THE INDUCTION 0F RECESSIVE
.SUPPRESSOR MUTATIONS AND SOMATIO
RECOMBINATION. IN THE COMMON-AB
DIPLOID‘ 0F SCHIZOPHYLLUM COMMUNE

‘E‘hesis for the Degree of Ph. D.
MtCHIGAN. STATE UNIVERSITY
DALUCE IVAN. MiLLS
1969

m4

LIBRARY "3
Michigan State I
1 University

V“ 'T

 

This is to certify that the

thesis entitled

The Induction of Recessive Suppressor Mutations and
Somatic Recombination in the Common-£12: Diploid
of Schizophjllum commune

 

presented by

Dallice Ivan Mills

has been accepted towards fulfillment
of the requirements for

Ph. D. degree in BotanX and Plant
Pathology

[Mia/[4

Major prof . or

Date June 12, 1969

0-169

       
     

BINDING IV

A & SBNS'
800K Bill)!” INC.

LIBRARY 01100188
”II-nun Italian

ABSTRACT

THE INDUCTION OF RECESSIVE SUPPRESSOR MUTATIONS AND
SOMATIC RECOMBINATION IN THE COMMON-Ag, DIPLOID or
SCHIZOPHYLLUM COMMUNE
BY

Dallice Ivan Mills

The objectives of this research were to: l) induce recessive
suppressors of auxotrophic mutations that would be effective for
detecting recombinational events, 2) develop techniques for synthe-
sizing stable diploid strains, and 3) use the recessive suppressors
for detecting recombinational events in diploid strains of Schizophyllum
commune.

Genetic analyses have been made to detect recessive
suppressor mutations in twelve prototrophic strains of _S_. commune
derived by treating an arginine dependent strain with either hydroxyl-
amine, nitrosoguanidine or acridine red. The results indicate that
one strain possesses a recessive suppressor, 322, which maps
outside the gig-_Z locus and is capable of suppressing auxotrophy
conferred by the 155:3 mutation. This suppressor is recessive in
dikaryons and diploids and is incapable of suppressing auxotrophy
conferred by eight other loci. Prototrophy in the remaining eleven

strains resulted from either intragenic suppression, reversion, or

Dallice Ivan Mills

from a suppressor mutation that is closely linked to the 212;; locus.
Heterokaryotic allelic tests with the eleven strains indicate that the
mutation to prototrophy is recessive. The mutation to prototrophy
in each of the twelve strains segregates as a single gene through at
least two generations of backcrossing to the parent auxotroph. No
recessive suppressor mutations were detected among 764 prototrophs
induced with ultraviolet light.

A technique was developed which allows for the recovery of
stable vegetative diploid strains from the common-A_B heterokaryon
of g. commune. The common-ALB diploid, which is heterozygous for
auxotrophic mutations, is prototrophic and resembles the homokaryon
in morphology. An analysis of the segregation of five biochemical
markers and mating-type factors from a diploid X haploid cross
closely fit the predicted values for triploid segregation. At least four
linkage groups appear to be present in_§. commune based on the
segregation of mutant markers in segregants of an aneuploid from the
diploid X haploid cross.

Somatic recombination in the common-A_B diploid was studied
using the recessive suppressor, £1.11» to detect recombinational
events. An analysis of spontaneous and ultraviolet induced recombi-
nants indicate that diploid, aneuploid and haploid individuals were
recovered. Results from preliminary experiments indicate crossing

over is rare and haploidization proceeds via stages of aneuploidy.

THE INDUCTION OF RECESSIVE SUPPRESSOR MUTATIONS AND
SOMATIC RECOMBINATION IN THE COMMON-fig DIPLOID OF

SCHIZOPHY LLUM COMM UNE

 

BY

Dallice Ivan Mills

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOC TOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1969

DEDICATION

To Mary

ii

ACKNOWLEDGMENTS

A special dept of gratitude is due Dr. Albert H. Ellingboe,
my major professor, for his guidance and encouragement during this
investigation and in the preparation of this manuscript.

Special thanks are also due Drs. D. Schoenhard, E. C. Cantino
and R. P. Scheffer for their assistance in the preparation of this
manuscript.

I wish to thank Dr. W. G. Fields for making available his
photographic equipment and for his many helpful suggestions during
the preparation of the photographs.

Appreciation is also due Judy Crawford, Judy Chen and
Cindy Daroci for technical assistance.

I am deeply greatful to my wife, Mary, for her understanding
and assistance during the course of this investigation.

The finantial support for this investigation was furnished by

the Atomic Energy Commission for which I am indebted.

iii

TABLE OF CONTENTS

 

Page
DEDICATION. ii
ACKNOWLEDGMENTS . . iii
LIST OF TABLES . vi
LIST OF FIGURES . ix
INTRODUCTION . . . 1
LITERATURE REVIEW . 3
MATERIALS AND METHODS. 19
Schizophyllum cultures . l9
Tester Strains 19
Media . . . 23
Single Spore Isolation and Determination of
Germination Percentage 23
Purification of Cultures. . . 24
Scoring for Nutritional Requirements 25
Heterokaryotic Allelic Tests . 25
Experiments with Ultraviolet Light . 26
Experiments Using Growth Factor Analogues 27

Induction of Mutations to Prototrophy with Hydroxylamine,
N- methyl- N'- nitro- N- nitrosoguanidine and
Acridine Red. .

Tests of the Prototrophs Recovered Following
Treatment with Mutagens .

Synthesis and Isolation of Diploid Strains

Diploid X Haploid Mating. . .

Testing for Mating- Type Factors of Progeny and
Segregants of the Diploid (D- 1) X Haploid Cross

Detection of Somatic Recombinational Events in
Common-_A__B Diploids

Statistical Analysis

iv

27
3O
31
31
32

33
34

Page
RESULTS.....................35

Growth Responses in the Presence of Growth Factor

Analogues. . . . . . . . . . . . . 35
Prototrophs Isolated Following Treatment with
Ultraviolet Light . . . . . . . . . . . . 35
Induction of Recessive Suppressor Mutations with
Acridine Red. . . . . . . . . . . . 39
Recessive Suppressor Mutations Induced with
N- --methyl N'- nitro- N- nitrosoguanidine. . . . . . 43
Recessive Suppressor Mutations Obtained Following
Treatment with Hydroxylamine. . . . . . . . . 47
Recombination Between the Suppressor Mutations and
the arg-Z locus. . . . . . . 53
Supersuppressibility Tests with su- -l and Eight
Different Auxotrophic Mutations . . . . . . . . 58
Recovery of Diploid Strains from Common-AB
Heterokaryons . . . . 65
Segregation of Auxotrophic Mutations and Mating- Type
Factors from a Diploid X Haploid Cross . . . . . 68
Synthesis of Multiply Auxotrophic Strains. . . . . . 77
Verification of Diploid Strains which Possess s__u- -l . . . 83
Somatic Recombination in Common- -1_A_B Diploids . . . . 85
Isolation and Characterization of Recombinants Derived
from Common-_A__B Diploids . . . . . . . . . . 87
DISCUSSION....................103
SUMMARY....................114
LITERATURE CITED . . . . . . . . . . . . . . . . 116

Table

1.

LIST OF TABLES

Growth of four auxotrophic strains and a wild type strain
on minimal medium supplemented with the following
growth factor analogues: A) pyridine-3-su1fonic acid;
B) picolinic acid HCl; C) acetyl pyridine; D) benzimid-
azole; E) 2, 6 diamino purine SO4 .

Total number of colonies isolated as prototrophs follow-
ing treatment of six auxotrophic cultures with UV
irradiation.

The number of colonies obtained following UV irradia-
tion that were tested in heterokaryotic allelic tests

Growth of cultures isolated form minimal medium
following treatment of mycelial fragments of an
arg-Z culture with acridine red.

Segregation of the mutations to prototrophy of two cul-
tures recovered following treatment of an arg-Z
culture with acridine red when backcrossed to an arg-Z
strain

Growth of cultures isolated from minimal medium
following treatment of mycelial fragments of six
auxotrophic cultures with nitrosoguanidine

Segregation of the mutations to prototrophy of two
cultures recovered following treatment of an arg-Z
culture with nitrosoguanidine when backcrossed to an

arg-Z strain .

Growth of cultures which were isolated from minimal
medium following treatment of mycelial fragments of
an arg-Z culture with hydroxylamine .

vi

Page

36

38

40

41

42

46

48

49

Table Page

9. Segregation of the mutation to prototrophy of eight
strains following treatment of an arg-Z culture with
hydroxylamine when backcrossed to an arg-Z strain . . 50

10. Recovery of arginine dependent progeny from crosses
of suppressed strains X wild type strains on minimal
medium...................54

11. Recovery of arginine dependent progeny from crosses
of suppressed strains X wild type strains on minimal
medium...................59

12. Progeny identified from crosses of su-l (arg-Z su-l)
with eight doubly auxotrophic strains, each of which
possessed arg-Z and one additional mutation . . . . . 61

 

13. Growth of dikaryons derived from crossing arginine
dependent progeny from two different crosses with
testerstrains. . . . . . . . . . . . . . . . . 62

14. The frequency of recovery of mutant markers in 153
progeny of the common-A_B diploid X haploid . . . . 69

15. The expected and observed frequencies of mating-type
factors among progeny from a diploid X haploid cross
based on trisomic segregation . . . . . . . . . . 71

16. Segregation of A and B mating-type factors among
progeny of the diploid X haploid cross . . . . . . . 72

17. Segregation for nutritional dependence among 67 progeny
derived from an A41/A42 B41/B42 diploid . . . . . . 73

 

18. Segregation of A mating-type factors with auxotrophic
markers among 153 progeny from the diploid X haploid
mating...................75

19. Segregation of I_3 mating-type factors with auxotrophic
markers among 153 progeny from the diploid X haploid
mating...................76

20. Recovery of mutations present in segregants of an
aneuploid derived from the diploid X haploid cross . . 78

vii

Table Page

21. Recombinant progeny derived from crossing strains
possessing two auxotrophic mutations with a
suppressed strain, arg-Z su—l . . . . . . . . . . 80

 

22. Recombinant progeny derived from crossing suppressed
strains, each of which possessed a single different
auxotrophic mutation . . . . . . . . . . . . . 82

23. Recombinant progeny derived from crossing suppressed
strains, each of which possessed two different
auxotrophic mutations . . . . . . . . . . . . . 84

24. Recovery of auxotrophic mutations present in the
progeny derived from diploid X haploid crosses . . . 86

25. Spontaneous recombinants recovered from rapidly
growing sectors of the common-£3 diploid, D-9 . . . 9O

26. Segregation of auxotrophic mutations in the progeny
derived from crossing haploid spontaneous recombi-
nants selected at random and one aneuploid recombi-
nant with a wild type strain . . . . . . . . . . . 92

27. Growth of primary and secondary segregants derived
fromtheD-9diploid. . . . . . . . . . . . .. 96

28. Segregation of auxotrophic mutations in the progeny
derived from crossing secondary segregants from a
prototrophic recombinant with a wild type strain . . . 97

29. Recombinants recovered from fast growing sectors
following treatment of strain D-9 with UV irradiation . 98

30. Nicotinic acid-requiring segregants from the proto-
trophic sectors that were isolated following treatment
of D-9 with UV irradiation . . . . . . . . . . . 100

31. Spontaneous recombinants derived from a common-fl
diploid, D-25, which were isolated from yeast medium
as pink sectors or in areas of the mycelium that
exhibited sparse growth . . . . . . . . . . . . 102

viii

Figure

LIST OF FIGURES

Genetic map of Schizophyllum commune .

 

Growth responses of suppressed strains in
heterokaryotic allelic tests .

Growth of suppressed homokaryons on migration-
complete (MC) and minimal (MM) media .

Growth of arginine requiring progeny, derived from
the cross su-l (HA 41 X 699 (wild type), as homo-
karyons and dikaryons with an arg-Z tester strain

Growth of dikaryons derived from crossing three
different arginine dependent progeny from two crosses

with three different testers .

Diploids of Schizophyllum commune

 

Sectors from common-_AB diploid, D-9, on arginine
drop-out medium .

ix

Page

20

44

51

56

63

66

88

INTRODUCTION

A considerable effort has been expended to elucidate the
mechanisms of somatic recombination in fungi (Pontecorvo 1956;
Ellingboe and Raper 1962a; Sweizynski 1963). At least three mechan-
isms have been described. Pontecorvo (1956) described a mechanism

of recombination in diploid strains of Aspergillus nidulans whereby

 

haploidization of the diploid nucleus proceeds via stages of aneuploidy,
with infrequent mitotic crossing over. Crossing over and haploidi—
zation are distinct in time and space and haploidization results in
recombination of genes on different chromosomes.

The methods for detecting recombinational events in

Aspergillus were not available to those studying somatic recombina-

 

tion in Schizophyllum commune and Coprinus spp. In the absence of

 

stable diploid strains and recessive suppressors (which had been used

to detect recombination in Aspergillus) studies of somatic recombi-

 

nation in g. commune and Coprinus spp. utilized the technique of in-
compatible dikaryotic -homokaryotic (di-mon) matings to detect re-
combinational events in vegetative mycelium. Since neither nucleus
of the dikaryon was compatible with the nucleus of the homokaryon,
dikaryotization of the homokaryon could result when recombination

l

2

between the two nuclei of the dikaryon produced a nucleus that was
compatible with the homokaryon. Recombinants were detected as
dikaryotic sectors in the homokaryotic mycelium.

The results obtained with S. commune using this technique
suggested two new mechanisms of somatic recombination (Ellingboe
and Raper 1962a; Ellingboe 1963). A meiosis-like mechanism of re-
combination in vegetative cells was postulated to account for the high
frequency of recombination of linked and unlinked genes. A second
mechanism, Specific Factor Transfer, was suggested to account for
the transfer of only an incompatibility factor specificity from each
of the two nuclei of the dikaryon into the nucleus of the homokaryon.
The role of the intervening homokaryon in determining the mechan—
ism of recombination in incompatible di-mon matings is not known.
The transfer of the incompatibility factor ,specificities was confound-
ed with the method used to detect recombinational events. New tech-
niques for detecting recombinational events, therefore, are needed.

The purpose of this research was to develop new techniques
for studying somatic recombination in S. commune. The specific
objectives of the research were to: l) induce recessive suppressors
of auxotrophic mutations that would be effective for detecting recom-
binational events; 2) develop techniques for synthesizing stable diploid
strains; and 3) use the recessive suppressors for detecting recombi-

national events in diploid strains of S. commune.

LITERATURE REVIEW

There are numerous examples in the literature where a
second mutation, at a site different from a primary mutation, par-
tially or completely restored the function lost by the primary muta-
tion. These secondary mutations were termed suppressor mutations.
Two types of suppressor mutations are known. Intragenic suppres-
sion is the restoration of a lost function by a second mutation which
is located within the same gene as the primary mutation. Intergenic
suppression is the restoration of a lost function by a secondary muta-
tion that maps outside the site of the primary mutation. The mech-
anisms of intra- and intergenic suppression may be very diverse
(Yanofsky, Horn and Thorpe 1964; Capecchi and Gussin 1965).

Suppressor mutations have been reported in bacteria and a

variety of fungi including Aspegillus (Lilly 1965; Weglenski 1966;

 

Gajewski and Litwinska 1968), Neurospora (Giles and Partridge 1953;

 

Davis 1961; McDougal and Woodward 1966; Seale 1968), Coprinus

(Lewis 1961; Morgan 1966) and Saccharomyces (Hawthorne and

 

Mortimer 1963; Kakar 1963; Manney 1964).. These investigations have
revealed that suppressors may be dominant, semidominant or reces-

sive (Lilly 1965; Weglenski 1966; Gajewski and Litwinska 1968). It

is not fully understood why suppressor mutations exhibit these differ-
ent phenotypic responses. The following is a brief review of the work
which has given some insight into how suppression of auxotrophy is
believed to occur in the systems investigated in bacteria, fungi, and
yeast

Suppression has been most rigorously studied with the alka-

line phosphatase gene of Escherichia coli (Garen and Siddiqi 1962;

 

Weigert and Garen 1965; Weigert, Lanka and Garen 1967) and with the
rII genes of the T4 bacteriophage (Benzer and Champe I962; Stretton
and Brenner 1965; Kaplan, Stretton and Brenner 1965). The amber
triplet, UAG, and the ochre triplet, UAA. have been identified as
nonsense triplets or chain terminating codons which do not code for
any amino acids in suppressor-negative strains of _E. SEE (Sarabhai,
Stretton, Brenner and Bolle 1964; Brenner, Stretton and Kaplan 1965;
Weigert, Lanka and Garen 1967). That amber mutations result in

the cessation of polypeptide chain propagation has been confirmed by
demonstrating that only fragments of coat protein were found in T4
bacteriophage possessing amber mutations. The length of the poly-
peptide chain was determined by the position of the amber mutation
from the end of the gene corresponding to. the N-terminal end of the
polypeptide chain (Sarabhai, Stretton, Brenner and Bolle 1964). Sup-
pression of amber mutants in the alkaline phosphatase gene and the

coat protein of T4 phage was by insertion of an amino acid at the site

of the amber mutation thus allowing for the propagation of the entire
polypeptide chain (Kaplan, Stretton and Brenner 1965). Subsequent
studies have implicated mutated transfer RNA in the mechanism of
suppression. In a cell free system where amber suppressible mes-
senger RNA is used as messenger, suppression would occur only
when t-RNA from a suppressor strain of E. 2313 was present
(Capecchi and Gussin 1965; Garen 1968). Amber suppressors which
map anywhere in the genome suppress the amber mutation regardless
of its location in the genome of the bacteria or bacteriophage. The
suppressors of the £11112 allele in the in (amination) locus, which
codes for the primary structure of the NADP-linked enzyme glutamate

dehydrogenase (GDH) in Neurospora crassa, were also capable of

 

suppressing mutations present in three unrelated genes (Seale 1968).
In this respect, the suppressors of £111 were analogous to the amber
suppressors in E. go_li and the allele specific super-suppressors in
yeast (Hawthorne and Mortimer 1963; Gilmore and Mortimer 1966;
Mortimer and Hawthorne 1966). Therefore, amber suppressors and
super-suppressors are said to be allele specific and not gene specific.
Some ochre suppressors reported in E. Q map at genet-
ically different loci from amber suppressors while others map very
close to or within the same loci (Signer, Beckwith and Brenner 1965;
Eggertsson and Adelberg 1965). The level of restored activity by

ochre suppressors is very low (1-5%) in contrast to the level of

suppression by amber suppressors (30-63%) (Gorini and Beckwith
1966). Some ochre and amber suppressors are dominant over their
wild type alleles (Eggertsson and Adelberg 1965; Gorini and Beckwith
1966; Seale 1968).

Mutations to missense result in the transformation of a
codon specifying one amino acid into a codon specifying another amino
acid. Suppression of a missense mutant, A36, in the structural
locus for the tryptophan synthetase A—protein in E. go_li, was by the
production of a transfer RNA which was capable of introducing glycine
into the polypeptide chain in response to the AGA triplet which nor-
mally codes for arginine (Carbon, Berg and Yanofsky 1966). Trans-
fer RNA from strains carrying the suppressor of mutant A78 substi-
tuted glycine for cysteine in poly UG directed synthesis of a copoly-
peptide chain (Gupta and Khorana 1966).

An entirely different mechanism of suppression was suggest—

ed for pyr-3 (pyrimidineless) mutants in Neurospora crassa, proline-

 

less mutants in Aspergillus nidulans and isoleucineless mutants in

 

yeast. Suppression of m} mutants appeared to be through induction
of an enzyme defect in the pathway leading to arginine synthesis
which resulted in the diversion of precursors of arginine synthesis

to the pool leading to pyrimidine synthesis (Davis 1961, 1962; Davis
and Woodward 1962). Suppression by recessive or dominant suppres-

sors of pro mutants in A. nidulans was suggested to result from the

alteration of an enzyme which permitted the synthesis of proline by
an alternative pathway (Weglenski 1967). A similar mechanism of
suppression was suggested for the nonspecific suppressors of iso—
leucine mutants of yeast (Kakar 1963).

The mechanism of suppression of methionine suppressors

and purple color suppressors in Coprinus lagopus (Lewis 1961;

 

Morgan 1966) and suppressors of the arg-Z locus in Schizophxllum

 

commune (Mills and Ellingboe 1968) is not known.
Recessive suppressors, regardless of their mechanism of
suppression, have been extremely useful tools for studying somatic

recombination in Aspergillus nidulans (Pontecorvo and Kafer 1958;

 

Kafer 1961) and Coprinus lagopus (Cowan and Lewis 1966). Diploid

 

strains of A. nidulans were synthesized which were heterozygous
for numerous auxotrophic mutations and the suppressor, but homo-
zygous for the mutation upon which the suppressor acted. When the
diploids were plated on a selective medium, either a recombinational
event which lead to the production of a diploid homozygous for the
suppressor or a haploidization event which yielded a haploid strain
with the suppressor allele would allow normal growth.

An extensive analysis of somatic recombinants obtained from
heterozygous diploids in _A.nidu1ans has resulted in the description
of the Parasexual Cycle (Pontecorvo 1956). The steps in the Para-

sexual Cycle are: 1) the fusion of unlike nuclei in the heterokaryon

2) mitotic crossing over may occur during the multiplication of the
diploid nuclei and 3) haploidization of diploid nuclei via stages of
aneuploidy (Pontecorvo 1956).

In the Parasexual Cycle, crossing over or haploidization of
the diploid nuclei via stages of aneuploidy may yield recombinant
nuclei. Crossing over occurs in approximately one out of 500 nuclear
divisions. The rarity of crossing over is evident in that a clear case
of crossing over in two different regions during the same division
has never been observed. If a diploid nucleus were heterozygous
for several markers on one chromosome, segregation in any one re-
combinational event would occur only for the markers distal to the
crossover. Markers proximal to the exchange are unaffected.
Because of these infrequent exchange crossovers, it was possible to
order the genes on chromosomes using a "mitotic map" by observing
the segregation of genes distal to the crossover. Markers that re-
mained heterozygous were proximal to the crossover (Pontecorvo
1956).

Since the haploidization process proceeded via stages of
aneuploidy, barring infrequent crossovers, whole chromosomes
assorted recombining genes only on different chromosomes. When
aneuploid strains were recovered as segregants from diploid strains,
some chromosomes were disomic and heterozygous for all markers.

Subsequent haploidization which yielded the chromosome monosomic

resulted in expression of the auxotrophic mutations (Pontecorvo 1956).
This method of haploidization provided a convenient method for es-
tablishing linkage groups in g. nidulans.

Recessive suppressors of r_n_e_t (methionineless) in 9. la 0 us
have been used to detect recombinational events in a dikaryon (Cowan
and Lewis 1966). Though only 6 recombinants were recovered and
analyzed, the recovery of haploid and disomic nuclei suggested a
mechanism of haploidization proceeding via stages of aneuploidy, as
expected with the Parasexual Cycle. 1

The methodology and techniques applied in the study of somatic
recombination in _A_. nidulans were not available for a study of somatic

recombination in Schizophyllum. In Schizophyllum commune and

 

 

Coprinus spp. , heterothallic basidiomycetes, compatibility is con-
trolled by two unlinked mating factors, _A and E. Matings between
two strains with dissimilar E and E factors (1. e. _A_lB_1 X £2 E)
results in the establishment of the dikaryon, the secondary mycelium,
which has two haploid nuclei per cell. The diploid phase is short in
duration and normally is confined to the basidium. Meiosis proceeds
immediately following the formation of the diploid nucleus. The four
haploid nuclei migrate into the four basidiospores that are formed on
the basidium (Raper 1966). Matings between strains with similar £1
factors (1. e. ALE} X El E2) or similar E factors (El _111 X £2 E)

result in the establishment of common-é or common-E heterokaryons,

10

respectively. A third type of heterokaryon, the common-_A_B hetero-
karyon, results from matings between strains with similar E and
E factors.

That transient diploid nuclei exist in vegetative mycelium in
higher fungi has been tacitly accepted since Quintanilha (1939) first
demonstrated nuclear exchange in Coprinus. However, only recently
have diploid strains been recovered in three different species of
higher fungi. They have been recovered from common-£1 heterokaryons

of Coprinus lagopus (Casselton 1965); from incompatible dikaryotic-

 

homokaryotic (di-mon) matings in (_3. radiatus (Prud'homme 1965);
and in S. commune from common-E heterokaryons (Parag and
Nachman 1966), compatible dikaryons (Koltin and Raper 1968) and
common-AE heterokaryons (Mills and Ellingboe 1969).

Casselton (1965) was able to obtain diploids from a common-_A
heterokaryon of 9.1agopus heterozygous for complementing auxo-
trophic mutations in the two nuclei. Since the oidia are uninucleate,
only diploid oidia which resulted from a fusion of two unlike nuclei
could germinate and produce a colony on a nonsupplemented medium.
The diploid strains were very stable. Only rare haploidization yielded
intermediate aneuploids. The diploid nucleus was apparently unstable
when in a dikaryon with a haploid nucleus and always underwent hap-
loidization before the dikaryon fruited.

A common-E diploid in S. commune was found to be stable

11

as a homokaryon but was unstable when mated to a compatible strain
(Parag and Nachman 1966). Germination and viability of progeny of
a diploid X haploid cross were both very low. Although all the mu-
tations present in the diploid and haploid were recovered in the prog-
eny of this mating, the segregation of these mutations was not in
accordance with triploid segregation.

Koltin and Raper (1968) discovered an unusual mating in
which the product of the interaction of two compatible strains, each
with different auxotrophic mutations, had the morphology of a haploid
rather than a dikaryon. The product of this mating was also proto-
trophic and compatible with either parent. The authors concluded
that the establishment of the dikaryon in S. commune is controlled
by a dominant gene, 213+. Interactions between strains with the re-
cessive allele, gli_k, results in the formation of a diploid mycelium.

In the absence of diploids and recessive suppressors, the
most complete analysis of somatic recombination in Coprinus spp.

and Schizophyllum has been done using dikaryotic-homokaryotic

 

(di-mon) matings for the detection of recombinational events
(Quintanilha 1939; Papazian 1954; Crowe 1960; Ellingboe and Raper
19623.; Ellingboe 1963, 1964; Swiezynski 1962, 1963).

The dikaryotization of a homokaryon by a dikaryon was first
studied in Coprinus lagopus by Buller in 1931 (Buller 1931). The

process was later termed the "Buller Phenomenon" by Quintanilha

12

(1937). Three kinds of matings were shown to occur. They may be
1) compatible, where both nuclei of the dikaryon are compatible with
the nucleus of the homokaryon (fig +_A_2 B_2) X _A_3 E2, 2) hemi-
compatible, where only one nucleus in the dikaryon is compatible
with the nucleus of the homokaryon (£1 E_l + £2 E_2) X 1&1 El, and
3) incompatible, in which neither nucleus of the dikaryon is compat-
ible with the nucleus of the homokaryon (El E1 + A_2 E_2) X El E2.
In the compatible and hemicompatible matings, a nucleus of the di-
karyon which is compatible with the nucleus of the homokaryon mi-
grates into the mycelium of the homokaryon and forms a new dikaryon
(A1_1+£-s2> X é§B_3-—>(A1§1+A§_B_3)°r(é_2.§§+A_313_31——>
and (El __1 + 1_A_2 E_2) X Al El——)(_A_2 E2 + __1 _1). The incompatible
mating is not expected to result in the dikaryotization of the homo-
karyon. An infrequent recombinational event leads to an "Illegitimate"
dikaryotization of the homokaryon (El _B_l + _A_2 E_2) X E_l E2
(_A_2 _l + __1 E2) (Quintanilha 1939; Papazian 1954). The mechanisms
of recombination which ultimately result in the formation of a recom-
binant nucleus have been the subject of considerable investigation.
The preliminary work (Quintanilha 1939; Papazian 1954)
demonstrated that there had been an exchange of genetic material
between the nuclei of the dikaryon. Further evidence (Papazian 1954)

for somatic recombination was derived from the fact that following

two successive incompatible di-mon matings, hyphal tip isolation

13

yielded a nucleus of a genotype which at no point had been introduced
into either of the di-mon matings. No evidence for disomy was ob-
served.

Additional evidence for somatic recombination in incompatible
di-mon matings has been presented (Ellingboe and Raper 1962a;
Ellingboe 1963, 1964). A number of different auxotrophic mutations
introduced into the cross in each of the three types of nuclei were
used to determine the mechanism of recombination. One hundred and
seventy eight recombinants of independent origin, selected only on the
basis of recombinant mating-type factors, were analyzed from a total
of 7,160 incompatible di-mon matings (Ellingboe and Raper 1962a).
Two classes of individuals were described. Class I consisted of those
individuals that possessed non-selective markers in various combina-
tions from the nuclei of the original dikaryon. Class II consisted of
those individuals that possessed, aside from the recombined mating-
type factors, only the non-selective markers from the homokaryon.
The recombination frequencies of linked and unlinked auxotrophic mu-
tations of class I individuals were indistinguishable from frequencies
observed via the standard sexual cycle, and was distinctly different
from recombination expected via the Parasexual Cycle. There were
no aneuploid recombinants observed and no indications that recombina-
tion and haploidization were separable in time and space. The mech-

anism of recombination of class II individuals involved some event

14

which transferred only incompatibility factors from the two nuclei of
the dikaryon into the nucleus of the homokaryon. Subsequent experi-
ments designed to study the mechanism of recombination of class II
individuals confirmed the uniqueness of a second mechanism of
somatic recombination in S. commune.

Ina comprehensive series of experiments, Ellingboe (1963)
demonstrated conclusively that incompatibility factor specificities
of the dikaryon could be recovered in the genome of the homokaryon
without concomitant transfer of closely linked auxotrophic mutations.
This novel mechanism of recombination was termed Specific Factor
Transfer. That only the incompatibility factors were transferred
was demonstrated in the following manner. The _A mating—type factor
consists of two closely linked subunits, _‘l and L (Raper, Baxter and
Ellingboe 1960). Using incompatible di-mon matings, where one
nucleus of the dikaryon possessed pa_b (para-aminobenzoic acid re-
quiring mutation) situated between the _oz__ and .8. subunits while the
other component possessed M (adenine-requiring mutation) which
mapped O. 5 units from. the El subunit, it was shown that without
concomitant transfer of these auxotrophic mutations, the g_ and L
specificities from one nucleus and the E factor specificity from the
other nucleus of the dikaryon were transferred into the genome of the
homokaryon (Ellingboe 1963). Further evidence for Specific Factor

Transfer was substantiated by the recovery of the £15) mutation

15

(unknown requirement), a mutation linked tofi and introduced into
the mating in the homokaryon, in all of the recombinants. Further-
more, the recombinants possessed the §1_1_ mutation (unknown require-
ment) not linked to mating-type factors that was introduced into the
cross only by the homokaryon. The author concluded that " The in-
corporation of specific genetic material into the nucleus [of the homo-
karyon] would have to assume a specificity of incorporation analogous
to the incorporation of a temperate phage into a bacterium and the
prophage's association with a specific site on a bacterial chromosome"
(Ellingboe 1963). This unique mechanism of somatic recombination
has not been observed in Coprinus spp. nor have these types of
experiments been performed.

Further evidence for a meiosis-like mechanism of somatic
recombination in S. commune was obtained with the recovery of a
larger sample of recombinants from a single incompatible di-mon
mating (Ellingboe 1964). The 61 recombinant nuclei detected on the
basis of recombinant mating-type factors comprised 43 genotypes.
The frequency of linked and unlinked genes was compatible with a
meiosis-like process of reduction of a diploid nucleus to a haploid
nucleus. That no class II recombinants were recovered among the
recombinants of this dikaryon suggests that Specific Factor Trans-
fer may be lacking .in some incompatible di-mon matings.

Neither of the two recombinational mechanisms described

16

in _S_. commune have been acknowledged in incompatible di-mon
matings in two species of Coprinus. Progeny analysis of derived
dikaryons from incompatible and hemicompatible di-mon matings
was a method used to detect somatic recombination in _(_I_. lagopus
(Swiezynski 1962, 1963). Germination of the basidiospores was low
in many derived dikaryons (2-71%) with 10 of the 18 derived dikaryons
having less than 10% germination. The viability was low when spores
did germinate and the segregation of mutant markers was abnormal
for most dikaryons. The results were interpreted as indicative of a
parasexual mechanism of recombination with haploidization proceed-
ing via stages of aneuploidy.

Other techniques have been employed to detect somatic
recombination in the Basidiomycetes. Somatic recombination has

been detected in Coprinus radiatus using a triheterokaryon, i. e. , a

 

mycelium with three nuclear types (Prud'homme 1965). Two of the
nuclear types, although dissimilar in mating-type factors, possessed
a morphological mutation, col, which prevented dikaryosis and,
therefore, none of the three possible combinations of two gave a di-
karyon. Dikaryotic sectors which arose via a recombinational event
were analyzed for their genetic constitution by progeny analysis and
dedikaryotization by maceration. The conclusions drawn were 1)

that haploidization proceeded via stages of aneuploidy since numerous

aneuploid and some diploid strains were recovered 2) recombination

17

via crossing over is rare (none observed in 21 recombinant nuclei)
and 3) there is in many cases, a parental association of independent
non-selected markers.

An analysis of 14 prototrophic hyphal tips of a common-_AE
heterokaryon of S. commune which was nutritionally forced on min-
imal medium by non-allelic auxotrophic mutations indicated recom-
bination in all 14 cases with evidence for disomy of certain chromo—
somes in 10 of 14 recombinants (Middleton 1964). The disomic
chromosomes were unstable as indicated by the recovery of different
markers in several crosses of each of the 14 isolates to wild type
tester strains. These results were interpreted to favor a mitotic
mechanism of haploidization as described in the Parasexual Cycle.

Recombinants were obtained from five prototrophic hyphal
tips isolated from an aged culture of a heterokaryon of S. commune
formed between compatible homokaryotic strains, each of which
possessed a modifier mutation (thereby yielding the heterokaryon
incapable of fruiting) (Raper and Raper 1964). Three were recombi-
nant homokaryotic prototrophs and two were recombinant dikaryons.
No evidence of aneuploidy was observed.

Somatic recombination has been observed in the Uredinales
(Watson 1957; Ellingboe 1961) and in the Ustilaginales (Holliday 1961)
using spore color, pathogenicity or nutritional markers to detect

recombinational events. When equal proportions of a urediospore

l8

mixture of 2 isolates of Puccinia gaminis tritici, one having red

 

spores and few genes for virulence, the other orange spores and
many genes for virulence, were used to inoculate wheat seedlings,
4 virulent red races were recovered as a result of somatic recom-
bination (Watson 1957). Fifteen recombinants were obtained from

mixtures of 2 dikaryons of Puccinia graminis tritici when spore

 

 

color and virulence were the selective techniques used to detect
recombination in a subsequent study (Ellingboe 1961).

Holliday (1961) studied induced mitotic crossing over in
Ustila o maydis using diploid strains heterozygous for auxotrophic
mutations. Two types of segregation were observed; crossing over
and haploidization. Following irradiation with ultraviolet light, in-
duced mitotic recombination was detected by the acquisition of aux-
otrophy by previously prototrophic diploid cultures. That these
auxotrophic recombinants were diploid was shown by subsequent
segregation of other auxotrophic markers following a second treat-
ment of UV. The results were best explained by a mechanism of
mitotic crossing over and haploidization as described in the Para-

sexual Cycle.

MATERIALS AND METHODS

SchinghLllum cultures: All cultures used in this study had

 

a common background genome, strain 699 (Ellingboe and Raper 1962a,
1962b). The auxotrophic mutations and their linkage relations as
determined from meiotic products, are given in Figure l. The sym-
bols used are as follows: ade-l, ade-Z, ade-3 and _a_de_-_4 (unlinked
adenine-requiring); m, m and 2&8 (unlinked arginine-re-
quiring); 21_c_:2_ and M (unlinked nicotinic acid-requiring); SE-l,

su-2 .... su-12 (recessive suppressors of arg-Z).

 

Tester strains: A large number of tester strains which

 

possessed a single auxotrophic mutation were synthesized by cross-
ing selected auxotrophs with strain 699. Tester strains which pos-
sessed two mutant markers were derived from crossing two compat-
ible strains, each of which possessed a different mutation. The
auxotrophic progeny from these crosses were subsequently tested

for the mating-type factors they possessed. A number of prototrophic
tester strains with different A and E factors were synthesized.

These strains were established by crossing strain 699 with a number
of other wild type strains that possessed different A and E factors.

The mating-type factors of the progeny from these crosses were

19

20

Figure 1. Genetic map of Schizophyllum commune. Linkage re-

 

lations adopted from Ellingboe and Raper (1962a) and

Middleton (19 64).

21

 

 

 

 

 

 

A
Cent. so 0‘ 16.3 B 28.5 x15
W
B
*

u ra-l no.5 ode-3 ”.0 n ic-2
My]
ode-2 25 org-2 I x11 col ode-l

17.5
m 33
(”9‘1 , ode-4
u
org-6
—
nic-3

 

 

22

determined by mating reactions with tester strains for which the A
and E factors were known. An example of how the mating types of
the progeny from the cross A41 B41 X A42 B42 are determined is

given below:

Testers

A B C. D
Expected
genotypes sass at}. s_1 51183.1 a1 2:12
_Ajl _B_4__l F + B +
ELI}. E32 F + + B
£42 _B_4_l + F B +
_A_4_2 E4_2 + F + B

+ = compatible reaction

F flat reaction

U3
ll

barrage reaction

A second series of prototrophic tester strains was synthe-
sized to detect intra-é factor recombinants. The 5 factor is com-
posed of two linked subunits, AOL and AB . From a cross of

A410 1- 81 X A4201 3- B 5, the recombinant progeny Aa 3-8 1 and

 

 

Au 1- B 5 are compatible with all four tester strains in the example
used above. Progeny that scored as having recombinant A factors
were mated to compatible stock tester strains known to possess

A 013- 31 or Aal-B 5. The ”flat" reaction with one of these testers

23

established the genotype of each of the recombinant progeny. Once
the intra-_A_ factor recombinants were identified they were mated to
compatible stock cultures which possessed a E factor (B_47) which
was different from all cultures used in this study, and recombinant
progeny were saved for stock cultures. These cultures were of

mating type Aal— 85 B47 or A 013- 81B47.

 

 

Media: All matings were made on migration-complete
medium (Snider and Raper 1958). Scoring for nutritional require-
ments was made either on minimal medium (Raper and Miles 1958)
or drop-out medium. Drop-out medium is minimal medium supple-
mented with all but one of the growth factors required by the cultures
being tested. The omitted growth factor is specified in the descrip-
tion of the media; e. g. , arginine drop-out medium contains no argi-
nine. All growth factors were supplemented at 10'4 M concentration.
The heterokaryotic allelic tests were performed on minimal medium.
Progeny from crosses were germinated on migration-complete medi-
um, yeast medium or yeast medium supplemented with arginine.
Yeast medium is made from minimal medium by substituting yeast
extract and bacto peptone at 2 g/ liter for asparagine. All stock
cultures were maintained on yeast medium in bottles at 40 C.

Single spore isolation and determination o_f germination

 

 

percentage: The progeny from all of the crosses were obtained by

 

removing an agar plug bearing the mature fruiting body from the

24

established dikaryon and placing it on the inside of the cover of a
petri dish containing migration-complete medium. When the cover
was placed back on the petri dish, the fruiting body assumed an in-
verted position over the agar in the dish. The spores abjected from
the basidia were collected on the agar. The basidiospores were
spread over the surface of the agar in a drop of sterile water with

a small bent glass rod. The spores were allowed to germinate at
220 C for 20-30 hr. The percentage of germination was estimated
from counts of a minimum of 100 spores in several fields of vision
in a dissecting microscope with magnification of 48X. The well
separated germlings of 20-30 hr were removed, together with a
small agar block, with the aid of a sharpened isolating needle. Each
germling was placed either in a stock bottle or in petri plates and
incubated at 32° C until macroscopic growth was evident.

Purification o_f cultures: A number of experiments in this

 

study involved the isolation of prototrophic strains from sectors that
arose in an auxotrophic culture. Inoculum from each prototrophic
sector was transferred to fresh medium identical to that from which
it was removed and incubated at 320 C for 2 days. With the aid of a
dissecting microscope and an isolating needle, 3 to 5 hyphal tips
consisting of 2-4 cells were removed from the growing edge of each
culture. The hyphal tips were transferred to migration-complete

medium and incubated at 320 C for two days.

25

Scoring £93 nutritional requirements: In crosses where a
single auxotrophic mutation was segregating, a small agar block
containing mycelium was transferred from each of the progeny to
migration-complete and minimal media and incubated at 220 C for
3 days. In crosses where more than one gene was segregating for
nutritional requirements, transfers were made to several types of
drop-out media that would identify the growth factors required by
each of the progeny. Scoring for nutritional requirements in homo-
karyons on minimal or drop-out media was done at 3 days.

Heterokaryotic allelic tests: The genotypes of progeny from

 

crosses where more than one gene was segregating for a particular
growth factor were inferred from heterokaryotic allelic tests. For

+
example, there could be three genotypes, ade-2 ade-4+, ade-2 ade-4,

 

 

and ade-2 ade-4 which would require adenine among progeny of a

 

cross involving a_<i_e;_2 and M. Each of the adenine requiring prog-
eny was mated to two compatible strains, each of which possessed a
single adenine mutation, either id_e:2 or 953:3. The matings were in-
cubated at 320 C for 3 to 4 days to establish dikaryons. Transfers
from each dikaryon were plated on migration-complete and minimal
media and incubated at 22° c for 5 days.

Dikaryons synthesized from progeny of unknown genetic con-
stitution and gig-_4 tester strains that failed to grow on minimal me-

dium must have been homoallelic for the mutation carried by the

26

tester strain. Since the tester strain carried the ade-4 mutation,
the other component of the dikaryon, the nucleus from the adenine-
requiring progeny, of necessity, must also have possessed the ade-4
mutation. Dikaryons derived from adenine-requiring progeny and
ade-4 testers that grew on minimal medium indicated that the wild
type allele was present at the ade-4 locus in the strain being tested.
Progeny that failed to produce dikaryons which would grow on mini-
mal medium with either ade-2 or ade-4 tester strains must have
had both adenine mutations.

Experiments with ultraviolet light: Cultures of six different

 

auxotrophic strains were transferred to petri dishes that contained
minimal medium plus the appropriate growth factor supplemented at
a concentration that would allow very limited growth. After incuba-
tion at 220 C for 5 days, cultures were irradiated for 10, 20, 30, 40,
50, or 60 sec with a General Electric G-3OT8 germicidal lamp (short
wave length, 254 mu) at a distance of 12 inches. Irradiated cultures
were incubated in the dark at 220 C for 3 days to minimize photore-
activation. After 10-14 days, sectors in which growth was more rapid
than the parent culture, were removed from the dishes, transferred
to migration-complete medium and incubated at 320 C for 2 days and
then transferred to minimal medium. Cultures which grew on mini-
mal medium were purified by hyphal tip isolation and transferred to

minimal and migration-complete media. Prototrophic strains that

27

possessed normal morphology were placed in stock bottles for sub-
sequent tests.

Experiments using growth factor analcgues: Two adenine-

 

requiring strains, two nicotinic acid-requiring strains and a wild
type strain, 699, were tested for their ability to grow in the pres-
ence of growth factor analogues. Five different kinds of media were
prepared, each consisting of minimal medium with one growth factor
analogue supplemented at 10-4 M concentration. Three nicotinic
acid analogues, pyridine-3-su1fonic acid, picolinic acid and acetyl
pyridine and two base analogues, benzimidazole and 2, 6 diamino
purine SO4 were tested. Growth responses on migration-complete
and minimal medium served as controls.

Induction 3f mutations t_o prototrophy with hydroxylamine,
N-methyj-N'-nitro-N-nitros_oguanidine and acridine red: Experi-

mentation with hydroxylamine (HA): Strain 10209 (A41 B41 arg-Z) was

 

grown for 5 days on migration-complete medium at 320 C and then
fragmented in a Waring Blendor with 25 m1 sterile distilled water for
one min. Two m1 of the suspension of mycelial fragments were added
to 25 m1 migration—complete liquid medium in a 300 ml Erlenmeyer
flask. Sterilized HA was added to a final concentration of l X 10-3
mg/ml. After incubation at 220 C for 48 hr, the contents of each

flask were fragmented in a Waring Blendor for 15 sec. One ml was

pipetted onto each of 25 plates containing minimal medium. The

28

plates were examined for prototrophic colonies 9 to 10 days later.
Each culture that developed on minimal medium was transferred to
migration-complete medium and incubated for 2 days at 320 C, then
transferred to minimal and migration-complete media. Hyphal tips
were removed from cultures that were growing on minimal medium
and transferred to stock bottles to be used in further tests.

Experimentation with nitrosoguanidine mg: Experiment I:

 

A culture of each of 6 auxotrophic strains grown on migration-complete
medium for 5 days at 320 C was fragmented in a Waring Blendor with
25 m1 sterile distilled water for one min. Five m1 of macerate of
each strain were pipetted into a 300 ml Erlenmeyer flask containing 20
ml of liquid migration-complete medium and incubated for 4 days at
320 C. The contents of each flask were fragmented in a Waring Blen-
dor for l min, centrifuged in a Servall Model ss-l clinical centrifuge
at 6, 500 X g for 20 min. The pellet was resuspended in 25 m1 of 0. 2
M citrate buffer pH 6.0, and nitrosoguanidine was added to a final con-
centration of 1 mg/ml. Following an exposure time of 45 min, the
fragments were centrifuged at 6, 500 g for 10 min and the pellet was
resuspended in 25 m1 sterile distilled water. One ml of the treated
fragments was pipetted onto each of 20 petri dishes containing minimal
medium. Prototrophic colonies were removed from the dishes 10-20
days after treatment

Experiment II: Cultures of each of the 6 strains grown on

 

29

migration-complete medium for 5 days at 320 C were fragmented

in a Waring Blendor for 1 min with 25 ml sterile distilled water.
Five ml aliquots of the fragments were pipetted into 300 ml Erlen-
meyer flasks containing 20 ml liquid migration-complete medium
and NC at a total concentration of 5 X 10.3 mg/ml. - After 3 days
incubation at 220 C, the contents of each flask were centrifuged in

a clinical centrifuge at 6, 500 X g. The pellet was resuspended in 25
ml sterile distilled water and fragmented in a Waring Blendor for

10 sec. One ml of the mycelial suspension was pipetted onto each

of 25 petri dishes containing minimal medium. Prototrophic colonies
were pruified and bottled for subsequent tests.

Experimentation with acridine red (AR): Strain 10204

 

(A47 B47 arg-2)was incubated for 5 days on migration-complete medi-

 

um at 320 C, fragmented in a Waring Blendor with 25 ml sterile dis-
tilled water, and 4 ml of the mycelial suspension from each culture
were pipetted into 300 ml Erlenmeyer flasks containing 20 ml migra-
tion-complete medium. One ml from a sterilized stock solution of
acridine red (AR) was added to bring the final concentration to 5 X 10-3
mg/ml. After 48 hr incubation at 220 C, the contents of each flask
were fragmented for 60 sec in a Waring Blendor. One ml portions
of the treated mycelial fragments were pipetted onto petri dishes

containing minimal medium. Prototrophic colonies were removed,

purified and transferred to stock bottles.

30

Tests o_f the prototrophs recovered following treatment with

 

mutagens: A11 prototrophs were mated to a strain which possessed
different mating-type factors and the a_1:_g-_2 mutation, e. g. , either
10204 (A_4_7 B_4:l arg-2) or 10201 (A_4_2 E42 arg—Z). After 4 days incuba-
tion at 320 C, small pieces of the newly synthesized dikaryons were
transferred to minimal and migration-complete media. If the mutation
to prototrophy were the result of the induction of either a dominant
suppressor or an g;g-_2 revertant, the dikaryon should grow on min-
imal medium. If the mutation to prototrophy were the result of the in-
duction of a recessive suppressor, the dikaryon formed by mating a
prototrophic culture with an arginine dependent culture should be homo-
allelic for m, heteroallelic for the suppressor and, therefore,
require arginine for growth on minimal medium. Progeny were col-
lected from those dikaryons that did not grow on minimal medium and
tested for the segregation of prototrophy. Prototrophic progeny that
had the normal morphology were mated to the parent auxotroph and the
newly synthesized dikaryon again was tested for arginine dependence.
After two or three generations of backcrossing to the auxotrophic par-
ent, prototrophic progeny were again mated to the auxotrophic parent
and the dikaryon was tested for arginine dependence. Failure of the
dikaryon to grow on minimal medium even though one of the two com-
ponents of the dikaryon has no requirement has been considered evi-

dence for the presence of a suppressor which is capable of suppressing

31

the arginine requirement in the homokaryon but which is recessive to
its homologous wild type allele in the other component of the dikaryon.
The cultures used in subsequent genetic tests were recovered as pro-
totrOphic progeny from at least two generations of backcrossing to the
auxotrophic parent. Similar procedures were used for the induction
of prototrophy in strains possessing mutations other than arg-Z.

Synthesis and isolation 9_f diploid strains: Common-AB

 

heterokaryons, heteroallelic for complementing auxotrophic muta-
tions, were synthesized on migration-complete medium at 320 C.
After 4 days, small pieces of inocula from the area of confluence
of the two mated cultures were transferred, one plug per plate, to
50 petri dishes containing minimal medium. Incubation was at 220 C.
Inocula from common-A_B heterokaryons which were heteroallelic
for £121 and complementing auxotrophic mutations were transferred
to minimal medium supplemented with arginine. Diploid strains
were recovered from dense, rapidly growing, white, fan-shaped
sectors that arose from a background of sparse, irregular mycelia
typical of the morphology of the common-_A_E heterokaryon. A11
suspected diploid strains were tested for nutritional dificiences on
minimal medium and drop-out medium.

Diploid 22$ haploid mating: One putative diploid strain, D-l,

 

was mated to a compatible wild type strain (A42 B42) to determine

if mating-type factors and nutritional markers would show trisomic

32

segregation. Spores were collected from fruiting bodies which de-
veloped on the haploid side of the mating. The single spore isolates
which were collected were plated one per plate, to migration-com-
plete medium. When growth was macroscopically visible on the
plate, the agar plug bearing the germling was transferred to a stock
bottle and stored at 40 C. Sectors which developed on each plate
were tested for nutritional requirements and mating-type factors.

All single spore isolates plus any sectors which developed on the
plates were first tested for nutritional requirements by plating on
drop-out media. Those progeny and sectors with nutritional require-
ments were subsequently mated to tester strains with the appropriate
mutations and the heterokaryotic allelic tests were performed to
determine which mutations conferred auxotrophy. The primary
sectors were subsequently plated to fresh migration-complete medium
and secondary or tertiary sectors were isolated and analyzed as
described above for primary sectors.

Testing fo_r matinktype factors o_f progeny and segregants

 

 

_<_)_f t_l_i§ diploid (D-l) )_( haploid cross: The mating-type factors of the

 

 

progeny and segregants from the D-1 X haploid cross were deter-
mined by mating with 6 tester strains. All progeny and segregants
were first mated to four tester strains with the following mating-

type factors: (Ail _B_41), (_A__4_2_ Efl), (£43 E4_2), and (£2 _B__4_2). All

cultures and sectors which scored compatible with A41 and A42

33

were tested further by mating them with recombinant A tester strains,

(A 013- 81 B47) and (A01 1-8 5 B47) (Raper, Baxter and Middleton 1958;

 

 

Raper, Baxter and Ellingboe 1960). Strains which possessed a re-
combinant _A_ factor on a monosomic chromosome were incompatible
with one of the recombinant A tester strains. Cultures and segregants
disomic and heterozygous for the E factor, i. e. , Ail/{:42 or

A 013- Bl/Aa l- B 5, were compatible with all A factors present in the

 

tester strains. Distinction between strains disomic and heterozygous
either for intra-E factor recombination or A41/£42 was made by

test mating haploid segregants from the cultures with the tester
strains.

Detection o_f somatic recombinational events i_r_1 common-AB

 

 

diploids: Method I: Inocula from a common-£3 diploid strain, homo-

 

zygous for 2&2 but heterozygous for _s_u_-_l and other auxotrophic
mutations were plated onto minimal medium supplemented with ade-
nine and nicotinic acid. The ensuing growth was sparse because the
diploids were heterozygous for iu_-l, homozygous for a_rg_i, a geno-
type that resulted in very limited suppression of auxotrophy conferred
by a_rg-_2. Recombinational events which yielded a diploid homozygous
for 311, or a haploidization event which yielded a haploid strain
which carried fig-_l were detected as dense white sectors.

Method 11: The appearance of pink colored sectors in a

background of white mycelium provided a second method for detecting

34

recombinational events. Haploid strains that carry the ad_e_—_4_
mutatuon in the absence of the M mutation, and dikaryons homo-
allelic for ade-4 and heteroallelic for $9.21, accumulate a pink
substance in their hyphae. Dikaryons heteroallelic and diploids
heterozygous for ade-4 do not accumulate this substance and remain
white.

Statistical analysis: Chi square tests were used with 5%

 

level of significanc e .

RESULTS

Growth responses _12 the presence _o_f growth factor analogues:

 

 

 

Recessive mutatuons to drug resistance have proved to be useful

tools for detecting recombinational events in Aspergillus nidulans

 

(Pontecorvo and Kafer 1958). A limited study was conducted to de-
termine whether growth of wild type strains would be inhibited in

the presence of growth factor analogues or if these analogues could
be substituted for the growth factors required by auxotrophic strains
of S. commune. The analogues were incorporated into minimal
medium at concentrations ranging from 4. 6-8. 4 X 10.4 M. The ade—
nine-requiring strains were unable to utilize the adenine analogues
tested and neither could the nicotinic acid-requiring strains utilize
the niacin analogues (Table l). The wild type strain was insensitive

to any of these growth factor analogues at the concentrations used.

Prototrophs isolated following treatment with ultraviolet

 

1_igll_t_: Six auxotrophic cultures were exposed to UV radiation from

a General Electric G-30T8 germicidal lamp at a distance of 12 inches.
No attempt was made to determine the per cent kill because of the
difficulty in obtaining accurate data from irradiated cultures grow—

ing on an argar surface. The experiments were designed to

35

36

Table 1. Growth of four auxotrophic strains and a wild type strain
on minimal medium supplemented with the following
growth factor analogues: A) pyridine-3-sulfonic acid;

B) picolinic acid HCl; C) acetyl pyridine; D) benzimid-
azole; E) 2, 6 diamino purine SO4.

 

 

 

 

 

Growth factor analoguea Controls
Culture A B C D E MC MIN
699 wild type +b + + + + + +
10505 nic-2 - - - + _
10810 nic-3 - - - + _
10915 3.23:2 - _ + -
101717 ade-4 - - + -

 

 

a A, B and C are analogues of niacin; D and E are
analogues of adenine.
b + = growth, - = no growth.

37

determine the exposure time most effective for inducing prototrophic
colonies that could be tested for the presence of recessive suppressors.
An exposure of 30 sec was found most effective for inducing proto-
trophic colonies (Table 2). Nearly twice as many prototrophs were
isolated following the 30 sec UV treatments as were isolated follow-
ing the 20 or 40 sec UV treatments. UV treatments of 60 sec appear-
ed to inhibit the formation of prototrophic colonies.

An examination of the number of colonies isolated as
prototrophs from each of the six cultures exposed to UV treatment
indicate that UV is extremely effective for inducing prototrophy in
cultures with the 3.33:3 mutation but relatively ineffective for in-
ducing mutations to prototrophy of the cultures with Ell—C2} or gig-:1.
One-half the total colonies isolated as prototrophs (815/1, 631) were
obtained following UV treatment of the 3387—2 auxotroph. As indicat-
ed by the number of prototrophic colonies isolated from strains

carrying ade-Z, arg-6 or nic-2, radiation is equally effective for

 

inducing prototrophy in these strains.

Upon purification of these 1, 631 colonies it was apparent
that many of the prototrophs merely represented adaptations be-
cause when the cultures were returned to minimal medium they
would not grow. The prototrophic strains that possessed good morph-
ology after single hyphal tip isolation were mated to the appropriate

tester strains and heterokaryotic allelic tests were performed to

38

Table 2. Total number of colonies isolated as prototrophs following
treatment of six auxotrophic cultures with UV irradiation.

 

Exposure time in seconds

 

Culture 10 20 30 4O 50 60 Total
10915 $1322 38 33 134 24 5 0 234
101716 g._c_1_e;4 13 9 42 13 2 0 79
10204 3&2 76 159 298 188 86 8 815
101422 818:2 25 51 53 47 46 o 222
10505 M 44 57 51 45 33 1 231
10810 21.9.22 5 9 21 11 4 0 50

 

Totals 201 318 599 328 176 9 1, 631

39

determine whether any of these strains possessed a recessive
suppressor (Table 3). None of these strains possessed a recessive
suppressor.

Induction _o_f recessive suppressor mutations with acridine

 

_I'Ecl: An attempt was made to recover prototrophs from an 33.3.3.2
strain following treatment with acridine red (AR). Twenty-one
arginine independent cultures isolated in one experiment were tested
for the presence of a recessive suppressor in heterokaryotic allelic
tests (Table 4). Two backcrosses to the parent auxotroph were
made in an effort to rid these two prototrophs of any additional un-
desirable mutations that may have been induced with AR and to de-
termine if the mutation to prototrophy segregated as a single gene.
Prototrophs with normal morphology were selected from the progeny
of each backcross to be used in subsequent crosses. The mutations
to prototrophy in strains AR 6 and AR 17 segregated as a single gene
through two generations of backcrossing (Table 5). Prototrophic
progeny were selected from the last backcross of AR 6 and AR 17
with the 223:3 culture and heterokaryotic allelic tests performed to
reaffirm the presence of a recessive suppressor.

Each prototroph was mated to 1) an gig-_Z culture, 2) an
M+ culture and 3) a compatible sister prototrophic culture.
Growth of these three dikaryons on minimal medium was compared

to the growth of a dikaryon homoallelic for the arg-2 mutation.

40

Table 3. The number of colonies obtained following UV irradiation
that were tested in heterokaryotic allelic tests.

 

Eyosure time in seconds

 

 

Culture 10 20 30 40 50 60 Total
109l5§_<_1_e_;_2_ ll 13 116 9 2 0 151
101716 22:2. 6 3 32 2 2 o 45
10204 35;; 45 79 132 64 67 2 389
101422 333$ 18 25 24 20 24 0 111
10505 313$ 11 ll 5 9 5 l 42
10810 pi_9_;2_ 2 4 16 4 0 0 26

 

Totals 93 135 325 108 100 3 764

41

Table 4. Growth of cultures isolated from minimal medium follow-
ing treatment of mycelial fragments of an arg-Z culture
with acridine red.

 

Growth of isolated cultures
on minimal medium

 

as homokaryons as dikaryons

 

 

No. of cultures (Mutant + arg-2)
tested _a + - +
22 l 21 2 19
a - = no growth on minimal medium; + = growth on min-

imal medium.

42

Table 5. Segregation of the mutations to prototrophy of two cultures
recovered following treatment of an arg-Z culture with
acridine red when backcrossed to an arg-Z strain.

 

 

Strain f1 b1 b2
AR 6 17/5081 17/50 24/50
AR 17 26/50 21/50 23/50

 

a Prototrophs /tota1 progeny.

43

The growth responses of AR 6 and AR 17 after 2 backcrosses to an
arg-Z parent are shown in Figure 2 D and E. The mutation confer-
ring prototrophy in the homokaryon is recessive since dikaryons
homoallelic for the mutation exhibit no nutritional requirement on
minimal medium while those dikaryons heteroallelic for the muta-
tion are arginine dependent.

Recessive suppressor mutations induced with N-methyl-

 

N'-Nitro-N—nitrosoguanidine: Six auxotrophic strains were treated

 

with nitrosoguanidine (NG) using two different methods of exposure.
With the first method, mycelial fragments from each of the 6 strains
were exposed to NC for 45 min, washed and pipetted onto minimal
medium. The second method allowed mycelial fragments of each
strain to grow in the presence of NG in liquid complete medium for
48 hr before plating onto minimal medium. Twenty-three proto-
trophic cultures were isolated from the nicotinic acid-requiring,
adenine-requiring and the 333$ auxotrophic strains using both
methods of exposure (Table 6). None of these 23 cultures possessed
a recessive suppressor mutation as indicated by heterokaryotic
allelic tests.

The Pig-_Z strain yielded 69 prototrophic cultures that were
tested for recessive suppressor mutations; 54 cultures following
treatment using Method I and 15 cultures following exposure by

Method II (Table 6). Two strains scored as having recessive

Figure 2.

44

Growth responses of suppressed strains in heterokaryotic
allelic tests. In each set of dishes, five day old dikaryons
growing on migration-complete (left) and minimal media
(right). In all dishes; top row, dikaryons homoallelic for
$123.12.: heteroallelic for suppressor; row two, homoallelic

for arg-Z; row three, homoallelic for arg-Z and suppres-

sor; bottom row, suppressor X wild type. A) su-l (HA41);

B) su-3 (HA 61); C) su-6 (HA 1034); D) su-9 (AR 6);

E) su-lO (AR 17); F) su-12 (NC 18).

 

 

 

‘M

45

 

46

Table 6. Growth of cultures isolated from minimal medium follow-
ing treatment of mycelial fragments of six auxotrophic
cultures with nitrosoguanidine.

 

Growth of isolated cultures
on minimal medium

 

 

 

Auxotroph as homokaryons as dikaryons
treated No. of cultures (Mutant + 11253—2.)
with NG tested _a + - +
10915 _a_.£1_e_-2 9 7 2 0 2
101716 M 11 7 4 0 4
101422 _a_1;g_—£ 9 3 6 0 6
10505 gig-i l4 l3 1 0 1
10810 M 35 25 10 0 10
10204 ESL;

Method I 168 144 54 l 53

Method II 18 3 15 1 14

a

- = no growth on minimal medium; + 2 growth on min-
imal medium.

47

suppressor mutations in heterokaryotic allelic tests. The mutation
to prototrophy in each of these strains (NC 18 and NG 21) segregated
as a single gene through two generations of backcrossing to the
auxotrophic parent (Table 7). Heterokaryotic allelic tests performed
with the progeny from the second backcros sing reaffirmed that each
strain carried a recessive suppressor of a_1;g_-_2. Growth responses
of NG 18 in heterokaryotic allelic tests are shown in Figure 2 F.

Recessive suppressor mutations obtained following treatment

 

with hydroxylamine: Two hundred thirty arginine independent cultures

 

were isolated from an _a_1;g_-_2 strain in four separate experiments
(Table 8). Each culture was mated with an Egg-_Z strain and the di-
karyon resulting therefrom was transferred to minimal medium.
Eight of the 230 dikaryons did not grow on medium lacking arginine.
The mutation conferring prototrophy in these eight strains segregated
as a single gene through at least two generations of backcrossing to
the parent auxotroph (Table 9). The heterokaryotic allelic tests
performed with progeny from the second and third backcross genera-
tions confirmed that each of these 8 strains carried a recessive
suppressor of the M mutation. Heterokaryotic allelic tests with
3 strains induced with HA are shown in Figure 2 A-C. The growth
responses of some of the suppressed strains as homokaryons that
were induced with either HA, AR or NG are given in Figure 3. The

morphology and growth characteristics of strain HA 41 on minimal

48

Table 7. Segregation of the mutations to prototrophy of two cultures
recovered following treatment of an arg-Z culture with
nitrosoguanidine when backcrossed to an arg-Z strain.

 

 

Strain f1 b1 b2
NG18 25/50a 21/50 22/50
NC 21 25/50 29/50 47/100

 

a Prototrophs /tota1 progeny.

49

Table 8. Growth of cultures isolated from minimal medium follow-
ing treatment of mycelial fragments of an arg-Z culture
with hydroxylamine.

 

Growth of isolated cultures
on minimal medium

 

as homokaryons as dikaryons

 

 

Experiment No. of cultures (Mutant + arg-Z)
No. tested _a + -— +
1 164 20 144 3 141
2 63 28 35 1 34
3 41 4 37 3 34
4 ' 15 l 14 1 13
Totals 283 53 230 8 222
a

- = no growth on minimal medium; + = growth on min-
imal medium.

50

Table 9. Segregation of the mutations to prototrophy of eight strains
following treatment of an arg-2 culture with hydroxylamine
when backcrossed to an arg-Z strain.

 

 

Strain f1 b1 b2 b3
HA 41 26/50 ----- 24/50 25/50
HA 31 18/50 21/50 25/50

HA 61 23/50 ----- 23/50 26/50
HA 132 27/50 26/50 23/50

HA 134 ----- 27/50 28/50

HA 1034 18/50 ----- 17/50

HA 1068 23/50 25/50 23/50

HA 1049 44/50 24/50 29/50

 

Exact number of prototrophs not recorded. One proto-

troph was selected for backcrossing to the arg-Z strain.

Figure 3.

51

Growth of suppressed homokaryons on migration-
complete (MC) and minimal (MM) media. A) _s_u_-_l
(HA 41) MC, left, and MM, right; B-F) top row of
plates is MC, bottom row is MM. B, _s_1__1_-_§ (HA 61);
C, 331) (HA 1034); D, 33:2 (AR 6); E, su_-l(_)

(AR 17); F, 511-12 (NG18).

 

 

 

53

medium as a homokaryon and in dikaryons homoallelic for arg-2
but either homoallelic or heteroallelic for the suppressor, were
considered ideal for the study of somatic recombination.

Prototrophy in the remaining 222 mutants tested was assum-
ed to be due either to back mutation at the arg-Z locus (to wild type)
or to a dominant suppressor mutation. Prototrophs obtained follow-
ing treatment with any mutagenic agent, including HA, which did not
possess a recessive suppressor were not tested further.

Recombination between £h_e suppressor mutations and £1_1_e

 

 

 

arg-2 locus: An attempt was made to recover the arg-Z mutation
from each of the 12 prototrophic strains which initially scored as
having a recessive suppressor mutation. Each strain was crossed
to a compatible wild type strain in an attempt to isolate recombinant
progeny which would possess the arg-Z mutation. With random re-
combination between the suppressor locus and arg-Z, one quarter of
the progeny should require arginine for growth. Linkage of the
suppressor to the arg-Z locus should result in fewer auxotrophic
progeny. The 8 prototrophs induced with HA were mated with a com-
patible wild type strain, the arg-2 mutation was recovered in the
progeny from only one of the 8 crosses (Table 10). Failure to recov-
er any arg-Z recombinants from the other 7 crosses indicated that
the mutation to prototrophy occurred either in close proximity to, or

at, the arg-Z locus. If the mutation to prototrophy did occur within

54

 

 

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0 o a: $-53 m2: «8

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.655 woodponm £023 >comonm .oZ ocflcwmnm .oZ HmuoH 3:3 pommono mcwmuum

 

6852068 388:8 co magnum 09$ 3?? N
mcwmhm pommonmmaflm mo mommono 80.5 cacowoum «concomop ocficwmum mo >Ho>ooom

.oH 3an

55

the fig locus, it did not score as a reverse mutation. The re-
cessive nature of two of these 7 strains when scored in the hetero-
karyotic allelic test is shown in Figure 2 B and C. All 7 of these
prototrophs score similarly to strain gu_-_l in the heterokaryotic
allelic tests. Therefore, either they possess a recessive suppressor
or a mutation within the m locus which confers the wild type
phenotype, but which is recessive to the mutant 33;? allele.

The 19 arginine requiring progeny collected from the cross
HA 41 (s_u_—_l) X 699 (wild type) were mated to a compatible a_rg;_2
tester strain. The newly synthesized dikaryons were plated on min-
imal and migration-complete media. Failure of these dikaryons to
grow on minimal medium was considered proof that the a_r_g_-2 muta-
tion was recovered (Table 10). Growth responses of 9 of the 19
progeny on migration-complete and minimal media are shown in
Figure 4 A-C. The 19 progeny are interesting because only 6 exhibit
a non-leaky requirement for arginine in the heterokaryotic allelic
tests whereas all 19 failed to grow on minimal medium as homokaryons.
Heterokaryotic allelic tests performed with progeny, selected at
random from the 19, are shown in Figure 4 D-F. A clear explanation
for these differences in scoring is not available at this time.

The recovery of arginine dependent progeny from crosses

of suppressed strains induced with AR or NG with wild type strains,

Figure 4.

56

Growth of arginine requiring progeny, derived from the
cross s_u;l (HA 41) X 699 (wild type), as homokaryons
and dikaryons with an a_rg_—_2 tester strain. A-C, Nine
progeny growing on migration-complete (MC), top row,
and minimal media (MM), bottom row. D-F, In each
photograph, first petri dish shows growth of dikaryons
derived from mating the two arginine requiring progeny
shown in the second and third dishes with an a_rg-_2 tester
strain. Top row, MC; bottom row, MM. The dikaryon
formed between homokaryon in the middle dish and the
3.3;; tester is plated on the right side of the first petri
dish in each photograph. The dikaryon formed with
homokaryon of petri dish on far right and the gag-E
tester is plated on the left side of first petri dish in each

photograph.

 

 

 

58

as determined by heterokaryotic allelic tests, was not obtained
(Table 11). Ten progeny from AR 6 X wild type exhibited a require-
ment for arginine when scored as homokaryons at 3 days. However,
growth was detected in 5-7 days as homokaryons and the gig-g muta-
tion was not recovered in heterokaryotic allelic tests. Similar re-
sults were obtained with one of the progeny from NC 18 X wild type
and 3 progeny from NG 21 X wild type.

Supersuppressibility tests with su-l and eight different

 

auxotrophic mutations: The ability of su-l to suppress auxotrophy

 

conferred by mutations other than _a_r_g_-_._2 was tested by examining
progeny of crosses s_u-_l by eight different doubly auxotrophic strains.
Each of the 8 doubly auxotrophic strains possessed the gig-_Z muta-
tion plus one other auxotrophic marker. For example, progeny were

obtained from a cross of su-l arg-Z ade-1+ X su-l+ arg-Z ade-l.

 

 

The four genotypes expected are: su-l+ arg-Z ade-l+, su-l+ arg-Z

 

ade-l, su-l arg-2 ade-l+ and su-l arg-2 ade-l. If su-l does not

 

 

suppress ade-l, all four classes of progeny should be identifiable by
plating on two media, one lacking arginine, the other lacking adenine.
The last two classes should be indistinguishable on these media if

su-l also suppresses ade-l. The progeny from crosses of su-l arg-2

 

with strains carrying the adenine or nicotinic acid mutations were
scored on adenine or nicotinic acid drop-out media. The data show

one recombinant class of individuals which has no requirement for

59

 

o a mo 72-53 m: oz

 

o m 5; 27:3 3 oz

0 0 NS 875...; E 5.

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mwoumou Nuwhm HE? mcormfip 0390.3 whoufldvoh >cowoum mohdfido 093 EC?
-0030 poodponm £033 unnowona .oZ ocwcfiwnm .oZ HmuoH 0“ p088 magnum

 

.83po8 38858 no magnum 09$ 2?». X
mafimfim commonmngm mo mommono 80.3 lanowoam unopcomop ocflcwmnm mo >Ho>ooom .: 3an

60

arginine but possessed requirements for either adenine or nicotinic
acid (Table 12). These data demonstrate that _s_1_1_-_l is incapable of
suppressing auxotrophy resulting from these adenine or nicotinic
acid mutations.

All progeny from crosses of strain su-l arg-2 by the double

 

auxotroph arg-Z arg-6 were tested for their ability to grow on mini-

 

mal medium. Those progeny that displayed a requirement were
subsequently mated to three tester strains which had the following

genetic constitutions: A) arg-Z su-l arg-6+, B) arg-2 su-l+ arg-6+

 

 

and C) arg-2+ su-l+ arg—6 (Table 13 A). The recombinant progeny

 

which possess only the 3.33:2 mutation should produce dikaryons with
testers A and B which will not grow on minimal medium (Figure 5 A).
Those progeny which possess only the 287—2 and £38 mutations
should produce dikaryons with testers A, B and C which will not
grow on minimal medium (Figure 5 B). Those progeny which possess
Eli and the 9.5.33 and Egg-_(i mutations should produce dikaryons
with testers B and C which will not grow..on minimal medium

(Figure 5 C). Only those auxotrophic progeny which possess §_1_I_-_l,
§£g_12 and a_rg_-_6 produce dikaryons with tester A that will grow on
minimal medium (Figure 5 C and Table 13 A). Similar results were
obtained when progeny were examined from crosses of strain s_u_—_1

by the double auxotroph 338i Egg-_l (Table 13 B). The three tester

strains used in the heterokaryotic allelic tests with these progeny

61

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«N Tom anum + + Numum + $me + Tom $me

 

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02303834.. 3390p “swam at“? 2-5m anumv Tom mo mommono So: poflficog >Comonnm

 

 

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62

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m

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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u u .. Twnm + Numfim
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7me Nuwnm X Tum ~-m.~m .m
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025 80.3 >cmwonm “GmeQOv madfimnm wcwmmOHu Scum vm>whmv mcoknhmxfiu mo "33090

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Figure 5.

63

Growth of dikaryons derived from crossing three different
arginine dependent progeny from two crosses with three
different testers. A-C. Progeny from the cross:

arg-Z su-l arg-é+ X arg-Z su-l+ arg-6. In each set of

 

 

plates, top row, tester A, arg-Z su-l arg-6+; bottom
row, tester B, arg-Z su-l+ arg-6+; middle row, tester

C, arg-Z+ su-l+ arg-6. D-F. Progeny from the cross:

 

 

arg-Z su-l arg-l+ X arg-Z su-l+ arg-l. In each set of
plates, top row, tester A, arg-Z su-l arg-1+; bottom row,

tester B, arg-Z su-1+ arklfl middle row, tester C,

 

ag-f’ su-l+ arg-l. For each set of plates, plate at left

 

contains migration-complete medium; plate at right con-

tains minimal medium .

 

 

64

 

65

were the same as those described above except that the tester strain
possessing 123:1) was replaced with a tester strain carrying a_rg;1.
Again the recessive nature of the suppressor and the three genotypic
classes of progeny that exist among progeny exhibiting a requirement
for arginine is evident (Figure 5 D-F and Table 13 B). Since the

frequency with which the recombinant genotypes arg-Z su-l arg-6

 

and arg-Z su-l arg-l is high (30/88 and 30/90 respectively, Table 12),

 

it is concluded that 12.-.1 does not suppress auxotrophy conferred by
either the argi or a_rg-_6 mutations.

Recovery o_f diploid strains from connon-AB heterokaryons:
Four rapidly growing sectors were isolated from common-fl hetero-
karyons, heteroallelic for 5 complementing auxotrophic mutations,
growing on minimal medium. These strains resembled the homo-
karyon in morphology, were prototrophic, and remained stable
through repeated transfers to migration-complete and minimal media
(Figure 6A-C). None of these strains has sectored or acquired a
nutritional requirement.

One presumptive diploid was mated with a haploid, allowed
to fruit, and 200 germinated basidiospores were collected and plated
onto individual petri plates. Growth from 140 germlings was macro-
scopically visible within 14 days. Colonial growth of 13 germlings
was detected only after one month of incubation at 220 C. Many of

the original germlings developed colonies with abnormal morphology

Figure 6.

66

Diploids of Schizophyllum commune. A. Fan-shaped

 

diploid sector arising from the common-fl hetero-
karyon on minimal medium. B. Diploid strain, D-l,
on migration-complete medium and C. on minimal
medium. D. Sectors arising from a presumptive
aneuploid derived from the diploid X haploid cross.
E. Fruiting bodies on a compatible diploid strain
isolated as a single spore from the diploid X haploid

CIOSS.

67

 

68

which eventually produced many sectors (Figure 6D). Many of the
colonies which sectored were later shown to be aneuploids, disomic
for some of the markers that were present in the common-AB heter-
okaryon. Some germlings developed colonies which never sectored
and resembled the homokaryon in morphology. Strains which scored
in the initial heterokaryotic allelic tests as auxotrophic, were not
considered auxotrophic if sectors were subsequently recovered which
possessed the wild type allele for the mutation in question. In these
cases the initial scoring was considered to be made on haploid segre-
gants from strains which were originally disomic for the chromosome
in question. All mutant markers present in the common-fl hetero-
karyon were recovered among the progeny of the diploid X haploid
cross. The expected values for triploid segregation assume that all
products of meiosis are recovered among the progeny of the diploid
X haploid cross and that each of the mutant markers should be ex-
pressed in one-sixth of the progeny. Progeny exhibiting a nutritional
requirement should be monosomic for the chromosome carrying that
particular auxotrophic mutation.

Segregation of auxotrophic mutations and mating-tyg factors

from _a; diploid )_( haploid cross: The frequency of recovery of three

 

mutations, ade-Z, arg-Z and arg-6, closely fit the frequency predict-
ed for trisomic segregation, while two mutations, ade-4 and nic-Z,

were recovered in excess (Table 14).

69

Table 14. The frequency of recovery of mutant markers in 153
progeny of the common-AB diploid X haploid.

 

Expected Observed Differencez/expected Z dZ/e

 

 

 

 

 

 

ade-Z 25.6 29 .5
+
ade-Z 127.4 124 .1
.6
ade-4 25.6 44 13.2
+
ade-4 127.4 109 2.7
15.9*
axg-Z 25.6 24 .1
arg-z+ 127.4 129 .02
.12
arg-6 25.6 26 .006
arg-6+ 127.4 127 .001
.007
nig-Z 25.6 42 10.5
mic-2+ 127.4 111 2.1
12.6*

 

* Significant deviation from expected at the 5% level.

70

The segregation of mating-type factors also shows a
reasonably close fit to the frequency predicted for a diploid X haploid
mating. The £4_2 and l_34_2 mating-type factors that were introduced
via the haploid component were recovered in excess of the expected
frequency whereas recovery of disomics A_41/£1§ and _Bfl/gfl was
less than expected (Table 15). Eight intra-é factor recombinants
were also detected among the progeny. Since the frequency of re-
combinant 5 factors is extremely variable, (Raper, Baxter and
Middleton 1958) no attempt was made to determine their frequency.
Analysis of the segregation of the 3’} factors with the _I_3_ factors is
given in Table 16. The results indicate trisomic segregation, with
the exception that the number of individuals Ail} B_42 is greater than

expected and individuals of mating type A41/A42 B41 is less than

 

expected.
Some progeny that were disomic for chromosomes carrying

dissimilar é and £3 mating-type factors, i. e. , A41/A42 B41/B42,

 

developed fruiting bodies (Figure 6 E). Basidiospores were collected
from one culture and were analyzed for nutritional deficiencies.
Viability was low (67%) but the results were compatible with two genes
segregating for adenine dependence, two genes for arginine depend-
ence, and one gene segregating for nicotinic acid dependence (Table 17).
Recovery of four progeny that had no requirements substantiates the

premise that this diploid was heterozygous for all markers. All

71

Table 15. The expected and observed frequencies of mating-type
factors among progeny from a diploid X haploid cross
based on trisomic segregation.

 

% among Number among progeny
Segregants progeny Expected Observed

 

I. The 5 factor A41 33. 3
77 77

A41/A41 16.7
A41/A42 33.3 51 37
A42 16.7 25 31

II. The E factor B41 33. 3
77 72

B41/B41 16.7
B41/B42 33.3 51 46
B42 16.7 25 35

III. Recombinant

A factor Ax):< ---- -- 8

 

Ax indicates an 1ntra-1_\ factor recombinant.

72

Table 16. Segregation of A and I; mating-type factors among progeny
of the diploid X haploid cross.

 

 

 

 

 

 

 

Mating types recovered Expected Observed
$141241 38. 2 37
£11121} 12.5 16
A4_2 211 12.5 14
A42 B42 4 3 9
A41 B41/B42 25.5 24
A42 B41/B42 8. 5 8
A41/A42 B41 25. 5 l6
A41/A42 B42 8. 5 7
A41/A42 B41/B42 17.0 14
Az§fl* 5
.4224:- 3
Asa/w o

 

Ax indicates an intra-A factor recombinant.

73

Table 17. Segregation for nutritional dependence among 67 progeny
derived from an A41/A42 B41/B42 diploid.

 

 

 

Nutritional No. expected No. observed
requirement among progeny among progeny
. >c<
adenine 50 47
*
arginine 50 49
. . . . **
n1cot1n1c ac1d 33. 5 41

 

Assum1ng segregation at two unlinked loc1. Assum1ng
segregation of one gene.

74

markers present in the common-_A__B heterokaryon have been recover-
ed in segregants of this compatible diploid, except for the ad_e:4_
mutation.

Segregation of the A mating-type factors with auxotrophic
markers is given in Table 18. The expected number of progeny of
Ag genotype includes both those disomic A_41/_A_4_l and those mono-
somic A41. The expected number of progeny of A42 is based on the
recovery of only monosomics, since only one dose of £112 entered
the cross.

Results of the segregation of nutritional markers with g
mating-type factors are presented in Table 19. The expected number
of progeny with the £41 and 1342 mating-type factors was determined
with the same method as used for the A factor in Table 18.

An analysis of segregants isolated from a sectoring aneuploid,
disomic for the A mating-type factor was used to determine linkage
between the markers present in the diploid X haploid cross. Segre-
gants from this strain possess only the _B_4_2 mating-type factor
believed to be carried on a monosomic chromosome. Since the _l_3_4_2
mating-type factor went into the cross in the haploid parent, a cross-
over between the centromere and _Bilg. plus proper alignment at
metaphase II would be necessary to yield a disomic chromosome
homozygous for _B_42. If the mechanism of recombination is similar

to the Parasexual Cycle (Pontecorvo 1956), mitotic crossing over

75

Table 18. Segregation of A mating-type factors with auxotrophic
markers among 153 progeny from the diploid X haploid

 

 

 

 

 

 

 

 

 

 

mating.
Genotype Expected Observed
A41ade-2 17.0 18
A41 ade-4 17.0 22
A41 arg-Z 17.0 9
A41 arg-6 17.0 14
A41 nic-Z 17.0 15
A42 ade-Z 4. 3 8
A42 ade-4 4. 3 8
A42 arg-Z 4. 3 5
£42. a_r_g-_6 4. 3 2

A42 nic-Z 4. 3 5

 

 

76

Table 19. Segregation of E mating-type factors with auxotrophic
markers among 153 progeny from the diploid X haploid

 

 

 

 

 

 

 

 

 

mating.
Genotype Expected Observed
B41ade-2 17.0 13
B41 ade-4 17.0 20
B41 arg-Z 17.0 11
B41 arg-6 17.0 14
321.1. M 17.0 18
B42 ade-Z 4. 3 9
B42 ade-4 4. 3 9
B42 arg-Z 4. 3 6
B42 arg-6 4. 3 6

 

B42 nic-Z 4. 3 8

 

 

77

would be infrequent and haploidization would proceed via stages of
aneuploidy recombining genes on nonhomologous chromosomes. The

data from these segregants indicate that ade-Z, ade-4 and arg-Z were

 

heterozygous in culture 144 (Table 20). Segregant 144A, A41 B42, is
very likely monosomic for chromosomes carrying mating-type factors,

but is apparently disomic for chromosomes carrying ade-Z, ade-4

 

and arg-Z since expression of these markers occurs in secondary
sectors 144Al and l44A2. Segregant 144C, A42 B42, is very likely
monosomic for chromosomes carrying the mating-type factors and

chromosomes carrying ade-4, arg-6 and nic-Z, but disomic for

 

chromosomes carrying ade-2 and arg-Z since the latter two mutant
markers are expressed in 144 C1, a secondary segregant. All seg-
regants possessed the arg-6 and nic-Z markers.

Synthesis o_f multiply auxotrophic strains: For a study of

 

somatic recombination in common-fl diploid strains of §_. commune,
there was a need for a large number of homokaryotic strains with
different mating-type factors and auxotrophic mutations. Various
combinations of these strains were used to synthesize common-Al?)
heterokaryons that were heteroallelic for a number of different,
complementing auxotrophic mutations. A systematic series of
crosses was made which ultimately resulted in two groups of multiply
mutant, auxotrophic, homokaryotic strains that possessed different

mating-type factors. The auxotrophic mutations present in each

78

Table 20. Recovery of mutations present in segregants of an
aneuploid derived from the diploid X haploid cross.

 

Mutations recovered in segregants

Segregant Mating type ade-Z ade—4 arg-Z arg’-6 nic-2

 

144A _A_flgfl yes yes
l44A1 £14121} yes yes yes
144A2 fl_B_4_2 yes yes yes yes yes
144B fig} Egg yes yes yes yes
144C 1334—2 §_4_§ yes yes yes
144Cl £12 _B_4_2 yes yes yes yes yes
144D A42 §_4_2 yes yes yes yes yes
144E 131:2 B42 yes yes yes yes yes

144F A42 B42 yes yes yes yes yes

 

79

group were unique to that group except that the _a_rg_-_2 mutation was
present in all strains of both groups and the recessive suppressor,
s21, was present in some strains of both groups.

The crosses were designed so that a minimum number of
auxotrophic mutations were introduced into each of the crosses.
This method enabled the detection of morphological changes which
may have occurred upon introducing two different mutations into one
strain. A second advantage was that the genotypes of the auxotrophic
progeny from many of the crosses could be determined on drop-out
media and did not necessitate the use of heterokaryotic allelic tests.

Four strains, each of which carried the a_rg-_2 mutation and
one other auxotrophic mutation were first mated with a strain which
possessed the recessive suppressor, ELL—_l, and a_rg-_2 (Table 21).
The recombinant progeny in the first three crosses were identified
on arginine drop-out medium and medium lacking the second growth
factor, e. g. , in the first cross, adenine. Recombinant progeny that
had no requirement for arginine but possessed a requirement for the
second growth factor were transferred to stock bottles for subsequent
experiments.

The genotype of the progeny from the cross involving 313:9
did require the use of heterokaryotic allelic tests. Those progeny
that displayed a requirement for arginine were subsequently mated

to three tester strains of the following constitutions:

80

Table 21. Recombinant progeny derived from crossing strains
possessing two auxotrophic mutations with a sup-
pressed strain, arg-Z su-l.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Desired recom- No. recombinants
Cross binant genotype total progeny

arg-Z su-l +

X arg-Z su-l ads-3 21/85
arg-Z + ade-3
arg-Z su-l +

X arg-Z su-l nic-3 22/78
arg-Z + nic—3
arg-Z su-l +

X arg-Z su-l ade-4 29/105
arg-Z + ade-4
arg-Z su-l +

X arg-Z su-l arg-6 30/92
arg-2 + arg-6
arg-2 su-l +

X arg-Z su-l ade-l 15/92
arg-Z + ade-l
arg-Z su-l +

X arg-Z su-l nic-Z 19/84
arg-Z + nic-2
arg-2 su-l +

X arg-Z su-l ade-2 24/92
arg-Z + ade-Z
arg-2 su-l +

X arg-Z su-l arg-l 30/91
arg-Z + arg-l

 

 

81

A) arg-Z su-l arg-6+, B) arg-2 su-l+ arg-6+, and C) arg-Z+ su-l+

 

 

 

25.3.1” Those progeny which possessed a_rg_-_2, s_u-_l and m
produced dikaryons with tester A that would grow on minimal medium
but dikaryons with testers B and C would not grow on minimal
medium (Figure 5 C).

The second group of multiply auxotrophic homokaryons was
synthesized using the same procedure. The initial matings (crosses
5-8) are given in Table 21. Heterokaryotic allelic tests were per-
formed on all arginine dependent progeny derived from the cross
where a_rg_—_l was present. The progeny were mated to the following

three tester strains: A) arg-Z su-l arg-l+, B) arg-Z su—l+ arg-l+

 

 

and C) arg-Z+ su-l+ arg-l. Only those progeny which were arg-2

 

 

su-l arg—l produced dikaryons with tester A that would grow on

 

minimal medium (Figure 5 F).

The next four crosses made between the 8 newly synthesized
strains are given in Table 22. The procedure eliminated the neces-
sity of using heterokaryotic allelic tests to distinguish between
strains carrying the M and £614 mutations of the first 2 crosses
and the 1513:} and ESL-2.2 mutations of crosses 3 and 4. The recom-
binant progeny in each cross were identified on adenine, nicotinic
acid or arginine drop-out media. Arginine dependency among
progeny from the second cross and the fourth cross resulted from

the arg-6 and arg-l mutations respectively. The progeny of each

82

 

 

 

 

arg-Z
2. arg-Z
arg-Z
3. arg—Z
arg-Z
4. arg-2

arg-Z

su-l +- nic-3

su-lade-4 +
X"
su-l + arg-6

su-lade—l +
X'
su-l + nic—Z

su-lade-Z +
X'
su-l + arg—l

Table 22. Recombinant progeny derived from crossing suppressed
strains, each of which possessed a single different
auxotrophic mutation.

Desired recombinant No. recomb.
Cross genotype total
L arg-2 su-lade-3 +-
X' arg-Z su-lade-3rfic-3 30/108

 

arg-Z su-lade-4 arg-6 13/86

arg-2 su-lade-lrfic-Z 20/98

arg-Z su-lade-Z arg-l 9/71

 

83

cross possessedaigi and _s_u_:_l. The cross made between recombi-
nant progeny derived from crosses l and 2 in Table 22 is shown in
Table 23A. Because the arg-Z mutation was suppressed by 114;) in
all of the progeny, any arginine requirement was attributed to the
11.23.28 mutation. The strains that possessed a requirement for
adenine, nicotinic acid and arginine were mated to two adenine-
requiring testers; one that possessed the gig-_3 mutation and one that
carried the 3M mutation. The strains which failed to grow as
dikaryons on minimal medium with both of these testers possessed
the £2 and a_d_e-_4 mutations. The multiply auxotrophic homokaryons
which comprised the second group were derived from crossing the
recombinants of crosses 3 and 4 in Table 22 and are presented in
Table 23 B. Heterokaryotic allelic tests were performed on all
progeny of each group to determine the mating-type factors of each.

A similar series of crosses was performed to yield strains
which possessed the auxotrophic mutations of each group; however,
none possessed the suppressor. Therefore, common-fl—B matings
between homokaryons from the different groups were heteroallelic
for all auxotrophic mutations except gig-_Z which was always homo-
allelic. The suppressor, s_u-_l, in these matings was either homo-
allelic or heteroallelic depending on the cross.

Verification o_f diploid strains which mssess su-l: To

 

 

circumvent the laborious task of demonstrating trisomic segregation

84

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

wmimfl Twnm N600 N130 + Tam Mulmm
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85

each time a putative diploid strain was isolated, two criteria were
adopted as evidence for the presence of diploidy. Strains that were
presumed to be diploid were plated onto arginine, adenine and
nicotinic acid drop-out media and minimal medium supplemented
with all growth factors. Strains which were prototrophic, except
that they grew very slowly on arginine drop-out media, were con-
sidered to be diploid. Strains that were subsequently used in the
study of somatic recombination were mated to a compatible wild type
strain. Spores were collected from a single fruiting body that de-
veloped on the haploid side of the mating, germinated on yeast me-
dium supplemented with arginine, and the viable germlings were
plated to drop-out media, where auxotrophic requirements were
determined. The mutations conferring auxotrophy in the progeny
were inferred from heterokaryotic allelic tests. The recovery of
all of the mutations present in the common-fl heterokaryon among
the progeny of a cross between the putative diploid and a haploid
wild type strain was considered evidence that the one parent was
diploid and that the diploid was heterozygous for all auxotrophic
mutations. The recovery of auxotrophic progeny derived from two
diploid X haploid crosses is given in Table 24. All mutant markers
present in each common-AB heterokaryon from which these diploid
strains were derived were recovered among the progeny of each cross.

Somatic recombination i_n common-AB diploids: One diploid

 

 

86

 

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SN X hNuQ

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Muofic N130 01wnm N-wnm 7me 0.000 Mu00m N100m 700m

”0ommommom £033 >Com0hm mo HonESZ

 

 

.mommono 033.0: N 03230 803 003.80
tncomonm of S.“ “Cowman 00033.98 00.303830 m0 >H0>000m

.0m 3an

87

strain, D-9, provided most of the recombinants that were analyzed
during this study. D-9 produced a mycelium which was very sparse
on arginine drop-out medium. Recombinants were derived from
dense, white sectors which were conspicuous within 10-20 days after
the plates were inoculated (Figure 7). Inocula obtained from these
sectors were plated to arginine drop-out medium and after 48 hr
growth at 320 C, hyphal tips were isolated. The cultures derived
from the hyphal tip isolations were plated onto adenine, arginine
and nicotinic acid drop-out media and to minimal medium supple-
mented with all growth factors. Cultures that possessed nutritional
requirements were mated to appropriate auxotrophic testers, and
the auxotrophic mutations were inferred from heterokaryotic allelic
tests. Cultures that exhibited no nutritional requirements were
transferred to either migration-complete or yeast medium and
examined periodically for sectors.

Isolation and characterization 9_f recombinants derived from

 

 

common-AB diploids: The results from three separate experiments

 

with D-9 are given in Table 25. The genetic constitution of D-9 with
established linkage groups is also given.

Recombinant progeny have been placed in three separate
classes based on their state of ploidy. The first class is comprised

of 129 recombinants which in all respects appear to be haploid. All

of these individuals possessed the nic-2 mutation. None of the

 

88

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89

 

90

Table 25. Spontaneous recombinants recovered from rapidly grow-
ing sectors of the common-AB diploid, D-9.

 

Common-AB Diploid, D-9

A41B41arg-2 + ade—Z + nic-2 arg-l + +
A41 B41 arg-Z su—l + ade-3 + + ade-4 nic-3

Number of
recombs.

 

I. Haploids:

arg-Z su-l ade-Z+ ade-3+nic-2 arg--l+ ade-4 nic-3 69

 

+
arg-2 su-l ade-2+ ade-3+nic-2 arg-l+ ade-4 nic-3 6O

 

 

II. Aneuploids:

 

 

 

 

 

+ +

arg-Z su-l ade-2+ ade-3 nic—Z arg-l + nic-3 2
+ ade-4
+ + . +
arg-Z su-l ade-2 ade-3 + arg-l ade-4 nic-3 l
ade-3 nic-2
. . *
III. D1p101ds:

arg-Z su-l ade-Z + nic-2 arg-l + + 12
arg-2 su-l + ade-3 + + ade-4 nic-3

\

 

* Assumed to be homozygous for su-l since none exhibit a
requirement for arginine; some may be homozygous for wild type
alleles at other loci.

91

recombinants possessed the argi mutation or the argi mutation
without 311:}, which is to be expected since recombinants were
selected on arginine drop—out medium. Furthermore, ad_e;2 which
is linked to a_rgLZ and is in repulsion with su_-l, was not recovered
among these recombinants. The n_i_c_:-_3 mutation segregated inde-
pendently of all auxotrophic mutations. N0 crossing over between
linked markers was detected in these strains. To demonstrate that
these individuals were haploid, three cultures were mated to a
compatible wild type strain and progeny were collected from single
fruiting bodies that developed on the wild type side of the cross.
The results indicated no evidence for triploid segregation (Table 26).
The aneuploid recombinants consisted of three strains.
Two exhibited no requirement for arginine or adenine although both
strains possessed the n_10_:_2 mutation (Table 25). These strains were

assumed to be aneuploid, at least for the arg-l ade-4 chromosome,

 

although their genetic constitution has not been ascertained. They
may be recombinant for these genes. They were plated onto yeast
medium and examined periodically for sectors. Neither strain has
sectored.

The third recombinant aneuploid was assumed to be aneu-

ploid for only the ade-3 nic-Z chromosome. The ade-3 mutation

 

was detected twice while the nic-2 mutation was detected only once

in two separate heterokaryotic allelic tests. The recombinant was

92

 

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93

mated to a compatible wild type strain and progeny were collected
from a single fruiting body. The observed number of nicotinic acid-
requiring progeny (19/102) was compatible with the expected number
for trisomic segregation (17/102) assuming only one dose of M
entered the cross. Segregation of the 53132.3 mutation was confounded
with the segregation of ad_e:_4. There are two possible genotypes

for the 51532;} mutation on a disomic chromosome. If ad_e_-_3 were
heterozygous, a mating between this recombinant and a wild type

strain, where only ade-3, ade-4 and nic—Z mutations are depicted

 

is given below:

 

 

ade-3 +
ade-4 + nic-Z
X'

+ + +

 

The expected number of adenine-requiring progeny would be
59/102. If ade—3 were homozygous and disomic, two doses of ade-3
and one dose of nic-Z would enter the cross as indicated below:

ade-3 +
ade-4 ade-3 nic-Z

 

 

X

+ + +

 

The expected number of adenine-requiring progeny would be

94

76. 5/102. The number of adenine-requiring progeny recovered was
75/102. A mitotic crossover between £12} and n_ic-_2 would be
required to yield a disomic homozygous for 3513;3’: since $2.3. and
r_.i_c_-_2 were in repulsion in the D-9 diploid. If the frequency of cross-

ing over in triploids of _S_. commune is reduced, as is the case in

Drosophila, (Bridges and Anderson 1925; Redfield 1930, 1932), the

 

mating between the aneuploid homozygous for a_cl§-_3 and a wild type
strain should yield progeny that always have the adenine requirement,
conferred by a_de_—3, when the nicotinic acid requirement is expressed.
However, if £22 were heterozygous and disomic, 8. 5/102 progeny
would be expected to have a nicotinic acid requirement in the ab-
sence of an adenine requirement. The observed number of progeny
which possessed only a nicotinic acid requirement was 8. These
data are compatible with £132 being heterozygous and disomic even
though the number of adenine-requiring progeny (75/102) was higher
than expected (59. 5/102, Table 26).

The third group of recombinants was also selected on the
ability to grow normally in the absence of arginine (Table 25). None
exhibited requirements for adenine or nicotinic acid. These strains
appeared to be homozygous for s_u;_l and Egg-_Z but heterozygous for
the remaining auxotrophic mutations. These strains were plated
onto yeast medium where some sectoring was observed. Segregants

of each strain were tested for nutritional requirements on drop-out

95

media and auxotrophic mutations were inferred from heterokaryotic
allelic tests. Two of the 12 strains of this recombinant group sectored
and an analysis of the segregation of biochemical mutations is given
in Table 27. The id_e_—_3 and a_rg_-_l mutations were not recovered
among secondary segregants of recombinant 128. Segregant 128f is
the only individual that possesses the file—12 mutation and the 31%;?
mutation in the absence of fly}. This would suggest that 128 was not
homozygous for _s_u_-l or that nuclei still heterozygous for Ewere
transferred with the nuclei homozygous for sud. in the original trans-
fer to yeast medium. The segregation of auxotrophic mutations in
the progeny of matings between 128e and 128f and a wild type strain
reaffirmed that these recombinants are haploid (Table 28).

Segregants obtained from recombinant 194 possessed only
the pig—_Z mutation (Table 27).

Recombinants were also recovered from diploid D-9,
following 10, 20 or 30 sec exposure to UV irradiation (Table 29).
One recombinant genotype differed from those obtained from spon-
taneous recombination. The suppressor was not prosent in this
recombinant and this was the only recombinant isolated that possess-
ed the flei mutation. Since 32122 and ELI were in repulsion, a
crossover between 33;]. and a_d_e-_2 was required to allow them to

segregate as a unit in any given nucleus. The results suggest that

this recombinant, isolated from an arginine deficient medium, was

96

Table 27. Growth of primary and secondary segregants derived
from the D-9 diploid.

 

Test media: Growth on minimal medium of dikaryons

Drop-out derived by mating the segregants with
and fully auxotrophic testers.

supple-

mented Tester strains:

a
Segregant A _Ii C 1_3_ ade-2 ade-3 ade—4 arg-l arg-Z nic-2 nic-3

 

 

128 +b+ + + + + + + + + +
128a T + + + + + +
128b + + - + + -
128C + J! + +
128d + + - + + -
128e - + - + + + - + t - +
128f - — - 1r - + - + - — -
128g + + - + + + + + + - +
194 + + + +
194a + + - + - +
194b + + — + — +
194C + + - + - +

 

a Test media: A = adenine drop-out; B = arginine drop-out;
C = nicotinic acid drop—out; D = fully supplemented media. + =
growth, - = no growth on the indicated medium.

97

 

 

 

 

 

 

 

 

 

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98

 

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99

probably carried over in the original inoculum transferred from the
rapidly growing sector. Hyphal tip isolation resulted in the purifi-
cation of a recombinant nuclear type that would not be expected to
be detected using these methods. The other two haploid genotypes
were common among spontaneous recombinants, and displayed a
pink color. The single recombinant that possessed the ad_e;2 as
well as the _a_dgil mutation was white.

The recombinants that exhibited dense growth on all drop-
out media were assumed to be diploids, homozygous for 3112. This
group consisted of 25 individuals (Table 29 II). These strains were
maintained on yeast medium and examined periodically for segre-
gants. Fifteen of the 25 strains sectored on yeast medium (Table 30).
A total of 27 sectors analyzed for nutritional requirements on drop-
out medium exhibited a requirement for nicotinic acid. Hetero-
karyotic allelic tests indicated that 26 possessed only the _n_ic_—2
mutation, one sector was a double mutant, having n_1_c;2 and ni_c_-_3,
but none of the sectors possessed only the n_i_c_-_3 mutation (Table 30).

The appearance of colored sectors in the common-A_B
diploid culture on yeast medium provided another method for the
detection of recombinational events. Only a limited number of
recombinants were recovered and analyzed from a single diploid
strain, D-25. Strain D-25 was assumed to be diploid and hetero-

zygous for all auxotrophic mutations since it was repeatedly

100

 

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101

transferred from the stock bottle to drop-out media. It possessed
only a requirement for arginine.

The recombinant sectors derived from D-25 are presented
in Table 31. Since there was no selection against arginine-requir-
ing recombinants, it should be possible to recover both the a_rg;2
and a_r_g;§ mutations. The second recombinant genotype listed in
Table 31 must have arisen as the result of a crossover between _s_u_:_l
and a_de;2 as only the _a_rg_-_2 mutation was recovered. The third
recombinant nuclear type listed possessed the 22% mutation.

312-} and n_ic_-_2 went into the diploid in repulsion and it is considered
significant that when Iail_e_-_3 was recovered, Mwas not. The
fourth recombinant possessed the £8.73 requirement and was isolated

from a sector of very restricted growth.

102

 

 

H enmuw muoflc 73541300 7me Nuowc mumpm Nnopm Tum Numnm
+ + + + + + +

 

and loan .0 m -0 .m umhm -08 .no .0 :0 m udm umhm
3 o +m.+~pvp+fi +N.mp+NpHN

 

H oumnm Mnowc 70pm vuopm Tmnm Nuowc muopm Nuopm 7.0m Nuwhm
+ + + + + + + + +

 

nan -08 .0 m .0 m ‘uwnm -08 .0 m .0 m nSm umh
3 +0 m.+~pvp+fi N.+mp+NpHNm

 

mucmcwficcooou 30 .OZ moguosmm “confinacooom

ouwnm m-08 + vnopm + + muopd + .730 Nuwnm 31m Nv<
+ + 70pm + 7me Nnowc + Nrmpm + Numnm Sum Nv<

 

3-0 .2295 m<éoeeoo

 

 

.fsvoum mmummm pofifiwsxo
was» 8330>8 we: 30 mmmnm :3 .Ho mucuuom Mafia mm 853008 ammo.» 803 @3203
0.83 :03? .mN-Q 6303930 .mlflucoEvcoo a 803 put/Cop m8d038000n mdoocmucomm Am 03mm.

DISCUSSION

Ultraviolet light and chemical mutagens were employed

in an intensive search for recessive suppressors in Schizophyllum

 

commune. Of 764 prototrophic colonies obtained by treatment of 6
auxotrophic strains with UV, none possessed a recessive suppressor.
If suppressors were present among these prototrophs, they were
either partially dominant or dominant and, were of no value for
detection of recombinational events in somatic cells.

Twelve cultures possessing suppressors of the a_rg-_2
mutation were isolated following treatment of mycelial fragments
with chemical mutagens. Eight strains were isolated following
treatment with hydroxylamine (HA), two strains were isolated follow-
ing treatment with nitrosoguanidine (NG) and two after treatment
with acridine red (AR). No suppressors were obtained for loci
other than alg:_2. The recovery of the 23$ mutation from one
prototrophic strain induced with HA indicated that the mutation to
prototrophy did not occur at the 33;; locus. The recovery of the
a_rg-_2 mutation from the other eleven suppressors has not been
accomplished although relatively small numbers of progeny have
been tested. The mutations could have occurred within the 2333—2

103

104

locus in each of the eleven strains as a result of intragenic suppres-
sion. However, dikaryons synthesized between these eleven proto-
trophic strains and an auxotroph possessing arg:_2 failed to grow on
minimal medium. The data suggest that if the mutations to proto-
trophy occurred at the a_rg_-_2 locus, they are recessive to the a_rg-_2
allele or that prototrophy may have resulted from a recessive sup-
pressor which is very closely linked to the £535.; locus.

The two mutations which suppress auxotrophy conferred by
a_rfl which were induced with either NO or AR appear to be differ-
ent from those induced with HA. In heterokaryotic allelic tests on
medium lacking arginine, comparisons were made between dikaryons
that were heteroallelic for. the .suppressor but homoallelic for Egi
and dikaryons that are homoallelic for both a_rg-_2 and the suppressor.
Differences in growth rates were easily detected up to 4 days but
were not discernible after 7-8 days. These suppressors may
correspond to the semidominant suppressors reported by other
investigators (Weglenski 1966; Gajewski and Litwinska 1968). In
contrast, dikaryons homoallelic for a_rg-_2 but heteroallelic for the
suppressors induced with HA are clearly distinguishable after
several weeks on arginine-less medium from dikaryons homoallelic
for the suppressor and gig-_Z.

It is unclear how EFL} suppresses auxotrophy conferred by

arg-Z. If su-l were analogous to amber suppressors in bacteria

105

(Sarabhai, Stretton, Brenner and Bolle 1964) or the super-suppressors
in yeast (Hawthorne and Mortimer 1963) it may have been possible

to suppress auxotrophy at other loci. No biochemical data are
available which would indicate the type of mutation present within
other auxotrophic loci in _S_. commune. It is entirely possible that
none of these loci possess amber mutations. That mutations to
prototrophy which map near to, or within, the a_rg—_2 mutation were
induced with HA, NG and AR suggests that the a_r_g-_2 locus does not
possess a nonsense mutation. Hydroxylamine appears to react
specifically with cytosine producing one-way base transitions where-
by the original guanine-cytosine base pair is replaced by an adenine-
thymine base pair (Brown and Schell 1961; Freese, Bsutz-Freese,
and Bautz 1961). In Neurospora, HA was shown to produce reversions
only in strains which revert by base-pair substitution (Malling 1966).
Reversion studies of amber and ochre mutations within the rII genes
of bacteriophage T4 (Champe and Benzer 1962; Brenner, Stretton

and Kaplan 1965) indicate that neither mutation can be induced to re-
vert by HA. Acridine dyes appear to cause deletions or insertions

of base-pairs in the DNA which result in frameshift mutations
(Brammar, Berger, and Yanofsky 1967). These frameshift mutations
have been postulated as a mechanism for intragenic suppression.

The specific action of NC is thought to be a methylation of guanine

which ultimately leads to mispairing and base substitution

106

(Singer, Fraenkel-Conrat, Greenberg and Michelson 1968). Because
all three mutagens induce mutations which map at, or very close to,
the a_rg_—_2 locus, they may act on the same mutated site. If the
mutation to auxotrophy at the 353:2 locus were by the induction of

an ochre or amber triplet, no transition of the type induced by HA
would restore prototrophy. All the 230 prototrophs recovered
following treatment with HA would have necessarily been suppressors
(Table 8). A more plausible interpretation would be that both NG
and HA are capable of causing base-pair substitutions within the
a_rg;2_ locus which lead to prototrophy and that the mutation which
had lead to auxotrophy was not a mutation to a nonsense codon.

The results shown in Table 10 and Figure 6 are surprising.
Only 6 of the 19 arginine-requiring progeny obtained from the cross
HA 41 (_si-_l) X 699, failed to grow as dikaryons with an gig-_Z tester.
These data suggest that 13 of these progeny carried a suppressor
but the arginine requirement was poorly suppressed in the homo-
karyotic state. Other examples of these differences in scoring were
observed in strains induced with NO and AR (Table 11).

Regardless of how ELI suppresses auxotrophy conferred by
£35.23 it is ideally suited for experiments on somatic recombination.
It is incapable of suppressing auxotrophy at 8 loci tested thus far and
it maps within one of the few established linkage groups in S. commune.

The technique used to obtain diploid strains was rewarding.

107

The experimental results clearly show that stable vegetative diploid
strains of S. commune have been isolated from a common-_A_B
heterokaryon. All mutant markers that are present in both com-
ponents of the common-AB heterokaryon can be recovered from
diploid strains (Table 14 and 24).

The segregation of mating-type factors and nutritional
markers among progeny of a diploid X haploid cross follow the
predicted ratios of triploid segregation assuming recovery of all
types of meiotic products.

Two mutant markers, 53M and M (each of which was
contributed by a different nucleus), were recovered with a frequency
greater than expected by trisomic segregation (Table 14). It is
conceivable that the diploid nucleus is not stable prior to the forma-
tion of the fusion nucleus in the basidium, as reported in Coprinus
1a 0 us (Casselton 1965). The fusion of an aneuploid nucleus with
the haploid nucleus prior to meiosis could account for the high fre-
quency of recovery of M and 119;?“ Another possible explanation
for the high frequencyof-recovery of 9.39:3 and M is that haploid-
ization involving chromosomes with these markers occurred very
soon after basidiospore germination, and that some strains originally
disomic for certain markers were scored as haploids. That haploid-
ization of some disomic chromosomes is occurring early is sub-

stantiated by results obtained for the segregation of mating-type

108

factors. Although the observed values agree reasonably well with
the expected for trisomic segregation (P= . 2-. 05), it can be seen
that disomic A and }_3 factors occur less frequently than expected
(Table 15 and 16). Another explanation would be that there is selec-
tive pressure against individuals disomic for chromosomes possess-
ing the M and n_iE_-_2 markers in the heterozygous condition, and
that these individuals were among those non-viable germlings.
Progeny from the diploid X haploid cross that have disomic
chromosomes apparently undergo haploidization via stages of
aneuploidy with reassociation of whole chromosomes, as in the
Parasexual Cycle (Pontecorvo 1956). From a limited number of
primary and secondary segregants, at least four linkage groups
are shown to occur. Recovery of a_rg-_6 and E23 among all segre-
gants of culture 144 indicates that these markers very probably are
not on chromosomes known to be disomic, i. e. , chromosomes
carrying the _A mating-type factors and chromosomes carrying

mutant markers, ade-2, ade-4 and arg-Z. The genetic map of S.

 

commune (Ellingboe and Raper 1962a) places Eli-_Z and 23:3 in the
same linkage group approximately 25 cross-over units apart. The
data obtained from segregants of culture 144 is consistant with this
linkage arrangement. Mutant markers a_de;2 and a_rgfi, entered the
diploid X haploid cross in the cis-arrangement and they segregated

as a unit among segregants of culture 144 (Table 20). For culture

109

144, recombinationhad not occurred between a;g-_2_ and 513:} in
meiosis. Segregation of age-_‘l was independent of ad_e-_2 and Egg-_Z
and the A mating-type factors and is interpreted as belonging to
another linkage group. It remains to be established whether M
and/or _r_1_i_c_-_2 are linked to the B factor or whether they belong to
separate linkage groups or to the same linkage group.

The recovery of strains which were disomic and homozygous
or heterozygous for mating-type factors, from the diploid X haploid
mating, provides a way for obtaining commom-A, common-S or
fully compatible diploids for a study of somatic recombination.

The stability of these diploids is uncertain but at least eight strains
which scored as compatible diploids have failed to fruit or sector.

The common-AB diploids heterozygous for §_u_-l provided a

method, analogous to that used in Aspergillus nidulans (Pontecorvo

 

and Kafer 1956), for studying somatic recombination. The greatest
number of spontaneous recombinants (129/154) obtained from D-9
were haploids (Table 25). None of the 129 haploid strains appear to
have recombined genes via crossing over. The failure to recover
Q_i_c_;2 and a_d_e__-_3 in equal frequency is not understood. The _n_i£_:_2
mutation which is in the same linkage group with 3512;} and in re-
pulsion in D-9, was recovered in all of the 129 haploid recombinants.
Since n_ic-_2 was in repulsion with s_u_-_1, their being in the same

linkage group is excluded since segregation as a unit would require

110

crossing over in each of the 129 recombinants. It is conceivable
that the high frequency of recovery of n_19_-_2 results from selective
pressure against haploid strains which carry the a_c_l£_-_3 mutation.
Since selection for recombination is based on detection of rapid
growth, differential growth rates between haploids carrying a_de-_3 or
_rl_'1_<_:-_2 may account for selection. There may be selective pressure
for strains carrying the £212 marker. There was also a higher
frequency of recovery of M in the diploid X haploid cross (Table 14).

The recovery of 339;} in all of the somatic segregants is
easier to explain. Linkage data obtained from fruiting bodies of
common-_A__B heterokaryons and compatible matings (Middleton 1964)
place M and gig—_l in the same linkage group, approximately 4
to 8 crossover units apart. Recombinants were recovered on an
arginine drop-out medium which excludes those recombinants that
possess an arginine requirement. Since a_r_g;1 and _a_<_i£-_4 were in
repulsion, (barring crossing over between the markers, which
could result in segregation of the wild type allele at each locus), all
haploid strains should carry the ad_e-_:l mutation.

None of these haploid recombinants show recombination
between linked genes. These results suggest that haploidization
proceeds via stages of aneuploidy, thereby recombining genes only
on different chromosomes.

The segregation of ade-3 and nic-2 in the progeny of a

lll

cross of a putative aneuploid X wild type strain was compatible
with trisomic segregation. The recovery of 8 progeny with the 2i_c_-_2
mutation in the absence of an adenine requirement was compatible
only with file—J and M being heterozygous and disomic.

Further evidence for haploidization via stages of aneuploidy
was observed in genotypes of secondary segregants derived from a
prototrophic primary segregant of D-9 (Table 27). The recovery of
the fiL-Z and M mutation apparently without s_u;1 indicates that
although the original prototrophic segregant, 128, appeared to grow
well on arginine drop—out medium, it may still have been hetero-
zygous for §u_-l.

Analysis of recombinants obtained following treatment with
UV light revealed no increase in the incidence of crossing over
(Table 29). One recombinant genotype was unique in that it was
isolated from arginine drop-out medium but it did not possess _s_u_-1.
Examination of the nicotinic acid-requiring segregants of prototrophic
sectors isolated following UV treatment indicated strong selection
pressure for the chromosome carrying Bic—'2 (Table 30).

Experiments designed to select for recombinants on the
basis of colored segregants provided a means whereby arginine-
dependent segregants could be recovered. This is possible because
pink color is present in haploid strains that carry a_de_-_4 in the

absence of ade-2, and in diploids homozygous for ade-4 but

112

heterozygous for M. Thus, any recombinational event leading
to either of these two situations is detectable on yeast medium.
Although no biochemical data are available about the product that
accumulates or the position of the block in $911 or ad_e__-_2 mutants,
it appears that the ad_e_-_2 mutation affects an earlier step in the
adenine biosynthetic pathway than 3.25:4. With this method, only

4 segregants were recovered and analyzed, a sample too small to
draw definite conclusions. The a_rg-_6 mutation was recovered in 2
of the 4 cases. Modifications of this technique may provide an
efficient way to obtain segregants.

The limited data on somatic recombination in common-AB
diploids indicate a parasexual mechanism. The techniques used
would not have detected Specific Factor Transfer since the A and
}_3 factors were similar. The methodology for detecting recombina-
tional events in diploids is now available for the critical experiments
that will test the role.of mating-type factors in somatic recombina-
tional events. It should now be possible to synthesize common-A,
common-B and compatible diploid strains for the purpose of examin-
ing somatic recombination.

In earlier experiments (Ellingboe and Raper 1962a;
Ellingboe 1963), the origin of class I recombinants was explained
by meiosis-like recombination within the dikaryon of the incompatible

di-mon mating. Using a compatible diploid heterozygous for the

113

recessive suppressor, experiments can be conducted to determine
whether the Parasexual Cycle, Specific Factor Transfer, or a
meiosis-like mechanism of recombination are indeed the mechanisms
by which genetic material is exchanged in somatic cells or if these
mechanisms are simply a reflection of the technique used to detect

recombination.

SUMMARY

Recessive suppressor mutations of the arg-Z locus have

been isolated in Schizophyllum commune by treating mycelial frag-

 

ments of an a_rg:2 strain with hydroxylamine, nitrosoguanidine or
acridine red and selecting for prototrophs. Each mutation to pro-
totrophy segregated as a single gene through two or three genera-
tions of backcrossing to the gig-_Z parent. One suppressor, _s_\_1_:_l,
maps outside the a£g_-2 locus and is incapable of suppressing aux-

otrophy conferred by mutations at eight other loci. su-l is recessive

 

in the dikaryon and common-_A_B diploid. Prototrophy in the remain-
ing eleven strains may have resulted from intragenic suppression

or from a mutation in a suppressor that is very closely linked to

the arg-2 locus. If the suppression is intragenic, all are recessive
to the a_rL-E allele.

A technique was developed which permitted the recovery of
stable vegetative diploid strains from common-A__B heterokaryons of
S. commune. Common-A_B diploid strains heterozygous for auxo-
trophic mutations are prototrophic and resemble the homokaryon in

morphology. Those strains homozygous for arg-Z but heterozygous

114

115

for $1.11 and other auxotrophic mutations exhibit very slow growth

on arginine drop-out medium. Segregation of five auxotrophic
mutations and mating-type factors of a diploid X haploid cross
closely fit the predicted values for triploid segregation. Segregation
of mutant markers among somatic segregants of an aneuploid derived
from the diploid X haploid cross indicated that at least four linkage
groups are present in S. commune.

The recessive suppressor, _s_u__-_l, was used to detect somatic
recombination in common-A_B diploids of S. commune. Recombinants
were recovered from dense fast-growing , sectors on arginine drop-
out medium. The majority of recombinants analyzed in this study
were haploid. Aneuploid and diploid strains were also recovered
and analyzed. Genetic analysis of spontaneous recombinants indicat-
ed that crossing over is rare and that haploidization proceeds via
stages of aneuploidy. Recombinants derived following treatment with
UV light showed no increase in the frequency of crossing over. The
preliminary results favor a parasexual mechanism of recombination

in common-_A_B diploids .

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