Genetics/lab

BIOL 3600 EV1 BIOL 3600 EV2

Winter 2012

Review of the syllabus

InstructorDr. Paula Faria Waziry

Text Pierce, Benjamin A 2010. Genetics: A Conceptual Approach 4th Edition, W.H. Freeman, New York, NY (ISBN number = 1429232501)

WebsiteAll course materials will be posted on Blackboard (when possible) or on SlideShare prior to the lectures.Videos of the lectures will be posted on Blackboard (when possible) or on my Youtube page under playlist: Genetics_Dr. Paula Waziryhttp://www.youtube.com/playlist?list=PL3CBA6D82E9B35226&feature=view_allThere are several other playlists related to genetics on my Youtube channel. I frequently add related videos to those playlists in order to enrich your learning experience.

Review of the syllabus

GradesThe final average will be calculated as follows: Four unit tests @ 12.5% each 25% Midterm exam 20% Final exam 20% Quizzes 10% Lab final 10% Lab activities 15% 100% The grading scale: 93% = A,

90-92%=A-, 87-89%=B+, 83-86%=B, 80-82%=B-, 77-79%=C+, 73-76%=C, 70-72%=C-, 60-69%=D, <60%=F

- Class grades will be curved if necessary

If having problems, address them as early as possible!!

Tips for success

1. Attendance is required at all lectures, labs, and exams - You are responsible for getting any missed info

2. Ask questions in class and participate in discussion

3. Don’t just read and highlight the handouts. Understand and practice questions.

4. You will be responsible for the conceptual material covered in class/ powerpoints.

5. Be considerate: no cell phones, texting, sleeping, snoring…

Chapter 1 Introduction to Genetics

People have understood the hereditary nature of traits and have practiced genetics for thousands of years

The rise in agriculture began when people started to apply genetic principles to the domestication of plants and animals

Pharmaceutical industry: Numerous drugs are synthesized by

fungi and bacteria. Ex: growth hormone, insulin

In medicine: Many diseases and disorders

have a hereditary component. Ex: hemophilia, sikcle-cell

anemia, diabetes, muscular distrophy,

EMD1: Emerin; Xq28; RecessiveEMD2: Lamin A/C; 1q21.2; DominantEMD3: SYNE1; 6q25; DominantEMD4: SYNE2; 14q23; DominantOther: Sporadic & Dominant

                      

         from A Kornberg MD

2 years-old Kristian

12 years-old Ashley and2-weeks old brother Evan

Hutchinson-Gilford Progeria

Defect in Lamina A processing

Hutchinson-Gilford Progeria

Lamin A/C (a)Lamin A (c)

DAPI

Control Patient

Samuel DalesSamuel Dales

mRNAmRNA

Ribosomal Ribosomal subunitssubunits

tRNAtRNA

snRNPssnRNPs

Proteins Proteins with NESwith NES

hnRNPshnRNPs

RibosomalRibosomalproteinsproteins

Proteins Proteins with NLSwith NLS

snRNPssnRNPs

TPR (translocated promoter region)

Nup98

Nup98

Nup98

The Nuclear Pore Complex

Fusion Transcript• Nup98-HOXA9• Nup98-HOXD13• Nup98-PMX1• Nup98-DDX10• Nup98-RAP1GDS1• Nup98-TOP1

• DEK-Nup214• SET-Nup214

Translocation

t(7; 11)(p15; p15)

t(2; 11)(q31; p15)

t(1; 11)(q23; p15)

inv(11)(p15; q22)

t(4; 11)(q21; p15)

t(11; 20)(p15; q11)

t(6; 9)(p23; q34)

inv(9)

Nucleoporin Translocations in Acute Leukemia

Genetic Diversity and evolution

Living organisms have an important feature in common:

all use similar genetic systems

A complete set of genetic instructions for any organism is its genome

All genomes are encoded in mucleic acid:

either DNA or RNA• Suggestive of a common ancestor

Since all organisms have similar genetic systems, the study of one organism’s genes eveals principles that apply to other organimsms

GAS motifSTAT 1

STAT 1P

PAccessory TF

motifs TATA

Accessory TFfactors

Nucleoporins and Nuclear Traffic Proteins are involved with Mitotic Spindle Checkpoints and Aging

Baker DJ, Jeganathan KB, Malureanu L, Perez-Terzic C, Terzic A, van Deursen JM.

J Cell Biol. 2006 Feb 13;172(4):529-40.

Enabling us to use animal models to study diseases or natural processes

- Influenza virus killed as many as 50 million people in a single year (1918-1919)- Extremely virulent - Fast, nasty, killed all ages- Up until 2006, had no idea why it was so virulent- Had no way to tell if a similar strain was forming

- In October 2005, scientists recreated the 1918 strain using various genetic techniques - Obtained samples of the virus from preserved wax specimen and from a Eskimo woman who had died from it- Able to characterize it and predict / prevent future outbreaks?)

Influenza A pandemic

Microarray study reveals changes in regulation of genesUpon influenza viral infection

Replication-dependent genes

Geiss G, J.Virol, May 2001,p4321-4331

Cells expressing low levels of Rae1are susceptible to influenza infection

Influenza virus targets the mRNA export machinery and the nuclear pore complex.

Satterly, Tsai, van Deursen, Nussenzveig, Wang, Faria, Levay, Levy, Fontoura.

Proc Natl Acad Sci U S A. 2007 Feb 6;104(6):1853-8.

GeneticsWhat is it?

http://www.youtube.com/watch?v=0OnwOKiMVb8&feature=channel_video_title

• Genetics = Study of HEREDITY and VARIATION

- Heredity = Passing down of traits from one generation to another

- Variation = Differences in inherited characteristics among members of a population

GeneticsWhat is it?

• Genetics has many subfields

1. Transmission genetics – Study of heredity - How traits are passed down between generations - Example: My mom has disease, spouse's uncle has same disease Will our kids will have it? - Is allele dom/rec, is it on X chr or autosome?

2. Molecular genetics – Structure and function of individual genes - Includes study of cancer (cancer genetics), genetic engineering (manipulation of genes), study of chromosome structure (cytogenetics)

3. Population/Evolutionary genetics – Study genetic variation in populations - Includes conservation genetics

History of genetics

• Early genetics was more philosophy than science - No experimentation

- Many concepts were incorrect - Pangenesis – traits collected from all over body and put into sperm/eggs

- Pre-formationism – little person inside of gametes (homunculus) - Blending inheritance – actual mixing of genetic information

“Blending”

History of genetics

• Technological and scientific developments changed genetics in 1800s

- Microscopes invented – direct observation of gametes

- Darwin and Mendel revolutionize genetics - Evolutionary and transmission genetics begin

- Chromosomes observed and discovered to carry genetic information (early 1900s)

Chapter 2Chromosomes and Cellular Reproduction

1. Nucleus vs nucleoid - Nucleus – Membrane-enclosed organelle inside of eukaryotic cells that holds the chromsomes - Nucleoid – Region of a prokaryotic cell cytoplasm in which the chromosome resides

2. Chromosome - Single piece of DNA + proteins - Can be linear (eukaryotes) or circular (prokaryotes) - Eukaryotes have many, prokaryotes have one - Divided into many genes

The Nucleus in Context

• Origin of the Nucleus: Most likely, from invagination of cell membrane of an ancient bacterium. The interior of the

nucleus is topologically equivalent of the cytoplasm.

• The ER is continuous with the nuclear membrane. Topologically equivalent to the extracellular space.

Evolution of the Eukaryotic Nucleus

Chromosome distribution is not random

Chromosome Territories

Good Review:

Meaburn and Misteli, Nature, Jan 25, 2007, vol445, pp379 - 381

Chromosome Territories

Chromosomes maintain their individuality they have fixed homes spatially limited volume Each chromosome of a specific cell type can be assigned a preferential position relative to the center of the nucleus

territories have highly branched and interconnected channels

Chromosome Structure

• Centromere: attachment point for spindle microtubules

• Telomeres: tips of a linear chromosome

• Origins of replication: where the DNA synthesis begins

At times a chromosomeConsists of one single chromatid

At other times it consists of 2 (sister) chromatids

The telomeres are the stable ends of chromosomes

The centromere is a constricted region of the chromosome whereThe kinetochores form and the Spindle microtubules attach

Centromeres can be located at

different sites on a chromosome

3. Gene - Defined segment of a chromosome that provides the instructions to make a single product (ct (a proteina protein)) - One chromosome may be subdivided into 1,000 different genes

Chromosomes are divided into genes, which are composed of DNA

4. Diploid vs. haploid - Most eukaryotic organisms have 2 copies of each chromosome in each of their somatic cells - 2 copies = 2n = DIPLOID - Gamete cells (e.g. sperm and eggs) contain only 1 copy - 1 copy = 1n = HAPLOID

1 chromosome

insulin ATPsynthase

actin hemoglobin

• Some more genetic terms (and how they relate to one another): 5. Alleles - Alternate forms of the same gene caused by minor differences in the DNA sequnce

6. Genotype vs phenotype - Genotype – The genetic makeup of an organism (what the genes look like) - Phenotype – The actual observable characteristics that we see - Influenced by the genotype and the environment

eye color

blue eyes

brown eyes

Quick review of Mitosis

http://www.youtube.com/watch?v=AhgRhXl7w_g&feature=fvst

Genetic consequences of the cell cycle

• Producing two cells that are genetically identical to each other and with the cell that gave rise to them.

• Newly formed cells contain a full complement of chromosomes.

• Each newly formed cell contains approximately half (but not necessarily identical) the cytoplasm and organelle content of the original parental cell.

Quick review of Meiosis

http://www.youtube.com/watch?v=iCL6d0OwKt8&feature=channel_video_title

Room for genetic variation

Room for genetic vatiation

Genetic Variation is Produced through the random distribution of chromosomes in meiosis

• Try Animation 2.3file:///E:/media/ch02/Animations/0203_genetic_var_meiosis.html

Genetic Variation is Produced through crossing over

Consequences of Meiosis and Genetic Variation

• Four cells are produced from each original cell.

• Chromosome number in each new cell is reduced by half. The new cells are haploid.

• Newly formed cells from meiosis are genetically different from one another and from the parental cell.

Concept Check

Which of the following events takes place in meiosis II, but not in meiosis I?

a. crossing over

b. contraction of chromosomes

c. separation of homologous chromosomes

d. separation of chromatids

Transmission geneticsMendel and beyond

Chapter 3Basic Principles of Heredity

Transmission geneticsOverview

• Transmission genetics Study of how genes/traits are passed down through generations - Wrong hypotheses in history: - Pangenesis: Traits absorbed from all over body sperm/eggs - Lemarckism: Kids inherit their parents acquired characteristics - Preformationism: Little people inside of gametes (homunculus) - Blending inheritance: Traits of parents blend passed on

• Gregor Mendel – 1850-60s - Experiments with peas disproved all above - Found discrete units of inheritance passed down - Provide instructions for brand new organism - No blending - Unique combination of genes provides unique traits in offspring

Transmission geneticsReview of terminology

• Genes = Short segments of chromosomes/DNA that provide instructions to make a polypeptide

• Alleles – Different forms (variations) of the same gene that result from minor differences in the DNA sequence - Example: Gene controlling melanin

• Homozygous vs. heterozygous Homozygous – Organism has 2 copies of the same allele for a given gene Heterozygous – Organism has 2 different alleles for a given gene

• Dominant vs. recessive - In a heterozygous individual, both alleles are expressed

(assuming no imprinting) - However, one allele often has a greater impact on the final observable trait - Those alleles that mask the effects of other alleles DOMINANT Those alleles that get masked RECESSIVE

Simple Mendelian inheritanceMonohybrid cross

• Simple Mendelian inheritance - Each trait is controlled by one gene, each gene controls one trait - Alleles have a clear dominant-recessive relationship - Individuals have expected phenotype - If examining more than 1 gene they are on separate chromosomes

• Monohybrid cross (1 gene) - Example: Human earlobe shape - Dominant allele Unattached (E) Recessive allele Attached (e)

- Example mating: Two heterozygotes (Ee x Ee) - Need to predict offspring genotypes/phenotypes - Theory of segregation – Two copies of the gene are segregated and packaged into separate gametes - 1 gets the "E" and the other the "e"

Predict a 3:1 ratio of dom:rec phenotypes - Only a probability – may not see exact ratio

eE

eeEe

EeEE

e

E

Transmission geneticsReview of terminology

• Genetic shorthand - Dominant allele Capital letter Recessive allele Lowercase letter A = normal melanin production AA (homozygous dominant) a = no melanin aa (homozygous recessive) Aa (heterozygous)

• Genotype vs. phenotype Genotype – Genetic background of a trait (AA, aa, Aa) Phenotype – Actual observable trait that we see (skin color) - Multiple genotypes can give rise to the same phenotype (e.g. AA and Aa) - Not all individuals with a given genotype will have the expected phenotype

• Generation shorthand - P = Parental generation - F1 = First filial generation (produced from mating two parents) - F2 = Second generation (produced from mating 2 F1 offspring)

Choose letter wisely!

• Why was Mendel so successful? 1) His choice of experimental subject, the pea plant (Pisum sativum) is easy to cultivate and grow relatively rapidly ( by 17th century standards);

2) Peas produce many offspring (seeds);

3) Large variety of traits and traits were genetically pure;

4) He focused on characteristics that have 2 forms;

5) He adopted an experimental approach and used math!

6) He kept careful records of his experiments and was very patient…

Simple Mendelian inheritanceMonohybrid and dihybrid crosses

• Simple Mendelian inheritance - Each trait is controlled by one gene, each gene controls one trait - Alleles have a clear dominant-recessive relationship - Individuals have expected phenotype - If examining more than 1 gene they are on separate chromosomes

• Monohybrid cross (1 gene) - Example: Human earlobe shape - Dominant allele Unattached (E) Recessive allele Attached (e)

- Example mating: Two heterozygotes (Ee x Ee) - Need to predict offspring genotypes/phenotypes - Theory of segregation – Two copies of the gene are segregated and packaged into separate gametes - 1 gets the "E" and the other the "e"

Predict a 3:1 ratio of dom:rec phenotypes - Only a probability – may not see exact ratio

eE

eeEe

EeEE

e

E

• Dihybrid cross (2 genes) - Mendel's original experiments: - Done to determine if inheritance of one gene

affects inheritance of another - Found that when genes are on different

(nonhomologous) chromosomes, they move independently during meiosis and don't affect one another

Principle of independent assortment - Example (assuming independent assortment): - Earlobe shape (gene E) and hairline shape (F – widow's peak, f – straight)

- If mate EeFf x EeFf, what phenotypic ratios in offspring?FIRST SEPARATE THE GENES…

eeEe

EeEE

eE

e

E

ffFf

FfFF

fF

f

F

Then do separate Punett squares…

eE

eeEe

EeEE

e

E

fF

ffFf

FfFF

f

F

E_F_ = double dom. = 3/4 x 3/4 = 9/16eeF_ = 1 rec, 1 dom = 1/4 x 3/4 = 3/16 E_ff = 1 dom, 1 rec = 3/4 x 1/4 = 3/16eeff = double rec. = 1/4 x 1/4 = 1/16

INDEPENDENT TERMS

9:3:3:1

9+3+3+1=16

Simple Mendelian inheritanceMonohybrid and dihybrid crosses

• Dihybrid cross (2 genes) - Example problem: - Imagine you mate a person who has attached earlobes and a smooth hairline with a person who is heterozygous for both genes. What is the probability of them having a child with unattached earlobes and a widow's peak hairline?

- Step 1: Convert to letters - Parent 1 = attached, smooth eeff - Parent 2 = EeFf - ? offspring = E_F_

(2nd letter for each doesn't matter)ee

eeee

EeEe

e

E

ff

ffff

FfFf

f

F - Step 2: Do two Punett squares - Split up the E's from the F's

- Step 3: Do the math - Multiply the individual probabilities - E_F_ = 1/2 x 1/2 = 1/4

Simple Mendelian inheritanceTheoretical Probability for Simple Events

ee

eeee

EeEe

e

E

ff

ffff

FfFf

f

F

P(A) =# of outcomes favorable to A

Total # of outcomes

• What is the probability of getting all 3 Heads is you flip 3 coins?

• All possible combinations• 8 outcomes• Only 1 is favorable

1/8 P(A) =

Simple Mendelian inheritanceTheoretical Probability for Simple Events

ee

eeee

EeEe

e

E

ff

ffff

FfFf

f

F

P(A) =# of outcomes favorable to A

Total # of outcomes

• What is the probability of getting all 3 Heads is you flip 3 coins?

OR

• Probability for each coin: 1/2

• Each coin is “independent”

½ X ½ X ½ = 1/8

Simple Mendelian inheritanceTheoretical Probability for Simple Events

• What is the probability of getting all 6 Heads is you flip 6 coins?

• Probability for each coin: 1/2

• Each coin is “independent”

½ X ½ X ½ X ½ X ½ X ½ = 1/64

• What is the probability of getting either heads or tails in all 6 coins?

Simple Mendelian inheritanceTheoretical Probability for Simple Events

Probability – tree diagrams

• All in a bag. Pick one, replace in the bag, pick another

B

R

B

B

R

R

BB

BR

RB

RR

7/10

7/10

7/10

3/10

3/10

3/10

(7/10) X (7/10) = 49/100

(7/10) X (3/10) = 21/100

(3/10) X (7/10) = 21/100

(3/10) X (3/10) = 9/100

100/100

Simple Mendelian inheritanceTheoretical Probability for Simple Events

Probability – tree diagrams

• All in a bag. Pick one, replace in the bag, pick another

BB

BR

RB

RR

(7/10) X (7/10) = 49/100

(7/10) X (3/10) = 21/100

(3/10) X (7/10) = 21/100

(3/10) X (3/10) = 9/100

100/100

• What is the probability of picking 2 reds

• What is the probability of picking no reds

• What is the probability of picking at least 1 B?

(49/100) + (21/100) + (21/100) = 91/100

• What is the probability of picking one of each?

(21/100) + (21/100) = 42/100

The binomial expansion and probability

aA

aaAa

AaAA

a

A

Revisiting out Punett square and taking a look at kids with albinism…

Heterozygous parents:

Chances of having albino child: 1/4

If couple has 3 children, what are the chances for all 3 with albinism:

aA

aaAa

AaAA

a

A

aA

aaAa

AaAA

a

A

and another albino child: 1/4

And another albino child: 1/4

(1/4) X (1/4) X (1/4) = 1/64

The binomial expansion and probability

Now… 3 kids: What are the chances of having one child with albinism?

aA

aaAa

AaAA

a

A

non

albino

3/4

1/4

aA

aaAa

AaAA

a

A

aA

aaAa

AaAA

a

A

non

non

albino

albino

3/4

3/4

1/4

1/4

(3/4) X (1/4) X (1/4) = 3/64albino

albino

albino

non

non

non

non

albino

3/4

3/4

3/4

3/4

1/4

1/4

1/4

1/4

Non, non, non

Non, non, albino

Non, albino, non

Non, albino, albino

albino, non, non

albino, non, albino

albino, albino, non

albino, albino, albino

64/64

(3/4) X (3/4) X (3/4) = 27/64

(3/4) X (3/4) X (1/4) = 9/64

(3/4) X (1/4) X (3/4) = 9/64

(1/4) X (3/4) X (3/4) = 9/64

(1/4) X (3/4) X (1/4) = 3/64

(1/4) X (1/4) X (3/4) = 3/64

(1/4) X (1/4) X (1/4) = 1/64

(9/64) + (9/64) + (9/4) = 27/64

The binomial expansion and probability

Now… 5 kids: What are the chances of having one child with albinism?

aA

aaAa

AaAA

a

A

aA

aaAa

AaAA

a

A

aA

aaAa

AaAA

a

A

aA

aaAa

AaAA

a

A

aA

aaAa

AaAA

a

A

Here is where the binomial equation is useful….

(p+q)n Where p = probability of one event = albino

q = probability of the other event

n = # times the event occurs

(p + q)5 = p5 + 5p4q + 10p3q2 + 10p2q3 + 5pq4 + q5

Now… 5 kids: What are the chances of having one child with albinism?

= 5pq4 = 5(1/4)(3/4)4 = 5(1/4)(81/256) = 405/ 1024 = 0.39

The binomial expansion and probability

aA

aaAa

AaAA

a

A

aA

aaAa

AaAA

a

A

aA

aaAa

AaAA

a

A

aA

aaAa

AaAA

a

A

aA

aaAa

AaAA

a

A

Here is where the Pascal’s triangle is useful….(p+q)n

(p+q)0 1

(p+q)1 1 p + 1 q

(p+q)2 1 p2 + 2 p1q1 + 1 q2

(p+q)3 1 p3 + 3p2q1 + 3p1q2 + 1 q3

(p+q)4 1 p4 + 4p3q1 + 6p2q2 + 4p1q3 + 1 q4

(p+q)5 1 p5 + 5p4q1 + 10p3q2 + 10p2q3 + 5 p1q4 + 1 q5

(p+q)6 1 p6 +6p5q1 + 15p4q2 + 20p3q3 + 15p2q4 + 6p1q5 + 1q6

• An individual has a dominant phenotype what is the genotype (AA or Aa)? - Do a testcross

• Testcross - Take your individual in question and mate them with a homozygous recessive individual (aa) - Predictions: 1) If the individual is AA

AA x aa all offspring should have DOMINANT pheno

2) If the individual is Aa Aa x aa 1/2 should have dom. pheno 1/2 should have rec. pheno.

Routinely done to determine the genotype of an individual

aA

aaAa

aaAa

a

a

AA

AaAa

AaAa

a

a

• Observed ratio of progeny may deviate from expected ratios by chance.

We expected a 1:1 ratio, but after counting Yellow and Brown roaches…

There were 22 Brown and 18 Yellow

Observed Ratios of ProgenyThe Goodness-of-Fit Chi-Square Test

So… when do we use the Chi-Square Test?

When what comes out is not what we expected!

To see how well observed values FIT the expected values

It indicates the probability that the difference between observed and expected values is due to chance.

• The hypothesis that chance alone is responsible for any deviation between observed and expected values is called the null hypothesis .

•Looking at the cats: Black (B) is dominant over Gray (b)

Observed Ratios of ProgenyThe Goodness-of-Fit Chi-Square Test

bB

bbBb

BbBB

b

B

• If we cross 2 heterozygous black (Bb X Bb), we would expect a 3:1 ratio:

• Now we have 50 kittens: 30 black and 20 gray

Observed Ratios of ProgenyThe Goodness-of-Fit Chi-Square Test

• If we cross 2 heterozygous black (Bb X Bb), we would expect a 3:1 ratio:

• Observed values: 50 kittens: 30 black and 20 gray

• First get the expected values:

bB

bbBb

BbBB

b

BBlack kittens expected: (3/4) of 50 = 37.5

Grey kittens expected: (1/4) of 50 = 12.5

(observed – expected) 2

expectedChi-Square value =X2 =

(30 – 37.5) 2

37.5+X2 =

(20 – 12.5) 2

12.5X2 = 6

Observed Ratios of ProgenyThe Goodness-of-Fit Chi-Square Test

• Then we figure-out the degrees of freedom = n-1n = the number of ways that things can vary

in the cats’ case: it’s “2 phenotypes”

X2 = 6

• degrees of freedom = 2-1 = 1

• Now we look at the CHI table and see where “6” isFor a degree of fredom = 1

The probability of the event due to chance decreases

When value is less than 0.05, chance is not responsible for this!

Solve Problem 35 at the end of chapter 3