Download Genetics - Fresno State

10/22/2013
Genetics:
from Mendel and mutations to
solutions to the Valley
Alejandro Calderón-Urrea, M.Sc., Ph.D., Chair
Department of Biology
California State University, Fresno
California State University, Fresno – Department of Biology
I.
II.
The roots of genetics in pre-WWI science
Mendel who?
a.
b.
III.
IV.
Laws, laws, laws…
From Mendel to molecular genetics
Mutations and diversity
How can we help?
a.
b.
c.
Genetic transformation of Dunaliella primolecta:
Improve natural capacity of algae
Diagnostics for all…
Death to nematodes
California State University, Fresno – Department of Biology
1
10/22/2013
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California State University, Fresno – Department of Biology
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California State University, Fresno – Department of Biology
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2
10/22/2013
I.
II.
The roots of genetics in pre-WWI science
Mendel who?
a.
b.
III.
IV.
Laws, laws, laws…
From Mendel to molecular genetics
Mutations and diversity
How can we help?
a.
b.
c.
Genetic transformation of Dunaliella primolecta:
Improve natural capacity of algae
Diagnostics for all…
Death to nematodes
California State University, Fresno – Department of Biology
Why do children have characteristics from parents and
grandparents?
Homunculus, gemmule,
pangenesis, and the
Preformation hypothesis
California State University, Fresno – Department of Biology
3
10/22/2013
Q
Today, thanks to the Preformation hypothesis,
we know that everything in the embryo is
preformed: it simply gets bigger.
A) True
B) False
California State University, Fresno – Department of Biology
How traits are transferred from parents to offspring?
The “blending” hypothesis is the idea that genetic
material from the two parents blends together (like blue
and yellow paint blend to make green)
The “particulate” hypothesis is the idea that parents
pass on discrete heritable units (traits or genes)
California State University, Fresno – Department of Biology
4
10/22/2013
I.
II.
The roots of genetics in pre-WWI science
Mendel who?
a.
b.
III.
IV.
Laws, laws, laws…
From Mendel to molecular genetics
Mutations and diversity
How can we help?
a.
b.
c.
Genetic transformation of Dunaliella primolecta:
Improve natural capacity of algae
Diagnostics for all…
Death to nematodes
California State University, Fresno – Department of Biology
California State University, Fresno – Department of Biology
5
10/22/2013
So, what do you know about
genetics?
California State University, Fresno – Department of Biology
Q
If a sexually reproducing organism has
two sets of chromosomes (one from the
mother and one from the father), this
organism is said to be:
A) Homozygous
B) Heterozygous
C) Haploid
D) Diploid
E) Polyploid
California State University, Fresno – Department of Biology
6
10/22/2013
Q
If a sexually reproducing organism has
two sets of chromosomes (one from the
mother and one from the father), this
organism is said to be:
A) Homozygous
B) Heterozygous
C) Haploid
D) Diploid
E) Polyploid
California State University, Fresno – Department of Biology
Q
One of the alternative forms of a gene is
called:
A) Allele
B) Phenotype
C) Genotype
D) Locus (plural loci)
E) Levi’s 505
California State University, Fresno – Department of Biology
7
10/22/2013
Q
One of the alternative forms of a gene is
called:
A) Allele
B) Phenotype
C) Genotype
D) Locus (plural loci)
E) Levi’s 505
California State University, Fresno – Department of Biology
Cells and heredity through DNA
Sperm cell
Nuclei
containing
DNA
Egg cell
Fertilized egg
with DNA from
both parents
Embryo’s cells with
copies of inherited DNA
Offspring with traits
inherited from
both parents
Cells contain chromosomes made partly of DNA (the substance of genes)
which program a cells’ production of proteins and transmit information
from parent to offspring
California State University, Fresno – Department of Biology
8
10/22/2013
Mendel used the scientific approach to
identify two laws of inheritance
1) The Law of Segregation
2) The Law of Independent Assortment
California State University, Fresno – Department of Biology
“The Scientific Method”
1) Observation
2) Hypothesis
3) Prediction
4) Testing
California State University, Fresno – Department of Biology
9
10/22/2013
Allele
(trait) for
purple
flowers
Allele
(trait) for
white
flowers
Locus for
flower-color
gene (character)
Allele
(trait) for
yellow
seeds
Allele
(trait) for
green
seeds
Locus for
seed-color gene
(character)
Homologous
pair of
chromosomes
Homologous
pair of
chromosomes
California State University, Fresno – Department of Biology
1) The Law of Segregation
Allele
(trait) for
purple
flowers
Allele
(trait) for
white
flowers
Locus for
flower-color
gene (character)
Allele
(trait) for
yellow
seeds
Homologous
pair of
chromosomes
Allele
(trait) for
green
seeds
Locus for
seed-color gene
(character)
Homologous
pair of
chromosomes
Law of segregation (alleles):
The two alleles for a heritable character
separate (segregate) during gamete
formation and end up in different gametes.
California State University, Fresno – Department of Biology
10
10/22/2013
2) The Law of Independent
Assortment
Allele
(trait) for
purple
flowers
Allele
(trait) for
white
flowers
Locus for
flower-color
gene (character)
Allele
(trait) for
yellow
seeds
Allele
(trait) for
green
seeds
Locus for
seed-color gene
(character)
Homologous
pair of
chromosomes
Homologous
pair of
chromosomes
The law of independent assortment
(genes):
Each pair of alleles segregates
independently of each other pair of alleles
during gamete formation
California State University, Fresno – Department of Biology
TECHNIQUE
1
Mendel’s Experimental,
Quantitative Approach
• Advantages of pea plants for genetic
study:
– Characters (such as flower color)
– Traits (such as purple or white flowers)
– Mating of plants can be controlled
– Each pea plant has sperm-producing
organs (stamens) and egg-producing
organs (carpels)
– Cross-pollination (fertilization
between different plants) can be
achieved by dusting one plant with
pollen from another
California State University, Fresno – Department of Biology
2
Parental
generation
(P)
Stamens
Carpel
3
4
RESULTS
First
filial
generation
offspring
(F1)
5
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10/22/2013
• Mendel chose to track only those characters that varied in an
either-or manner
• He also used varieties that were true-breeding (plants that
produce offspring of the same variety when they selfpollinate)
• In a typical experiment, Mendel mated two contrasting, truebreeding varieties, a process called hybridization
• The true-breeding parents are the P generation
• The hybrid offspring of the P generation are called the F1
generation
• When F1 individuals self-pollinate, the F2 generation is
produced
California State University, Fresno – Department of Biology
EXPERIMENT
P Generation
(true-breeding
parents)
The Law of Segregation
• When Mendel crossed
contrasting, true-breeding
white and purple flowered
pea plants, all of the F1
hybrids were purple
• When Mendel crossed the
F1 hybrids, many of the F2
plants had purple flowers,
but some had white
• Mendel discovered a ratio
of about three to one,
purple to white flowers, in
the F2 generation
California State University, Fresno – Department of Biology
Purple
flowers
White
flowers
F1 Generation
(hybrids)
All plants had
purple flowers
F2 Generation
705 purple-flowered
plants
224 white-flowered
plants
12
10/22/2013
• Mendel reasoned that only the
purple flower factor was
affecting flower color in the F1
hybrids
• Mendel called the purple
flower color a dominant trait
and the white flower color a
recessive trait
• Mendel observed the same
pattern of inheritance in six
other pea plant characters,
each represented by two traits
• What Mendel called a
“heritable factor” is what we
now call a gene
California State University, Fresno – Department of Biology
So, how did Mendel explained
these amazing results?
California State University, Fresno – Department of Biology
13
10/22/2013
P Generation
1= alternative versions of
genes account for
1
variations in inherited Appearance:
Purple flowers White flowers
characters
Genetic makeup:
PP
pp
2= for each character an
Gametes:
p
P
organism inherits two
• Mendel’s segregation
alleles (traits), one from
model accounts for the 3:1
2
each parent
ratio he observed in the F2
two alleles (traits)
generation of his numerous 3=atifathe
locus differ, then one F1 Generation
3
crosses
(the dominant allele or
trait) determines the
• The possible combinations
organism’s appearance, Appearance:
Purple flowers
of sperm and egg can be
and the other (the
Genetic makeup:
Pp
recessive
allele
or
trait)
shown using a Punnett
1/
has no noticeable effect Gametes:
4 1/2 p
P
2
square, a diagram for
on appearance
predicting the results of a
Sperm
genetic cross between
4= law of segregation: the
p
P
individuals of known genetic two alleles for a heritable F2 Generation
character separate
makeup
(segregate) during gamete
P
• A capital letter represents a formation and end up in
PP
Pp
different gametes
dominant allele, and a
Eggs
lowercase letter represents
p
a recessive allele
pp
Pp
3
1
California State University, Fresno – Department of Biology
The Law of Independent Assortment
• Mendel identified his second law of inheritance (the law
of independent assortment) by following two characters
at the same time
• Crossing two true-breeding parents differing in two
characters produces dihybrids in the F1 generation,
heterozygous for both characters
• A dihybrid cross, a cross between F1 dihybrids, can
determine whether two characters are transmitted to
offspring as a package or independently
California State University, Fresno – Department of Biology
14
10/22/2013
Law of segregation:
The two alleles for a single heritable
character separate (segregate)
during gamete formation and end up
in different gametes.
Do pairs of alleles of two loci separate (segregate)
independently during gamete production?
Is it this: Y R
or this: Y R
Y R
Y R
Experiment to test if dependent vs. independent assortment:
EXPERIMENT
YYRR
P Generation
yyrr
Gametes YR
yr
F1 Generation
Predictions
YyRr
Hypothesis of
dependent assortment
Hypothesis of
independent assortment
Sperm
or
1/
Sperm
Predicted gametes produced by
F1 generation
1/
2
1/
YR
2
YR
4
1/
4
Yr
1/
4
yR
1/
4
yr
yr
California State University, Fresno – Department of Biology
EXPERIMENT
YyRr
F1 Generation
Hypothesis of
dependent assortment
Predictions
Hypothesis of
independent assortment
Sperm
or
Predicted
offspring of
F2 generation
1/
Sperm
1/
2
YR
1/
2
2
YyRr
Eggs
2
1/
4
1/
Yr
4
yR
1/
4
yr
4
YR
YYRR
YYRr
YyRR
YyRr
YYRr
YYrr
YyRr
Yyrr
YyRR
YyRr
yyRR
yyRr
YyRr
Yyrr
YR
YYRR
1/
YR
yr
1/
1/
4
1/
4
Yr
4
yR
Eggs
yr
YyRr
3/
yyrr
1/
4
1/
4
Phenotypic ratio 3:1
1/
yr
4
9/
16
3/
16
yyRr
3/
16
yyrr
1/
16
Phenotypic ratio 9:3:3:1
RESULTS
315
108
101
32
Phenotypic ratio approximately 9:3:3:1
California State University, Fresno – Department of Biology
15
10/22/2013
EXPERIMENT
YyRr
F1 Generation
Hypothesis of
dependent assortment
Predictions
Hypothesis of
independent assortment
Sperm
or
Predicted
offspring of
F2 generation
1/
Sperm
1/
2
YR
1/
2
2
YyRr
YYRR
2
1/
4
1/
Yr
4
yR
1/
4
yr
4
YR
YYRR
YYRr
YyRR
YyRr
YYRr
YYrr
YyRr
Yyrr
YyRR
YyRr
yyRR
yyRr
YyRr
Yyrr
YR
1/
Eggs
1/
YR
yr
1/
1/
4
4
Yr
4
yR
Eggs
yr
YyRr
3/
yyrr
1/
4
1/
4
Phenotypic ratio 3:1
1/
yr
4
9/
16
3/
16
yyRr
3/
16
yyrr
1/
16
Phenotypic ratio 9:3:3:1
RESULTS
315
108
101
32
Phenotypic ratio approximately 9:3:3:1
California State University, Fresno – Department of Biology
1) The Law of Segregation
Allele
(trait) for
purple
flowers
Allele
(trait) for
white
flowers
Locus for
flower-color
gene (character)
Allele
(trait) for
yellow
seeds
Homologous
pair of
chromosomes
Allele
(trait) for
green
seeds
Locus for
seed-color gene
(character)
Homologous
pair of
chromosomes
Law of segregation (alleles):
The two alleles for a heritable character
separate (segregate) during gamete
formation and end up in different gametes.
California State University, Fresno – Department of Biology
16
10/22/2013
2) The Law of Independent
Assortment
Allele
(trait) for
white
flowers
Allele
(trait) for
purple
flowers
Locus for
flower-color
gene (character)
Allele
(trait) for
yellow
seeds
Homologous
pair of
chromosomes
Allele
(trait) for
green
seeds
Locus for
seed-color gene
(character)
Homologous
pair of
chromosomes
The law of independent assortment
(genes):
Each pair of alleles segregates
independently of each other pair of alleles
during gamete formation
California State University, Fresno – Department of Biology
I.
II.
The roots of genetics in pre-WWI science
Mendel who?
a.
b.
III.
IV.
Laws, laws, laws…
From Mendel to molecular genetics
Mutations and diversity
How can we help?
a.
b.
c.
Genetic transformation of Dunaliella primolecta:
Improve natural capacity of algae
Diagnostics for all…
Death to nematodes
California State University, Fresno – Department of Biology
17
10/22/2013
Q
What are major types of macro-molecules
found in living organisms?
A) DNA
B) Proteins
C) Carbohydrates
D) Lipids
E) All of the above
California State University, Fresno – Department of Biology
A
C
Nucleotide
The genetic material: Mendel’s
traits are in the DNA!
Nucleus
DNA
T
T
A
R
A
R
A
T
C
T
A
C
G
A
R
T
Cell
Genome: The entire library
of genetic instructions that
an organism inherits
The Human Genome: 3
billion nucleotides (about
25,000 genes), 75,000
kinds of proteins
T
A
G
T
A
(a) DNA double helix
T
G
A
T
A
T
G
G
C
A
T
C
A
T
(b) Single strand of DNA
California State University, Fresno – Department of Biology
18
10/22/2013
The Basic Principle: Base Pairing to a Template Strand
•
•
Since the two strands of DNA are complementary, each strand acts as a
template for building a new strand in replication
In DNA replication, the parent molecule unwinds, and two new daughter
strands are built based on base-pairing rules
A
T
A
T
A
T
A
T
C
G
C
G
C
G
C
G
T
A
T
A
T
A
T
A
A
T
A
T
A
T
A
T
G
C
G
C
G
C
G
C
(a) Parent molecule
(b) Separation of
strands
(c) “Daughter” DNA molecules,
each consisting of one parental
strand and one new strand
California State University, Fresno – Department of Biology
Fig. 16-21a
Nucleosome
(10 nm in diameter)
DNA
double helix
(2 nm in diameter)
H1
Histones
DNA, the double helix
Histones
Histone tail
Nucleosomes, or “beads
on a string” (10-nm fiber)
Nucleosome: octamer of histone
proteins (2X each of H2A, H2B, H3, H4)
California State University, Fresno – Department of Biology
19
10/22/2013
Chromatid
(700 nm)
30-nm fiber
Loops
Scaffold
300-nm fiber
30-nm fiber
Looped domains
(300-nm fiber)
California State University, Fresno – Department of Biology
Replicated
chromosome
(1,400 nm)
Metaphase
chromosome
The Products of Gene Expression: A Developing Story
• Some proteins aren’t enzymes, so researchers later revised the
hypothesis: one gene–one protein
• Many proteins are composed of several polypeptides, each of
which has its own gene
• Therefore, Beadle and Tatum’s hypothesis is now restated as the
one gene–one polypeptide hypothesis
• Note that it is common to refer to gene products as proteins
rather than polypeptides
• A gene can be defined as a region of DNA that can be expressed to
produce a final functional product, either a polypeptide or an RNA
molecule.
California State University, Fresno – Department of Biology
20
10/22/2013
DNA
TRANSCRIPTION
•
•
•
•
•
In prokaryotes, mRNA produced by
transcription is immediately translated
without more processing
In a eukaryotic cell, the nuclear envelope
separates transcription from translation
Eukaryotic RNA transcripts are modified
through RNA processing to yield finished
mRNA
A primary transcript is the initial RNA
transcript from any gene
The central dogma is the concept that cells
are governed by a cellular chain of
command: DNA → RNA → protein
mRNA
Ribosome
TRANSLATION
Polypeptide
(a) Bacterial cell
Nuclear
envelope
DNA
TRANSCRIPTION
Pre-mRNA
RNA PROCESSING
mRNA
TRANSLATION
Ribosome
Polypeptide
California State University, Fresno – Department of Biology
(b) Eukaryotic cell
Genomics: Large-Scale Analysis of DNA
Sequences
• An organism’s genome is its entire set of genetic instructions
- Genomics is the study of sets of genes within and between
species
• The human genome and those of many other organisms have
been sequenced using DNA-sequencing machines. Genomics
requires
– “High-throughput” technology, which yields enormous
amounts of data
– Bioinformatics, which is the use of computational tools to
process a large volume of data
– Interdisciplinary research teams
California State University, Fresno – Department of Biology
21
10/22/2013
Evolution of the Genetic Code
• The genetic code is nearly universal, shared by the simplest bacteria
to the most complex animals
• Genes can be transcribed and translated after being transplanted
from one species to another
(a) Tobacco plant
expressing a
firefly gene
(b) Pig
expressing a
jellyfish gene
California State University, Fresno – Department of Biology
I.
II.
The roots of genetics in pre-WWI science
Mendel who?
a.
b.
III.
IV.
Laws, laws, laws…
From Mendel to molecular genetics
Mutations and diversity
How can we help?
a.
b.
c.
Genetic transformation of Dunaliella primolecta:
Improve natural capacity of algae
Diagnostics for all…
Death to nematodes
California State University, Fresno – Department of Biology
22
10/22/2013
Mutations are changes in the
genetic material of a cell or virus
Point mutations
Deletions
Insertions
California State University, Fresno – Department of Biology
Point mutations can affect
protein structure and function
•
•
Point mutations are chemical changes in just one base pair of a gene
The change of a single nucleotide in a DNA template strand can lead to the
production of an abnormal protein
3′′
5′′
Wild-type hemoglobin DNA
C T T
G A A
5′′ 3′′
3′′ 5′′
mRNA
5′′
Mutant hemoglobin DNA
C A T
G T A
5′′
3′′
mRNA
G A A
Normal hemoglobin
Glu
3′′ 5′′
G U A
3′′
Sickle-cell hemoglobin
Val
California State University, Fresno – Department of Biology
23
10/22/2013
Insertions and Deletions
•Insertions and deletions are additions or losses of
nucleotide pairs (or larger sequences) in a gene
•These mutations have a disastrous effect on the
resulting protein more often than substitutions do
California State University, Fresno – Department of Biology
Mutagens
• Spontaneous mutations can occur during DNA
replication, recombination, or repair
• Mutagens are physical or chemical agents that can
cause mutations
California State University, Fresno – Department of Biology
24
10/22/2013
I.
II.
The roots of genetics in pre-WWI science
Mendel who?
a.
b.
III.
IV.
Laws, laws, laws…
From Mendel to molecular genetics
Mutations and diversity
How can we help?
a.
b.
c.
Genetic transformation of Dunaliella primolecta:
Improve natural capacity of algae
Diagnostics for all…
Death to nematodes
California State University, Fresno – Department of Biology
Research on the microalgae Dunaliella
primolecta at Fresno State
California State University, Fresno – Department of Biology
25
10/22/2013
Algae and their uses
Algae are a diverse, polyphyletic assemblage of
photosynthetic eukaryotes unified primarily by their lack
of roots, leaves, and the other organs that characterize
higher plants.
Algae include macroalgae and microalgae
Microalgae, a large and diverse group of unicellular photoand heterotrophic organisms
California State University, Fresno – Department of Biology
EUKARYA
Dinoflagellates
Forams
Ciliates Diatoms
Red algae
Land plants
Green algae
Amoebas
Cellular slime molds
Euglena
Trypanosomes
Leishmania
Animals
Fungi
Sulfolobus
Green nonsulfur bacteria
Thermophiles
Halophiles
(Mitochondrion)
COMMON
ANCESTOR
OF ALL
LIFE
Methanobacterium
ARCHAEA
Spirochetes
Chlamydia
Green
sulfur bacteria
BACTERIA
Cyanobacteria
(Plastids, including
chloroplasts)
California State University, Fresno – Department of Biology
26
10/22/2013
Green microalgae:
β-carotene, Biofuels, Polyunsaturated
fatty acids (PUFA) , Anticancer drugs
Diatomes (silica based cell wall-frustule):
feeds in aquaculture, nanotechnology, major
contributors to CO2 fixation and O2
production in oceans.
From:
Parker et al., Annu. Rev. Genet. 2008. 42: 619–45
California State University, Fresno – Department of Biology
What do microalgae need?
Water
Sunlight
CO2
Or other C source
Nitrogen
Phosphorous
What do they produce?
β-carotene
Polyunsaturated fatty acids (PUFA)
Biofuels
Therapeutic proteins
Anticancer drugs
Astaxanthin
California State University, Fresno – Department of Biology
27
10/22/2013
Oil content of some microalgae
Production cost of biodisel from algae:
$2.80/liter (2006)
Production cost of petrodiesel:
$0.49/liter (2006)
How to reduce production cost?
Improving large scale production
Biodiesel production through Transesterification
Couple production to cleanup
Improve natural capacity of algae
California State University, Fresno – Department of Biology
From:
Y. Chisti. Biotechnology Advances. 2007. 25: 294–306
Oil content of some microalgae
Production cost of biodisel from algae:
$2.80/liter (2006)
Production cost of petrodiesel:
$0.49/liter (2006)
How to reduce production cost?
Improving large scale production
Biodiesel production through Transesterification
Couple production to cleanup
Improve natural capacity of algae
California State University, Fresno – Department of Biology
From:
Y. Chisti. Biotechnology Advances. 2007. 25: 294–306
28
10/22/2013
A
B
C
D
1
Developmental profile of D.
primolecta from younger cells (1)
through older cells (5). The various
panels (A though D) show cells
visualized with Nomarski
microscopy (A), chlorophyll autofluorescence (B), lipid
accumulation as visualized by
BODIPY staining (C), and overlay of
Nomarski, chlorophyll, and lipid
images (D).
2
3
4
5
California State University, Fresno – Department of Biology
Genetic transformation of Dunaliella primolecta:
Use genetic markers to test transformation
35S poly
HygR
35S pro
LB
Lac Z
35S pro
pCAMBIA1301
GUS
np
RB
Use Agrobacterium tumefaciens
Plate on MSS medium 10 mL106
cells/mL (Dp cells)
Wash Dp cells and plate on
selection medium
ON culture A. tumefaciens
3 days
MSS medium
48 hours
MSS medium
Isolate transgenic lines
MSS medium+ cefotaxime+ hygromycin
Cell colonies appear in 10-20 days
California State University, Fresno – Department of Biology
MSS medium+ cefotaxime+ hygromycin
29
10/22/2013
Features of our transformation protocol:
• No inducer molecule, such as acetosyringone, was used for
the transfer of genes from Agrobacterium to the algae cells
• We observed high efficiency of transformation, as compared
with other methods
• Although we consistently obtained an average of 1500 Hygr
transformed algae cells (concentration of 100 mgL-1), 96
were selected and develop into cell lines to study their
transgenic status.
California State University, Fresno – Department of Biology
Questions:
• Are the inserted genes stable?
• Do the genes insert in multiple sites in the
genome?
• Can this transformation protocol be used as an
“insertional mutagenesis” protocol?
California State University, Fresno – Department of Biology
30
10/22/2013
Are the inserted genes stable?
PCR amplification of GUS gene in selected HygR lines. The expected
450 bp fragment is amplified in all lines. C+ is the pCAMBIA1301
vector and C- is untransformed DNA.
MM G1
G2 G3 G4 G5
G6
G7 G8
G9 G10 G11 G12 C+
C-
1.0KB
0.45KB
California State University, Fresno – Department of Biology
Do the genes insert in multiple sites in the genome?
GUS expression of selected transformed lines. Cell lines farther away
from the “Y” axis (circled) are cell lines expressing GUS at high levels.
3500000
Number of cells/ml
3000000
Control
2500000
Row A
Row B
2000000
Row C
1500000
Row D
1000000
Row E
Row F
500000
Row G
0
Row H
0
50
100
150
200
250
300
350
RFU
California State University, Fresno – Department of Biology
31
10/22/2013
Can this transformation protocol be used as an
“insertional mutagenesis” protocol?
California State University, Fresno – Department of Biology
Can this transformation protocol be used as an
“insertional mutagenesis” protocol?
( )
gene A
( )
gene B
( )
gene C
( )
gene D
( )
gene E
etc.
California State University, Fresno – Department of Biology
32
10/22/2013
Can this transformation protocol be used as an
“insertional mutagenesis” protocol?
( )
gene A
( )
gene B
( )
gene C
( )
gene D
( )
gene E
etc.
What if one of this genes controls lipid biosynthesis? A mutation will have increased lipid content…
California State University, Fresno – Department of Biology
Can this transformation protocol be used as an
“insertional mutagenesis” protocol?
( )
gene A
( )
gene B
( )
gene C
( )
gene D
( )
gene E
etc.
What if one of this genes is involved in lipid biosynthesis? A mutation will have lower lipid content…
California State University, Fresno – Department of Biology
33
10/22/2013
Neutral lipid content measured by BODIPY fluorescence as it binds to
lipids. Cell lines with RFU values below the control line are cell lines
with high levels of lipid content. Conversely cell lines with RFU values
above the control line are cell lines with low levels of lipid content.
3500000
A5
Number of cells/ml
3000000
A8
2500000
2000000
B5
1500000
B7
1000000
500000
B12
0
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
F7
RFU
California State University, Fresno – Department of Biology
Comparison of lipids and concentration between untransformed and
transformed D. primolecta lines. Lines A5, B5, F7, and H5 have lower
lipid content compared to untransformed; Lines A8, B7, and B12 cells
lines have higher lipid content compared to untransformed lines.
Amount of lipid in ng
600
500
DPI
400
DPII
F7
300
H5
200
B5
A5
100
B12
0
B7
A8
LIPIDS
California State University, Fresno – Department of Biology
34
10/22/2013
Conclusions:
Agrobacterium–mediated transformation of D.
primolecta is simple and reproducible.
PCR amplification of the GUS gene and activity of
β-glucuronidase confirmed the transgenic status of
the HygR lines generated.
BODIPY assay showed that the lipid content in the
transformed cell lines varied; this may indicate that
the introduced genes are integrated at random
positions in the genome of D. primolecta.
California State University, Fresno – Department of Biology
Acknowledgements:
Preethi Sarvabhowman
Alex Guzzetta
Sabrina Romero
Rakesh Kumar
Raj Patidar
Funding for the research:
CSM trough various programs provided funds for the research in A. C-U.’s Lab
Materials provided:
UTEX and Wawona Frozen Foods, Clovis, CA
California State University, Fresno – Department of Biology
35
10/22/2013
I.
II.
The roots of genetics in pre-WWI science
Mendel who?
a.
b.
III.
IV.
Laws, laws, laws…
From Mendel to molecular genetics
Mutations and diversity
How can we help?
a.
b.
c.
Genetic transformation of Dunalliela primolecta:
Improve natural capacity of algae
Diagnostics for all…
Death to nematodes
California State University, Fresno – Department of Biology
Q
I learned something new from this
presentation:
A) Yes
B) No
California State University, Fresno – Department of Biology
36
10/22/2013
Thank you
Alejandro Calderón-Urrea, M.Sc., Ph.D.
Department of Biology
California State University, Fresno
California State University, Fresno – Department of Biology
10 / 21 / 2013
37