becture 4. Columbia University. April 15, 1964
Gene~Control Mechanisms in Higher Organisms. Part II.

I. Review of previous lecture:

1.Attempt to show the relation of cytoplasmic-gene interactions in

control of cell differentiation as a consequence of control of
specific genes.

2. Control of action of alleles: only one active at a time; of duplicate
genes and multiples of such genes: only one active at a time; of
regulators that determine the patterns of this; of two-element
systems regulatin chromosome behavior with B-type chromosomes;
of regulatory mechanisms controlling pattern of distribution of
one product of gene action - elytra patterns in Yady Beetles.

3. the built-in Genetic Timers: in control of patterns of expression of
particular genes; of phase variation -- active and inactive
regulators and their cycles.

II. Levels of control mechanisms:

1. The modihe of Yacob and Monod: basic mechaism of control of gene action.

2. The higher levels: Consider controls wé&spohsible for several different
xky types of organisms from one germplasm:

a). uarvae - adult in Drosophila; catipettar - moth;
Parasites - several different org:nisms from one germplasm-
How controlied? Hajor "swithh" genes known.

b). Polymorphism in insects: Levels: From altered patterns of
pigments, as in Lady Beetle, to mimicry. Mimicry can
involve a series of changes in structure. Genetic analysis,
indicates co-trol by one or two "Switch" genes.

3. Genetic comppnents in chromosomes that do nothing else but
regulate gene action: Regulate when a gene system is active; Regulate
the kind of product of gene action. Yo this through system of two elements.

a). These systems of regulator: Not like "operator-regulator" systems
in bacteria. They have "built-in" mechanisms that modulate
the transcription of the gene: this resembles gene mutation

changes. Type of change controlled. Several types may
be produced during development: mechanism cortrols type at
any one time.

b). These control mechanisms discovered in maize many yesrs ago.

III. Discovery of pattern controlling mecha:isms in maize: when a gene

will be xzctive and kind of product resulting from its action during
development.

1. Initial experiment: chromosome type of b.f.b.cycle. Reason for exp.
2. How experiment conducted: at a oh

  

3, Results of experiment: The selfed progeny - seedlings. The modified
action of genes in somatic tissue: Slides 1 to 5.

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4. Conclusions from observations: mechanism controlling how a gene
acts and how changes in this occur: related to some event occurring during
a mitotic cycle: questiom What is this mechanism? Why did the b.f.b cycle
reveal these mechanisms? What is present in the maize genome that can
become altered in such a manner that it will produce such types of control
of gene action?

 
-2.

5. Answers to questions obtained: Vyele uncovered presence of
controlling elements: Two elements to a control system. These behave
like Operators and Regulators:

1) Consider situation of phages: Can be inserted at various loci in
chromosome complement. When inserted, vecome a part of the bacterial
chromosome.

2) Consider controlling elements, something like phages, can transpose
from one location to another in the chromosome compleuwent. This occurs
at the time of chromosome replication.

3). Consider phage-like elements belonging to a system: one can become
inserted at locus of gene: then, gene comes under control of this element.
Other element, located elsewhere, acts like regulator to which element,
adjacent to gene will respond by turning on-or -off action of the gene or
modulating action of the gene.

4). The controlling elements: transposible elements; each in system of
two : an operator component and a regulator component.

5). When op. element transposed to any gene, this gene now onder control
of the system to which the operator element belongs.

6). These two-element systems of gene control named after their regulators
Each regulator of a system readily identifiable.

7) Examples of meaning of this: Take Single "wild-type" gene:

One plant, one cell: Op-l inserted at locus of gene A =~
Gene under control of op-reg system 1

Another plant, one cell: Op-2 inserted at locus of gene A -
This gene now under control of Op-reg- system 2

When Op-1 at gene locus, control of gene action only by Reg 1;
Regs. 2, 3, 4, do not alter op-1l; Each op-reg- system
highly specific.

Ili. The super~imposing Op-Reg systems, above, present in maize from all
over the world. Not recognized as such in early genetic studies.

1. The first case leading to puzzlement, not clarity: The a Dt system.

2. The A, gene;located in chr. 3; in bhosynthetic pathway leading to
anthocyanin in kernel and plant.

3. Standard recessive, a,3; very old mutant. Stable with mutagenic agents:
Tests of this.

4. Discovery of "gene" that would make it mutate: Dominant, initially
located at end of short arm of chromosome 9s

>» Effect produced by this gene: caused a, to mutate in specific
way: small A, streaks and few dots of AL in kernels,

Alleles: stable after mutation process: No like original -
various different levels of gene activity among alléles,
some completely recessive. Gene

Alleles: mostly completely stable with Dt. ,teleased from control

by the Dt. system.
6. Studies of Nuffer:

a). Hunt for Dt in strains of maize from S. America: Two stains with
Dt:
bv). Location of these Dt in chromosomes: One in chr. 6, Other in
chromosonme 7. Neither at end of short arm of chromosome 9.

e).Later, found transposition of the Dt element to new locations.
d). Found new case of Dt controll of A gene: pattern of mutation
not the same. “hows in slides.
e). Must consider development of kernel:
Embryosac Pollen grain _
| . f

Hi

Embryo and endosperm: twins, genetically.

Mature kernel: Slides 6 to 8.

Ear gee

Cell lineages during development of kernel: Slides 9 to ll.

f) Nuffer: standard a, and new Dt co:trolled a - Nuffer, Slide 12
New a, = mutations in anther wen Dt present. “lide 13
Standard a, and new ay alleles in plant: Slide 14

Mutants; change in state: +erneis, Slide 15.

IV. Harlier test of presence of inactive Dt in maize not showing Dt regulation
1. From b.f.b. experiment, decided inactive regulators present.
2. On this premise, should be able to recreate Dt by b.f.b.cycle
3. fhe experiment: Cross

Female constitution Male constitution an
G ry co TTT
re 3 ay/ ay tk 8) / ay a
wa ES aan
Chromosome 9: normal ~=—y¢= es insq B vai
Gametes produced by male: Functional male gametes: with
Gs @ a3 broken end.
JO yoo OS
( _ } ( Pon} } /
a 5 ; . — | pian;
ae 7 SS it a ©. By 95% aT alls nad
Pukey! - Pak Ee ‘

4, Observations of kernels on ears from this type of cross: Found
sectors in which Dt present: Slides 16 - 18

5. Reason why only dotts produced in kernel, and their spacing:
State of operator at standard a locus: will give late mutations
with Dt only.

6. Review of Dt system: standard a: completely stable; cant cause mutatio
by X-ray, U.V., or mutagenic agents. when Dt not present.
b). Dt present in inactive phase in maize plants not showing it.
~4-

c) Type of response to Dt: depends on the state of the operator

at a-~standard locus.

a). This shown by tests of new Dt controlled a, locus: Original
response to Dt; mutant of operator - changed response like
standard kx a-

e). Dt, the regulator, can transpose from one location to another

in chromosome complement without losing identity in process.

V. The Spm system: The Ac system
1. Similar basic mechanism: two elements, an operator and a regulator.

2. Insertion of op af this system at Ay locus: Now, gene under control
of system to which operator belongs:

3. Isolation of cases of insertion of operators at normal A, locus:
independent occurrences:

Dt system Ac system Spm system
Standard a a m3 | a, m4 m=) m-2 m=5
1 1 1 ay » Ay a
New cases; : ye.
. McClintock A
One by Rhoades McClintock
Onde by Nuffer m

ay Peterson.

4. Lypes of genes that have come under control of these and other
systems: All types; Those selected for study. Reason

5. Other systems isolated, not yet defined.

VI. The Spm system -- examine By again.

1. Spm, the Regulator: Reason for name: Given in first studies:

Reason: Gene, op of Spm system adjacent: Spm present, no gean
action until change occurred, in response to Spm, that
gave particular pattern of gene action. These Wmutations"

as operator removed at time of occurrence of mutation.

Spm absent: Gene active. Degree depends on state of operator.

2. States selected as described above: Illustration, same two ears
shown on Monday: Slide 19

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3. Other illustrations of states: Pigments in kernels: Slides 20, 21

The independent control by each operator of time and type of
gene action during development: Like “lytra of ~ady Beetle.

State B¥x9ax 5719A-1 “lide 22; State5720 Slide 23
Cross: Slide 24. sh —
Both states in same k@rnel with Spm: Slide 25

VII. Methods of testing for Spm and for response of gene locus to Spm

1. To test for Spm: with any change at A, locus: Tester stock:
State:5719A-1 Slide 26: Pr. Appearance with pr. pale

SRN tm easter

eG VA ye . sp
~5-

Slide 27 : to show deep red with normal pr and Ay

2. Get stock with no Spm (completely inactive) this a,” sh, pr, ete.
h

3. Plant: near colorless ,kefnels on ear of an a,"'Sh,/a, sh, Spm plant
a) Question: is Spm present inkernel and embyyo?
if not, would a gene respond to Spm? A changed state?
a stable mutant?
b) The tests: Grow bene from kernel. Cross ears:
(1) by a, sh, no Spm For control of other tests.
(2) by ay sh, Spm To test response to Spm

(3) by a," sh, pr, no Spm: To test for presence of Spm-

Slide 28: ‘Three ears of plant 8424C-1 (Pr/Pr)

4, Take variegated kernels on ear in middle: have Spm.

a). Grow plants from these kernels: Test for Spm distribution in
progeny: he test:

(1) Ear x a, sh, - for control

(2) Ear x ajmsh, pr
Expect segregation for Pr and pr in test (2):
Slide: 29 Two ears, plant 8939C~4,

NRHN ee ccc nb eot

5. Constitution of the plant being tested: Wx Wx standard.(no repponse to
Spm). Vonstotution of tester with a = wx-m8: responds to Spm
Gives series of alleles of Wx gene action.

The Wx gene action: I-KI solutions (Demonstration)

6. Pigment: in aleurone layer; starch, in cells under this layer.
Expect two types of kernels with respect to wx gene action:
No Spm: near wx in staining.
Spm: sectors showing alleles of Wx gene;

The Test: Slides 30, 31.

7. Illustration: Control of action of two very different genes by same
system.

VIII Activation cycles of Spm:
1. How seen easily: Selected state: Operator fixed in location:

no losses to locus -- no mutations -- no stabilizztion.

2. Effect produced: Normal wild-type action when no Spm present or when
“pm inactive. When Spm present, gene inactive.

3. Shows changes in phase of activity of Spm: Active, colorless, inactive
pigmented. Patterns expected from inactivation cycles:

fi . . tdi) We ha
Sted agp > egtucir welt ~7 Bete ws -
a Auscboe spay WOMESAR St sage

 
Ix, Reversal of response of operator to Spm: One case isolated:
Spm active: gene active; Spm inactive’ gene inactive.

Activation cycles: Slides 35; Test with wx™®; Viide 36.

Reason for dots of deep color: represent transpositions of Op away
from locus of gene? release from control by Spm system.

Start with fully active Spm: Pattern observed: Slide 34: Time of
transposition of Op from locus of # gene. ~~
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