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MOLECULAR GENETICS 681502 Spring 2007 Tuesdays
and Thursday, 10-1130 AM Waksman Institute
Auditorium Dr. Andrew Vershon, Waksman
Institute, Rm 234 Phone [phone] E-mail
vershon_at_waksman.rutgers.edu Course Web
Site http//mmg.rutgers.edu/502.html Username
502 Password 502
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• Course structure
• Lectures -
• Reading - General genetics/ Mol. Biol. texts for
  review
• Modern Genetic Analysis 7th Ed , Griffiths et
  al., Freeman Co, 1999 (is available online)
• Assigned Papers - to help with understanding and
  provide perspective
• Problem sets - For practice, some will be
  collected/graded
• Exams - Three closed book - UMDNJ East Lecture
  Hall

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• Genetic approaches used in different model
  systems
• Will discuss methods of classic and molecular
  genetics
• Will learn genetic approaches in different
  systems
• bacteria and phages, used to establish the
  paradigms
• yeast - simple eukaryote
• worms - model systems for studying development,
  
• flies - complex development, short generation
  time
• plants - compare differences with animals,
  crops
• mice - mammalian systems
• humans - ultimate goal
• c. Goal is to be able to read papers in these
  systems
• Have a background in the genetic approaches
  used

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1. What is genetics? - Study and manipulation of
heredity a. Genetic manipulation - b. Genetic
analysis started with Mendel c. Genetic
analysis is not just used to study heredity
2. The Genetic Approach - The Salvation of
Doug How to use genetics to dissect a biological
process
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3. Genotype vs. Phenotype a. Genotype -
describes the complete set of genes inherited
by an individual b. Phenotype - (derived
from Greek - the form that is shown) describes
the aspects of individuals morphology,
physiology, behavior, etc. c. No two
individuals have the same genotype- Always
some slight differences, even in bacteria DNA
Pol 1/107 mutation rate d. If referring to same
genotype or phenotype then referring to a
subset of traits that are of interest
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4. Mendel a) Pick the right organism for the
research - pea plants Can cross strains- cross
pollinate Fast generation time Relatively cheap
to grow Produce many offspring Have markers
(phenotypes) that can score, Can follow
traits pea color (yellow/green), flower color
(white/purple) pea characteristic
(round/wrinkled)
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b. The Experiment
P
W
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c. Segregation of the markers
P
W
AA
aa
Two alleles of the gene
W
P
AA
aa
Parent
P
F1
Aa
P
W
P
P
F2
aa
AA
Aa
Aa
P
3PW
W
F3
AA
aa
AAAaaa 121
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d. What would happen if Mendel did not breed his
strains true?
P
W
W
P
Aa
aa
Parent
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e. What would happen if the phenotype is caused
by a dosage effect of the gene?
P
W
AA
aa
W
P
AA
aa
Parent
R
F1
Aa
P
W
R
R
F2
aa
Aa
AA
Aa
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f. Test Cross - Used to determine the genotype of
a strain Mate strains with a recessive tester
strain
W
P
AA
aa
Parent
P
F1
Aa
P
W
P
P
F2
aa
AA
Aa
Aa
P
F3
Aa
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6. Segregation of two traits
RRyy (ROUND, green)
rrYY (wrinkled, YELLOW)
Homozygous
RryY (ROUND, YELLOW)
Punnett Square
Phenotype Ratio RYRyrYry 9331
Rr - 124 - 31 Yy - 124 - 31
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• 7. Methods for calculating frequencies
• Product rule probability of independent traits
  occurring (r,y) is the product of the individual
  events r or y
• Ex If probability of rr is 1/4 and yy is 1/4
• then ry phenotype (rryy) is 1/4 X 1/4 1/16
• Ry 3/4 x 1/4 3/16
• RY 3/4 x 3/4 9/16
• 9331 ratio of the phenotypes

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c. Ex Cross two strains AabbCcDd X AaBbccDd
Want abcd phenotype (aabbccdd) How many
colonies would you have to screen? then Aa X
Aa 1/4 aa bb X Bb 1/2 bb Cc X cc 1/2
cc Dd x Dd 1/4 dd 1/4 x 1/2 x 1/2 x 1/4
1/64 Want AbCd (A-bbC-dd) 3/4 x 1/2 x 1/2 x
1/4 3/64
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d) Can also be used to calculate probabilities of
a disease (xx). XX and Xx are healthy If X
and x alleles are present at the same frequency,
then 1/4 of population will be xx However if
carriers (Xx) are 1/25 of the population Gettin
g the disease allele has a frequency of 1/50,
Probability to get the disease (xx) is
therefore 1/2500
1/25
1/25
Xx
Xx
1/2
1/2
xx
(1/25 x 1/2)(1/25 x 1/2) 1/2500
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8. Lethality- If homozygous marker is lethal
Aa X Aa
AA
Aa
aa
Genotype
1
2
1
Phenotype
WT
No Tail
Gene is pleiotropic - has more than one distinct
phenotype Mutant must be recessive
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9. Relationship between phenotype and genotype
a) Characteristics of an organism is determined
by the phenotype of its parts, Ultimately
decided by which proteins are expressed in which
cell types Frequently those that are studied
have to have clear and distinct phenotypes, A
given phenotype indicates a particular
genotype b) One-to-one relationship dominates
genetics examples but rare in real life However
not always the case curly mutant with curled
wings at 25C is WT at 19 purple has different
color when young but WT when adult
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c. How can mutations affect a biological process?
Enz1
A
B
If you screened for mutants that would not grow
in the absence of B, what types of mutations
would you get?
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• Mutations - process where genes change from one
  allelic form to another
• Forward changes away from WT

Reverse mutation rate is less than a forward
mutation Because you need a specific change
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12. Types of mutations a) Single base pair
substitutions transition A-gtG, G-gtA, C-gtT,
T-gtC transversion, A-gtC or T, G-gtC/T WT UAU -gt
Tyr Wild type Silent UAC -gt Tyr Wild
type Misense UCU -gt Ser Non-conserved Neutral
UUU -gt Phe Conserved Nonsense UAG -gt
Stop Truncated
Mutant or WT?
Mutant or WT?
Mutant or WT?
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b) Small deletions or insertions Leu Lys Arg Leu
CUC AAG CGC UUA A CUA AGC GCU UAA Leu Ser Ala S
TOP CUA AGC AGC UUA A Leu Ser Ala Leu
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c) Large deletions or insertions
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d) Reverse mutations WT UUA Leu UAU
Tyr Mutant GUA Val UAA Stop True UUA
Leu UAU Tyr Equivalent CUA Leu UAC Tyr
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e) Intragenic Suppressors Compensating mutation
within protein
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How can you tell between a true revertant and a
suppressor? Back cross vs WT. If revertant
only get WT If mutant/suppressor will get mutants
Phenotype

R
E
WT

Mut/Supp
R
E
-
-
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f) Extrageneic suppressors i) Nonsense
suppressors - mutations in tRNA
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ii) Suppressor mutations in associated proteins
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iii) Mutations that over expresses protein, or
with higher activity
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g) Null vs leaky or conditional mutations
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i) Conditional mutations - Phenotype is only
observable under specific conditions i)
Temperature Sensitive
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ii) Protein Dependent
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13. Making Mutations a) Spontaneous mutations -
very rare naturally occurring due to errors in
replication or repair b) Induced mutations -
Use of base analogs, 5 bromouracil,
2-amino-purine Alkylating agents - EMS,
nitrosoguanidine Hydoxylamine- GC-gtAT Nitrous
Acid - deaminate C's Intercalating agents - slip
between bases and mimic bases, Cause
insertions/deletions Activating SOS repair - UV,
aflatoxin B Mutator strains - MutS, MutY, MutT,
Contain mutants in proteins involved in
repair mutY - GgtT mutT - AgtC Transposon
mediated - Tn etc
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A -gt G Mutation by deamination of adenine
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c) Mutation frequency- number of mutants found
in population d) Mutation rate- number of
mutations that occur over time usually the
organismal generation span number of mutations
per cell division
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14. Genetic interactions between genes
a) None - 4 distinct phenotypes -
1 2 3 4 9 3 3 1
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14. Genetic interactions between genes
b) Complementation - Need a WT copy of both genes
- A B AAbb - - X----gt Y----gt Z aaBB
- 9 7
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14. Genetic interactions between genes
c) Duplication- multiple genes, aabb to obtain a
phenotype A or B - X---------gtY 15 1
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14. Genetic interactions between genes
d) Suppressors - cancel the effects of a mutant
phenotype, aa restore the WT phenotype on
bb - 13 3