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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).

Reading for lectures 15-17 (We F27, Fr F29, We M5)

What do we mean (what have we meant) by "a gene":

How do we recognize ifA 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

A definition of what we mean by "gene": the thing that exists in two different forms (alleles)

….different alleles (forms) of one gene ….a "monogenic traite"

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))

Green's/Lewis'/Benzers' "complementation test"

The gene as the cis-acting unit of function --- phenotype of the hybrid

Recall that a conflict arose

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

lz(BS)

lz(g)

hybrid looks mutant(same defective unit of function = functional alleles)

(breeding behavior of hybrid)

wt

…and yet:

rare wildtype gametes

hence can't be segregational alleles

lz(BS)

lz(g)wt

wt

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

wt

lz(BS)

lz(g)

hybrid looks mutant(phenotype - function)

…and yet:

(breeding behavior of hybrid)

rare wildtype gametes

hence can't be segregational alleles

wt wt

lz(g)lz(BS)(also: )

recombination

2

Are mutant a and mutant b alleles (i.e. genetic alternatives)?

Do we go with Mendel or (Green/Lewis)/Benzer?high-resolution

segregation testfor recombination

breedingbehavior

of hybrids

complementation testfor functionphenotypeof hybrids

mutant amutant b

mutant amutant b

ORa ++ bab ? OR

a ++ b

ab ?

Are a & b alternativeunits of segregation

during meiosis?

i.e. are they segregational alleles?

Are a & b alternativecis-acting units of function

during development?

i.e. are they functional alleles?

Genes as defined as cis-acting units of function have multiple parts that are separable by recombination

The complementation test is generally favored:

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)

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)?

complementation testfor functionphenotypeof hybrids

mutant amutant b

ORa ++ bab ?

mutantphenotype

(don’t complement)

wildtypephenotype(complement)

allelesnot alleles

--- could we ever be misledby the apparent failure of theserecessive mutants to complement in trans?

Are mutant a and mutant b alleles (i.e. genetic alternatives)?

complementation testfor functionphenotypeof hybrids

mutant amutant b

ORa ++ bab ?

mutantphenotype

(don’t complement)

wildtypephenotype(complement)

allelesnot alleles

…could mutants be recessive individually and in different cis-acting units of genetic function (i.e. not be functional alleles),but interact in combination toappear dominant together)

a+

+b

a ++ b

wildtype wildtype mutant

?

Are mutant a and mutant b alleles (i.e. genetic alternatives)?

complementation testfor functionphenotypeof hybrids

mutant amutant b

ORa ++ bab ?

mutantphenotype

(don’t complement)

wildtypephenotype(complement)

allelesnot alleles

When alleles FAIL TO COMPLEMENTwe may need the

cis control:

mutant a mutant b + +

BECAUSE:If the two mutants are individuallyrecessive alleles of different genes,

but dominant in combination

the cis control will also be mutant!

Are mutant a and mutant b alleles (i.e. genetic alternatives)?

complementation test for function

phenotypeof hybrids

wildtypephenotype

mutant a mutant b + +

ANDsame recessivemutants in CIS

mutantphenotype

don’t appear to complement

mutant amutant b

IF:recessive mutants

in TRANS

trans cis, hence a & b are functional alleles of the same gene …orientation of the genetic information matters for genes

… then we can trust the trans result

3

Are mutant a and mutant b alleles (i.e. genetic alternatives)?

complementation test for function

phenotypeof hybrids

mutant amutant b

mutantphenotype

don’t appear to complement

recessive mutantsin TRANS

wildtypePhenotype

… we can trust the trans result

mutant a mutant b + +

same recessivemutants in CIS

CIS/TRANS test

But nobody bothers with the cis control for recessive mutantswhen doing complementation tests

despite what all the textbooks say (not even Benzer did it, as we will see)

Are mutant a and mutant b alleles (i.e. genetic alternatives)?

complementation test for function

phenotypeof hybrids

CIS/TRANS test

…then why am I wasting your time with this?

Cis test is too hard in most cases,and in most cases unnecessary.

Are mutant a and mutant b alleles (i.e. genetic alternatives)?

CIS/TRANS testfor

functional allelism

CIS phenotype TRANS phenotype, hence functional alleles

The complete cis/trans test will allow us to determine allelismeven if one or both of the mutants are not recessive!

…and all that matters is whether the cis vs. trans phenotypes arethe same or different, not what the phenotypes of each actually are!

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 = TRANS phenotype, hence NOT functional alleles

high-resolutionsegregation test

for recombination:alleles as alternativeunits of segregation

(recombination)

complementation testfor function

alleles as alternativecis-acting units of function

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)?

Intuitively, a better operationaldefinition of alternative forms of genes

Conflictfavored

Raises questions:

Fig. 7.20p228

Phage kill bacteria for a living (and replicate in the process). geneticists count “centers of klling” (plaques)

4

“multiplicity of infection”Must have a phenotypic difference between pure-breeding lines

to do genetics

(centers ofinfection)

Phage phenotypes (differences): based on growth

plaque morphologylarge/small (growth rate)clear/turbidsmooth edged/rough edged

growth conditionsbacterial host rangetemperature 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

The young S. Benzer: (1950s)phage T4

as his genetic workhorse

An older Benzer: (1967-present)fruit fly (D.melanogaster)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

•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

P.N.A.S.-US v41p344 (1955)

5

Need a selective genetic system(one with high resolving power for small map distances)

Rfa-b = NP pfu from hybrid / total pfu from hybridpfu = “plaque-forming units”

hybrid = mixed infection

Benzer’s system made measuring 0.0001 cM (1x10-6) easy

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 concentrationwithout complication from the much larger number of P pfu

…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 ratemeasured 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)

Among Hershey’s T4 collectionwere rapid lysis (r) mutants

--- produced distinctivelylarge plaques.

They arose spontaneously(1:10-4) and fell into severaldifferent complementation groupsthat mapped at different places

One complementation groupwas rII

and Benzer discovered somethingspecial about it that made

it perfect for studying genetic fine structure.

(1) rII vs. rII+ easily distinguished based on plaque morphology

(2) extremely rare (

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Yes, but extremely tedious(almost never do)

p.229Fig. 7.20 (still)

(for complementation test)

(1) Characterize mutants: fell into two complementation groups,

rIIA & rIIBvery close on the genetic map.

Make recombinantsby mixed infectionof the permissive

host

Assay progenypfu concentration

on permissive (=total)and nonpermissive

(=50% of nonparental)hosts

p.229Fig. 7.20 (still)

(can be either a quantitative testor a qualitative test)

(“unchanged in the hybrid”)

…or NO: segregational alleles, so only…

rIIA1 & rIIA2

no progenylyse K(λ)

YES