CHAPTER 10 GENETIC MATERIAL

I. Discovery of DNA

 Discovery of genetic material can be traced back to late 1920’s (1928)

1. Frederick Griffith (1928)

British Trying to develop a vaccine for pneumonia Used two strains of bacteria in his experiment There are 2 strains of streptococcus bacteria One strain called S strain is disease causing (virulent- vir-yoo-lent) It is called S strain because the smooth edged colonies it forms when cultured

2nd strain is called R strain, and is non disease strain. Called R strain because of the rough colonies it froms when it is cultured.

EXPERIMENT 1 & 2 Griffith injected either live R or live S cells into mice. Found that only S cells killed mice

EXPERIMENT 3

To see if mucous coat caused the disease he heated strain S. the heat killed the bacteria but did not destroy the mucous coat. He then injected this into the mice – mice okay CONCLUSION: mucous coat alone does not cause the disease

EXPERIMENT 4

Combined the harmless strain(R strain) and heated killed strain –he hypothesized that this strain would be harmless, but mice died of pneumonia. When they examined dead mice, found live strain S bacteria and mucous coats in blood.

Conclusion: some material passed from dead S strain to live R strain changing R bacteria into S bacteria, He called this transformation. This factor called the TRANSFORMING FACTOR.

So guessed that this transforming factor was genetic since new interited traits were seen in the new cells. (in blood) Griffith called this transformation- the process during which bacteria changed by absorbing genetic material from outside source. 2. 1940’s – Oswald Avery

American Wanted to figure out what this transforming factor actually was Suspected it was either protein, RNA or DNA

3 Experiments

1 st Experiment

 Added Protease (enzyme that destroys protein) in the heat killed S strain, mixed it with R strain

 Result – all died

 Conclusion – not the transforming factor (this is because the transforming factor is in fact still there because it is killing the mice

Experiment 2

 Added RNase (destroys RNA) in the heat killed S strain, mixed with R strain

 Result – all died

 Conclusion – RNA not the transforming factor, because whatever is transforming is still there and killing the mice

Experiment 3

 Added DNase (destroys DNA) into the heat killed S strain, mixed it with R strain

 Result – All lived

 Conclusion – DNA is the transforming factor. The transforming factor was destroyed which allowed the mice to live. 3. Hershey and Chase (1952)

American Scientists Many scientists up to this point did not know enough about DNA to even think that DNA was a transforming factor Hershey and Chase believed that either protein or DNA was in fact the transforming factor. Devised an experiment to confirm the belief of Oswald Avery, that DNA was in fact the transforming factor.

In their experiment they used Bacteriophages Bacteriophage is creted when a virus is allowed to attach to a bacteris.

The virus attached to the bacteria, infects and destroys bacteria.

Experiment 1:

 Labeled / tagged the protein of the virus (T2 virus) with radioactive sulfur  Allowed the virus to attach to the bacteria (e. coli)

 Suspended the bacteriophages in a liquid and agitated the culture in a blender to shake loose any parts of the phages that remained outside of the bacterial cells (separated the virus on the outside of the bacteria from the bacteria itself)

 They then spun the sample in a centrifuge separating the virus and the bacteria (virus smaller, so when centrifuged it goes to the top of the sample)

 Results - found NO radioactive sulfur in the bacterial cells  Conclusion – protein did not get into the bacterial cell (not the transforming factor)

Experiment 2 :

 Tagged viral DNA with radioactive phosphorus  Allowed the virus to attach to the bacteria to create a bacteriophage  Suspended the bacteriophages in liquid and agitated the culture in a blender to shake loose any parts of the phages that ramined attached to the bacterial cells (separated virus from bacteria)

 Spun sample in centrifuge, separating virus and bacteria

 Results – when tested the bacterial sample, found radioactive phosphorus inside the bacterial cells

 Conclusion – the viral DNA had been injected into the bacterial cell. This was the transforming factor they were looking for.

 So concluded that DNA must be genetic material and the key to transformation. DNA STRUCTURE

Many scientists help to develop the structure of DNA

1. 1950’s – Linus Pauling, (California) Maurice Wilkins and Rosalind Franklin (London) took x-ray crystallographic image of DNA this allowed Watson and Crick to summize shape of DNA

2. Edwin Chargaff- studied nitrogen bases of DNA realized that for every A there was an equal amount of T and every C there was an equal amount of G

3. 1962 - Watson and Crick –

Used lots of information from other scientists including all of the above and Erwin Chargoff, who developed Chargoff’s rule that the amount of Adenine always equaled the amount of Thymine in DNA molecule to construct 3-D model of DNA

Due to all of the scientific research on DNA in the mid 1900’s and current research, we now know the structure and function of DNA

II. Structure of DNA / RNA

 The discovery of DNA and modern research has allowed us to know the following about DNA

DNA = deoxyribonucleic acid

Structure: 1. nucleotides – monomers of nucleic acids 2. nucleotides made of  phosphate  5 carbon sugar (deoxyribose – DNA or ribose -RNA)  nitrogen base (A, T, C, G) 3. double helix backbone = phosphate and 5 carbon sugar rungs= nitrogen bases (A, T, C, G ) 4. A and G are purines (double ring structure, so larger) C and T are pyrmidines (single rings, smaller) RNA = ribonucleic acid

Structure

Nucleotides  Ribose (5 carbon sugar)  Phosphate  Nitrogen base (A,U,C,G)  U replaces the T that is present in DNA

III. DNA Replication

Semi-conservative model – uses a strand ( half) of its DNA to make a new strand of DNA Occurs S phase of interphase Occurs to assure that each cell going through cell division has the identical DNA so can perform identical function.

Enzymes:

DNA polymerase –  There are few different types of this  Attaches complementary bases to template strand

DNA ligase;  Joins Okazaki fragments (O-kah-zocki) together

Primase-  Creates “starting point” for nucleotides to be added onto strand

Helicase  Enzyme that breaks “weak bonds” and unwinds DNA

Proteins  Other enzymes

STEPS OF DNA REPLICATION:

1. Helicase breaks bonds, unwinds and unzips. This creates a replication bubble.

2. Strands separated at replication fork into a leading strand and lagging strand. 3. Binding proteins prevent the strands from reattaching to each other.

4. Leading strand

 RNA primer attaches to leading strand to give it a “starting point”  DNA polymerase adds complementary nucleotides (A-T and C-G) from 3 to 5 prime end of leading parent strand (so is really creating the opposite or a 5 prime strand)  The DNA polymerase can only add bases to the 3’ end of the strand, never to the 5’end  So really creating a 5-3 strand (opposite of the leading strand template)

*** 3’ end refers to where the OH is attached to the deoxyribose (attached to 3rd carbon) while 5’ end the phophate group is attached to the 5th carbon.

Adds bases in direction toward replication fork.

5. Lagging strand

 RNA primer attaches to strand close to the replication fork to create a starting point (3 end of the template lagging strand)  DNA polymerase attaches complementary bases beginning at the RNA primer – adding bases toward the 5 end of the strand (away from the replication fork)

 Creating an opposite strand from the template or a 3 – 5 strand

When completed, RNA primase is replaced by DNA and ligase bonds all DNA together. 6. DNA binding proteins then go through each strand and correct any mistakes on DNA strands – “cleans it up”

7. End result is TWO identical strands of DNA.

Show stolaf.edu video 1st and explain the steps again

Video steps: Watch leading strand first:

1. helicase (green) unwinds and unzips DNA 2. primase (red) creates a starting point 3. DNA polymerase attaches complementary bases (A-T and C-G) – adds bases toward replication fork.

Now watch the lagging strand:

1. Helicase unwinds and unzips DNA 2. Primase creates starting point 3. DNA polymerase attaches complementary gases (A-T and C-G) 4. Adds bases from primase away from the replication fork. 5. Primase creates another starting point – DNA polymerase adds bases away from replication fork and continues doing this until completed 6. When strand copied, polymerase replaces the RNA primase 7. Ligase connects DNA 8. Enzymes (proteins) check DNA for mistakes

END RESULT – TWO STRANDS OF IDENTICAL DNA. SHOW MCGRAW VIDEO

DNA strands need to be identical because each strand is going into a different cell. You want the cells to do the same function as cells they are replacing, so need same DNA.

IV. Expression of genes

DNA gives instructions to make proteins which give individuals certain traits.

Proteins are the link between DNA and phenotype

DNA Replication

V. Protein Synthesis

Chromosomes are made of genes which are made of nucleotides. Genes = small region of chromosomes with a particular sequence of nucleotides Sequence of nucleotides is very important because tis the recipe to make a particular protein

Genetic code equated to Rosetta Stone Rosetta Stone contained Egyptian hieroglyphics, Egyptian script and Greek translation. It was like a key to the hieroglyphics meaning.

In 1961, Marshal Nirenberg discovered codons. Codons are triplets or 3 nitrogen bases that code for an amino acid. He decoded the triplets of RNA molecule into AAA they produced.

Now have a chart decoding all of the codons and all the AA they code for (pg. 192) The produced the triplets of RNA molecule Protein Synthesis

1. Transcription: 4 stages : initiation, RNA elongation, termination, modification

1. initiation – RNA polymerase attaches to a promoter to signal the start of protein synthesis

2. RNA elongation complemntary bases matched up

3. Termination o mRNA polymerase eaches terminator codon o mRNA poluerase detaches from DNA o now have mRNA chain that decoded DNA information

4. Modification – o mRNA modified o introns spiced out – “junk DNA” and exons squished together to make functional mRNA that can exit the nucleus through the nuclear pores to the ribosomes in the cytoplasm

o before leaving - add nucleotides at tail ( 50 – 250 A’s) and to the cap (G nucleotides) of RNA molecule 2. Translation

3 phases of translation – initiation, elongation, terminiation

1. initiation mRNA bonds to small ribosome tRNA initiator binds to the start mRNA codon to initiate translation tRNA fits into p-site A site which is next to p site, is where the next tRNA fits into

2. elongation tRNA’s add AA to polypeptide chain as mRNA is moved through ribosome one codon at a time moves from A to p site and continues until hits stop codon

3. Termination stop codon, polypeptide released ribosome comes apart and tRNA and mRNA released

This is a very fast process, less than a minute to make protein

Polypeptide coils and folds into 3D shape (tertiary structure) several may come together forming a quaternary structure. The protein determines the appearance of capabilities of the cell and organism.

Changes in sequence of DNA can cause mutations

If a codon or nucleotide is missing or added – it causes a frameshift – which effects everything else downstream because codons regrouped in a different order

These muations can be caused by 1. DNA replication 2. mutagens in the environment MICROBIAL GENES

Virus – nonliving – genes in a box Need host to survive Composed of DNA and protein Capsid – protein coat that encloses DNA

Reproductive cycle of virus

2 cycles lytic cycle lysogenic cycle

Both cycles start like this:

Virus injects DNA into host Viral DNA forms circle inside host cell Enters lytic or lysogenic cycle Then enters either lytic or lysogenic cycle

Lytic cycle –

 Viral DNA produces new viral DNA and protein  Virus assembled inside host cell  Host cell lyses (explodes) releasing new viruses

Lysogenic cycle

 DNA becomes part of host DNA (prophage)  Host cell (with viral DNA) undergoes mitosis and now viral DNA inside more cells  Can reproduce without destroying host cell  They can continue and lay dormant indefinitely  When conditions are right, chemicals trigger to enter lytic cycle, make virus in the new cells  New cells explode and viruses released throughout the body

Viruses in Animals

 Variety of shapes  Some have DNA, some have RNA (retro-viruses)  Some have glycoprotein spikes  May effect different types of cells (target different cells)

Emerging Viruses

 HIV  Ebola – RBC hemorrhage – massive bleeding and vomiting  Encephalitis  West niles  SARS – severe acute respiratory syndrome  H1N1 – swine flu  Hanta virus – rodent droppings

VIRUS AND CANCER

 Some viruses can cause cancer  Can be permanent residents in host cells by inserting RNA or DNA into host’s DNA  When cancer causing genes inserted into host make cell cancerous  Gene that can cause cancer = oncogene  A normal gene that has the potential to become an oncogene = proto-oncogene

 Many proto-oncogenes control rate of cell division

 Tumor suppessor gene  Codes for proteins that help prevent uncontrolled cell division  If mutated – cells divide uncontrollably and eventually form a tumor

Cell can get oncogene from a virus For protooncogene to become oncogene – a mutation must occur in cell’s DNA BACTERIA AND DNA

 Most bacterial DNA = single chromosome (prokaryotic organism)  Bacteria divide by binary fission (asexual)  But can still produce new combination of genes 1 of 3 ways

1. transformation 2. transduction 3. conjugation

1. Transformation takes up foreign DNA from environment (other dead cells) and integrated it inot own DNA. (like Griffith’s experiment)

2. Transduction fragment of DNA of a phage accidentally packaged within

3. Conjugation a sex pili attaches to recipient cell and fuses to this cell to create a “bridge” between them DNA replicated and passed through bridge part of this DNA integrats into cells DNA to create genetic diversity GENE REGULATION (PART OF CHAPTER 11)

Gene regulation Turning on and off genes Helps respond to environment changes A gene “turned on” means it is being transcribed into a protein or the genotype is being expressed as the phenotype

Turning on and off of the transcription is main way of gene expression

Prokaryotes regulate genes differently then eukaryotic

Prokaryotic cells 3 areas that regulate genes

1. Promoter where RNA polymerase attaches to start process

2. Operator acts as a switch determines whether RNA polymerase can attach to promote to transcribe genes

3. Operon cluster of genes combo of promoter and operator and genes to make the protein

Repressor Binds to operator and blocks any attachment of RNA polumerase Turns genes off

A protein made by regulatory gene which is located outside the operon expressed continually so cell makes small supply of suppessor protein Operon turned on by a molecule that interferes with attachment of repessor to operator by binding to repressor and changing its shape. If repressor changes shape won’t fit on operator and won’t block RNA polumerase so gene “turned on”

Eukaryotic Gene expression

DNA coiled and packed because histones Histones = proteins account for about half mass of DNA Histones “fold and pack” DNA so gene expression doesn’t occur.

Gene Expression Use regulatory proteins that bind to DNA and turn transcription genes on or off

Transcription factors Proteins assist in polymerase

Activators Bind to enhancers (DNA sequence) Usually far from gene they regulate

 Binding of activators to enhancers binds DNA  Once DNA bent, the activators interact with factor proteins which then interact with promoter and initiate transcription.

 Repressor Proteins (AKA silencers)  Inhibit and start of transcription  Bind to DNA to inhibit start of transcription

So between repressor proteins and activators it turns genes on and off when needed Cellular differntiation results from selective turning on and off of genes

THERAPUTIC CLONING

Producers embroyonic stem cells which can give rist to all types of cells in body Can divide independentaly Right conditions – induce changes in gene expression that cause differentiation into a particular cell type Scientists trying to find this “right condition” so can “grow organs” and use them for transplants

Pbs.com did movie on this, find this on pbs.

Key developmental genes

Homeotic genes Help direct embryonic development Contains nucleotide sequence about 60 AA long Binds to specific sequence of DNA enabling homeotic proteins to turn genes on and off during developtmen

Scientists found the homeotic genes are in a lot of other organisms not just humans which suggests unity in diversity.