Download recessive lozenge-shaped-fly-eye "alleles" in trans: recessive

What do we mean (what have we meant) by "a gene":
Reading for lectures 15-17 (We F27, Fr F29, We M5)
Chp 8: from 258 (Nonoverlapping...) to 261 (…Cracking) &
from 285 (8.6) to 293 (end of "essential concepts)
Chp 14: Using deletions to locate genes (496-498)
Chp 20: all except (for the time being) 724-25 (cloning) &
RNA Interference (726-728).
How do we recognize if
A given heritable difference in phenotype (appearance)
is caused by the
simplest possible difference in genotype (breeding behavior)
SIMPLEST DIFFERENCE:
….a difference with respect to one gene
….different alleles (forms) of one gene
….a "monogenic traite"
A definition of what we mean by "gene":
the thing that exists in two different forms (alleles)
Two different operational tests for allelism:
Mendel's "segregational test"
The gene as the unit of segregation
-- breeding behavior of the hybrid (1:2:1)
(=genotype of the hybrid))
Recall that a conflict arose
Green's/Lewis'/Benzers' "complementation test"
The gene as the cis-acting unit of function
--- phenotype of the hybrid
recessive lozenge-shaped-fly-eye "alleles"
in trans:
recessive lozenge-shaped-fly-eye "alleles"
in trans:
recombination
lz(g)
lz(g)
lz(BS)
lz(BS)
hybrid looks mutant
(same defective unit of function = functional alleles)
…and yet:
wt
lz(g)
lz(BS)
wt
hybrid looks mutant
(phenotype - function)
…and yet:
wt
wt
rare wildtype gametes
rare wildtype gametes
hence can't be segregational alleles
(breeding behavior of hybrid)
(also:
wt
wt
lz(BS)
lz(g)
)
hence can't be segregational alleles
(breeding behavior of hybrid)
1
Are mutant a and mutant b alleles (i.e. genetic alternatives)?
Do we go with Mendel or
high-resolution
segregation test
for recombination
a +
+ b
OR
a
b
Genes as defined as cis-acting units of function
have multiple parts that are separable by recombination
complementation test
for function
phenotype
of hybrids
breeding
behavior
of hybrids
mutant a
mutant b
The complementation test is generally favored:
(Green/Lewis) /Benzer?
mutant a
mutant b
a +
a
OR
+ b
b
?
Different alleles of the same gene may be defective
for different reasons
Segregation test is a useful preliminary test
(especially when we can't make the hybrids we would like)
?
Are a & b alternative
units of segregation
during meiosis?
Are a & b alternative
cis-acting units of function
during development?
i.e. are they segregational alleles?
i.e. are they functional alleles?
If: mutant alleles with similar phenotypic effects
map to approximately the same point
Then: those mutants are likely to be alleles of the same gene
Are mutant a and mutant b alleles (i.e. genetic alternatives)?
Are mutant a and mutant b alleles (i.e. genetic alternatives)?
complementation test
for function
phenotype
of hybrids
complementation test
for function
phenotype
of hybrids
--- could we ever be misled
by the apparent failure of these
recessive mutants to complement in trans?
mutant a
mutant b
a +
+ b
OR
a
b
?
mutant
phenotype
wildtype
phenotype
(complement) (don’t complement)
not alleles
alleles
wildtype
Are mutant a and mutant b alleles (i.e. genetic alternatives)?
When alleles FAIL TO COMPLEMENT
we may need the
cis control:
BECAUSE:
If the two mutants are individually
recessive alleles of different genes,
but dominant in combination
the cis control will also be mutant!
OR
wildtype
phenotype
a
b
?
mutant
phenotype
(complement) (don’t complement)
not alleles
alleles
mutant
AND
same recessive
mutants in CIS
IF:
recessive mutants
in TRANS
mutant a
mutant b
mutant a mutant b
+
+
mutant a
mutant b
a +
+ b
wildtype
Are mutant a and mutant b alleles (i.e. genetic alternatives)?
complementation test
for function
phenotype
of hybrids
mutant a mutant b
+
+
…could mutants be recessive individually
mutant a
and in different cis-acting
mutant b
units of genetic function
(i.e. not be functional alleles),
a +
a
?
OR
but interact in combination to
+ b
b
appear dominant together)
mutant
wildtype
phenotype phenotype
a
+
a +
(complement) (don’t complement)
+
b
+ b
?
not alleles
alleles
wildtype
phenotype
complementation
test for function
phenotype
of hybrids
mutant
phenotype
don’t appear to complement
… then we can trust the trans result
trans 䍫㻃cis,
hence a & b are functional alleles of the same gene
…orientation of the genetic information matters for genes
2
Are mutant a and mutant b alleles (i.e. genetic alternatives)?
recessive mutants
in TRANS
same recessive
mutants in CIS
mutant a mutant b
+
+
wildtype
Phenotype
… we can trust the trans result
mutant a
mutant b
Are mutant a and mutant b alleles (i.e. genetic alternatives)?
CIS/TRANS test
CIS/TRANS test
complementation
test for function
phenotype
of hybrids
complementation
test for function
phenotype
of hybrids
mutant
phenotype
don’t appear to complement
But nobody bothers with the cis control for recessive mutants
when doing complementation tests
despite what all the textbooks say (not even Benzer did it, as we will see)
Cis test is too hard in most cases,
and in most cases unnecessary.
…then why am I wasting your time with this?
Are mutant a and mutant b alleles (i.e. genetic alternatives)?
CIS/TRANS test
for
functional allelism
The complete cis/trans test will allow us to determine allelism
even if one or both of the mutants are not recessive!
Remember: the “complementation test” per se is limited to recessive mutants.
Most mutants are recessive, but some of the most useful & interesting are not.
CIS phenotype
CIS phenotype
䍫㻃TRANS phenotype, hence functional alleles
= TRANS phenotype, hence NOT functional alleles
…and all that matters is whether the cis vs. trans phenotypes are
the same or different, not what the phenotypes of each actually are!
complementation test
for function
alleles as alternative
cis-acting units of function
Conflict
favored
Intuitively, a better operational
definition of alternative forms of genes
high-resolution
segregation test
for recombination:
alleles as alternative
units of segregation
(recombination)
Raises questions:
If one can get recombination between functional alleles (alternative forms of a gene),
then how do the genetic maps one can therefore construct within single genes
compare to the genetic maps that can be (had been) constructed between genes?
--- WHAT IS THE NATURE OF GENETIC FINE STRUCTURE?
…and what is the basic (minimal) unit of recombination
(i.e. what are true segregational alleles)?
Fig. 7.20
p228
Phage kill bacteria for a living (and replicate in the process).
geneticists count “centers of klling” (plaques)
3
(centers of
infection)
“multiplicity of infection”
Must have a phenotypic difference between pure-breeding lines
to do genetics
Phage phenotypes (differences): based on growth
plaque morphology
large/small (growth rate)
clear/turbid
smooth edged/rough edged
growth conditions
bacterial host range
temperature range (T4: 25-42 C)
By early 1950s,
Alfred Hershy had isolated a number of phage T4 mutants
and constructed a (circular) genetic map
based on
(1) complementation (to group the mutants into genes)
and
(2) recombination to organize those genes on a map.
…and along came physicist Seymour Benzer
P.N.A.S.-US v41p344 (1955)
The young S. Benzer: (1950s)
phage T4
as his genetic workhorse
• reconciled the segregrational
and functional definitions of the gene
•set up the experimental system
used in the 2nd most elegant
genetics paper in history
An older Benzer: (1967-present)
fruit fly (D.melanogaster)
as his genetic workhorse
•founded many of the most
interesting areas of modern
behavioral genetics
(clocks & learning, etc.)
•set up the experimental system most
effective for studying fly development:
the compound eye
4
Need a selective genetic system
(one with high resolving power for small map distances)
Rfa-b = NP pfu from hybrid
/ total pfu from hybrid
pfu = “plaque-forming units”
hybrid = mixed infection
Phage are small, but plaques are often larger than fruit flies!
How do phage help with measuring small Rfs?
Use selective systems to easily measure NP pfu concentration
without complication from the much larger number of P pfu
Benzer’s system made measuring 0.0001 cM (1x10-6) easy
…and by the way,
as an added bonus for mapping,
phage happen to have a MUCH
greater rate of recombination per unit DNA
than fruit flies or garden peas
In rII, smallest non-0 recombination rate
measured was 0.02 cM (mutants 1 bp apart)
(2 NP/10,000 total)
In my first effort at fine-structure mapping in flies,
I measured 0.007 cM (one recombinant) for a
distance (I only later found out to be) 3,100 bp
(1 NP/14,286 total)
(1) rII vs. rII+ easily distinguished based on plaque morphology
Among Hershey’s T4 collection
were rapid lysis (r) mutants
--- produced distinctively
large plaques.
(2) extremely rare (<10-7) rII+ recombinants easy to recover
They arose spontaneously
(1:10-4) and fell into several
different complementation groups
that mapped at different places
One complementation group
was rII
and Benzer discovered something
special about it that made
it perfect for studying genetic fine structure.
Fig. 7.20
p.229
Fig. 7.20
(1) Characterize mutants:
fell into two complementation groups,
rIIA & rIIB
very close on a genetic map.
infect 1:1 mixture
at “multiplicity of infection”
of ~3 phage:1 bacterium
(still)
NON-permissive
host
mutants 1&2
in same “gene”
alternatives
mutants 1&2
in different “genes”
Complementation test: phenotype of the hybrid
Question: did he test by
plaque morphology
or
ability to growth on K(λ)?
No growth
growth
(AND no recombination!)
BUT WHAT did Benzer do FIRST?!
determine that mutants are recessive
5
(for complementation test)
(1) Characterize mutants:
fell into two complementation groups,
rIIA & rIIB
very close on the genetic map.
Yes, but extremely tedious
(almost never do)
p.229
Fig. 7.20
(still)
p.229
Fig. 7.20
(still)
Make recombinants
by mixed infection
of the permissive
host
Assay progeny
pfu concentration
on permissive (=total)
and nonpermissive
(=50% of nonparental)
hosts
YES
…or NO: segregational
alleles, so only…
rIIA1 & rIIA2
(“unchanged in the hybrid”)
(can be either
a quantitative test
or a qualitative test)
no progeny
lyse K(λ)
6