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DNA Replication & Mitosis (Applications of DNA Replication (Gel…
DNA Replication & Mitosis
The Cell Cycle
Chromosome Dynamics During Mitosis of Eukaryotic Cells
Metaphase
Alignment of chromosomes at cell center
Metaphase plate
Kinetochore microtubules are attached at kinetochores of sister chromatid
Facilitate metaphase plate alignment
Anaphase
Kinetochore microtubules shorten
Sister chromatids shorten
Made into individual chromosomes
Pulled toward opposite spindle poles of cell
Polar microtubules push against each other, to elongate cell
Prometaphase
Fragmentation of nuclear envelope
Breaks down microtubes
Attaches them to centromeres of chromosome
Kinetochores
Specialized protein structures
Microtubes can directly attach from centrosome
Pull chromosomes to poles
Polar microtubules
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Prophase
First stage in mitosis
Each chromosome, appears ad identical sister chromatin, joined at centromeres
Centrosomes begin forming mitotic spindle
Centrosomes positioned at opposite ends of cell
Mitotic spindle separates chromosomes into cells
Telophase
Equal segregation of chromosomes at 2 ends of dividing cell
2 new daughter nuceli form in cell
Nuclear envelope reforms around chromosomes
Chromosomes decondense
Spindle microtubules depolymerized
Cytokinesis
Division of cytoplasm, and the cell
Formation of contractile ring
Formation of cleavage furrow
Separates cell
Controlling Progression Through Cell Cycle
Cyclins Proteins
Gel electrophoresis
Protein separation
Protein bands became darker as cell division progressed
Protein increased and decreased with cell division in gel electrophoresis
Cyclins Control Kinase Proteins
Mitosis promoting factor
Cyclin protein
Cyclin-dependent kinase (CDK)
Activity of kinase is dependent on being bound by cyclins
Control progression of cell cycle
Kinase
Enzymes that activate/inactivate proteins by phosphorylating amino acids on proteins
Inactive until activated by binding to cyclin proteins
Cyclin-CDK
Triggers changed during cell cycle
Multiple Cyclin-CDK Kinases
CDK activity changes in concentration of activating cyclin protein
Cyclin-CDK
G1/S complex
transition from G1 to S phase
Prepares cell for DNA replication
S complex
Initiate DNA synthesis
M complex
Initiates mitosis process
Checkpoint
Cellular surveillance
Block activity that has gone wrong
Pause cell division until next stage preparation is complete or damage is repaired
DNA damage checkpoint
End of G1 phase
Structural damage to DNA
Double-strand breaks in phosphodiester backbone
Kinases can phosphorylate p53
Inhibit cell cycle when turned on
Production of CDK inhibitor protein
Bind & block G1-S cyclin-CDK complex
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DNA replication checkpoint
End of G2 phase
Spindle assembly checkpoint
Before anaphase, during mitosis
Prometaphase in mitosis
Monitor degree of which sister chromatids attach to microtubules of mitotic spindle at kinetochore region
Unattached kinetochores create "wait" signal
Activate checkpoint
Lack of tension in centrosome area
Prokaryotic Cell Division
Binary Fission Process
Asexual reproduction
Initiated when DNA of chromosomes is attached by proteins, to inside of plasma membrane
DNA replication begins along origin of replication region of chromosome
Cells elongate
Newly synthesized DNA anchored to plasma membrane
Cells elongate until 2 DNA attachment sites are at opposite end of elongated cell
Cell constricts at midpoint
Synthesis of new membrane wall
Division of 2 halves into daughter cells
Bacterial Cell Division
Cell division is reproduction
Make exact copies of genome, segregating a copy, to make daughter cells
Replication
DNA Replication
Initiating Replication
Template strand is copied from 3' end to 5' end
Produces daughter strand, elongated in 5' - 3' direction
DNA Synthesis Occurs 5'-3'
Replication forks
Region of separation of parental strands during DNA double helix unwinding
Origin of replication
Continuous & Discontinuous Replication
Leading strand
Replication is continuous from primer
1 primer required for DNA polymerase to add nucleotides to daughter strand as replication fork progresses
Antiparallel parent strand
Discontinuous
Replications occur
Lagging strand
Produced daughter strand
DNA polymerase replicate DNA in direction away from replication fork
Okazaki fragments
Contains segments of DNA
Both Strands are Templates
Initiation
Requires short stretch of primer (5-10 nucleotides long) be synthesized, and a base pair with template DNA strands
DNA can be replicated on either strand, due to antiparallel complementary fashion
Elongation progression
Polymerization of newly replicated daughter strand, is catalyzed by DNA polymerase enzyme in 5' - 3' direction
DNA polymerase
Synthesizes replicated DNA strand from primers, that heat template strand
Replication Complex
Proteins that participate in DNA replication form a single large complex
Unwound DNA tends to rejoin, single-stranded binding proteins bind and stabilize parental strands until elongation begins
Topoisomerase
Minimize torsional strain occurring at replication fork
Initiating proteins triggering unwinding process
RNA primase synthesizes
short RNA stretches of nucleotides, complementary to parental strands, that DNA polymerase elongate from
DNA polymerase III
Does most of elongation work
DNA polymerase I
Removes RNA primer after DNA replication
Removes short sequences with DNA nucleotides
Replicating Chromosomes
Ends of Linear DNA Molecules
DNA replication machinery cannot complete 5' ends of daughter strands when DNA is linear
Lagging strand does not loop
Shorter and shorter DNA molecules are produced with uneven ends with repeated rounds of DNA replication
Telomerase
Elongates telomere regions
Synthesize DNA from RNA template
Has RNA template in complex
Elongating linear chromosomes
Semiconservative Model
DNA consists of a pair of template chains, complementary to each other
Prior to replication, hydrogen bonds broken down between complementary strands
Unwinding and separation
Complimentary strands
Each strand is template for new nucleotides into new complementary strand
DNA double helix replicates, produces 2 strands; 1 old from parent molecule, 1 new
Applications of DNA Replication
In Vitro Replication
Polymerase Chain Reaction (PCR)
Denaturing
Unwinding of double stranded DNA into two individual strands (thermocycler). These strands become template sequences.
Annealing
Primers attach to their complementary sequences on the template strands (thermocycler cooling)
Extension
Heat stable DNA polymerase polymerizes the daughter strands using the 4 dNTPs (starting from the primers, in the 5' to 3' direction)
DNA sample in buffer solution with essential ions, 4 dNTPs, a pair of primers, and DNA polymerase
Each round of amplification creates 2^n copies of DNA target sequence)
Gel electrophoresis
separates DNA fragments in an agarose gel in an electrical field, anode (-) to cathode (+)
agarose gel acts like a sieve/net - allows smaller fragments of DNA to travel farther than larger fragments
load wells (at anode) with DNA sample (undetermined number of base pairs) and DNA ladder (predetermined number of base pairs - measurement method)
special dyes are used to stain the separated DNA fragments - makes them easy to visualize
DNA sequencing
shotgun sequencing
break genome into different sized pieces
GENOME ASSEMBLY
multiple DNA sequences combined into a consensus region of DNA (aka contigs)
Phase 1
randomly sequence DNA in each fragment
Phase 2
assemble sequence: identify and assemble regions of overlap (between the fragments that were generated) to make one continuous sequence of nucleotides
Phase 3
annotate the sequence: identify the functions of each region of genomic DNA
establish correct reading frame, only one correct RF to code for a protein
Sanger (dideoxy) sequencing & chain terminators
modified deoxynucleotides
normal dNTP: (-OH) group on 3' end that allows for elongation
makes covalent bond with 5' phosphate of adjacent nucleotide
dideoxynucleotides (ddNTP): lack (-OH) group, acts as a chain terminator
additional nucleotides cannot be incorporated
incorporated less frequently than dNTP
allows for a variety of different sized fragments
4 types: T, A, C, G
fluorescently dyed to label the chain terminators in the replicated DNA fragments
both incorporated randomly
once a daughter strand sequence is known, the template strand sequence can be determined
Genome ANNOTATION
identifying functional sequences
sequence motifs
protein coding regions of DNA
RNA from these regions contain open reading frames, consisting of triplets of nucleotides that specify amino acids
binding sites for transcription factors that regulate gene expression
DNA sequences that bind transcription factors are often short sequences present in multiple copies near a protein-coding gene
can be identified from the hypothetical RNA molecule that is inferred from the sequenced DNA molecule
DNA Mutations
Somatic vs. Germline Mutations
Somatic
Occur in non-germline cells
Can't be inherited
Mutation on specific cells in specific tissues
Germline
Occur in germline cells
Inherited
All cells in offspring inherit mutation
DNA Repair Mechanisms
Mismatch Repair
Identifying Mistake
Mismatching of single nucleotide pairs during DNA replication
Kink created by mismatched nucleotide pair
Single-stranded cleavage
Repairing Gap
Cleaved backbone, is removed nucleotides from DNA strand
DNA polymerase and DNA ligase induce DNA synthesis to close gap
Base Excision Reapir
Uracil in DNA signals need for repair
Detected by DNA uracil glycosylase enzyme
Cleaves uracil from sugar DNA backbone
AP endonuclease detects lack of nitrogenous base
DNA polymerase and DNA ligase fill gap with nucleotides
Nucleotide Excision Repair
Remove & replace 1+ damaged nucleotide bases
Corrects single nucleotide pair mismatch
Damaged bases signal enzymes to cleave DNA backbone to cleave either side of damage/mismatch
DNA synthesis fills gap with complimentary nucleotides
Nucleotide Sequence vs. Chromosomal Mutations
Nucleotide sequence
Small Point Mutation
Single nucleotide pair changes in DNA sequence
Arise during DNA replication
Single nucleotide pair substitution
Most common
One base pair incorrectly placed
SNPs
Single nucleotide polymorphisms
Effects
Can have no effect on structure/function of protein
Can be fatal
EX: sickle cell anemia
Single-nucleotide substitution
Non-synonymous mutation of single amino acid in B-globin protein subunits of hemoglobin molecules
Translate valine amino acid instead of glutamate
Synonymous/silent mutations
Redundant genetic code leads to ability for some mutations to still code for same amino acid
Deletions Removing Amino Acids
Insertion mutations
1+ nucleotide inserted into replicating DNA
Skipping or removal of 1}+ nucleotides during DNA replication
CF
Deletion of 3 nucleotides in protein coding region of chloride channel
CFTR transporter
Nonsense Mutations
Point mutations
Substitutions usually lead to missense mutations
Can change amino acid into stop codon
Translation of protein to stop early
Shorter polypeptide sequence
Nonfunctional proteins
Frameshift Mutations
Insertion or deletion is not a multiple of 3
Improper grouping of nucleotides
Leads to missense mutations and is terminated
Nonfunctional proteins
Chromosomal mutations
Duplication & Deletions
Deletion
Chromosomal fragment lost
Genes can be lost
Centromere
Chromosome dies within a few duplications
Diploid organism
2 homologous chromosomes
Delete one, if other can survive to produce enough, it will survive
Embryo
Death
Birth defects
Duplications
Divergence
New gene formed from gene being of advantage
Reciprocal Translocations
Portion of one chromosome is able to attach to non-homologous chromosome
2 non-homologous chromosomes exchange terminal chromosomal fragments
Noncoding regions
Gamete may inherit mutations leading to developmental abnormalities
Affect larger regions of DNA
Visile changes of chromosomal structure
Chromosomal Inversions
Normal order of chomosome is reversed
Chromosome fragment breaks off and reattaches to same chromosome in reverse order
Not harmful, all genes still present
Gamete formation can cause problems if breaks are on genes
Variation and Gene Evolution
Gene families arise from mutations
B-globin
Codes for hemoglobin
Expressed differently throughout development
1 duplication from 1 copy
Y-globin
2 genes expressed during fetus growth, expressed at different times
Globin genes are all similar, with small mutations
Possibility of divergence
Applied Lecture
Telomeres & Immorality
Tissue homeostasis
Tissues die naturally
No net change in cell numbers
Tissue degeneration
Cells die and are not replaced
Cancer
Increase in cell number
Cells proliferate and don't die
Hayflick limit
Limited capacity for cells to divide
Programmed cell death
Telomeres don't code for anything
Solution
Telomerase
Expressed in germ cells, embryonic stem cells, cancer cells
Most somatic cells do not make telomerase
Telomeres get eroded
Cellular aging loses telomeres
Extends DNA template
Length decreases with division
Aging
Length decreases
Pre-mature aging syndromes
Telomeres shorten at much higher rate than normal somatic cells
Stem cell theory
Shorten telomeres
New cells can't be made fast enough to replace dying cells
leads to aging
Can't replace differentiated cells quickly
Can't maintain homeostasis
Cancer cells can reactive telomerase
Replicate indefinitely
Progeria
Premature aging
Large decrease in telomere length in children (ages 9-14)
Gene therapy
Mice injected with telomerase live 20% longer
Applied Lecture
Mutations & Gene Evolution
Hemoglobin
B-Globin gene family
A-Globin gene family
Genes evolve from duplication and mutations
Random crossing over
Duplication
Replication
Sickle cell anemia
Inherited disease
Homozygous
Will get malaria
Malfunction of B-globin gene
A gene replaced with T
Codon 6, exon 1
Don't have donut shaped red blood cells
Symptoms
Reduced red blood cells
Anemia
Reduced oxygen throughout body
Cell death
Tissue degeneration
Pain
Death
Affects
Sickled cells block capillaries
Fragile cells
Selective advantage to places with malaria
Some blood cells will be affected, but most won't
Only if heterozygous
Treatment
No cure
Deal with symptoms
Frequent blood transfusions
Medications
Possible Cure
Gene duplication
Turn back on fetal gamma globin
CRISPR
Disrupting gene function
Mutations can be lost if they serve a disadvantage
Malaria
Damaged red blood cells
Breaks cells to release disease
Heterozygous sickle cell patients have advantage