genetics: from mendel and mutations to solutions to the … academic... · 2013-10-22 · from...

10/22/2013

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California State University, Fresno – Department of Biology

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


I. The roots of genetics in pre-WWI science

II. Mendel who?

a. Laws, laws, laws…

b. From Mendel to molecular genetics

III. Mutations and diversity

IV. How can we help?

a. Genetic transformation of Dunaliella primolecta: Improve natural capacity of algae

b. Diagnostics for all…

c. Death to nematodes

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= A

= B

= C

= D

= E

Fold the clicker


= A

= B

= C

= D= I have no idea what

you are talking about

Fold the clicker

= E

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II. Mendel who?









Why do children have characteristics from parents and

grandparents?

� Homunculus, gemmule,

pangenesis, and the

Preformation hypothesis

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QToday, thanks to the Preformation hypothesis,

we know that everything in the embryo is

preformed: it simply gets bigger.

A) TrueB) False


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)

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II. Mendel who?









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So, what do you know about

genetics?


QIf 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) HomozygousB) HeterozygousC) HaploidD) DiploidE) Polyploid

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QIf 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) HomozygousB) HeterozygousC) HaploidD) DiploidE) Polyploid


QOne of the alternative forms of a gene is called:

A) AlleleB) PhenotypeC) GenotypeD) Locus (plural loci)E) Levi’s 505

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QOne of the alternative forms of a gene is called:

A) AlleleB) PhenotypeC) GenotypeD) Locus (plural loci)E) Levi’s 505


Sperm cell

Nuclei

containing

DNA

Egg cell

Fertilized egg

with DNA from

both parents

Embryo’s cells with

copies of inherited DNAOffspring with traits

inherited from

both parents

Cells and heredity through DNA

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

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Mendel used the scientific approach to

identify two laws of inheritance

1) The Law of Segregation

2) The Law of Independent Assortment


“The Scientific Method”1) Observation

2) Hypothesis

3) Prediction

4) Testing

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Allele (trait) for

white flowers

Allele (trait) for

purple flowers

Locus for flower-color

gene (character)

Homologous pair of

chromosomes

Allele (trait) for

green seeds

Allele (trait) for

yellow seeds

Locus for seed-color gene

(character)

Homologous pair of

chromosomes


Allele (trait) for

white flowers

Allele (trait) for

purple flowers


gene (character)

Homologous pair of

chromosomes

Allele (trait) for

green seeds

Allele (trait) for

yellow seeds


(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.


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Allele (trait) for

white flowers

Allele (trait) for

purple flowers


gene (character)

Homologous pair of

chromosomes

Allele (trait) for

green seeds

Allele (trait) for

yellow seeds


(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



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

TECHNIQUE

RESULTS

Parentalgeneration(P) Stamens

Carpel

1

2

3

4

Firstfilialgener-

ationoffspring(F1)

5

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• 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 self-pollinate)

• In a typical experiment, Mendel mated two contrasting, true-breeding varieties, a process called hybridization

• The true-breeding parents are the P generation

• The hybrid offspring of the P generation are called the F1generation

• When F1 individuals self-pollinate, the F2 generation is produced


The Law of Segregation

• When Mendel crossed contrasting, true-breeding white and purple flowered pea plants, all of the F1hybrids were purple

• When Mendel crossed the F1 hybrids, many of the F2plants had purple flowers, but some had white

• Mendel discovered a ratio of about three to one, purple to white flowers, in the F2 generation

EXPERIMENT

P Generation

(true-breeding

parents)

Purpleflowers

Whiteflowers

F1 Generation

(hybrids)

All plants hadpurple flowers

F2 Generation

705 purple-floweredplants

224 white-floweredplants

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• 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


So, how did Mendel explained

these amazing results?

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• Mendel’s segregation model accounts for the 3:1 ratio he observed in the F2generation of his numerous crosses

• The possible combinations of sperm and egg can be shown using a Punnettsquare, a diagram for predicting the results of a genetic cross between individuals of known genetic makeup

• A capital letter represents a dominant allele, and a lowercase letter represents a recessive allele

P Generation

Appearance:Genetic makeup:

Gametes:

Purple flowers White flowersPP

P

pp

p

F1 Generation

Gametes:

Genetic makeup:Appearance: Purple flowers

Pp

P p1/21/2

F2 Generation

Sperm

Eggs

P

PPP Pp

p

p

Pp pp

3 1

1= alternative versions of

genes account for

variations in inherited characters

2= for each character an

organism inherits two

alleles (traits), one from

each parent

3= if the two alleles (traits)

at a locus differ, then one

(the dominant allele or

trait) determines the

organism’’’’s appearance,

and the other (the

recessive allele or trait)

has no noticeable effect

on appearance

4= law of segregation: the two alleles for a heritable

character separate (segregate) during gamete

formation and end up in

different gametes

1

2

3

4


• 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

The Law of Independent Assortment

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Y R

Y R

Y R

Y R

Do pairs of alleles of two loci separate (segregate) independently during gamete production?

Is it this: or this:

P Generation

F1 Generation

Predictions

Gametes

EXPERIMENTYYRR yyrr

yrYR

YyRr

Hypothesis ofdependent assortment

Hypothesis ofindependent assortment

Predicted gametes produced byF1 generation

Sperm

Spermor

1/21/2

1/41/4

1/41/4

YR

YR

yr

yrYr yR

Law of segregation: The two alleles for a single heritable

character separate (segregate)

during gamete formation and end up

in different gametes.

Experiment to test if dependent vs. independent assortment:


F1 Generation

Predictions

EXPERIMENT

RESULTS

YyRr



Predictedoffspring ofF2 generation

Sperm

Spermor

Eggs

Eggs

Phenotypic ratio 3:1

Phenotypic ratio 9:3:3:1

Phenotypic ratio approximately 9:3:3:1315 108 101 32

1/21/2

1/2

1/2

1/41/4

1/41/4

1/4

1/4

1/4

1/4

9/163/16

3/161/16

YR

YR

YR

YR

yr

yr

yr

yr

1/43/4

Yr

Yr

yR

yR

YYRR YyRr

YyRr yyrr

YYRR YYRr YyRR YyRr

YYRr YYrr YyRr Yyrr

YyRR YyRr yyRR yyRr

YyRr Yyrr yyRr yyrr

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F1 Generation

Predictions

EXPERIMENT

RESULTS

YyRr



Predictedoffspring ofF2 generation

Sperm

Spermor

Eggs

Eggs

Phenotypic ratio 3:1

Phenotypic ratio 9:3:3:1

Phenotypic ratio approximately 9:3:3:1315 108 101 32

1/21/2

1/2

1/2

1/41/4

1/41/4

1/4

1/4

1/4

1/4

9/163/16

3/161/16

YR

YR

YR

YR

yr

yr

yr

yr

1/43/4

Yr

Yr

yR

yR

YYRR YyRr

YyRr yyrr

YYRR YYRr YyRR YyRr

YYRr YYrr YyRr Yyrr

YyRR YyRr yyRR yyRr

YyRr Yyrr yyRr yyrr


Allele (trait) for

white flowers

Allele (trait) for

purple flowers


gene (character)

Homologous pair of

chromosomes

Allele (trait) for

green seeds

Allele (trait) for

yellow seeds


(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.


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Allele (trait) for

white flowers

Allele (trait) for

purple flowers


gene (character)

Homologous pair of

chromosomes

Allele (trait) for

green seeds

Allele (trait) for

yellow seeds


(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




II. Mendel who?








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Q What are major types of macro-molecules found in living organisms?

A) DNAB) ProteinsC) CarbohydratesD) LipidsE) All of the above


Nucleotide

(b) Single strand of DNA

A

C

T

T

A

A

T

C

C

G

T

A

G

T

(a) DNA double helix

AGenome: 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

G

A

T

A

T

G

G

C

A

T

C

A

T

T

A

R

R

A

T

A

R

T

Nucleus

DNA

Cell

The genetic material: Mendel’s

traits are in the DNA!

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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

GC

T A

TA

G C

(a) Parent molecule

A T

GC

T A

TA

G C

(c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand

(b) Separation of strands

A T

GC

T A

TA

G C

A T

GC

T A

TA

G C


Fig. 16-21a

DNA double helix (2 nm in diameter)

Nucleosome(10 nm in diameter)

HistonesHistone tail

H1

DNA, the double helix Histones Nucleosomes, or “beads

on a string” (10-nm fiber)

Nucleosome: octamer of histone proteins (2X each of H2A, H2B, H3, H4)

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30-nm fiber

Chromatid (700 nm)

Loops Scaffold

300-nm fiber

Replicated chromosome (1,400 nm)

30-nm fiber

Looped domains

(300-nm fiber)

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.

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• 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

TRANSCRIPTION

TRANSLATION

DNA

mRNA

Ribosome

Polypeptide

(a) Bacterial cell

Nuclearenvelope

TRANSCRIPTION

RNA PROCESSINGPre-mRNA

DNA

mRNA

TRANSLATION Ribosome

Polypeptide

(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

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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



II. Mendel who?








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Point mutations

Deletions

Insertions

Mutations are changes in the

genetic material of a cell or virus


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

Wild-type hemoglobin DNA

mRNA

Mutant hemoglobin DNA

mRNA

3′′′′3′′′′

3′′′′

3′′′′

3′′′′

3′′′′

5′′′′5′′′′

5′′′′

5′′′′5′′′′

5′′′′

C CT T TTG GA A AA

A A AGG U

Normal hemoglobin Sickle-cell hemoglobin

Glu Val

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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


Mutagens

• Spontaneous mutations can occur during DNA

replication, recombination, or repair

• Mutagens are physical or chemical agents that can

cause mutations

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II. Mendel who?









Research on the microalgae Dunaliella

primolecta at Fresno State

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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 photo-

and heterotrophic organisms

Algae and their uses


Fungi

EUKARYA

Trypanosomes

Green algaeLand plants

Red algae

ForamsCiliates

Dinoflagellates

Diatoms

Animals

AmoebasCellular slime molds

Leishmania

Euglena

Green nonsulfur bacteria

Thermophiles

Halophiles

Methanobacterium

Sulfolobus

ARCHAEA

COMMONANCESTOR

OF ALLLIFE

BACTERIA

(Plastids, includingchloroplasts)

Greensulfur bacteria

(Mitochondrion)

Cyanobacteria

Chlamydia

Spirochetes

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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


What do microalgae need?

What do they produce?

CO2

Or other C source

β-carotene BiofuelsTherapeutic proteins

Anticancer drugsPolyunsaturated fatty acids (PUFA) Astaxanthin

Sunlight

Nitrogen

Phosphorous

Water

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From:

Y. Chisti. Biotechnology Advances. 2007. 25: 294–306

Biodiesel production through Transesterification

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

Couple production to cleanup

Improve natural capacity of algae


From:

Y. Chisti. Biotechnology Advances. 2007. 25: 294–306

Biodiesel production through Transesterification

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

Couple production to cleanup

Improve natural capacity of algae

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1

C

2

A B D

3

4

5

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 auto-

fluorescence (B), lipid

accumulation as visualized by

BODIPY staining (C), and overlay of

Nomarski, chlorophyll, and lipid

images (D).


Genetic transformation of Dunaliella primolecta:

Use genetic markers to test transformation

Use Agrobacterium tumefaciens

Isolate transgenic lines

LB RB

35S poly HygR 35S pro 35S proLac Z GUS np

pCAMBIA1301

Plate on MSS medium 10 mL106

cells/mL (Dp cells)

3 days

ON culture A. tumefaciens

48 hours

MSS medium MSS mediumMSS medium+ cefotaxime+ hygromycin

Wash Dp cells and plate on

selection medium

MSS medium+ cefotaxime+ hygromycin

Cell colonies appear in 10-20 days

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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.


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?

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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-

0.45KB

1.0KB

Are the inserted genes stable?


GUS expression of selected transformed lines. Cell lines farther away

from the “Y” axis (circled) are cell lines expressing GUS at high levels.

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

0 50 100 150 200 250 300 350

Nu

mb

er

of

cell

s/m

l

RFU

Control

Row A

Row B

Row C

Row D

Row E

Row F

Row G

Row H

Do the genes insert in multiple sites in the genome?

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Can this transformation protocol be used as an





( )

gene A

( )

gene B

( )

gene C

( )

gene D

( )

gene E

etc.

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( )

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…




( )

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…

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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.

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000

Nu

mb

er

of

cell

s/m

l

RFU

A5

A8

B5

B7

B12

F7


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.

0

100

200

300

400

500

600

Am

ou

nt

of

lip

id i

n n

g

LIPIDS

DPI

DPII

F7

H5

B5

A5

B12

B7

A8

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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.


Acknowledgements:

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

Preethi Sarvabhowman Rakesh Kumar

Raj PatidarSabrina Romero

Alex Guzzetta

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II. Mendel who?





a. Genetic transformation of Dunalliela primolecta: Improve natural capacity of algae




Q I learned something new from this presentation:

A) YesB) No

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Thank you

Alejandro Calderón-Urrea, M.Sc., Ph.D.

Department of Biology

California State University, Fresno

10 / 21 / 2013

genetics: from mendel and mutations to solutions to the … academic... · 2013-10-22 · from...

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