Lecture 3, Columbia University. April 13, 1964
Title Peg 1.) qboakUa

\r. Gene control mechanisms in Higher Organisms}, Two common types and several
special types.

Hill, The inducible systems: Enzyme needed to break-down a product from
the enviponment: some carbohydrate, as example. Example, galactose.

a). The Lac. locus: Req ‘ ad \ Oy Pa
b). Gene active when substrate, ‘g actose, ie meat om Gene very low

in activity when no substrate. Substrate contols degree of
activity of the gene.

 

c). this substrate, called the inducer; induces formation of the enzyme.

ad). Related substances act as inducers; enzyme, beta-galactosidase can

not act on these related substances. These useful in study of
enzyme formation and thus, rate of gene action with substrate.

2. The Model for control of gene action: Regulator and Operator

 

relationship:
a). Inducible system: Reg. SG-1_ 4, _SG-2 ,
AA VPIVUUOCV IANA . VAAN ee .
\ m EWR
Repressor F(effector molecule, small).
pupo itl molecule

R+ F ===" RF. Substrate -galactose: Attaches to R. R kept
from sitting on operator region and blocking gene action.

Substrate, small molecule, low or absent; R is not bound. Sits
on operator region of gene: no gene transpcription.

3. The "repressible" systems: Genes in biosynthetic pathwy. End product
controls the degree of action of the genes;

Same model: Now, R and F(small molecule) when combined, sit on
operator region of first enzyme: no gene transcription.
When F substance limited, R is freed of F; this
results in unbinding with the segment, operator, at
peginning of operon.

4, The mRNA in bacteria: examined cases, short lived; functions several
times and is distroyed. Must keep making mRNA.

In higher organisms, some mRNA, such as that for haemoblogin, long-
lived.

Mechanism of breakdown or protection from breakdown not known.

Advantage to bacteria of breakdown of mRNA and replacements of mRNA.
Constant change in environment; constant change in life cycle
when food readily available. Requires changes in action of
different genes to accommodate this.

5. Detection of the regulator genes: Through mutations of several types:
Gene action no longer controlled by substrate: Locus of change
at region not in structural gene. Other mutations: block

transcription altogether: site of mutation at same locus.
-~ 2 —

6. Detection of changes in operator region: Gene transcribes all the time,
regardless of substrate concentrations: Locate site of mutation
anh end of first gene in operon.

Genes not transcribing at all: Site of mutation: at beginning of the
operon3, i.e., a&% same position.

7. Studies of inducible and repressible enzyme-gene relationships: show
that cytoplasmic constitution and gene action intimately related.
A cycle of events from cytoplasm to gene and to cytoplasm.

This type of gene control very prominent in all organisms. Substances
in cytoplawmcontrolling specific genes very different, in many
cases, than substrate inducers or end-product of bio-synthetic
pathway controls. Will consider.

8. A Special Type of Control of gene action in Bacteria: Not cytoplasmical]l
controlled. Reflects a strictly genetic control system.

Example: The duplicate genes for flagella proteins: flagella
antigens:

H-1 op H-2 Op, Reg.

a ere ror Tye
Mutant sites: antigen type.
Only one gene active at any one time; other gene repressed.

 

>

Control of which gene active, resides in the Regulator:

Regulator undergoes changes in phase of activity: active - inactive-~
active.

When Regulator is active: H, gene active. When Regulator gene is
inactive; H-2 gene reSressed, H-1 gene activated.

Duration of cycle of activity of the regulator: Surprising results:

Long duration of inactive phase: Monophase-l. Change to
active phase in single bacterium: its progeny now whow change
from action of H-1 gene to action of H~2 gene; H-1 gene repressed

Long duration of active phase: Monophase-2. Changes, same as above

Bacterium: undergoes frequent chantes in phase in progeny:
Diphase,

Duration of phases: quite different after isolation following a
particular phase.

Control of duration of phase: locked into the regulator:
The regulator is a GENETIC CLOCK , GENETIC TIMER.

II. Examples of control of duplicate genes or of alleles, alternately,
in higher organisms.
Lode -carlydu cuter. .

1. lactic -dehydrogenase enzyme: a muscle emzyme. In mature individual,
this enzyme differs in muscles: Skeletal muscles, quick acting,
have M enzyme, produced by one gene. Heart, rythmic, slower action,
have H enzyme, produced by another gene.

2. Embryos in organisms with both types of muscles: Enzyme in early
development from H gene only; M gene repressed.
- 3-

3. Transition period in skeletal muscles, both xxum products present in
cell - H gene product and M gene product. In heart, only H type
as M gene permanently s#repressed,.

4, The lactic-dehydrogenase: composed of 4 sub-units.

In Heart: H,; In mature skeletal muscles: My

In transition tissues ~ skeletal muscle- find three other types:
Composition: H 1 H My» HM. This shows relationship of
Hgene and M gene au units?

5. Physicab and chemical properties of Hy and My» nevertheless, not the
samee

6. Modifications of control of these genes by addition of hormone:
Estradiol. When introduced into uterus of female with young
embryos, embryos develop some M subunits: activation of M gene.
When hormone removed, return to production of H sub-units

Estradiol: to chicken heart cells in culture: Force them to
produce some M sub-units. When removed, this gene repressed
and only H subunits produced.

7. Relation of muscular-dystrophy to action of H and M genes: Change to
M gene action disturbed; ineffective M gene product or not
enough. Either gene change or control mechanism change.

IML The place of action of hormones:

1. Hormones, known to activate certain gene products specifically in
only certain cells.

2. Discovery of relation of hormones to activation of specific genes:
Discovered in Chironomous: ecdgsone, activating genes related
to pupation process: (Has been discussed here earlier.)

3. The place where the hommone acts: First evidence very recent:
This shows: hormone goes into the nucleus. In one case tested,
hormone goes into nucleus or resides at periphery of nucleus:

4, The test: Toad bladder epithelium: controls passage of speciric
substances, one of them Na.

a). Discovered: control of Na-pump, that is, control of passage of
sodium is by ephthelium cells of bladder.

b). Can be made to start this by adding hommone Aldersterone

ce). Test of where hormone goes when added to toad bladder cells:
1) Hormone made radioactive.
2) Cells autogadiographed: Found hormone entered only the
epithelium cellw; only in nucleus; and at periphery of
nucleus: Slide l.
5. Reason why hormones now being studied activily for relation to
activation of gene: relation in structure to bases in DNA.
Expect very great increase in knowledge of this control very soon.
IV. Mechanisms of control of gene action in differentiation; continued.
A. Mosat# Cytoplasms.
1. Control of gene action by differentiation of the cytoplasm tinto
regions, each with different composition: At varies different
times in development, this must occur.

2. Example: the mosac egg cytoplasms: used in last part of last centruy
and early part of this century in “hay-day" of embrydlogy.

(a) Nucleus of egg, before meiosis: manufactures many different
substances; places them in specific parts of the cytoplasm.

(ob) Some eggs: these different substances very conspicuous, visually.
Egg is visually a mosaic.

(c) Sthdg of development: could relate aifferent parts of egg to
development of different parts of organism after cleavage
difisions following fertilization.

3 Visible mosaic cytoplasm at later stages in development: Development
of stomata in leaves of plants:

(a) Function of stomata: to co:trol intake of gases and loss of water
when water is low, or transpiration high.

(ob). Function controlled by two celis: Diagram: Open and close
an air space by their movement:

(c) Development of stomata: Appearance of stomata in maize leaf:
Slide 2 Found in rows: parachemya cell between earh.

(d) Rows of cells: laid down very early in embryo development:

Note pattern of laying down of cells. Cran
The development of the stomata and the subsidiary cells: 7
Slide 3. Diagram of events: Slide 4

 

(a). The cytoplasmic differentiation of each cell in row: lp

(2). The guard-mother cells formed after division: and the vi
parenchyma.
(3) Formation of the two guard cells

(4) “ormation of the subsidiary cells: Induction from the
G.M.C. Differences in cytoplasm and nuclei after
division.

4. (5). Abnormal types of e¥Yents: part of study of causes: Slide 5.
4, The lessons from study of development of stomata: —

(1). Pattern: cells in embryo: certain ones selected in precise pattern
to form row of cells with quart-cell future.

(2). Visible mosaic cytoplasm before division of cells in this row:
Unequal deivison of cytoplasm: two different cells as consequence

Mosaic cytoplasm during development. Unequal divisions of cytoplasm

(3) Induction of cytoplasmic change from GMC to adjacent cell in
adjacent row. Control of division plane; result of mosaic
cytoplasm. Induction: like hormone control.

 
-5-

V. The Built-In Genetic Timegs.in higher organisms.

1. This clearly shown in gene control in Paramoecium and Tetrahymena.

Genes for: Mating type protein; célia protein - antigens;
esterases; phosphotases.

2. The life cycle of Paramoecium: Diagram:

Micronucleus: the germ line; Macronucleus: the soma; dies. (Dissolved
5 during mtiotic process.

3. P. aurelia: 12 known genes for cilia protein type. Only one gene
active at a time; all others repressed.
Environmental alterations - temperature - can change from one
gene to another in action: active gene repressed, mawther gene
activated; all other genes remain repressed.

4. Tetrahymena - mating type protein: 7 mating types; only one active
in heterozygous individuals. Only one locus for 7 mating types.

a). The mating type process: Slide 6

ce

 

b). Process: from adhesions of special cilia membrans: Slide 7.

ce). Time of setting of which mating type will be expressed: In
fourth cell after meiosis and fertilization: Diagram.

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a). After setting occurs, no known means of altering this setting in

the Tetrahymena studied, while organisms reppoducing by
fission.

e). After meiosis and fertilization: process begins all over again.
Macronuclei discarded; resetting from genes in the micronucleus-
unmodified by macronuclear type in parents undergoing
conjugation and fertilization.

5. Above: Examples of control of action of only one of several
different genes for same end producte; and only one of
two alleles in same nucleus.

6.The control mechanisms: Can compare with the setting of the X chromosou
in mammals: In Females XX : One chromosonefor part of chromosae)
inactivated by structural process: Timing early, probably;
Which gkromosome ~ varies frm in different cells at time of setting:

7 xt x In same organism:7\ Mary Lyon, mouse: Slide 4

1
xX” X
‘Photo, ear evelopment, rabbit Slide >

Pen NOS
-6-

Only one X active when more than two present:
XX XXX XXX COOK: Like repression of all but one gene, when
duplicates present; Suppression of one allele, similar.

Conclude: Behavior of X chromosxe mm control mechanism and that in
Paramoecium and tetrahymena: Now, not so strange to consider
mechanisms of this type.

7.- Control mechanism and phase variation in Paramoecium:

a). B. multimicronucleatum: Diurnal change in mating type, without
fission of organism.
Gene found: Sets mating type rigidly, as
described above. Blocks diurnal phase variation.

b). Must consider control mechanisms that could do this: maize types
have some aspects related to all of this.

VL. Controlling elements:
1. Controlling elements in X chromosome of Sciara: Reviewed.
a). Emphasis, at this point, on setting of the controlling element:
Germ line of male:

Resett ng in germ line of female.
b). Controlling element exists in at least two alternating "states",

2. Other examples of control systems affecting chromosome behavior:
The B-type chromosomes in plants.

a). B-chromosomes: not part of normal chromosome complement; No
known gene action produced by them. Origins unknown.

b). The piling-up of B-type chromosomes: Maize - 30 and effect.
Grasses
c).The appearance of the B-type chromosome in several types of plants:

Slides 10 - 15.

PLANAR AE TTL TTS

a

3. Behaybbr of B-type chromosomes in microspore-pollen development:
a). Yiagram

b). Control of this: Maize: B-autosome translocations: Diagrams:

ec). Control of non-disyjunction: requires distal part. How shown.

ad). Similar control in Rye, Festuca, Briza, Holcus, Phlem: when
centromere fragments present: some require distal part
for non-disjunction control: Diagram.
- * - lecture 3

g). Must assume that a controling element - a regulator is present in
one position. Not yet identified as with Sciara.

Vo Begulator genes in higher organisms: ‘the pattern controlling regulator
genes,

1. Examples: Distrigution of pigment in flowers: Same pigment in
ditferent parts of flower; or different pigments, but each
shows a particular pattern of distribution.

Distribution of one pigment in plants: Anthocyanin - red -
pihgment’. Present in certain parts, not present in others.
Intensity differs. Alleles that control both distribution and

intensity.

2. Control of end product of gene action - is @istribution.in developmental
process. Not regulated by cell contents. Like setting genes in
paramoeciun.

3. Excellent example of pattern regulator: Elytra of lady beetle:
Pigment patterns: black on yellow. Series of alleles at one locus.
Both alleles operate when together. Each allele determines
particular pattern. Slide. %

sera

Behaves as if Regulator at gene: xe Reg.

this may not be so: maize cases: One regulator; differences in
pattern due to state of operator at gene locus: Slides (D &

 

YA eee lad EER TERME RLM AN

A gene; each with operator in different state.
One regulator controlling pattern of ahtion of this gene.

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VI. Vrigin of studies of control systems in maize.
“~
1. Initial experiment: chromosome type of b.f.b. cycle.
2. Experted results -
3. How experiment conducted.
4, The b.f.b. cycle seedlings: self of recovered branches.
5. The seedlings: Many selfed ears gave altered gene action; unstable.
6. The gsranplanted seedlings: plants; chlorophyll types. Vhanges in
rate of gene action: Slides. ips a
—Iww
7. Conélusions: controlling elements responsible for altered gene action.
8. Types of change in chromosomes of complement as result of b.f.b. cycle.
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