Biology 1A03 Theme 4

Binary Fission

a form of asexual division that makes two identical copies of the original organism. usually in prokaryote it is also reproduction

Cell divison

ability of preexisting cells to give rise to another cell

The process of binary fission

The cell elongates and new DNA is anchored to the plasma membrane

the bactera doubles in size

constriction occurs along the midpoint

new cell is formed with a new wall and plasma membrane

Stem cells

unspecialized cells that can reproduce indefinaetly under proper conditions and specialze into various cell types

Adult stem cells

stem cells that are not able to find rise to all cell types but are able to replace non reproducing specailzed cells

G0 phase

pause in the cell cycle to allow for growth

G1 phase

Prepare cell for DNA synthesis

S phase

DNA synthesis

G2 phase

Prepares cell for mitosis

Before mitosis what happens ?

DNA sequences are replicated and the newly synthezied molecules are assocaited with histones and other chromosomal proteins for tight comactoion

Centromere

fully repilicated and highly compacted that phat the paired centromeres appear fused

Sister chromatids

chromsome that is duplicated into identical copies

23 distinct chromosome pairs

46 chromosomes...22 homologous pairs and 2 sex chromsomes

M phase

mitosis and cytokinesis occurs

Prophase

Each chromosome will appear as identical sister chromatids that are joined at their centromeres

• Centrosomes (duplicated cellular microtubule organizing centers) begin to radiate long microtubules, forming a mitotic spindle (crucial for separating the chromosomes into the two daughter cells)

• Centrosomes become positioned at opposite poles of the cell

mitotic spindle

long microtubules that help with mitosis

Prometaphase

breaking down of the nuclear envelope

kinetochore microtubules radiate from centrosome and attach to the kinetochore regions to pull chromosome to poles of the cell during mitosis

polar microtubules radiate from centrosome and interact with each other to push the poles of cell away from eachother during mitosis

kinetochores

specialized protein structures that associate with one of the sister chromatids on either side of centromere

kinetochore region

areas on the chromosome that kinetochore proteins attach to to pull chromosome

polar microtubules

they raadiate from centrosome to help push poles of cell away from eachother

Metaphase

alignment of chromosome at the centre of the cell in the metaphase plate

metaphase plate

center of the cell where chromosomes align

Anaphase

Kinetochore microtubules begin to shorten

sister chromatids seperate into individual chromosomes that are pulled towards the opposite spindle poles of the cell

Polar microtubules push against eachother and help elongage the cell

Equal segregation of chromosomes at the two ends of the dividing cell

Cytokinesis

Division of cytoplasm and therefore of the cell

cytokinesis in animal cells

• Animal cells: begins with the formation of a contractile ring made up of motor proteins that contract bundles of actin fibers along the midline of the cell

• Cleavage furrow separate the cell into two distinct and separate daughter cells

cytokinesis in plant cells

• Plant cells: lay down a newly developed cell wall along a cell plate region in the middle of the dividing cell (complete once the forming cell wall fuses with the original)

Cyclin protein

Mitosis promoting factor combined with a cyclin dependant kinase (CDK) that control the progression of the cell cycle

Cyclin dependant kinase (CDK)

with the cyclin protein form the CDK that control the cell cycle progression

G1/S cyclin -CDK complex

needed for the transition from G1 to S phase and helps prepare cell for DNA replication by increasing expression of histones and unwinding DNA

S cyclin CDK complex

helps initiate DNA synthesis

M cyclin CDK complex

Initiates process of mitosis

Checkpoints

points in cell division that can pause cell division untill the preparation for the next stage is completed and serve as an opportunity for damage to be repaired

DNA damage checkpoint

located at the end of the G1 phase and only allows undamaged DNA to enter the S phase for replication

How does the DNA damage checkpoint work?

kinases phophorylate p53 that can inhibit the cell cycle when turned on

p53

protein that can inhibit the cell cycle when turned on (usually present at low levels but upon phosphorylation can accumulate and act as a transcription factor that turns on genes that inhibit the cell cycle

CDK inhibitor

p53 leads to the production of a CDK inhibitor that can bind to and block the activity of the G1-S cyclin CDK complex and stop the cell cycle in G1

DNA replication Checkpoint

At the end of G2 pase and only allows progression when ALL DNA is replicated

Spindle assembly checkpoint

before anaphase and during mitosis

cell only completes mitosis

cell

Regualatory proteins can monitor the degree to which the sister chromatids are attached to the microtubules and unattached kinetochores create a "wait" signal that recruits assembly checkpoints that are activated by a lack of tension in the centromere area

seperase

enzyme that splits sister chromatids

Semiconservative model

model of DNA replication that says DNA replication consists of the parental DNA splitting and forming two new DNA double helices each with 1 parental and one daughter DNA strand

Conservative model

two daughter strands

dispersive model

fragments from the parental and daughter strands

proof of the semiconservative model

DNA was added to a medium of 15N after being grown in 14N and the result was DNA with one 15N containing strand and one 14N containing strand

Where does DNA replication begin?

S phase of the cell cycle at specific regions along DNA

Origins of Replication

Where DNA replication begins

What direction is DNA copied and elongated?

copied 3' to 5 and elongates 5' to 3'

Replication forms

region of DNA where it is being unfound

RNA primer

5-10 nucleotides long and is required since the DNA polymerase can only begin to elongate from an existing peice

DNA polymerase

enzyme that synthesizes the replciated DNA stran from primers that anneal to template strand

Leading strand

requires only one primer and is continually replicated

lagging strand

has discontinuous replication forks containing okazaki fragments that needs a separete primer for each fragment

What replaces the RNA primers then?

another DNA polymerase comes into to repalce them

okazaki fragments

fragments of dna on the lagging strand

DNA Helicase

enzyme that can unwind the DNA double helix by breaking the hydrogen bonds between base pairs

Single stranded binding proteins

bind to and stabelize each parental strands untill elongation can begin so the strands don't rejoin

Topoisomerases

able to bind upstream of replication form to minimize torsional strain brought about from the unwinding

RNA primase

synthezies the short RNA primers that are needed to begin replication

DNA polymerase III

does most of the elongation work in prokaryotes

DNA polymerase I

responsible for removing RNA primers after DNA replication and replacing them with DNA nucleotides

DNA ligase

joins the 3' end of a fragment to adjacent DNA nucleotide by catalyzing the phosphodiester bond which brings the okazaki fragments together

Proof Reading DNA

during replication errors can occur but DNA polymerase proof reads each nucleotide but some errors can even avoid that so enzymes also help with correction

Why do the ends become shorter in linear Eukaryotic DNA

the DNA becomes shorter and shorter because once RNA primers removed, the DNA polymerase can not add nucleotides onto it

Telomeres

contain six nucleotide sequences between hundreds to thousands of times leading to many tandem repeats of GT that serve as a buffer zone so coding regions are protected

Telomerase

specific type of reverse transcriptase that is able to synthesize DNA from an RNA template and plays a key role in aging and cancer progression

PCR

Polymerase chain reaction a means of amlifying or replicating DNA in a tube

How is PCR set up

The sample DNA is put into a tube with essential ions and salts and dna primers, dNTPs and DNA polymerase (taq polymerase) and the tubes are places in a thermocylcer

dNTP

deoxyribonucleotides

Taq Polymerase

heat tolerant DNA polymerase

Denaturation

heating to break hydrogen bonds

Annealing

Cooling to allow primers to anneal

Extension

DNA polymerase extends and polymerizes the daughter strands using four dNTPs starting from primers and extending 5' to 3'

Thermocylcer

machine that goes from heating and cooling cycles to facilitate replication

Gel Electrophoresis

seperated molecules based on size . used to seperate DNA fragments from other sources to visualize DNA molecules and since DNA is negatively charged it is attracted to the positive end of the gel

Small molecules in gel

they travel faster and longer

large molecules in gel

slower and shorter distance

Sandard Ladder

the DNA is compared to this which has all the DNA sizes

DNA sequencing

developed by Fredrick Sanger that could sequence small fragments

Sangar Sequencing Technique

Dideoxy chain termination method, and required all key components of DNA replication Dideoxynucleotides (ddNTPs) are missing the -OH group at the 3' position and would not allow for further elongation of a growing DNA strand sing the -OH group is required for the attachment of the next nucleotide

o Leads to a series of interrupted daughter strands, each terminating DNA replication at the site where the ddNTP is incorporated

Shot Gun method

Could sequence larger phases

breaks the entire genome into different sized peices and proceeds with three phases

Shot gun phase 1

random sequencing of the DNA in each fragment

Shot gun phase 2

identifies the regions of overlap between the fragments and assembles the continous sequence of nucleotides in the DNA molecule that make up the chromosome

the overlapping regions helped to identify the order of the seqeuence

Shot gun Phase 3

annotating the sequences to best identify the regions of genomic DNA that encodes genes (regulatory and non-coding). usually done by a computer

contigs

the overlapping regions of the DNA

DNA mutations

errors in the DNA that can be corrected but sometimes cannot be thus allow the mutations to propogate

What creates DNA mutations?

environmental factors, spontaneous muations, errors during replication, etc.

Somatic mutation

occur in non germline cells and cannot be inherited

Germline cells

the cells that come together to produce offspring (egg and sperm)

germline mutation

occur in germline cells and can be inherited

What happens to a mutation in a non dividing or G0 phase cell?

the effect of the mutation is negligble

How can we prove that mutations are random?

Joshua and Esther Lederberg in 1952 showed that mutations such as those of antibiotic resistance in bacteria are random and not directed

• Stamping a plate with a cloth and then stamping a new plate to transfer bacteria is referred to as replica plating, and preserves the relative arrangement of colonies on the new plate relative to the first agar (non-selective supplemented nutrients) plate

Replica Plating

Stamping a plate with a cloth and then stamping a new plate to transfer bacteria is referred to as replica plating and it preserves the arrangement of te colonies on a new plate

Joshua and Esther Lederberg Experiment

showed that mutations are random through replica plating

○ Whether the mutation occurred in the dish with penicillin or when it was without penicillin

- They took bacteria from the same colony from the non - selective agar and cultured them in a medium with penicillin

○ Led to a culture of bure antibiotic resistant bacteria

- Since the colonies were never exposed to penicillin before isolation, it can be concluded that the mutation was already existing prior to extraction, thus proving that mutations are random.

Mutagens

things that cause mutations such as radiation or chemicals

DNA ligase

enzyme to repair breakage in the DNA backbone

Mismatch Repair

Mismatching of a single nucleotide during replication can be corrected by the DNA polymerase and other proofreading proteins

What happens when DNA base pairs are mismatched?

they create a kink in the DNA that is recognized by proteins that scan the DNA for damage

What is the sign of a mismatch in DNA?

A kink in the molecule

How are mismatches fixed?

the strang with the wrong base is cleaved by nuclease (dna cutting enzyme_a distance away from the mismatch and another enzyme removes the nucleotides including the mismatched one

other enzymes such as DNA polymerase and DNA ligase intorduce synthesis to close the gap to form an intact DNA

nuclease

enzyme that cuts DNA

Base excision Repair

occurs when there is a Uracil that might end up in DNA