1. Injected mice with live, encapsulated virulent pneumonococcus - mice died
2. Injected mice with dead virulent pneumonococcus - mice lived
3. Injected mice with heat killed virulent pneumonococcus - mice lived
4. Mixed heat killed virulent pneumonococcus and live, nonvirulent pneumonococcus - mice died
5. Extracted DNA from heat killed pneumonoccocus and mixed with live, nonvirulent pneumonoccocus - mice died

Conclusion - DNA transformed the nonvirulent strain into a virulent strain

Was DNA or protein the genetic material?

1. Protein has sulfur while DNA does not - protein was radioactively labeled with isotope 35S
2. DNA has phosphorus while most protein do not - DNA was radioactively labeled with isotope 32P
3. Infected 35S and 32P bacteriophages into bacteria and centrifuged
4. Found that most 35S viruses were found in the supernatent while 32P viruses were in the pellet
5. Concluded that DNA contains genetic material

1. Treated some bacteria with heavy 15N isotope and others with a lighter 14N isotope
2. After growing in 15N for some time, bacteria was transferred to the 14N medium

First generation - an intermediate 'band' (one strand from the 15N isotope and another from the 14N)

Second generation - an intermediate 'band' and a light density band on the growth medium

Light density continues to grow proportionally

Conclusion - DNA replicates semi-conservatively

• Requires RNA primer for a 3' OH group
• Reads template strand in 3' to 5' direction
• Codes new strand in 5' to 3' direction
• Adds nucleoside triphosphates to a growing strand

• Synthesizes RNA primer
• Primer donates the 3' OH group needed to start replication

Helicase - unwinds DNA to individual single strands through the hydrolysis of ATP

SSBP - keeps each template strand single binding onto each strand near the replication fork

• β2 subunit (sliding clamp) forms a loop on the lagging strand and holds it
• Primase adds a primer
• Polymerase adds about 1000 nucleotides (Okazaki fragment)
• Polymerase lets go of the β2 subunit and it slides down to form another loop

• Two metal ions held by two aspartic acids hold the new nucleoside triphosphate and the existing chain in place
• The 3' OH group on the existing chain nucleophilicly attacks the inner most phosphate group on the NTP
• The new nucleotide is added to the chain and the pyrophosphates are escorted out by the metal ions

• DNaB helicase
• Clamp loader
• 2 polymerase cores - 3' to 5' exonuclease, 2 beta subunit sliding clamps, proteins that interact with SSBP's

• 5' to 3' polymerase
• Removes primer with its 5' to 3' exonuclease (not in III) and adds DNA (with 5' to 3' polymerase)
• Very slow replication speed
• 3' to 5' exonuclease

Covalently bonds "nicks" between Okazaki fragments using ATP

Binds the 5' phosphate group to the next 3' hydroxyl group

1. All DNA have replication origins - E. coli is oriC

• OriC consists of consensus sequence = DNA unwinding element (AT rich) and DNaA binding sequences

2.   DNaA monomers bind to binding sites (and can interact with each other through ATPase in their C terminal regions) - hexamer

3.  Binding of DNaA promotes DNaB (helicase) to bind to DNA to start replication

1) Eukaryotes have many origins of replication while prokaryotes only have 1

2) Eukaryotes have linear DNA strands while prokaryotes are circular

Since eukaryotic DNA is linear, lagging strand would become shorter and shorter as there is no way to synthesize new bases at the 5' end when the primer is removed

• Telomerase - RNA and protein complex reverse transcriptase that adds DNA primer to 3' end of template strand using internal RNA template

• Telomers - TTGGGG rich sequence that is an "overhang" on the 3' end of chromosomal strand

1. Telomerase first adds DNA primer to chromosome end
2. Primase +rNTP's binds primer to newly elongated end
3. Conventional DNA replication on new strand with polymerase and NTP's to fill the gap
4. DNA ligase and removal of RNA primer

• Germ cells have longer telomers than somatic cells
• Somatic cells don't have a telomerase - why DNA sequence gets shorter and shorter and eventually cell dies (20 - 30 times of replication)
• Some cells - gametes/bone marrow cells have telomerases and can divide indefinitely

1. DNA is replicated
2. Growth of cell wall and membranes separates DNA strands
3. Growth of cell wall and membranes into cytoplasm
4. Two separate identical prokaryotes

Location - somatic cells

Mitosis allows cells to:

• Grow
• Repair tissue damage
• Synthesize into specialized cells
• Reproduce asexually

• Chromosomes begin to condense
• Centrosomes begin to separate (consists of 2 centromers at right angles from each other)
• Microtubules start to grow

• Chromosomes continue to become more dense
• Nuclear envelope begins to break down
• Spindles start to form

• Nuclear envelope has completely broken down
• Centrosomes are on opposite ends of the cell
• Kinetechore microtubules grow and attach to kinetechore polar ends of each chromatid
• Chromosomes have condensed completely

• Chromosomes are lined up so that each sister chromatid is facing a different pole

Telophase - the chromosomes are at the each pole and the nuclear envelope reforms

Cytokinesis - division of cytoplasm through pinching of microfilaments (animals) and chromosome uncoils

Plants - vesicles (that have materials that form cell wall) form during telophase and fuse during cytokinesis. Cell wall is laid down to make two different cells

G1 - growth of cell

S - replication of DNA

G2 - cell gets ready for division and centrioles replicate

M - mitosis

R point - between G1 and S phase

Between G2 and mitotic phase

Between metaphase and anaphase

1. Inactive cdk (without cyclin)
2. Cyclin binds to cdk, activating the cdk enzyme
3. Cdk phosphrylates target protein, activating the protein and continuing the cycle

G1/S cyclin - allows cell to continue pass R restriction point and into S phase (produced late in G1 phase)

S cyclin - stimulates DNA replication and controls some early mitotic stages (produced at beginning of S phase)

G2/M cylin - allows cell to go from G2 to M phase (produced between G2/M)

1. Phosphorylation of the cdk/cyclin complex

2.   Inhibitory proteins that inactivate the cdk/cyclin complex by changing its conformation

• It is a noncellular infectious agent that contains nucleic acid embedded with a protein coat
• Can only replicate itself using host's bio-synthetic machinery

• Helps protect virus when not in a host cell
• May have receptors that help bind virus to receptors on host cell
• Can mutate rapidly, which is why it is so difficult to find effective vaccines

1. Virus binds to receptors on host cell
2. Injects DNA into bacterium
3. DNA takes over host's bacterial machinery and makes copies of its own DNA
4. Takes over transcription/translation of viral proteins/capsids
5. Adds glycolipids and other proteins to make up new viruses
6. Could add viral envelope from the nuclear membrane
7. When there are around 100 new virulent cells, bacterium ruptures

1. Virus bonds to receptors on cell and injects its DNA
2. DNA integrates itself with bacterium's chromosome and becomes dormant = provirus
3. If cell is stressed/viral DNA is removed from the bacterial cycle, lytic cycle ensues

Examples

Herpes simplex

Herpes zoster - chicken pox (causes shingles many years later)

Some viruses have an envelope that is made from the plasma membrane of the host cell

Directs the synthesis of viral proteins that get integrated into host cell membrane

Integrase - enables HIV DNA to be integrated into host DNA

Protease - cleaves long polypeptide chain to make active smaller proteins

Reverse transcriptase

Viral RNA coated with proteins

gp120 - receptor that binds to receptor on host cells

Envelope (lipid membrane), glycoproteins etc

1. gp120 binds to CD4 receptor on host cell
2. HIV injects viral RNA and viral proteins into cell
3. Reverse transcriptase makes a cDNA from viral RNA
4. A complimentary strand of cDNA is made
5. Double helical cDNA is integrated into host's chromosome
6. Transcription of viral mRNA occurs
7. Translation of viral proteins/capsids occurs
8. Glycoproteins and protein coat are added to viral proteins
9. Budding gives new HIV nuclear envelope

Early stage - HIV can only bind to CD4 receptors on macrophages (M-tropic HIV)

Middle stage - some HIV have altered gp120's that can now also bind to CCR5 receptors on T cells (Dual tropic HIV)

Late stage - all HIV have altered gp120' and can only bind to CCR5 receptors on T cells

• Has no DNA intermediate - viral RNA (- strand) can code for mRNA (+ strand)
• Has a couple membrane proteins - hemaglutinin and neuraminidase

• Genetic material = RNA + Nucleoprotein + polymerases

M1 protein = capsid

M2 protein = hydrogen pump

1. Hemoglutinin binds to cellular surface
2. Virus enters the cell via endocytosis
3. As the vesicle holding the virus becomes more acidic, M2 protein pumps H+ into capsid, also making it acidic
4. When capsid is sufficiently acidic, RNP's is released into host cell
5. Uses RNA dependent RNA polymerase to make mRNA (transcription/translation) and cRNA (template to make more viral RNA)
6. After both of these occur, new viruses are assembled in the cytoplasm
7. Virus leaves by cell budding

Has 5' end cap and poly A tail on the 3' end

Genome = a single + sense RNA

Replication:

1) Translate genome to make viral RNA polymerase

2) Viral RNA polymerase creates (-) sense strands from + sense

3) Uses (-) sense strands to create more + sense strands (which will be used in genomes of new viruses)

4) Produces mRNAs that direct synthesis of viral proteins

Nested mRNA's

• Growth factors
• Receptors for growth factors
• Proteins in the cell signaling cascade
• Proteins that control progression through the cell cycle

• Protein that permits progression through Restriction point R

• Is inactivated when phosphrylated by G1/S cdk

• Is an example of tumor repressor gene

When DNA is damaged by radiation, p21 is synthesized to not allow activation of G1/S cyclin Cdk and S Cdk

In the absence of p21, damaged cell will continue to S phase replication will be incomplete and cell will rupture

Nucleosome - DNA wrapped around histone octamers

150 bp of a left handed double helix wrapped around the core

Histones - octamer, 2 copies of 4 histone proteins

H2A, H2B, H3, H4

Basic amino acids (lyscine, arginine) - 102-135 amino acids

Structure - N terminal fold and alpha helices at histone fold

1. H3 and H4 bind together to form a dimer, then bind to another H3-H4 dimer to form a tetramer
2. DNA wraps around tetramer
3. H2A and H2B bind to the complex

• Made up of 6 nucleosomes in each helical turn with a thin hollow coil from the 10nm fiber
• Forms a left handed helix
• Linker Histone (H1) keeps DNA bound to histones
• Helps condense nucleosomes to pack more DNA in

• In most biological systems, DNA is underwound (helical twist of 34 degrees is ideal) - negatively supercoiled
• Facilitates the packaging of DNA and makes replication and transcription easier

• Cuts 1 strand of DNA by nucleophilicly attacking the phosphate group on the backbone of DNA chain by Try 723 in central cavity
• There is controlled rotation on the noncut strand to relax supercoiling
• Cut strand is resealed when the free hydroxyl group attacks the tyrosine residue on enzyme and releases tyrosine

1. Binds DNA to C terminal domain of enzyme
2. ATP binds to both N terminal domains
3. Interaction with ATP changes N terminal domain conformation and brings the two together
4. This change traps part of the DNA and cuts boths strands and allows one DNA to go through the other
5. Hydrolysis of ATP and return to original confirmation

Adds supercoiling to DNA

As DNA gets denatured, bases absorb UV light

Tm - when half the bases of a strand have been dissociated

Tm is affected by # of GC base pairs in a strand (harder to denature than AT)

• Capable of autonomous DNA replication after introduction to host cell
• Must have a restriction site and be able to carry foreign DNA
• Must contain at least one marker to indicate vector's presence in host

1. Extract RNA from a sequence
2. Hybridize polyA tail by adding deoxythymine and use as primer and mRNA as template
3. Use reverse transcriptase to create a sequence for cDNA
4. Add a poly (G) tail that serves as primer
5. Use first cDNA strand to create a complimentary second strand