The action of suppressors of a tryptophan synthetase mutant of neurospora in heterocaryons

Vol. 18, No. 5-6, 1965

BIOCHEMICAL AND BIOPHYSKAL RESEARCH COMMUNICATIONS

TRE ACTION OF SUPPRESSORS OF A TRYPTOPHAN SWTRETASEMUTANTOF NEUROSPORA IN UETEROCARYONS* Patricia St. Lawrence, Richard Naish, and Benjamin Burr Department of Genetics, University of California 94720 Berkeley, California

Received January 25, 1965 Experiments

with heterocaryons

performed some years ago in the labora-

tory of David M. Bonner suggested that the effect is observed only when the suppressor same nucleus.

However, according

ing,

suppressors

that

tion

of the primary

restore structure

tem in the cytoplasm

sible

to our present

the activity

stimulate

are in the and understsnd-

protein

by altera-

operate through the protein-synthesizing

Gilbert,

to be reported

and suggest that

nuclei

show that

this

type

and the suppres-

of a heterocaryon,

in some cases this

the wild type allele

the growth of some heterocaryons

sys-

1962; Brody

1964). Therefore,

and rfirini

should be expressed whether the suppressor

experiments

is correct

information

of a defective

mutant are in the same or in different

preliminary tion

and the mutant affected

(Benzer and Chsmpe 1962; Garen and Siddiqi

and Ysnofsky 1963; Davies, of suppression

of a suppressor mutation

The expecta-

of the suppressor may

which carry

a suppressor

in one

of the component nuclei. DESCRIPTIONOF THE SUPPRESSORS Genetic analyses: recurrences

Yourno and Suskind (1@4a and b) have described

of a suppressor of the tryptophan

synthetase

mutant, s-201

1960). They have located this

suppressor

in linkage

and Catcheside

and have examined the effect of tryptophan lit

synthetase.,

or closely

*Supported

linked

of these mutations In our laboratory,

to those investigated

in part by U. S. Public

(Ahmad

group VIIR

on both normal and mutant forms five

suppressors

that

are alle-

by Yourno and Suskind have been

Health Service

868

seven

Grant ~~-07664

Vol. 18, No. 5-6, 1965

recovered

after

We shall

as E-YSl,

We shall

Suppressed strains

conidia

of as-201

and from nitrous

The 10 suppressed%-201 were established the s-201

from single

possible

combinations

The constitution tures

ascospores.

from un-

or -su-R genes.

of the suppressor

and

from a cross of each suppressed one ascus containing

all

four

and suppressor genes was chosen for

of the ~wild type, B+;suppressor,

from each ascus was verified

as z-Rl,

the suppressors

the E-YS

Segregation

From each cross,

of the z-201

linked

were crossed to wild type and stocks

mutant was observed in asci derived

JXJ-201 stock to wild type.

(or closely

all

light.

Five additional

have also been obtained

of either

cultures

etc.

to these suppressors

acid administration;

so far examined appear to be mutations

with ultraviolet

are alleles

refer

of u-201

strain s3YS2,

in the same experiments

at another locus.

etc.

treated

of conidia

to these suppressors

isolated

mutations) -su-R2,

treatment

refer

suppressors

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

study.

and t&-2Ol;suppressor

cul-

by study of the progeny from appropriate

back-crosses. In order to determine the specificity mutants at the tryptophan

synthetase

of the suppressors

locus,

suppressor was crossed to the following z-24,

-td-71.

a J.cJ-201 culture

strains:

I&I,

for other carrying

~a-2,

I&3,

a t-cc-16,

The spores of 5-10 asci from each cross were germinated.

of the tryptophan

synthetase

for growth;

of the segregants of these asci on minimal medium and medium

tests

supplemented with indole nation

mutants employed except -Cl-201 can utilize

None

showed that

with the -M-201 mutant.

the suppressors were active

these suppressors

are absolutely

argues for caution

specific

for the s-201

Germinants from 35 complete tetrads t&2Ol;E-YS2

by t+201;~-R2

-su-Rl by -td-201;=-YS These studies

(a strain

demonstrated that

in concluding allele.

were analysed from a cross of

and from 30 asci from a cross of t&201; kindly

supplied

by Dr. S. R. Suskind).

the -su-R and -su-YS genes are linked.

Other evidence suggests that the -su-R locus is closer than the z-YS

only in combi-

However, the large number of tryptophan

tase mutants that have not been tested

locus. 869

indole

to the centromere

synthethat

Vol. 18, No. 5-6, 1965

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1.

Genotype

TRE GROWTR RATE OF E-201

SUPPRESSORS

Medium

z>&

min m-P

5.12 0.1

min tryp

5.12 0.1 5.22 0.1

td-201;su-Rl

su-RP

min tryp

;*g --

su-R3

min W-P

su-R4

wild

type

I

5.2~ 0.1

Genotype

Rate mm/hr

Medium

td-201

min trn

5.0; 0.1

min tryp

4.0+ 0.1

td-201;su-R2

min tryp

3.4+ 0.1 4.7? 0.1

4.7+ 0.04 5.0? 0.1

td-201;su-R3

min tryp

3.6+ 0.1 4.G 0.1

min tryp

4.6+ 0.1 5.0? 0.1

td-201;su-R4

min -mT

3.5+ 0.1 4.8? 0.1

su-R5

min tme

4.9; 0.1 4.% 0.1

td-201;su-R5

min tryp

3.2+ 0.2 2.87 0.1

su-YSl

min tryp

4.4T 0.1 4.3* 0.1

td-201;su-YSl

min trm

3.3r 0.1 4.R 0.1

su-YS3

min tryp

4.7; 0.1

td-201;su-YS3

4.9;

min trm

3.7F 0.1 4.57 0.1

min tryp

4.7; 0.1 4.7; 0.1

td-20l;su-YS5

min tryp

4.2;

su-Rl

su-YS5

Growth studies: the effect

;.; *

0.1

Two segregants

of each suppressor

(Ryan, Beadle,

0.1

4.57 0.1

from the same ascus were used to study

on growth in the presence and absence of the

The most satisfactory

-td-201 gene.

5.1? 0.1

and Tatum 1943).

results

were obtained

by the tube method

The growth tubes contained

Vogel's

minimal

medium (Vogel 1956) with 2$ sucrose and 1.5% sgar and were incubated

34%.

Supplement was supplied

as L-tryptophan

of the rate of growth were initiated cate tubes.

Ro studies

was established.

12-24 hours after

were made of the lag period

and the standard error

tubes.

Measurements

inoculation

before

A second set of tubes was inoculated

mycelium from the end of the first

gression

(O.&g/ml).

at

of dupli-

a constant

with transfers

The mean growth rate

rate of

in mm/hr

of the mean were computed from measurements of pro-

through both sets of tubes.

The data given in Table 1 are based on

a minimum of 16 observations.

870

Vol. 18, No. 5-6, 1965

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Considerable

heterogeneity In all

the data in Table 1. Hith a -su-R allele, to that

strains

carrying

Growth of the tcJ+;~-R3

minimal medium differed apparent stimulation

significantly

medium appeared equivalent

and _4d';s-R4

from xild

by tryptophan

mutants can be seen from

the -td+ gene in combination

the growth rate on tryptophan

of :\‘ild type.

is difficult

among the five s-R

cultures

on

The importance

type.

3f the

of the grov%h rate of these two strains Possibly,

to assess at present.

the inclusion

of modifiers

ab-

sent from the genome of the other &u-R stocks could account for the observations. None of the 2-R

mutants restored

rate on minimal medium to cultures the -td-20l;su-Rl substantial carrying

either

in Table 1.

as wild

the g-201

allele,

stock is probably

were obtained

Stocks carrying

is evident

characteristic

of that

one of three members of the c-YS

60-VI& of wild type.

Generally

b) in studies

A few determinations

tures

is probably

suppressor;

locus grew more slowly ?lhether the -td'

or -td-201

effect

similar of their

results E-YS

Enzyme analyses:

as

cultures

only

one allele

mutants.

of each locus.

of the suppressors.

is present

was

were obtained by Yourno and

isolates

The results

In fact,

comparable to the growth attained

of the two suppressors

medium, but,

have been made of the growth oftd-201

two suppressors:

cate any additive

medium

of the same genotype.

the E-YS;td'stocks was equivalent to growth on tryptophan in the case of the 2-R mutants, the growth of E-YS;td-201 -

carrying

strains

from the data

in the growth on tryptophan

with another culture

A

in the genome. On minimal medium, the rate of growth of

was present

Suskind (l+ta,

medium.

medium of z-201

than wild type on medium supplemented with tryptophsn allele

and only

Qpe on tryptophan

the -su-R2, -su-R3, or -su-R4 alleles

The absence of an increase

results

to grow at a wild type

in the growth rate on tryptophan

of the t&-2Ol;su-R5 similar

carrying

stock grew as well

increase

the ability

do not indi-

the growth of such cul-

when the least

effective

alone.

The extensive

enzymological

Yourno and Suskind (196413) of the tryptophan 871

and physical

synthetase

studies

by

(TSase) from -td-201;

Vol. 18, No. 5-6, 1965

z-YS

strains

teristic

8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

suggest that,

of the s-201

from the wild an otherwise physical

mutant,

bly two proteins)

purified

extracts

one altered

of a -su-YS mutant into

on the enzymic, antigenic,

The restored

is associated

with a protein

Extracts

also be formed by this

that

or

properties

strain.

show

to TSase (and possi-

of the t3-201;su-RI distinguishable

indicate

but physically

that

from

an altered

different

species identical

The other E-R

culture

(Rachmeler and St. Lawrence

is physically culture

A molecular

type enzyme is produced.

strain

related

enzymatic activity

of a I&;=-Rl

with normal catalytic

of a td7201;z-Rl protein

are also formed by this

1964 and unpublished).

tein

Introduction

indistinguishable

of the normal TSase formed by such a strain.

of partially

normal TSase.

enzyme (CRM) charac-

a small amount of protein

wild type genome has no effect

the presence of CRM; at least

strain

to the altered

type enzyme is produced.

properties

Studies

in addition

pro-

from the wild

to normal TSase may

mutants have not yet been ex-

amined. STUDIES OF HETEROCARYONS The g-16

allele

is devoid of the enzymatic activities

normal TSase and some CRMs, does not form a protein neutralizing

antiserum

despite

and is unaffected

No suppressor of the -ta-16

numerous attempts

lowing treatment

cross-reacts

with with

to the wild type enzyme, does not complement the .t&

201 mutant (Rachmeler and YanofskY l$l), of the -M-201 allele.

that

associated

of -ta-16

to discover strains

one.

by suppressors

mutant has been obtained

The recovery

of reversions

with a mutagen show that this

fol-

mutation

is

not a large deletion. In order to force the formation -td-201 alleles,

two stable markers,

thenate requirement a +2Ol;nic-2

vigorous inoculate

niacin

(p&n-z) were used.

strain

type on the surface

of heterocaryons

in contact

heterocaryon growth tubes.

requirement

Heterocaryons

with a qd’16;pa11-2

of an agar plate

containing

had been established, The conditions 872

between the z-16 (nit-2) --

and

and panto-

were formed by placing strain

tryptophsn

of the same mating medium.

a massive transfer

Once a

was used to

of growth and determinations

of

Vol. 18, No. 5-6, 1965

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

growth rate for the heterocsryons individual test

At the end of the growth period,

strains.

and plated

on suitably

stained,

ants in the conidia

of the conidial

per conidium was

Crosses to a -ta-16 stock to detect -td+ revert-

are in progress.

As can be seen in Table 2, each of the four suppressors cluded in the s-16

All

studied

2, 5, 8, ll),

nucleus only (heterocaryons

nucleus only (heterocaryons

7, 10, 13).

were madeto

tubes was filtered

A portion

conidia.

and the number of nuclei

(Ruebschman 1952).

determined

3, 6, 9, 12), or in both nuclei

the heterocaryons

heterocaryon

(heteroceryons

The inclusion

-su-R2 mutant in the same nucleus as the -td-201 allele 6 in Table 2) gave, as might be expected, prisingly,

equally

the growth rate of heterocaryons

the su-YS1 or z-YS3 significantly

allele

compatible

of nuclear

rapid

inbreeding

in conidia

Sur-

and either

media.

of cultures

9 and 12 may be re-

of satisfactory

that preceeded selection

stocks were exceptional

ratio

growth rates.

formed one component (9 end 12 in Table 2) was

to problems encountered in the formation

fully

3 and

(heterocaryons

reduced on both minimal and trvptophsn

Despite the extensive

achieved

of the -su-Rl or

in which mutant t&201

The reason for the unexpected behavior latea

heterocaryons.

of our final

among the isolates.

frequent.

strains,

Determinations

produced by four of the most vigorous

ons suggest that both components are equal&

The nuclei

heteroc~ymust be

well mixed in the cytoplasm because few or no homocaryons were detected conidia

having an average number of 2-3 nuclei

nuclear

ratios

tion

to obtain nit-2

of heterocaryon

of the conidia vigorous

heterocsxyons either

were plated.

9 (Table 2) were unequal,

were homocaryotic.

nucleus csrrying

4,

in the -ta-16 nucleus

with the suppressor

(1 in Table 2).

was in-

or in the s-201

grew at the rate of wild type on both media and exceeded the rate by the control

for

supplemented sorbose media to estimate

the number of homo- and heterocaryotic suspension was fixed,

described

transfers

A suspension from the test

tubes of the same media.

through cotton

were the same as previously

Many unsuccessful

of the t&IL6;Em-_2

the=-YSl,

873

B:r contrast,

when the

and a large proporattempts

were made

component with the -td-201;

-su-YS3, or -su-YS5 allele.

Whether

Vol. 18, No. 5-6, 1965

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

TABLE 2.

THE GROWTH RATE OF HETEROCARYONS OF -td-201 SUPPRESSORS

No.

Genotype of components of heterocaryons

1

ta-16;pm-2 td-201;nic-2

Rate mm/hr

Medium

+ tryp

4.6 2 0.1

min tme

5.0 + 0.1 5.17 0.1

min tryp

5.1 + 0.1 4.92 0.1

+

min tryp

2.6 + 0.1 4.6 z 0.03

su-R2 +

min trYP

5.0 + 0.1 5.02 0.1

min trxe

5.2 + 0.1 5.02 0.1

min W-P

3.8 + 0.1 4.4 2 0.1

min trxe

5.0 _+ 0.1 5.12 0.1

min tr:rp

3.0 + 0.1 4.5 T 0.1

2

td-16;pan-2;su-R1 td-201;nic-2

3

td-16;pan-2 + td-20l;nic-2su-Rl

4

td-16;pan-2;su-Rl td-20l;nic-2;su-Rl

5

td-lb;pan-2; td-201;nic-2

6

td-16;pan- 2 + td-201;nic-2;su-R2

7

td-16;pan-2;su-R2 td-201;nic-2;su-R2

8

td-l6;pan-2;su-Ysl ta-20l;nic-2

9

td-16;pan- 2 + td-201;nic-2;su-YSl

10

ta-16;pan-2;su-YS~ td-201;nic-2;su-YSl

+

mi.n WJlJ

3.5 + 0.1 4.2 z 0.1

11

td-16;pan-2;su-YS3 td-201;nic-2;su-YS3 td-16;psn-2 + td-201;nic-2;su-YS3

+

min trn min tr:fp

5.5 + 5.2 2 3.7 + 4.72

td-l6;pan-2;su-YS3 td-201;nic-2;su-YS3

+

min trap

3.1 + 0.1 4.7 i 0.03

12 13

the inability a function or an effect

to maintain

+

+

the correct

balance of nuclei

0.1 0.04 0.1 0.1

for maximum growth is

of the -su-YS mutants when in the same nucleus as the -td-201 allele, of extraneous

The greatly

increased

to the suppressed i&201 suppressor was present -td alleles

+

genes, is unclear

at present.

growth rate of the vigorous homocaryons requires

in each.

in the heterocaryons

an explanation

There was no indication reverted, 874

heterocarJons

but this

that

possibility

compared

because a single either

of the

is difficult

Vol. 18, No. 5-6, 1965

to eliminate.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Mitotic

recombination

but no evidence that

it

could presumably produce -id+ alleles,

occurs in Neurospora has been obtained

(Case and Giles

1962). It lated

that the -ta-16 allele

is possible

to TSase but so altered

methods usually

employed.

sensitive

has revealed

tests

previously

classified

The application

of recently

TSase protein,

rate were the modified

products

at the -su-R or s-YS

ons) could be related

if

the structure

in the translation activating

heterocaryons

subject

to such errors,

information

and the over-all

could be more or less deleterious. gene in a cell translation

might,

errors

enough "mistakes"

de-

Clearly

a wild

Instead,

effect.

(compared to the homocary-

allele-specific

locus, suppres-

by causing rare "mistakes"

at the level

the proteins

of amino acid

in the cell

would be

consequences of suppressor action

The presence of one wild type suppressor

however, reduce the probability

mediated by the suppressor.

normal and mutated suppressor loci

Such

in a hetero-

of a normal gene at a suppressor

enzymes or acceptor RNA. All

suppres-

factors

of a suppressor

of a mutant protein

of the genetic

contain

of the -td-201 and -ta-16 alleles.

It has been suggested (Benzer and Chsmpe 1962) that sors may rectify

that

the only critical

locus does not have this

to the action

is considered

growth on minimal medium.

the augmented growth of the vigorous

strains

4, 7, 10, 13 Table 2).

Complete dominance of the wild type allele caryon would be expected to prevent

the suppres-

some weakly

This possibility

might be expected to grow very well

type allele

and Bonner

of growth on minimal medium but could contribute

sors in both of the components (heterocaryons

termining

in many -td mutants

Ensign,

because of the poor growth of the heterocaryons

cultures

highly

The suppressed s-16

to some extent.

CRM to the growth of a heterocaryon.

unlikely

developed,

the presence of such a protein

product

which is re-

by the immunological

mutant does produce an altered

sors might modify this

active

as to escape detection

as devoid of CRM (Kaplan, Mills,

1964). If the g-16

are incapable

may produce a protein

Simultaneous

in a heterocsxyon

to produce an amount of active

875

of the occurrence action

could provide

TSase sufficient

of

of the just for growth

Vol. 18, No. 5-6, 1965

8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and a minimum of inactive

molecules

the tryptophan

and other proteins

synthetase

date the nature of the action pressor

of other proteins.

An investigation

of the heterocaryons

of

may eluci-

of the wild type and mutant forms of the sup-

genes.

Ahmad, M., end D. G. Catcheside, 1960. Heredity 15:55-64. Benzer, S., and S. Chsmpe, l.962. Proc. Natl. Acx Sci. U.S. 48:l114-1121. Brody, S., and C. YanofskJ, 1963. Proc. Natl. Acad. Sci. u.s.?o:~-16. Case, M. E., and N. H. Giles, 1962. Neurospora Newsletter 2:6-K Davies, J., W. Gilbert, and L. Gorini, 1964. Proc. Natl. A&d. Sci. U.S.

f&883-890.

Garen, A., and 0. Siddiq?, 1962. Proc. Natl. Acad. Sci. U.S. -48:1121-1126. Huebschman, C., 1952. Mycologia%:59+604, Kaplan, S., S. E. Mills, S. Ensign, and D. Bonner, 1964. J. Mol. Biol.

8:801-813.

Rachmel
P. St. Lawrence, 1964. Fed. Proc. !3:2, 378. C. Yanofsky, 1961. J. Bacterial. B:955-963. Beadle, and E. L. Tatum, 1943. Am. J. Botany 1_0:784-799. S. S. Suskind, 19&a. Genetics ~~803-816. , 196413. Genetics z:817-828. Microbial Genet. Bull. 3:42.

876