Bio 1A03 Test 3

Binary Fission steps

-Initiated when proteins attach DNA to plasma membrane

-Begins along ORI region

-Newly synthesized DNA also anchored to membrane

-Cell elongates until ~2 times its original size and two has two chromosomes on opposite ends of the cell

-Cell splits in two

Quiescent stem cells, function

Permanently arrested in G0 phase. In adults: can replace non-reproducing specialized cells) ex adult skeletal muscle/myofiber)

Steps of quiescent stem cell to myofiber

-Activated (reenter cell cycle)

-Proliferative (look over this one)

-Commitment to differentiation

-Fusion into myotube (precursor of myofiber)

-Myofiber

steps of cell cycle

G1 phase

S phase

G2 phase

M phase

~G0 phase~ perhaps

G1 phase

growth phase, prepares cell for dna synthesis

s phase

cell undergoes dna replication

g2 phase

growth phase, cell prepares for division

M phase

mitosis and cytokinesis

cells in permanent g0 phase

eye lens, nerve, mature muscle cells

binary fission is common to

bacteria, archaea, mitochondria, chloroplasts

Satellite Stem Cells

quiescent stem cells that specifically are activated to replace injured muscle fibers

Walter Flemming

Discovered stages of mitosis from salamanders

During interphase, the genetic material of a typical eukaryotic cell is

chromatin fiber

genetic material during s phase

sister chromatids are synthesized (including centromere) but chromosomes don't actually compact until M phase (46 X-shaped not linear chromosomes / 92 chromatids)

chromatids

chromosome in duplicated state

# chromosomes and chromatids in human before & after mitosis, meiosis I, and meiosis II

Mitosis

Before: 46, 92

After: 46, 46

Meiosis I

Before: 46, 92,

After: 23, 46

Meiosis II

Before: 23, 46

After: 23, 23

https://datbootcamp.com/biology-strategy/chromosome-and-chromatid-numbers-during-mitosis-and-meiosis/

genetic material during interphase

chromatin fiber, can't identify specific chromosomes

prophase (mitosis)

identical sister chromatids apparent

centrosomes (microtubules) form mitotic spindle and positioned at opposite ends of the cell

centromere vs centrosome

centromere: holds together two sister chromatids into 1 chromosome

centrosome: hold together mitotic spindles

prometaphase

fragmentation of nuclear envelope allows mitotic spindle to attach to kinetochores on centromeres

kinetochores

The structures on sister chromatids where microtubules attach

1) some microtubules attach directly and pull

2) some k.c are polar and repel the sides of the cell from each other

metaphase (mitosis)

chromosomes line up along middle of metaphase plate, facilitated by kinetochore microtubules attaching to the kinetochores of each sister chromatid

anaphase (mitosis)

kinetochore microtubules shorten and pull apart chromosomes

polar microtubules repel each other and help elongate the cell

equal segregation of chromosomes (or stop)

telophase (mitosis)

nuclear envelope reforms giving 2 new daughter nuclei within the cell

chromosomes decondense

spindle microtubules depolymerized

end of mitosis

cytokinesis

division of the cytoplasm

animals: begins with contractile ring, cleavage furrow separates cell

plants: new cell wall formed on cell plate region

MPFs

mitosis promoting factors : cyclin + CDK (cyclin dependent factors)

how does cyclin-cdk regulate mitosis?

cyclin-cdk binds to kinases (constant concentration) in order to activate them. these kinases then phosphorylate and activate proteins needed to break down the nuclear envelope, regulate assembly of microtubules, etc

S cyclin-CDK

helps initiate DNA synthesis (cyclin A + cdk 2)

G1/S cyclin-CDK complex

prepares cell for DNA replication

Mitosis checkpoints

G1/S checkpoint, if cell is ready for DNA synthesis (DNA Damage checkpoint)

G2 Checkpoint: Is everything replicated/cell ready for mitosis?

Pre-Anaphase checkpoint: All chromosomes attached to spindle fibers?

How does the G1/S checkpoint work?

If damage to DNA is present, kinases phosphorylate the gene for p53, increasing the production of its protein which accumulates in the nucleus and acts a transcription factor for the inhibitory proteins. These proteins bind to and block G1/S cyclin-CDK complex so that the cell can repair DNA.

How does the spindle-assembly checkpoint work?

Unattached kinetochores release a "wait" signal from the lack of tension that recruits checkpoint proteins. These remain until all kinetochores are attached to micotubules. Seperase breaks apart sister chromatids

semiconservative replication

each new DNA molecule consists of one new strand and one old strand

conservative replication

2 parental strands rejoin after replication

dispersive replication

all 4 strands mix together

How was the semiconservative model confirmed?

Meselsohn & Stahl created DNA strand out of radioactive isotope of Nitrogen, replicated DNA, used centrifuge to separate the strands and used the placements of the new bands compared to the original strand to prove this model

prokaryotic vs eukaryotic DNA replication

Prokaryotes: only 1 ORI, replication moves in both directions due to circular DNA, no gaps in DNA

Eukaryotes: multiple ORI, okazaki fragments/dna ligase, free phosphate on 3' end of strands, telomeres (DNA shortening)

RNA primer

5-10 nucleotide sequence that binds to BP with template DNA strands so DNA Pol can begin replication (can only add to an existing strand)

After fragment/strand is complete, another DNA pol replaces the primer with DNA nucleotides

leading strand

3'-5', only req's 1 primer

lagging strand

5'-3', multiple primers (one per okazaki fragment), replicates away from replication fork

Helicase

breaks Hydrogen bonds between base pairs

topoisomerase II

relieves the stress of unwinding the DNA for helicase by uncoiling/straightening it

single stranded binding proteins (SSBs)

bind to each side of the DNA ladder keeping it from bonding back together during DNA replication

RNA primase

synthesizes RNA primers for DNA replication

Initiator proteins for DNA replixation

Helicase, SSBs, topoisomerase II

DNA polymerase I & III

dna pol I : elongation/synthesis/adding nucleotides

dna pol III : removes RNA primer & replaces with DNA

(prokaryotes only; eukaryotes different)

Reason for Telomeres and Telomere shortening

the last RNA primer on the lagging strand (3' end) has no 3' hydroxyl for DNA pol to attach to and replaxe these nucleotides once they are removed (unlike in prokaryotes) leading to shorter and shorter ends of DNA

What are telomeres?

ends of chromosomes designed to protect from DNA shortening. Made of of 100s-1000s of TTAGGG tandem repeats. get increasingly short with each division especially in somatic cells

telomere shortening does not occur in which types of cell?

gametes and stem cells. they contain telomerase, a reverse transcriptase that binds to the ends of the tails and adds repeats. potentially useful for aging/cancer

karyotype

A display of the chromosome pairs of a cell arranged by size and shape.

ploidy

number of sets of chromosomes in a cell

haploid vs diploid

one set of chromosomes vs two sets of chromosomes

mitotic spindle

a structure made of microtubules that controls chromosome movement during mitosis

unlike animals, during cell division, plant cells do not contain ____

centrosomes (though they do have mitotic spindle). final location defines the poles.

phragmoplast

In dividing plant cells, a structure formed by overlapping microtubules that guide vesicles containing cell wall components to the middle of the cell.

cyclin A

helps activate DNA synth

cyclin B

helps prepare for mitosis

Cyclin D & E

helps prepare for DNA replication (before A)

If one DNA strand is damaged during replication, what occurs in the other strand?

It slows down. DNA replication occurs simultaneously and the DNA polymerases interact together.

Amount of nucleotides lost per division

About 100. Adult cells can only divide under 50 times on average because of this, telomerase is inactive almost everywhere. Cells typically stop dividing when under 100 TTAGGG repeats are left. Telomerase reactivation has been associated with cancer.

Components needed for PCR (in vitro replication)

-Pair of primers to bind to specific point of DNA

-DNA polymerase (usually Taq)

-Template DNA

-essential ions and salts (buffered solution)

-free deoxyribonucleotides (dNTPs): dATP, dGTP, dCTP, dTTP

-topoisomerase and helicase not required due to thermocycler

Taq polymerase

heat-tolerant up to 95°C comes from thermus aquaticus

Stages of PCR with explanation

-Denaturation: unwinds DNA through heating

-Annealing: primers bind to DNA strands

-extension: DNA polymerase and dNTPS extends (/amplifies) DNA

gel electrophoresis

visualize DNA fragments, separate and compare lengths

shot gun sequencing phases

1. Random sequencing of DNA in each fragment (10-50 copies of each region)

2. Identifying the regions of overlap and inferring / assembling each chromosome

3. annotating the sequences to best identify regions of coding/regulatory/non coding regions

sanger sequencing (dideoxy chain termination method)

-Same requirements as PCR

-work on the basis of modified fluorescent dNTPs missing 3' hydroxyl (ddNTPs) which stop elongation of DNA strand. DNA added to tubes with different ddNTPs leading to series of interrupted daughter strands with varying lengths.

-Only works on small fragments (few hundred nucleotides)

spectogram trace

compiles fragments from sanger sequencing into complimentary (because of the binding) sequence to be read left to right

contigs

Overlapping DNA segments that share the same sequence.

consensus regions

Regions of DNA having similar structure and function in different organisms.

Created from overlapping sequences/contigs

How many reading frames are there in a double stranded DNA and how do you decide which to use?

6 possible reading frames.

Look for long periods without stop codons, a start codon, possibly promoter or other regulatory site (usually done by a computer). easier in prokaryotes

sequence motif

A pattern of nucleotides or amino acids shared by different genes or proteins that often have related functions. (ex transcription factors, hairpin loops, ORFs)

____ % of eukaryotic genome is non coding

50%

What type of genetic information is most likely to undergo mutations and why?

RNA viruses due to delicate RNA backbone, possible lack of proofreading

probability of a new mutation ________ with __________

decreases with increasing genome size

somatic mutations

occur only in cells of certain tissues and cannot be passed on (ex most cancers)

germline mutations

occurs in germ (sex) cells, can be inherited and cause mutation in all cells of offspring (ex, haemophilia)

joshua and esther lederberg experiment

"mutations are random"

1- bacteria grown on non selective agar plate

2- bacteria colonies brought to selective agar plate 2 containing penicillin -> can only grow if resistant (stamping)

3- only some colonies survive - contain mutation resistant to penicillin

mutagens

increase the likelihood of mutations in DNA at certain points (radiation, chemicals, smoking)

Types of DNA polymerase II proofreading methods

Mismatch repair

Base excision repair

Nucleotide excision repair

mismatch repair

kink in DNA from mismatch detected by proteins

Single stranded cleavage of DNA backbone by enzyme (usually nuclease) some distance away

Another enzyme removes successive nucleotides

DNA pol, ligase close the gap by redoing DNA synth

Base excision repair

-fixing incorporation of Uracil into DNA

-DNA uracil glycosylase cleaves uracil from backbone

-lack of nitrogenous base detected by AP endonuclease, cleaves backbone on either side of spot lacking a base

-DNA synth occurs at base (DNA pol, ligase)

nucleotide excision repair

-similar to mimatch repair but with multiple NTPs

-damaged bases excised and replaced with re-DNA synthesis

single nucleotide polymorphism (SNP)

point mutation

synonymous mutation

silent mutation

non-synonymous mutation

missense mutation

Types of Large Scale Mutations

deletion, translocation, duplication, inversion

deletion of a centromere leads to

deletion of a chromosome (diploids might be able to make up for it with homologous chromosomes)

Duplication

aka amplification

little harm due to homologous chromosome

can be beneficial, give rise to similar but new gene (duplication & divergence)

Inversion

not harmful in somatic cells; harmful in germ if it causes gene breakage

-small inversions common in large populations, explains variation in gene order, long term genetic evolution/diversity

reciprocal translocation

two-way exchange of segments between the chromosomes

-mainly in non coding regions

-generally don't disrupt gene function

-can cause problems in gametes if homologous chromosomes don't pair during cell division, leading to vital genes missing

beta-globin gene family

-5 genes that arose from duplication and divergence event 200MYA

tandem repeats

up to 1000s of nucleotides in length, next to each other, identical or close

simple sequence repeats

short 2-3 base sequences repeated thousands of times

locus

position of a gene on a chromosome

DNA markers

Sequence variations among individuals in a specific region of DNA (usually non-coding)

Detected via microarray analysis, PCR, Southern Blot, DNA sequencing, used for identification/relatedness

DNA microarray analysis of SNPs

-Common allele + SNPs of oglionucleotide (single strand, ofc) attached to glass

-Only matching alleles light up

-Identify homo vs heterozygous

variable number tandem repeats (VNTR) + identification

differences in copy number (I think?, identified using PCR and gel electrophoresis

DNA fingerprinting use

forensics

relatedness

large scale population analyses

autosome

Any chromosome that is not a sex chromosome (somatic refers to cells)

sickle cell anemia alleles + symptoms

HbA: normal

HbS: sickle (glutamine substituted for valine)

aggregation of beta-globin, blockage of capillaries, anemia, acute body pain, organ/tissue damage

Heterozygotes will express both versions of the protein but will not display symptoms of sickle cell anemia

Provides advantage in fighting off malaria