From Genotype to Phenotype

1
IGP Genetics Development Section
From Genotype to Phenotype
2
IGP Genetics Development Section
3
Daniela Drummond- Barbosa
David Greenstein Scott Williams
Bruce Appel
Kathy Friedman
Chris Wright
David Miller
4
Flextimes
Oct 12 Review of Genetics Problems Oct 14
Yeast Paper Review Oct 17 Cell Death Video Oct
19 C. elegans Paper Review Oct 21 C. elegans
Paper Group Presentations-Red Group Oct 24
Drosophila Paper Review Oct 26 Drosophila Paper
Group Presentations-Green Group Oct 31
Zebrafish Group Presentations-Blue Group November
8 Mouse Group Presentations-Purple Group
5
Special Events
Oct 21, 2PM Review Session for Exam 1 Oct 21,
430 Take-home Exam (due Oct 24 830 AM) Nov 7
2PM Review Session for Exam 2 Nov 7, 430
Take-home Exam (due Nov 9 830 AM)
October 28, 9 AM Genetics Symposium Laura
Lee Josh Gamse Matt Tyska
6
Genetics Development
Evolutionary Conservation
Central dogma Genetic code Cell structure Core
machinery Developmental mechanisms
7
IGP Genetics Development Section
From Genotype to Phenotype
8
Mendelian Genetics
Lectures 1-3, October 10, 2005
Text Genetics From Genes to Genomes, Hartwell
et al.,) Chapters 1, 3, 4 (2000 ed.) or
Chapters 1, 2, 4, 5 (2004 ed.) Problems Set See
handout (Answers Due October 14) References Wels
h, M. J. and Smith, A. E. (1995) Cystic Fibrosis.
Scientific American, December, 52 -
59. Champion, M. D. and Hawley, R. S.
(2002). Playing for half the deck the
molecular biology of meiosis. Nature Cell
Biology, 4 Suppl, s50-56. Internet Sites of
Interest Cystic Fibrosis Foundation
http//www.cff.org/home/ Online Inheritance in
Man (OMIM) http//www.ncbi.nlm.nih.gov/entrez/quer
y.fcgi?dbOMIM Translation of Mendels original
paper http//www.mendelweb.org/Mendel.html
David.greenstein_at_vanderbilt.edu
9
Mendelian Genetics
E-mail David.greenstein_at_vanderbilt.edu
Online office hours 9-11 PM
Office 3160 MRBIII
Help sessions as needed
10
Mendels Experiments
Controlled crosses in peas
Pure-breeding lines
Quantitation of results
11
Mendel established true-breeding lines
Then crossed lines differing in a single
character, forming hybrids (F1)
12
One character was transmitted entirely in the
F1 dominant vs. recessive
13
X
Round (RR) R dominant
Rinkled (rr) r recessive
100 Round F1 (hybrids) Rr
Mendels Explanations For every character (e.g.
seed shape) an individual has two instruction
sets (alleles) One allele derived from the sperm
(pollen) and oocyte (ovule) True-breeding
individuals both alleles are the same Hybrids
have one of each allele
14
X
Round (RR) R dominant
Rinkled (rr) r recessive
100 Round F1 (hybrids) Rr
Why is the R allele dominant to r allele? R is
the wild-type allele r is a mutant
allele Most mutations are loss-of-function Excep
tions--dominant gain-of-function mutations (will
talk about later)
15
X
Round (RR) R dominant
Rinkled (rr) r recessive
100 Round F1 (hybrids) Rr
Molecular cloning of the r (rugosus) locus
R allele wild-type SBEI gene Enzyme produced
Insertion in r allele results in frame-shift
r allele mutant SBEI gene No enzyme produced
Bhattacharyya et al. 1990. Cell 60, 115-122
16
X
Round R dominant
Rinkled r recessive
100 Round F1 (hybrids) Rr
17
F1 Intercross
X
X
Rr
Rr
Mendels data
336
107
31
Mendels Explanation When a hybrid forms
gametes, only one of the two parental alleles
ends up in a gamete But, both alleles posses an
equal chance of ending up in a gamete
18
Punnett Square
X
X
Rr
Rr
Oocytes
336
107
R
r
31
R
RR
Rr
Round rinkled 31
Round
Round
Sperm
r
Rr
rr
Round
rinkled
19
3
1
Round
Round
rinkled
2
1
1
20
Can we tell these apart?
21
Data
565
193
372
12
Round
Round
rinkled
22
Mendels Contributions
Determinants of heredity are discrete and subject
to quantitative analysis
Law of Segregation In the formation of
gametes, two members of a gene pair (alleles)
segregate into different haploid gametes with
equal probability
Law of Independent Assortment During
gamete formation, the segregation of alleles of
one gene is independent of the segregation of
alleles of another gene
(provided these genes are not linked, as
described in Lecture 3)
23
Human Metaphase Chromosomes Stained to produce
unique banding patterns
24
Chromosome Painting A Normal Karyotype
Spectral Karyotyping a new method
25
Karyotyping in Human Genetics
Translocations, Deletions, Insertions,
Duplications, Trisomies, Monosomies
Translocation in a Patient with Ataxia
26
Mitosis DNA replicated to produce two identical
copies of each chromosome prior to cell division
metaphase
cytokinesis
DNA replication (S-Phase)
27
DNA Sequence requirements for replication and
segregation of chromosomes
S-phase
Metaphase
28
Metaphase Chromosome
Tightly wound complex of DNA and protein
(chromatin) DNA content of each sister chromatid
is identical Cohesion between sisters established
in S-phase
29
Sister chromatids segregate in mitosis
30
Direct Observation of Mitosis
MOVIE 1
MOVIE 2
31
MOVIE 3
Mitosis of a Lily Cell (note plant cells organize
spindles with microtubules but dont have
centrosomes)
Cohesion between sister chromatids broken at
metaphase to anaphase transition
32
Human Karyotype
Homologous chromosomes arranged in pairs 22 pairs
of autosomes, 1 pair of sex chromosomes TOTAL
46 human chromosomes
Homologs Diploid (2C)
33
Homologous Chromosomes
Note similar size, banding patterns of each
homologous pair
34
Homologous Chromosomes
35
Homologous Chromosomes
36
Weissmans Theory of the Germ Line
Immortality of the Germ Line
How to avoid doubling of ploidy in each
generation?
From E.B Wilson, 1897
37
Each parent contributes an equal number of
chromosomes Sperm and egg contribute
one-half the diploid amount There must be a
process to halve the ploidy of gametes
Female and male pronuclei contain two chromosomes
Ascaris has two chromosomes. A diploid cell has
two copies of each.
T. Boveri, 1888
38
MEIOSIS Two successive nuclear divisions No DNA
replication intervening Homologs
segregate Gametes are haploid (1C)
39
Oocyte Meiosis
Sperm Meiosis
All meiotic products used for sperm
One meiotic product used in oocyte Others form
polar bodies
40
2nd Meiotic Division Resembles a Mitotic
Division Sister Chromatids Segregate from
One-Another
41
MOVIE 4
MOVIE 5
42
Meiosis II and Mitosis Sister chromatids segregate
Meiosis I and Mitosis are Different Pairing and
segregation of homologs
Haploid gametes result
Chiasmata hold homologs together Resolved at
metaphase of meiosis I
43
Chiasmata Cohesion A physical link between
homologs
44
Understanding meiosis
Pair Them
Join Them
Move Them
45
Reassortment of Genetic Material in Meiosis
Independent Assortment
Crossing-over
46
Origin of Downs Syndrome
Karyotype (1800)
True nondisjunction
Achiasmate nondisjunction
Premature separation of sisters
Trisomies Downs (21) Edwards (18) Patau (13)
XXX XXY XYY Monosomies X0
47
(No Transcript)
48
Sequence differences between homologous
chromosomes are small ( 1/1000 nucleotides)
Small differences in nucleotide sequence Some
differences are silent polymorphisms Some are
not human genetic diseases
49
Cystic Fibrosis
Most common genetic disease in people of northern
European descent
Thick mucous accumulates in lungs, colon, large
intestine. Pancreatic dysfunction, Sterility.
Defective epithelial chloride transport
Life expectancy about 30 Respiratory disease from
chronic P. aeroginosa infection
50
CFTR protein Cystic Fibrosis Transmembrane
conductance Regulator
Large membrane protein (1480 amino acids)
CFTR amino acid sequence deduced from DNA
sequence of CFTR gene
Regulates chloride efflux from sweat glands
mucus-secreting cells
51
CFTR protein
52
70 of CF patients carry the DF508 deletion
Normal
53
HOMEWORK QUESTIONS (Turn in October 14, 2005)
How does the deltaF508 mutation affect CFTR
function?
The deltaF508 mutation can be traced to a single
Founder mutation. What accounts for the current
prevalence of this deleterious allele?
54
Allele One of the different forms of a gene that
can exist at a single locus
Hundreds of different CFTR alleles
POINT MUTATIONS
G
U
Missense mutation
Asp
Tyr
55
FRAME SHIFT MUTATION
wild type
56
Some CFTR mutations disrupt mRNA splicing
57
CFTR mRNA splicing mutation
G
A
DNA
58
CFTR gene contains 24 exons
exons
250 kb
CFTR gene
CFTR mRNA
6.5 kb
1 kb 1000 nts
59
CFTR was first major human disease gene cloned
(1989) Isolated largely by Linkage Analysis
before biochemical identity of CFTR protein known
60
Genetic Terms
Genotype
CFTR()
CFTR()
Heterozygous Normal
CFTR()
CFTR(-)
Homozygous CF disease
CFTR(-)
CFTR(-)
CFTR(-) is recessive to CFTR () or,
CFTR() is dominant to CFTR (-)
61
Why is CFTR() is dominant to CFTR (-)?
Active CFTR Produced
Genotype
CFTR()
CFTR()
Most genes are haplosufficient A single gene
copy is sufficient for normal function
CFTR()
CFTR(-)
CFTR(-)
CFTR(-)
G
U
Nonsense mutation
Glu
Stop
62
CF is one of most common human genetic diseases
What accounts for 100x difference in frequency of
CF mutation (1/25) vs frequency of CF disease
(1/2500)?
63
carrier-2
carrier-1
64
Assume random matings!
carrier-1
carrier-2
1/25
1/25
Homozygous CFTR(-) offspring
1/4
Probability of CF 1/25 x 1/25 x 1/4
1/2500
65
Chronic respiratory infection, pancreatic and
colonic dysfunction, sterility. All of these
traits can be attributed to a defect in a single
genetic locus and are observed to segregate
together in successive generations of CF families.
Law of Independent Assortment During
gamete formation, the segregation of alleles of
one gene is independent of the segregation of
alleles of another gene.
(provided these genes are not linked!)
66
Two sets of homologous chromosomes
Heterozygous Parent (2N)
Gametes (1N)
67
Test Cross Deduce genotypes of gametes from
phenotypes of offspring.
68
Original Test Cross by Mendel with Garden peas
round
rinkled
R dominant to r
1
RrYy
1
Rryy
yellow
green
rrYy
1
Y dominant to y
1
rryy
69
Dihybrid Cross
X
RrYy
RrYy
round
rinkled
R dominant to r
?
yellow
green
On board!
Y dominant to y
70
(No Transcript)