affect its function.

Questions

How did researchers crack the code for what each codon represents? DNA is a triplet code because this provides enough possibilities for 4 bases to code for 20

different amino acids.

Artificial RNA was added to a medium containing bacterial ribosomes and a mixture of aminoacids. By comparing the bases in the RNA with the polypeptide produced, investigators were able

to decipher the code.

Thinking question: at which codon position (2 or 3) do you think the most missense mutations occur?What about silent mutations? Explain your answers.

I think the most missense mutations occur at the 2nd codon position, while the most silentmutations occur at the 3rdcodon position.

Looking at a chart relating base sequence and amino acid sequence, one can see that some aminoacids have more than one codon. With the exceptions of leucine and arginine, these codons only

differ by the base in the 3rdcodon position, so changing this might still yield the same amino acid.

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On the contrary, changing the base in the 2ndposition often results in a different amino acid than

the one intended, potentially affecting the function of the protein.

Discuss with a friend the causes and types of mutations. Errors in replication only occur in 1 out of 1 000 000 000 replicated nucleotides, since DNA

proofreads the new strand against the old strand and corrects mismatched pairs.

Mutagens include environmental influences such as radiation from radioactive elements andisotopes, X-rays, UV light, and gamma rays; as well as certain organic chemicals found in

pesticides or cigarette smoke. DNA repair enzymes have such a great repair rate and efficiency

that the mutation rate ends up being low.

Transposons are segments of DNA that have the ability to move within and betweenchromosomes. Their movement affects neighbouring genes by increasing their expression (up-

regulating) or decreasing it (down-regulating). Jumping genes which have been up- or down-

regulated due to transposons are responsible for certain genetic conditions such as Charcot-Marie-

Tooth disease in humans, and white corn kernels in plants.

Frameshift mutations occur when one or more nucleotides are inserted or deleted from asequence. This results in an incorrect mRNA reading, leading to an incorrect amino acid sequence

in the polypeptide.

Point mutationsinvolve the substitution of one nucleotide for another. Silent Mutations do notcause a change in the amino acid. Nonsense Mutations result in a premature stop codon and a

shortened polypeptide. Missense Mutations produce a different amino acid, which may or may

not affect the function of the final protein.

Explain again in detail transcription and translation Transcription: The nucleotide base sequence of a DNA template is copied letter by letter into

mRNA. It begins when RNA polymerase binds to the promoter region, a region containing a

special sequence of DNA that signals the start of transcription. RNA polymerase unzips the

helical DNA to allow CBP in just one specific region, and it also joins the complementary base

pairs to form an RNA sequence and subsequently, an mRNA molecule. Before entering the cytoplasm, primary mRNA must undergo processing to remove any introns.

Splicing is done by a complex of RNA and protein, called a spliceosome.

Translation in 3 steps: Chain Initiation: A small ribosomal subunit composed of rRNA and proteins attaches to a

mature mRNA strand around the start codon, AUG. An initiator tRNA-methionine complex

with the complementary anticodon, UAC, binds to the mRNA codon. A large ribosomal

subunit then joins with the small ribosomal subunit to being elongation.

Chain Elongation: Ribosomes have 2 binding sites for tRNA. New tRNA arrive at Site 2 andreceive a peptide from the tRNA at Site 1, which then leaves. In a process known as

translocation, the ribosome moves down the mRNA molecule, so the tRNA at Site 2 is now at

Site 1, making room for another tRNA at Site 2. Ribozymes attach the growing polypeptide

chain to the newly arrived amino acid at Site 2 by forming a peptide bond. This occurs over

and over again as protein is synthesized.

Chain Termination: Protein synthesis ends when the ribosome reaches a stop codon. Thenew polypeptide is cleaved enzymatically from the last tRNA by a release factor. tRNA and

the polypeptide leave, while the ribosome dissociates into its 2 subunits.

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DNA Biotechnology

Cloning of a Gene

Cloning is the production of identical copies of an organism. This occurs naturally in new plant shoots, bacterial colonies, and identical human twins. Gene cloning is the production of identical copies of a gene.Video: How to Clone Animals .

Using Recombinant DNA Technology

Recombinant DNA (rDNA do NOT get thismixed up with rRNA) contains DNA from two

or more different sources.

A vector plasmid (a small accessory ring ofDNA in bacteria) or virus is necessary to insert

foreign DNA into a host cell.

Restriction enzymescleave the vector DNA andthe source DNA at a specific sequence, leaving

sticky ends that allow a portion of source

DNA to be inserted into the vector DNA.

DNA ligase then seals the openings andrecombinant DNA is formed.

Cloning

After recombinant DNA enters a host cell, itmay be copied.

If successful, some particular gene(s) have beencloned and can be recovered.

Bacterial cells take up recombinant plasmidsand clone the new DNA.

Reverse Transcription

When a viral vector is used, the cloned DNA is inside newly formed bacteriophages. To express a human gene in a bacterium, it must not have introns. Reverse transcriptasecan be used to make a DNA copy of mRNA; this complementary DNA does

not contain introns. (see picture on the next page)

Using the Polymerase Chain Reaction

The Polymerase Chain Reaction(PCR) produces many copies of a single gene or piece of DNA. PCR requires DNA polymerase and a supply of nucleotides for the new DNA strands. Artificially cycles through a) unzipping DNA; b) copying DNA; and c) rezipping DNA. Cycle repeats 30-36 times so that with 1 starting copy, up to 235new copies may be made.

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DNA Fingerprinting

The entire genome of an individual can be cut byrestriction enzymes to yield variable fragment lengths.

Gel electrophoresis separates fragment lengths.

Use of probes results in a pattern unique to theindividual (sometimes called aDNA fingerprint).

CSI

You can identify a person who has committed acrime, or tell who is related to whom by doing a DNA

fingerprint.

Since PCR can amplify the smallest amount ofDNA, a single sperm, or one cell on a cigarette butt,

provides enough DNA to be identified by comparison

with sample DNA.

Video: How does DNA fingerprinting work? .

Biotechnology

Using natural biological systems to create a product or to achieve an end desired by human beings. Transgenic organismshave had a foreign gene inserted into them.Video: Fincasters Episode 8 GloFish .

Transgenic bacteria are used to produce biotechnology products such as insulin, human growthhormone, t-PA, and hepatitis B vaccine. They also add insecticidal toxins to plants, reduce frostdamage, degrade wastes, produce chemicals, and help mine metals.

Transgenic plants: Foreign genes are added to protoplasts, plant cells that lack a cell wall, using anelectric current. Foreign genes in cotton, corn, and potatoes have given them pest resistance; soybeans

are made resistant to herbicide for no-till farming. Transgenic plants produce human hormones,

clotting factors, and antibodies in their seed. One weed has even been engineered to produce plastic

granules.

Video: What is Genetically Modified Food? .

Transgenic animals: Foreign genes can be inserted into animal eggs by hand or by vortex mixing.Gene pharming uses transgenic farm animals to produce pharmaceuticals in their milk. There are

plans to use animals to produce drugs for the treatment of cystic fibrosis, cancer, blood diseases, etc.

In vitro fertilization is necessary.

Cloning of Animals

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Cloning of mammals was once considered impossible, but has now been accomplished with sheep,calves, and goats. A 2N nucleus from a bioengineered animal is inserted into enucleated (original

nucleus removed) eggs from a donor. A surrogate mother gives birth to the cloned animals.

Animal Organs as Biotechnology Products

Scientists are genetically engineering pigs to serve as organ donors for humans who need transplants.Transplants of organs across species isxenotransplantation.

Video: BioBytes Genetically Engineered Animals Xenotransplant concerns: Researchers are trying to make organs less antigenic to humans. One

concern is whether pig organs might carry animal viruses into humans. HIV is a virus that jumpedfrom monkeys into humans. Tissue engineering is an alternative method of securing transplant

material from culturing human tissue from a mixture of cells and synthetic polymers.