Download APDC Unit IX CC DNA Bio

Unit IX- Cell Cycle, DNA &
Biotechnology
Chapters 9 & 13
The Cell Cycle CH 9
Why make new cells at all?:
Functions of Cell Division:
Reproduction
Functions of Cell Division:
Growth & Development
Function of Cell Division:
Tissue Renewal
Eukaryotic Chromosomes
What is a
“chromosome”?
How related to
a “gene”?
How related to
“DNA”?
Cookbook
analogy – go!
Eukaryotic
Chromosomes
Chromosomes
must duplicate so
that each new cell
has an exact copy
of all of the code
The two “copies”
are called “sister
chromatids”
The Cell Cycle
I-P-M-A-T-C
I pee mainly after the commercials
Interphase (90% of time)
The DNA is in the THIN form
called CHROMATIN during
interphase
•G1 – cell grows
•S – sisters form;
chromosomes copy
•G2 – cell grows
Mitosis (10% of time)
• Pro – chromosomes form;
nucleus goes away
• Meta - middle
• Ana – sisters apart
• Telo – two nuclei form
(on far sides)
Cytokinesis – (NOT part of mitosis!)
cytoplasm splits; cell membrane
pinches in
I-P-M-A-T-C
I pee mainly after the commercials
I-P-M-A-T-C
I pee mainly after the commercials
Which Phase?
Which Phase?
Hint: nuclear membrane dissipating
Which Phase?
Hint: cannot see the chromosomes
Which Phase?
Hint: “late ___”
The Mitotic Spindle
Kinetochore Attachment site
between spindle
fiber and sister
chromatid
The Mitotic Spindle
Microtubules shorten from the kinetochore side
Cleavage Furrow in Animal Cell
Cell Plate in Plant Cells
Which phases do you see?
A
D
B
E
C
Name the Labeled Structure:
D
A
E
B
C
F
Name the Labeled Structure:
microtubule
sister
chromatids
metaphase
plate
kinetochore
centrosome
centriole
Binary Fission
(bacteria)
Mitosis in
eukaryotes may
have evolved from
binary fission in
bacteria
Evidence for Evolution
of Mitosis
Nucleus intact; spindle
passes thru
Nucleus intact; spindle
within
Spindle forms outside
nucleus; nucleus breaks
down
The End – Part 1
Evidence for Chemical Signals to
Control Cell Cycle
2 cells fuse…if one in M, other induced to start M
Cell Cycle Control System
Cell cycle proceeds
step-wise
But there are
several regulation
points
(shown in red)
Cell Cycle Control
• Signals transmitted by “transduction pathways” (cell
signaling!!!)
• Animal cells tend to have built in stop signals …must wait
for reports on cellular surveillance mechanisms
• If do not pass G1…goes to G0 (most cells actually here)
– nerve & muscle cells that generally don’t divide
– some can be called back up for service
2 Molecules that Regulate Cell Cycle:
• Protein kinases
– Activate other proteins by phosphorylating them
– Involved in both G1 & G2 checkpoints
– Present at constant concentration in cells, but only
activated when attached to a cyclin
– Called “cyclin-dependent kinases” (Cdks)
• Cyclins
– Concentration fluctuates cyclically within cell
– Concentration rises in interphase…drops in M
Molecular Control at G2 Checkpoint
MPF = “maturation-promoting
factor” or “M-Phase promoting
factor”
Cyclins join up with Cdk’s; the MPF
allows the start of mitosis
Also causes break down of nuclear
envelope (by phosphorylating its
proteins)
Later, MPF turns itself off by
destroying the cyclins
Cdk stays in cell inactive
Internal Signals
• Messages from Kinetochores
– M checkpoint
– Anaphase does not start until all chromosomes
properly attached…why?
– Signal from kinetochores delays anaphase
– When all attached, “anaphase-promoting
complex” (APC – a different one!) activates
• Triggers breakdown of proteins holding sister
chromatids together
• Triggers breakdown of cyclin
External
Signals
• Growth Factors
– Proteins from certain cells that signal others to divide
– Example: platelet-derived growth factor (PDGF)
• Required to make fibroblasts (connective tissue)
• At an injury, PDGF released…more tissue made
– Probably specific GF’s for each cell type
External Signals
• Growth Factors
– Density-dependent inhibition
• Crowded cells stop dividing…(lack GF & nutrients)
– Most animal cells exhibit anchorage dependence
• Must be attached to lower layer to divide
Effect of Growth Factor
on Cell Division
Describe how each regulates the cell cycle…
• Cyclins & Cdks
• Kinetochore Messages
• Growth Factors
Cancer Cells
Normally, damaged
cells undergo
apoptosis…
programmed cell
death
Cancer cells just
stay damaged
Cancer Cells Ignore Controls
Cancer cells do not respond to
normal control mechanisms
(divide excessively)
Perhaps have own GF
or do not need GF
or have short-circuit in
pathway
Cancer Cells
• Cancer cells stop dividing at random stages in cycle
• Can divide indefinitely if have nutrients
• General process:
– Transformation- change normal to cancer cell
• Alteration of genes that control cell cycle
– Tumor – if evades immune system; grows
– Metastasis – malignant tumor; travels to other sites
p53 (tumor suppressor gene)
• Signals repair processes
OR
• Signals apoptosis
Metastasis of a Malignant Tumor
The End – Part 2
Lab - Mitosis Rate
I. Purpose
Gibberellic acid
Phosphorus
Willow extract
Auxin
Nitrate
Aspirin
– How does ____ affect the rate
of mitosis in onion roots?
II. Background
– Discuss the chemical that your
group will test.
• You will likely need to look up info
on your chemical.
– Explain how root length is
related to mitosis rate.
Lab - Mitosis Rate
Chemicals Tested
– Gibberellic acid
– Auxin
– Phosphorus
– Nitrate
– Willow extract
– Aspirin
Lab - Mitosis Rate
II. Background
– Discuss the chemical that your group will test.
• You will likely need to look up info on your chemical.
– Explain how root length is related to mitosis rate.
III. Hypotheses
– State your null hypothesis.
– Then, list each of the 6 chemicals being tested
and rank from 1-6 in terms of which roots will
have the most growth.
(1 = most growth)
Gibberellic acid
Phosphorus
Willow extract
Auxin
Nitrate
Aspirin
Lab - Mitosis Rate
III. Hypotheses
– State your null hypothesis.
– Then, list each of the 6 chemicals being tested
and rank from 1-6 in terms of which roots will
have the most growth.
(1 = most growth)
IV. Procedure
Gibberellic acid
Phosphorus
Willow extract
– State your IV and DV.
– Describe how you will obtain your data.
– Sketch and label the set-up.
• It should be clear enough that any random person
could look at it and tell what you did.
• NO step-by-step instructions!!!
Auxin
Nitrate
Aspirin
Lab - Mitosis Rate
V. Data
– Record root lengths (in mm) on data table
– Show your calculations for both SD and SEM
• no credit without work shown
• Record all values to hundredths place
• May skip “middle column” (x-x)
– Create summary data table for your group data
• Use proper IV/DV format
Lab - Mitosis Rate
V. Data
Measure at least 8 roots, no more than 10
Be consistent with your cuts & measurements
You may measure on the plant, then slice off root,
or slice root first followed by measuring
Record to nearest whole mm (no decimals)
Place onions back in containers when finished (do
not throw onions or growth medium out)
Values for Roots in Water
x
5
6
5
5
8
4
5
7
6
6
(x-mean)2
Mean = 5.7
.49
.09
.49
.49
5.29
2.89
.49
1.69
.09
.09
n = 10
Sum (x-
)2 = 12.10
You calculate…
SD and SEM
Lab - Mitosis Rate
V. Data
– Create summary data table…use proper IV/DV format
– Calculate SD for both. Show work.
– Calculate SEM for both. Show work.
– Graph mean growth for each medium. Include the
error bars (2xSEM up & down)
Lab - Mitosis Rate
VI. Conclusion
– Follow the “normal” format
– 5 things!
Write as many factual sentences as
possible based on this information.
• Start all sentences with
“Cdk…” (use Cdk as the
subject of your
sentences)
• Number each sentence.
Warm-Up in your notes:
1. Draw and label a nucleotide.
2. Why is DNA a double helix?
3. What is the complementary DNA strand to:
DNA: A T C C G T A T G A A C
Warm-Up
1. What was the contribution made to science by these
people:
A.Hershey and Chase
B.Franklin
C.Watson and Crick
2. Chargaff’s Rules: If cytosine makes up 22% of
the nucleotides, then adenine would make up
___ % ?
3. Explain the semiconservative model of DNA
replication.
Warm-Up
1. What is the function of the following:
A. Helicase
B. DNA Ligase
C. DNA Polymerase (I and III)
D. Primase
E. Nuclease
2. How does DNA solve the problem of slow replication on the
lagging strand?
3. Code the complementary DNA strand:
1.
3’ T A G C T A A G C T A C 5’
4. What is the function of telomeres?
THE MOLECULAR BASIS OF
INHERITANCE
Chapter 13
What you must know
• The structure of DNA.
• The major steps to replication.
• The difference between replication,
transcription, and translation.
• The general differences between the bacterial
chromosome and eukaryotic chromosomes.
• How DNA is packaged into a chromosome.
PROBLEM:
Is the genetic material of organisms made of
DNA or proteins?
Frederick Griffith (1928)
Frederick Griffith (1928)
Conclusion: living R bacteria transformed into
deadly S bacteria by unknown, heritable
substance
Oswald Avery, et al. (1944)
– Discovered that the transforming agent was
DNA
Hershey and Chase (1952)
• Bacteriophages: virus that infects bacteria; composed of
DNA and protein
Protein =
radiolabel S
DNA = radiolabel
P
Hershey and Chase (1952)
Conclusion: DNA entered infected bacteria  DNA must be
the genetic material!
Edwin Chargaff (1947)
Chargaff’s Rules:
• DNA composition varies
between species
• Ratios:
– %A = %T and %G = %C
Rosalind Franklin (1950’s)
• Worked with Maurice Wilkins
• X-ray crystallography = images of DNA
• Provided measurements on chemistry of DNA
James Watson & Francis Crick (1953)
• Discovered the
double helix by
building models to
conform to
Franklin’s X-ray
data and
Chargaff’s Rules.
Structure of DNA
DNA = double helix
– “Backbone” = sugar +
phosphate
– “Rungs” = nitrogenous
bases
Structure of DNA
Nitrogenous Bases
–
–
–
–
Adenine (A)
Guanine (G)
Thymine (T)
Cytosine (C)
purine
pyrimidine
• Pairing:
– purine + pyrimidine
– A=T
– GΞC
Structure of DNA
Hydrogen bonds between base pairs of the two strands
hold the molecule together like a zipper.
Structure of DNA
Antiparallel: one strand (5’ 3’), other strand runs in
opposite, upside-down direction (3’  5’)
DNA Comparison
Prokaryotic DNA
Eukaryotic DNA
•
•
•
•
•
•
•
•
•
•
•
Double-stranded
Circular
One chromosome
In cytoplasm
No histones
Supercoiled DNA
Double-stranded
Linear
Usually 1+ chromosomes
In nucleus
DNA wrapped around histones
(proteins)
• Forms chromatin
PROBLEM:
How does DNA replicate?
Replication: Making DNA from existing DNA
3 alternative
models of DNA
replication
Meselson & Stahl
Meselson & Stahl
Replication is semiconservative
Major Steps of Replication:
1. Helicase: unwinds DNA at origins of replication and creates
replication forks
2. Initiation proteins separate 2 strands  forms replication
bubble
3. Primase: puts down RNA primer to start replication
4. DNA polymerase III: adds complimentary bases to leading
strand (new DNA is made 5’  3’)
5. Lagging strand grows in 3’5’ direction by the addition of
Okazaki fragments
6. DNA polymerase I: replaces RNA primers with DNA
7. DNA ligase: seals fragments together
1. Helicase unwinds DNA at origins of
replication and creates replication forks
3. Primase adds RNA primer
4. DNA polymerase III adds nucleotides in 5’3’
direction on leading strand
Replication on leading strand
Leading strand vs. Lagging strand
Okazaki
Fragments: Short
segments of DNA
that grow 5’3’ that
are added onto the
Lagging Strand
DNA Ligase: seals
together fragments
Proofreading and Repair
• DNA polymerases proofread as bases added
• Mismatch repair: special enzymes fix
incorrect pairings
• Nucleotide excision repair:
– Nucleases cut damaged DNA
– DNA poly and ligase fill in gaps
Nucleotide Excision Repair
Errors:
– Pairing errors: 1 in 100,000
nucleotides
– Complete DNA: 1 in 10
billion nucleotides
Problem at the 5’ End
• DNA poly only adds
nucleotides to 3’ end
• No way to complete 5’
ends of daughter strands
• Over many replications,
DNA strands will grow
shorter and shorter
Telomeres: repeated units of short nucleotide sequences
(TTAGGG) at ends of DNA
• Telomeres “cap” ends of DNA to postpone erosion of genes
at ends (TTAGGG)
• Telomerase: enzyme that adds to telomeres
– Eukaryotic germ cells, cancer cells
Telomeres stained
orange at the ends of
mouse chromosomes
Telomeres &
Telomerase
http://media.pearsoncmg.com/bc/bc_0media_bio/bioflix/bioflix.htm?8a
pdnarep
BIOFLIX: DNA REPLICATION
Closing Questions
1.
2.
3.
4.
What is recombinant DNA?
What are plasmids?
What are restriction enzymes (RE)?
When DNA is cut using an RE, describe the ends
of the DNA fragments.
Closing Question
A bacterial plasmid is 100 kb in length. The plasmid DNA was digested to
completion with 2 restriction enzymes in 3 separate treatments: EcoRI, HaeIII, and
EcoRI + HaeIII (double-digest). The fragments were separated by gel
electrophoresis below.
Draw a circle to represent the plasmid. On the circle, construct a labeled
diagram of the restriction map of the plasmid.
Closing Questions
1. Describe how a plasmid can be
genetically modified to include a piece
of foreign DNA that alters the
phenotype of bacterial cells transformed
with the modified plasmid.
2. How can a genetically modified
organism provide a benefit for humans
and at the same time pose a threat to a
population or ecosystem?
Biotechnology
Terms you Must Know
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Genetic engineering
Biotechnology
Recombinant DNA
Gene cloning
Plasmid
Restriction enzymes
Sticky ends
DNA ligase
Cloning vector
Nucleic acid hybridization
PCR
12.Gel electrophoresis
13.RFLPs
14.Genomic library
15.cDNA library
16.DNA microarray assays
17.SNPs
18.Stem cells
19.Gene therapy
20.Transgenic animals
21.GMO (genetically modified
organism)
What You Must Know:
• The terminology of biotechnology.
• How plasmids are used in bacterial transformation to
clone genes.
• The key ideas that make PCR possible and applications of
this technology.
• How gel electrophoresis can be used to separate DNA
fragments or protein molecules.
• Information that can be determined from DNA gel results,
such as fragment sizes and RFLP analysis.
Terminology
• Genetic Engineering: process of manipulating genes
and genomes
• Biotechnology: process of manipulating organisms
or their components for the purpose of making
useful products.
• Recombinant DNA: DNA that has been artificially made,
using DNA from different sources
– eg. Human gene inserted into E.coli
• Gene cloning: process by which scientists can product
multiple copies of specific segments of DNA that they can
then work with in the lab
Tools of Genetic Engineering
• Restriction enzymes (restriction
endonucleases): used to cut strands of DNA at
specific locations (restriction sites)
• Restriction Fragments: have at least 1
sticky end (single-stranded end)
• DNA ligase: joins DNA fragments
• Cloning vector: carries the DNA sequence to
be cloned (eg. bacterial plasmid)
Using a restriction
enzyme (RE) and DNA
ligase to make
recombinant DNA
Gene Cloning
Applications of Gene Cloning
Techniques of Genetic
Engineering
Techniques of Genetic
Engineering
 Transformation: bacteria takes up plasmid (w/gene
of interest)
 PCR (Polymerase Chain Reaction): amplify (copy)
piece of DNA without use of cells
 Gel electrophoresis: used to separate DNA molecules
on basis of size and charge using an electrical current
(DNA  + pole)
 DNA microarray assays: study many genes at same
time
PCR (Polymerase Chain
Reaction): amplify (copy)
piece of DNA without use of
cells
Gel Electrophoresis:
used to separate DNA
molecules on basis of
size and charge using
an electrical current
(DNA  (+) pole)
Gel Electrophoresis
Microarray Assay: used to study gene
expression of many different genes
DNA microarray that reveals expression
levels of 2,400 human genes
Cloning Organisms
• Nuclear transplantation: nucleus of egg is
removed and replaced with nucleus of body cell
Nuclear Transplantation
Problems with Reproductive Cloning
• Cloned embryos exhibited various defects
• DNA of fully differentiated cell have epigenetic
changes
Stem Cells
• Stem cells: can reproduce itself indefinitely and
produce other specialized cells
– Zygote = totipotent (any type of cell)
– Embryonic stem cells = pluripotent (many cell
types)
– Adult stem cells = multipotent (a few cell
types) or induced pluripotent, iPS (forced to be
pluripotent)
Embryonic
vs. Adult
stem cells
Using stem cells for disease treatment
http://learn.genetics.utah.edu/content/stemcells/sctypes/
Interactive: Go Go Stem Cells
Applications of DNA
Technology
1.
2.
3.
4.
5.
6.
Diagnosis of disease – identify alleles, viral DNA
Gene therapy – alter afflicted genes
Production of pharmaceuticals
Forensic applications – DNA profiling
Environmental cleanup – use microorganisms
Agricultural applications - GMOs
Gene therapy using a retroviral vector
“Pharm” animal: produce human protein
secreted in milk for medical use
DNA Fingerprinting
RFLPs (“rif-lips”)
• Restriction Fragment Length Polymorphism
• Cut DNA with different restriction enzymes
• Each person has different #s of DNA fragments
created
• Analyze DNA samples on a gel for disease diagnosis
• Outdated method of DNA profiling (required a
quarter-sized sample of blood)
RFLPs – Disease Diagnosis
VIDEO: INTRODUCTION TO DNA
FINGERPRINTING
Naked Science Scrapbook
STR Analysis
• STR = Short Tandem Repeats
• Non-coding DNA has regions with sequences
(2-5 base length) that are repeated
• Each person has different # of repeats at
different locations (loci)
• Current method of DNA fingerprinting used –
only need 20 cells for analysis
STR Analysis
STR Analysis
Genetically Modified (GM)
organisms
• Organisms altered through recombinant DNA
technology
• Insert foreign DNA into genome or combine
DNA from different genomes
Biotechnology Techniques
How to make
Recombinant DNA
Summarize: What is
this technique?
Draw and label a
diagram to show this
technique
What are the main
tools or materials
involved?
Applications: What is
this being used for?
Gel Electrophoresis
PCR