Unit 7

Fredrich Miescher

* 1869
* First isolated or discovered DNA
* Called it “nuclein” or nucleic acids

Fredrick Griffith

* 1928
* Discovered “transforming factor” in bacteria
* Harmless bacteria were transformed into harmful bacteria when introduced to dead, harmful bacteria

Oswald Avery

* 1944
* Repeated Griffiths experiments
* Discovered that the transforming factor in bacteria was DNA

Erwin Chargaff

* 1950
* Determined
* DNA differs between species
* Pairing of bases in DNA
* A = T and G = C
* This is known as “Chargaff’s Rule”

Hershey & Chase

* 1952
* Worked with bacteriophage viruses to prove viruses to prove DNA is the genetic material
* Experiment Summary:
* Radioactive sulfur stained viral protein coat
* Radioactive phosphorus stained viral DNA
* When virus infected bacteria, only radioactive phosphorus was found in the bacteria
* Means DNA is the genetic material

Rosalind Franklin

* 1952
* Used X-ray diffraction to produce “Photo 51”
* Discovered DNA was double helix
* Also worked with scientist Maurice Wilkins, who was the head of lab

Watson & Crick

* 1953
* Also worked with scientist Maurice Wilkins, who was the head of the lab
* Published work won Nobel Prize

Prokaryotic cells have…

1 circular DNA molecule

Eukaryotic cells have…

Strands of DNA in the nucleus

* These strands become chromosomes

Chromosomes are made of…

Pieces of DNA coiled around proteins

* These proteins are called histones

DNA meaning

Deoxyribonucleic Acid

DNA

* Stores genetic information
* Made of nucleotide
* Shaped like double helix
* Looks like twisted ladder
* Sides are made of the sugar phosphate backbone
* Alternating sugars & phosphates
* Held together by covalent bonds
* (Think of the backbone like the sides of the ladder)
* The middle of the DNA molecule is made up of the nitrogenous bases
* Like the rungs/steps of the ladder
* Bases held together by hydrogen bonds

Nucleotide Parts

1. Sugar (deoxyribose)
2. Phosphate
3. 1 of 4 nitrogenous bases


1. Adenine, Thymine, Guanine, Cytosine

2 sides of DNA molecule

* Sides known as anti-parallel
* Strands run from 5’ → 3’ direction
* 5’ always starts with a phosphate
* Complementary strands run in opposite directions

2 types of nitrogenous bases

Purines and Pyrimaidines

Purine

* Have 1 ring
* Adenine and Guanine

Chargaff’s Rule

A = T and C = G

Because of this base pairing rule, DNA is said to have complimentary strands

About DNA

* Chromosomes are made of DNA which contain sequences called genes
* Genes code for various proteins
* These proteins determine your traits
* DNA is the code that provides instructions to tell your cells how to make proteins
* The sequence of nitrogenous bases is the code that makes the particular protein for a given trait

Pyrimidine

* Have 2 rings
* Include Cytosine & Thymine
* Uracil in RNA only
* Always pairs with a pyrimidine

DNA Replication

The process by which DNA makes a copy of itself and in turn its chromosomes

Amount of Nucleotides in Human Body Cells

\~6 billion pairs

How Many Hours to Copy DNA

6 hours

DNA Replication produces…

2 identical strands of DNA

The Base Pairing Rule Allows Each Strand…

To serve as a template

What Do The 2 Strands of DNA Contain

Complimentary info

Watson & Crick suggested that…

The DNA double helix separates and each new strand was copied

The Proposed 3 Models of DNA Replication

Conservative Model, Dispersive Model, Semi-Conservative Model

Conservative Model

1 original helix and 1 brand new helix produced after replication

Dispersive Model

Sections of original DNA mixed together with brand new DNA

Semi - Conservative

1 strand of original DNA and 1 strand of new DNA

Steps of Replication

1. The DNA unzips
* Begins at specific sites called origins of replication
* Copying moves out in both directions creating replication bubbles
* Within each bubble there is a replication fork
* Bubbles enlarge in both directions & eventually merge/meet, forming duplicate strands
* This step uses helicase
* Does this by breaking the hydrogen bonds between the nitrogenous bases
* Found at the replication fork
2. Bases are Paired
* At the replication fork, enzymes add new complementary nitrogenous bases to parent/original DNA strands
* This step uses DNA polymerase
* If there is a mismatch the DNA polymerase can backtrack and fix the mistake
* There is a challenge with this step DNA polymerase can only add bases (build the complimentary strand) in the 5’ → 3’ direction
* No problem for original 3’ strand; the new complementary side starts with 5’ so bases can be added 5’ → 3’
* Replication is continuous on this side; known as the leading strand
* Bases can be added one after another
* The original 5’ strand has the issue… bases cannot be added 3’ → 5"‘ direction
* Replication on this side is discontinuous; known has the lagging strand
* New complementary bases are added in segments/chunks in the 5’ → 3’ direction
* These segments are called Okazaki fragments
3. Strands Linked Together
* This step uses ligase
* Each copy has 1 original strand and 1 now strand so it is semi conservative

Replication Fork

Site where the DNA is splitting

Helicase

Enzyme that “unzips”/”unwinds” the DNA

* Does this by breaking the hydrogen bonds between the nitrogenous bases

DNA Polymerase

Enzyme that pairs & proofreads the nitrogenous bases

Ligase

* Links Okazaki fragment together
* Hydrogen bonds reform bonds reform between the new base pairs, leaving 2 identical copies of the original DNA molecule
* Each copy has 1 original strand and 1 new strand

Correct DNA Replication Model

Semi - Conservative

What is the instructions to make proteins in DNA based on?

Based on the order of nitrogenous bases

Where are proteins made at

Ribosomes

Ribosomes monomers

Amino Acids

How many types of amino acids

20

The different arrangement of amino acids make…

Different proteins

The order of nitrogenous bases determines…

Which amino acid is added in what order, so ultimately

* The order of nucleotides → order of amino acids
* The order of amino acids → a specific protein

This process is called Protein Synthesis

RNA

RiboNucleic Acid

* One single strand/helix
* Found inside and outside the nucleus

RNA made of

Made of nucleotides, but different from DNA

* Phosphate
* Ribose (Sugar)
* Nitrogenous Bases (Adenine, Uracil, Guanine, Cytosine)

Types of RNA

mRNA, tRNA, rRNA

mRNA

Messenger RNA

* Copies info from DNA & brings it to ribosome
* Located in nucleus, cytoplasm, ribosome

tRNA

Transport RNA

* Brings amino acids to ribosome & pairs them with mRNA
* Located in cytoplasm and ribosome

rRNA

Ribosome RNA

* Directs functions of mRNA & tRNA
* Located in ribosome

2 phases of protein synthesis

1. Transcription
2. Translation

Transcription

Takes info from DNA and transfers it to a molecule of mRNA

* Occurs in the nucleus
* Only uses RNA polymerase = enzyme that adds and links complementary RNA nucleotides during transcription
* Does the job of helicase, DNA polymerase and ligase all in one
* Only uses the leading strand of DNA so new RNA strand can build 5’ → 3’

3 Steps to Transcription

1. Initiation
* RNA polymerase binds to genes promoter region (the start)
2. Elongation
* RNA polymerase unwinds & separates the 2 strands of the DNA helix & adds new RNA bases
* This continues until a stop sequence at the end of the gene is reached
3. Termination
* RNA polymerase lands at the stop sequence
* mRNA is released and the DNA rezips
* In eukaryotic cells, the mRNA has to be modified before it can move to the next phase of protein synthesis
* DNA is mixed with 2 types of nucleotide sequence, introns and exons
* Process called RNA splicing occurs
* Introns are removed/cut out
* Exons are stitched together in 1 seamless strand

mRNA is now ready for the next phase of protein synthesis

Base Pairing for RNA

* T = A
* A = U
* C = G & G = C

mRNA bases are added…

in groups of 3

Codons

Sets of 3 mRNA bases that code for an amino acid

Introns

Non-coding segment of nucleotide sequences

Exons

Coding segment of DNA; the parts that will be translated and expressed

Translation

* Occurs at the ribosome
* During this phase, tRNA carrying amino acids match up with mRNA to interpret the message & build proteins
* tRNA is folded into a compact shape
* Each tRNA has an anticodon
* tRNA anticodons determine the particular amino acid the tRNA carries/transports to ribosome
* This specific amino acid will therefore also correspond to the mRNA codon

Ribosomes are made up of what 2 parts

Large subunit and small subunit

* mRNA enters and binds on the small subunit
* There are 3 slots in the ribosome where the mRNA & tRNA bond and translation occurs

The 3 Slots where mRNA & tRNA Bond

Attachment Site (A Site), Peptide Bond Site (P Site), Exit Site (E Site)

Attachment Site (A Site)

Where tRNA enters ribosome & meets with mRNA

Peptide Bond Site (P Site)

Peptide bonds from between amino acid s of 2 tRNAs

* Where the protein chain forms

Exit Site (E Site)

“Naked” tRNA leaves the ribosome & goes off to pick up another corresponding amino acid

Steps of Translation

1. mRNA leaves the nucleus & enters the cytoplasm
* At the same tRNA picks up an amino acid and brings it to the ribosome
2. The mRNA “start codon”(AUG) enters the A site, then moves into the P Site, signaling the start of the protein
* Allows a tRNA carrying amino acid “methionine” to bind to the start codon
3. Next mRNA codon matches with next tRNA anticodon & amino acid in the A Site
4. Amino acid from 2nd forms a peptide bond with methionine
5. New tRNA enters A Site & first tRNA moves into E Site, where it leaves & goes off into cytoplasm to find another corresponding amino acid
6. Repeat steps 3 - 5 until the end of the mRNA strand is reached
* The last code on mRNA is the “stop codon”

Biotechnology

The use of organisms to perform practical tasks for humans

* This is accomplished through Genetic Engineering
* Through years of experiments, scientists observed bacteria & viruses can swap or absorb new genes from other bacteria & viruses or from their surrounding environment

Recombinant DNA

DNA made from 2 or more different organisms

Plasmids

* Many bacteria contain this
* Small, circular DNA molecules that are seperate from the main bacterial chromosome
* They carry genes & can replicate independently

When a plasmid replicates…

One copy can pass from one generation to another

* Results in gene “sharing” among bacteria

This is how antibiotic resistance can form

Can also be used in genetic engineering through gene cloning

Steps to Gene Cloning

1. Plasmid DNA is removed from a bacteria cell & desired gene is inserted into the plasma
2. Plasmid is now a combo of original DNA & new DNA - recombinant DNA
3. Recombinant plasmid is put back in the bacteria cell & replicates, making as many copies as needed

The piece of DNA is cut out of a larger DNA molecule by what enzyme?

Restriction enzymes

Restriction Enzymes

Bacterial enzymes that recognize & bind to short sequences of DNA, then cut the DNA between specific nucleotides within the sequence

* Cut the sugar phosphate backbone

Most restriction enzymes make staggered cuts cause…

DNA hanging off the ends of the fragments, known as sticky ends

* They are available to bind to any complementary sequence

DNA ligase “pastes” the sticky ends together in the recombinant DNA, repairing the sugar phosphate backbone

* The recombinant DNA can now go through gene cloning

One challenge of genetic engineering is…

Finding & isolating the cells that contain the gene of interest

* Cells that have taken up the plasmid DNA are identified by growing the bacteria is petri dishes with an an antibiotic
* Only the cells that survive the antibiotic exposure have the plasmid DNA
* The surviving cells reproduce & form a bacterial colony

Confirmation of the cloned gene can be accomplished by what

Gel electrophoresis

Gel Electrophoresis

A technique used to sort DNA fragments by length

Gel Electrophoresis Steps

1. DNA from the sample is cut into fragments by

restriction enzymes. The fragments are different sizes


2. Drops of the sample are placed in wells or slots at one

end of a thin slab of gel (made from agar)


3. An electric charge / field is applied & the DNA begins to move Because DNA has a negative charge, it migrates toward the positive end of the gel

\

4. The DNA separates into fragments according to size, producing a banded pattern. The larger fragments move more slowly & will be closer to the end of the gel with the wells. The smaller fragments are lighter and will travel farther across the gel