Copyright 0 1983 by the Genetics Society of America
MITOCHONDRIAL FOUR-POINT CROSSES IN ASPERGILLUS
NIDULANS: MAPPING O F A SUPPRESSOR OF A
MITOCHONDRIALLY INHERITED COLD-SENSITIVE MUTATION
RICHARD B. WARING’
AND
CLAUDIO SCAZZOCCHIO
Department of Biology, University of Essex, Colchester, United Kingdom
Manuscript received August 1,1980
Revised copy accepted November 12, 1982
ABSTRACT
Four-point mitochondrial crosses were conducted in heterokaryons of Aspergillus nidulans. The mutations used were (oliAZ), conferring resistance to
oligomycin, (camAll2), conferring resistance to chloramphenicol; (cs-67), conferring cold-sensitivity, and (sumD16), a suppressor of (cs-67). Initially, the
crosses were conducted by observing the segregation of extranuclear markers
in heterokaryotic sectors emerging from the original point of heterokaryosis.
This showed that (camAZl2), (cs-67) and (sumD16) were linked but were
probably all unlinked to (oIiAl). Second, four-point crosses were conducted
using a double marker selection technique, in which (camAl12) and (oliAl)
were always set in repulsion and the frequency of the phenotypes produced by
the segregation of the mutant and wild-type alleles of (cs-67) and (sumD) were
observed in (camAll2 oliAl) recombinants. From these results we concluded
that (camAllZ), (cs-67) and (sumD16) were linked and probably mapped in the
order given. It was observed that the two nuclear types of conidia from a
heterokaryon often had a dissimilar frequency distribution of the segregants of
a mitochondrial cross.
LTHOUGH mitochondrial recombination has been shown in Podospora
A
anserina (BELCOUR
and BEGEL1977), Schizosaccharomyces pombe (SEITZMAYR,WOLFand KAUDEWITZ
1978), Kluyveromyces lactis (BRUNNER,
DE COBOS
and GRIFFITHS
1977), Neurospora crassa (MANNELLA
and LAMBOWITZ1978) and
1975), extensive formal genetics
Aspergillus nidulans (ROWLANDSand TURNER
has only been carried out with the mitochondrial genome of Saccharomyces
cerevisiae. In A. nidulans the establishment of a genetic map has been hindered
by a lack of genetic markers and a reliable way of identifying the independent
events leading to segregation in a mitochondrial cross. To date four mitochondrially inherited phenotypes have been described: oligomycin resistance mapping at (oliA) and (oliB) (ROWLANDS
and TURNER
1973; TURNER
and WATSON
1976), chloramphenicol resistance mapping at (camA) and (camB) (GUNATILLEKE, SCAZZOCCHIO
and ARST1975; LAZARUSand TURNER
1977), cold sensitivity
mapping at (cs-67) (WALDRON
and ROBERTS1973) and suppression of (CS-67)
mapping at the (sumD) locus (WARING
and SCAZZOCCHIO
1980). (oliA) and (oliB)
Present address: Department of Biochemistry, University of Manchester, Instltute of Sclence and Technology,
P 0 Box 88, Manchester, M60 IQD Unlted Kingdom.
Genetics 103 409-428 March, 1983
410
R.
B. W A R I N G AND C. SCAZZOCCHIO
are difficult to separate by recombination and probably map in the same gene.
1977).
The same is true for (camA) and (camB) (LAZARUSand TURNER
Four different methods have been used to study the formal mitochondrial
genetics of A. nidulans: (1)random spore analysis in which heterokaryons are
set between strains carrying different mitochondrial markers and then haploid
conidiospores are collected, plated out, grown up and scored for the markers
segregating. Results obtained by this method are very inconsistent because in
reality the hyphae of heterokaryons often contain a high proportion of homokaryotic sections. ROWLANDand TURNER
(1975) partially reduced this variability
by defining a modified recombination frequency, in which they included in
their analysis only those colonies with a reassorted mitochondrial genotype,
that is, with either a recombinant genotype or showing reassortment of nuclear
and mitochondrial genomes. (2) cleistothecial analysis, whereby mitochondrial
crosses are scored using the fruiting bodies, produced during the sexual cycle of
A. nidulans. This method, however, did not give very consistent results (MASON
and TURNER
1975). (3) heterokaryon sector analysis based on the observation
that heterokaryons produce sectors which usually become rapidly homogeneous
with respect to their mitochondrial genome (i.e., the phenotype of the conidia
from a single sector is uniform and completely stable with respect to any
mitochondrial markers). One can, therefore, treat each sector as representing
the outcome of a single independent segregational event (ROWLANDS
and TURNER
1973, 1974; GUNATILLEKE
1974). (4) ROWLANDSand TURNER
(1975) analyzed
mitochondrial crosses using a marker selection technique but found that selecting for different markers in the same conidial sample from a heterokaryon
suggested dissimilar linkage maps. We considered that their method was possibly prone to biased sampling and so attempted to use this method with a
number of modifications described later.
In this paper four-point mitochondrial crosses have been conducted using
heterokaryon sector analysis and a marker selection technique. These showed
that (camAIlP), (cs-67) and (sumD26) are linked, probably mapping in the order
given, and that all three are probably unlinked to (oliA1). Although the crosses
were set for mapping purposes, the results obtained suggested some preliminary
conclusions about the formal mitochondrial genetics of A. nidulans.
Since this work was completed we have sequenced a considerable portion of
the mitochondrial genome of A. nidulans. A number of genes have been
sequenced and located on the physical map (DAVIESet al. 1982). Some of these
have intervening sequences that are likely to require the expression of mitochondrial genes in order to be correctly spliced. We have also identified a
number of unassigned open reading frames, some of which are probably bona
fide genes. New mitochondrial mutations and the mapping techniques developed
here will help to identify the function of the unknown genes and to investigate
the mechanism of mRNA splicing.
MATERIALS AND METHODS
Strains: All strains used in this work are derived from the original Glasgow wild-type strain and
are essentially isogenic except for the defined markers. To carry out mitochondrial crosses,
MITOCHONDRIAL CROSSES IN ASPERGILLUS
411
extranuclear markers were kept in the following two nuclear backgrounds: biAl IuAi cnxH5 and
yA2 pyroA4 cnxC3. biAl, luA1 and pyroA4 have nutritional requirements for biotin, leucine and
pyridoxine, respectively; yA2 produces yellow conidial color; cnxC3 and cnxH5 are both unable to
use either nitrate or hypoxanthine as a nitrogen source. On medium containing oligomycin, strains
carrying luA1 or IuAl (oIiAl) grew much slower than strains carrying luA+ or IuA+ (oIiAl),
respectively. This effect was partially alleviated by supplementing the media with twice the usual
amount of leucine. To date, only strains carrying the aforementioned two nuclear backgrounds
produced well-balanced heterokaryons without having to transfer the heterokaryons to fresh
medium.
In vivo techniques: These are described in WARING
and SCAZZOCCHIO
(1980). Chloramphenicol
and oligomycin resistance were routinely scored on media containing, respectively, 2.5 mg/ml and
3.0 pg/ml of the appropriate inhibitor. The cold-sensitive and suppressed phenotypes, conferred
respectively by the mitochondrial genotypes (cs-67) and (cs-67 sumDl6), were scored by their
reduced growth at 20' on complete medium. A strain with the mitochondrial genotype (cs-67+
sumD16) was indistinguishable from the wild type when grown at 20'. The degree of growth at 20'
on an arbitrary scale from 0-10 was 10 for the wild type, 3 for (cs-67), 8 for (cs-67 sumD16) and 10
for (sumD16).
Heterokaryon formation: Strains carrying the markers biAl luAl cnxH5 and yA2 pyroA4 cnxC3
were inoculated at exactly the same point on minimal medium, containing sodium nitrate as the
sole nitrogen source (REVER1965). After 3 days' growth at 37' a heterokaryon core appeared in
which good cytoplasmic mixing had usually occurred.
To analyze mitochondrial crosses in heterokaryons, good cytoplasmic mixing must be achieved
by establishing well-balanced heterokaryons. However, this encourages the production of sectors,
which are homogenous for their mitochondrial genome. When conidia are collected from a
heterokaryon beginning to sector, a comparatively small number of independent segregational
events are being analyzed. Each of these independent events will be represented by a different
number of conidia, particularly as sectoring promotes rapid radial growth. Thus, a biased conidial
sample will result. Because ROWLANDS
and TURNER
(1974, 1975) only sampled single young heterokaryons, we suspected that some of the variability they encountered, particularly with the marker
selection technique, was due to biased sampling. During the course of this work it became clear to
us that sampling from a number of heterokaryons gave more reproducible results, and we believe
that a bias is present during the initiation of sectors before they are even visible.
(1973) and GUNATILLEKE
(1974) include a transfer stage in establishBoth ROWLANDSand TURNER
ing heterokaryons. As we felt that this might produce a bias by promoting early sectoring, through
growing out onto fresh medium, heterokaryons were set using strains carrying markers such that
no transfer stage was required.
(1974,
Random spore analysis: This was a modification of the method of ROWLANDSand TURNER
1975). Heterokaryons were set as described and incubated at 37' until heterokaryotic cores had just
begun to appear. Conidia were collected from 20 cores for each cross and plated out on two types
of media which selected separately for the two parental nuclear backgrounds. The mitochondrial
genotype of the resulting colonies was then deduced by replicating them onto appropriate test
media, using a velvet replicating technique. The frequency of recombination between two extranuclear markers was expressed as: number of colonies with an extranuclear recombinant genotype/
number of colonies in which either of the two extranuclear markers being analyzed have reassorted
with respect to the nuclear genomes, or more concisely: number of recombinants/number of
reassorted. The number of reassorted/total number of colonies was known as the reassortment and
indicated the degree of cytoplasmic mixing that had occurred. The two nuclear backgrounds were
treated separately for the calculation of reassortment and recombination frequency values.
(1974) and
Heterokaryon sector analysis: The method is based on that of ROWLANDSand TURNER
GUNATILLEKE
1974). The two parent strains were set in heterokaryons and incubated at 37O until
sectors had grown out which were then velvet replicated onto appropriate test media. Individual
sectors could only be identified after analysis of the test plates, as it was not possible to define
these from the original heterokaryon plates. A sample of conidia from each sector, taken from the
original heterokaryon plates, was streakedout on two different types of media, selecting for the
two parental nuclear backgrounds. Master plates were made by taking up to five colonies per sector
per nuclear background, and the extranuclear phenotype of each sector was identified on test
412
R. B. WARINC AND C. SCAZZOCCHIO
FIGURE1.-Scoring mitochondrial crosses by heterokaryon sector analysis. In this example
heterokaryons were set between the strains yA2 pyroA4 cnxC3 (oliA1) and biA1 luAl cnxH5
(comAII2 cs-67). The phenotypes of each sector were derived as described in MATERIALS AND
MFTHODS by velvet replication onto the following media, incubated at 37' unless otherwise indicated:
Top left, master plate: middle left, synthetic complete (S.C.) + chloramphenicol: middle right,
complete medium (20'): bottom left, (S.C.) + oligomycin: bottom right, (S.C.). "Synthetic complete"
indicates minimal medium (COVE1966) plus all the necessary supplements (pyridoxine, leucine and
et al. (1953). Sectors are
biotin). "Complete medium" is a rich medium described in PONTECORVO
marked with their mitochondrial genotype (+ indicates wild-type allele, indicates mutant allele).
the markers always being written in the following order: (camAI12 oliAl cs-67). hetpl = a
heteroplasmic sector.
-
plates. If all of the colonies derived from a single sector did not have identical phenotypes this
sector was considered heteroplasmic and discarded. An example of this technique is shown in
Figure 1. Defining a sector in this manner means that two independent events producing juxtapositioned sectors with identical phenotypes will be scored as a single event. Conversely. a single
event, producing two sectors, that are separated by another sector will be scored twice. These two
sources of error make it impossible to identify independent events, but if it is assumed that these
events act independently of the mitochondrial markers present in the cross and in opposite
directions they should approximately compensate for one another. To define what constituted an
MITOCHONDRIAL CROSSES IN ASPERGILLUS
413
independent sector, the following principle was adopted. If two sectors of similar phenotype were
separated on both sides by a sector of a different phenotype, they were considered to be two
independent sectors (e.g., sectors C and D, or E and F in Figure 1).If this was not the case, they
were considered to represent only one segregational event (e.g., A and B in Figure 1).
Double marker selection analysis: The two selected extranuclear markers were (camA112) and
(oliA1) were set in the repulsion configuration in all crosses so that (camAI12 oliA1) recombinants
could be selected for. For each cross, 20 heterokaryons of the two parents (nuclear backgrounds:
yA2 pyroA4 cnxC3 and biA1 luA1 cnxH5) were set on minimal medium plus nitrate. After 3 days,
conidia were collected from the center of all of the heterokaryon cores. (camAI12 oliA1) recombinants were isolated by plating out the conidia on two types of media that selected separately for the
two nuclear backgrounds but contained both chloramphenicol and oligomycin to select for the
(camAll2 oliA1) recombinants. Some of these putative recombinants proved to be heteroplasmons
(ROWLANDS
and TURNER
1974, 1975). The only locus that was heteroplasmic at a significant
frequency was (oliAl),probably because the selection of the (oliA1) allele by oligomycin was being
counteracted by selection for [oliA+), due to the pleiotropic slow growth effect of (oliA1). Such
heteroplasmons were detected by testing the putative (camA112 oliA1) colonies on nonselective
medium at 37" and looking for sectoring which indicated the presence of a genome carrying the
(oliA+)marker. All heteroplasmons were discarded. The (cs-67) and (sumD16) markers were scored
by replicating the (camAI12 oliAI) recombinants onto two test plates: complete medium alone and
complete medium containing chloramphenicol and oligomycin. These were incubated at 20" and
scored for the three phenotypes: wild type, suppressed and cold-sensitive (the phenotype of strains
with a (cs-67+ sumDI6) genotype is the same as that of the wild type). Occasionally, a colony was
found to have a different phenotype on the two test media or to have a phenotype unlike any of the
three expected phenotypes, indicating that the colony was heteroplasmic. Such colonies were also
discarded. (comAll2 oliA1) recombinants in the yA2 pyroA4 cnxC3 background were scored very
reliably using the velvet replication technique. However, a master plate/wire replicator technique
was used for colonies having the biA1 luA1 cnxH5 background because strains carrying (oliA1) and
luA1 grew particularly slowly on oligomycin, and the colonies had to be checked carefully to ensure
that they were homoplasmic at the (oliA1) locus.
RESULTS
Transmission frequencies in heterokaryon sectors
The frequency with which a mitochondrial marker is transmitted in the
segregants of a mitochondrial cross is known as the transmission frequency
(DUJON, SLONIMSKI
and WEILL
1974). This can be best studied in A. nidulans
using heterokaryon sector analysis. The transmission frequencies of (camAZZZ),
(oliAZ) and (cs-67) in four four-point crosses are shown in Table 1. There was
bias against (oliAl) in all four crosses, whereas the transmission frequencies
for (camAZZ2) and (cs-67) were approximately 50%. The transmission frequencies for (sumDZ6) are not presented, as the mitochondrial genotype (sumDZ6)
cannot be distinguished phenotypically from the wild type. In other crosses in
which both parents carried the mutation (cs-67), (sumDl6) gave transmission
frequencies varying between 45 and 55% (data not shown).
There are three possible contributing causes to the low transmission frequency of (oliAZ). First, the mutation might act prior to cytoplasmic mixing by
lowering the input ratio of its own mitochondrial genome. Second, the mutation
SLONIMSKI
and WEILL1974) such that it acts at
could have a polar effect (DUJON,
the level of recombination, affecting a process such as gene conversion. Third,
the mutation might be selected against after cytoplasmic mixing during vegetative segregation. Given that (oliAl) is probably a point mutation similar to
414
R. B. WARING AND C. SCAZZOCCHIO
TABLE 1
Frequency of transmission of the mitochondrial markers camA112, OliAl and
cs-67 in four-point crosses using heterokoryon sector analysis
TransTransmission
No. of
mission
Total NO. of sectors fresectors
freno. of - quency ____ quency
sectors Corn+ corn(w) olj' o1j(W)
+
+ + )
cs-67 sumD)
sectors
-~
Transmission
fiequency
CS+
CS-
@I
86
41
45
52
61
25
29
37
49
57
59
30
29
49
37
22
37
31
28
47
+ )
67 sumD)
64
33
31
48
44
20
31
37
27
42
y (camA olla +
+ )
+ cs-67 sumD)
gr(
73
39
34
47
45
28
38
35
38
52
(
gr(camA
oliA
No. of
+
( +
+
gr(camA oliA
+
y (camA +
oliA
gr( +
+
+
+
)
67 sumD)
y and gr represent the nuclear backgrounds yA2 pyroA4 cnxC3 and biA1 luAl cnxH5, respectively.
oIi2 in S. cerevisiae (MACINO
and TZAGOLOFF
1980), we consider a polar effect
unlikely. Thus far, mutations causing polarity have been shown to be deletions
or insertions (STRAUSBERG
and BUTOW1981; DUJON1980). One can decide which
of the other two possibilities are more likely by considering those sectors in
which recombination has taken place between (camA) and (oliA). A decrease
in the relative input of (oliA1) would only alter the frequency of recombination
between (camA) and (&A) but not the ratio of the two possible recombinant
genotypes (see also DUJON,SLONIMSKI
and WEILL1974).In fact, the recombinant
class containing (oliA2) was less frequent in all crosses than the one containing
(oliA') (data not shown). This is what one would expect if (oJiAZ) acts after
recombination has taken place and is consistent with the observation that
strains carrying (oliAl) have a reduced growth rate.
Heterokaryon sector analysis of recombination in four-point crosses
The frequency of recombination between each pair of markers in each cross
is presented in Table 2. Each pair of markers was in coupling in two crosses
and in repulsion in two crosses except for (cs-67) and (sumDZ6) which were
always in coupling. The four crosses gave reasonably comparable results and
so average recombination frequencies were calculated. Equal weight was given
to each cross because it could not be assumed that they were strictly equivalent.
Although the recombinant genotype (cs-67+ sumDZ6) could not be identified
phenotypically, its reciprocal recombinant class (cs-67 sumD+) clearly occurred
rarely in each cross. As there is no phenotypic selection against the (cs-67
SumD') genotype, we have estimated the recombination frequency of (Cs-67)
with (sumDzf-3) by assuming that reciprocal recombinant classes occur with
equal frequency at the population level and doubling the frequency of the (CS67 sumD+) recombinant class.
415
MITOCHONDRIAL CROSSES IN ASPERGILLUS
_ -
U
na z0
-
- w
> w
m
r
h
N
'3g.
N
v
z:
V
se
h
E
TIE
2 m
?s
sg
:-g
9
h
N
m
- Q
gs
U
5
c z
m
Jc
$2
$4
v)
.-
v)
5
v)h
3
E
S E
26
h
4
Y
%
st
2
h
m
v
V
Em
R
X
!!
+2
c
01
.v)
h
ri
m
-
h
r(
w
v
A!=
2.2
,vJ
N
E
N
.m
25
0-0
52
&
24
ri
h
E
Q'S2
5
$
N
N
22
z '0
-c
v)
N
2s
'G CT
%
Y E
3 .5-
ab
3
b
;
.+
v
E .,o
6
23
zz
ri
0
C
h
h
9
,v
E
..-0
2 2
v)
G d
m m
5
$
2
S- mE
.O
5;;;
CkU
5
.9 ,o
42
E
E2
Y
s
c m
U
.&Q ,gl
(0
-r
2
$E
h -
9
E
+ 2
(0
h
--
0
Z E
d o
- h
9
E
+:,
3-
h
g::
+ 2
h
(0
ac,
9
E
+ & + & +;
%Z
7
+%
p
7
E
+z
w
v
h &
+7
4
0
-s +
0
7
5+
Y V
A &
2:
E 2
I
W
g
:
b
z:
m
gz
0
9
gzcx
:2
w
:=
B Ti
416
R.
B. WARING AND
C.
SCAZZOCCHIO
(oJiA1) showed an average recombination frequency of 33% with (camA112)
and 28% with (cs-67). (cs-67) showed an average recombination frequency of
14% with (camAZZ2) and approximately 8% with (sumD16). In preliminary
three-point crosses involving (camA112), (cs-67) and (sumDZ6), including ones
with (cs-67) and (sumDZ6) in repulsion, recombination frequencies were consistent with the four-point crosses presented here. The average of the estimated
recombination frequencies for (cs-67) - (sumD16) was 12%.These crosses were
not strictly comparable to the four-point crosses as the (oJiA1) marker was not
included, nor were the nuclear backgrounds identical.
Is (camAZ12) more closely linked to (cs-67) or (sumD26)? Of a total of 282
sectors analyzed in the four crosses, 11 could be identified as showing recombination between (cs-67) and (sumD16), and these belong to the (cs-67 sumD+)
recombinant class. Five of these were also recombinants for the alleles of
(camA) and (cs-67), whereas six were also recombinants for the alleles of
(camA) and (sumD). This meant that we were not able to deduce the relative
positions of (camA), (cs-67) and (sumDZ6) using heterokaryon sector analysis.
Double marker selection analysis
ROWLANDSand TURNER
(1975) concluded that a double marker selection
technique could not be used for mapping mitochondrial markers because of
irreproducibility and the apparent presence of nonreciprocal recombination.
We considered that both of these effects may have resulted from biased
sampling due to the way in which the crosses were conducted and so made
four modifications. First, we collected conidia from 20 identically set heterokaryons, rather than a single heterokaryon. Second, we used strains with nuclear
backgrounds such that heterokaryons could be established directly on forcing
medium because we thought transferring heterokaryons to fresh medium might
promote early sectoring and, hence, biased samples. Third, we analyzed much
larger sample sizes. Fourth, we analyzed each mitochondrial cross separately in
the two types of conidia present, one type having one parental nuclear genotype
and the second the other parental nuclear genotype. This was because we had
observed that this was an important variable in the random spore analysis of
heterokaryons, as is shown in Table 3 for two mitochondrial crosses. A x2
analysis showed that in both crosses the frequency of recombination between
(cs-67) and (camAZZ2) was significantly different in the two nuclear backgrounds.
To guard against any unidentified effect of the mitochondrial markers or their
nuclear backgrounds, eight crosses were performed with the full permutations
of the following three variables: coupled and repulsion configuration of (cs-67)
with (sumD16); coupled and repulsion configuration of both (cs-67) and
(sumDZ6) with both (camA112) and (oliA2); and interchanging the parental
nuclear backgrounds for each mitochondrial cross.
This method makes the assumption that reciprocal recombinational events
will have the same number of representatives in the total sample analyzed.
Work on yeast indicates that recombination is not reciprocal at the molecular
level (VANWINKLE-SWIFT
and BIRKY1978). However, except for polar crosses
417
MITOCHONDRIAL CROSSES IN ASPERGILLUS
TABLE 3
Dissimilar frequency of recombination of mitochondrial markers in the two
conidial nuclear types in heterokaryons
Colonies reassorted for (comA) or
(cs-67)
Total
colonies
scored
_
Cross
A
B
ye
_
_
gr
667 168
786 791
Total
_
y
~
gr
134 43
280 111
_
Nonrecombinants
_
_
y
gr
121 28
220 70
Recombinants
_
_
y
gr
13
15
41
60
Reassort-
Recombination
frequency
ment
70
_
y
gr
0.20 0.26
0.36 0.14
y
gr
x2
Level of
significance
10
21
35
37
13.49
9.19
0.001
0.01
Cross A was yA2 pyroA4 cnxC3 to biAl luA2 cnxH5 (camA112 cs-67); cross B was yA2 pyroA4
cnxC3 (cs-67 sumD16) to biA1 luAl cnxH5 (camA112). The frequency of recombination between
(camA122) and (cs-67) was measured in bath crosses using random spare analysis (MATERIALS AND
METHODS). This was done separately in the two nuclear backgrounds. x2 analysis consisted of
comparing the number of recombinants and the number of nonrecombinant reassorted colonies in
one nuclear background with those in the other, giving a 2 X 2 contingency table with one degree
of freedom.
” y and gr, see Table 1.
and crosses in which segregants are selected against at the phenotypic levels,
reciprocal recombinant classes are produced with approximately equal frequencies at the population level. An exception to this has been noted for reciprocal
recombinant genotypes of chloroplast loci of Chlamydomonas (HARRIS
et al.
1977). Heterokaryon sector analysis showed that only (oJiA1) could possibly
have a polar effect. It also showed that (oliAZ) is probably selected against at
the phenotypic level. Neither of these should affect the assumption because
(oliA1) was always one of the selected markers.
Heterokaryons were set to carry out four-point mitochondrial crosses in
which (camA112) and (oliAl) were always in repulsion. (camAI12 oliA1)
recombinants were selected for and these then scored for the segregation of the
two remaining markers, (cs-67) and (sumD16), with a view to establishing the
linkage relationship of all four markers. As a strain with a (sumD16) genotype
has the same phenotype as the wild type, one cannot determine the relative
frequencies of the various types of crossover event in a single cross alone, and,
therefore, all of the crosses must be analyzed as a unit. The validity of such a
procedure will now be considered.
The mitochondrial crosses fell into two groups on the basis of whether (cs67) and (oliA2) were originally in repulsion or in coupling (Table 4). At first
sight, the latter group appeared to give reasonably consistent results. In the four
crosses in which (oliA1) and (cs-67) were in repulsion there was significant
heterogeneity, particularly in the biAl luA1 cnxH5 nuclear background. A
reason for some of the variability could be seen by calculating the ratio of the
(cs-67) allele to the (cs-67+) allele in the (camA122 oliA1) recombinants (Table
4). The allele that appeared to be present in relative excess in the colonies with
a biAl luA1 cnxH5 background was also the allele that originated from the biA2
luAZ cnxH5 parent. A consideration of all of the data in Table 4 revealed that
418
R. B. WARING AND C. SCAZZOCCHIO
A
-
a??"
6%
mmt-
2-
cc99
*-*
0 t . N
"0
9 5
E$
4qh
" . t
&G$$
2.e s
D 'Z
gm
m m m
h.t;m
**
U
y?fi
LD??
C O N
h
22%
Fm
h
e cm!
? ? 4
"?@!
3"
g;'$zfb
Il-y""Yj
"3g 2
z.c2 g c
s?-v
W N
zc my my my 33 - 1
;%e"
g 2 8.E
2sm13us
Z-??
t
.
k
&?+
2:;
t.N
i?Zh
?3l 3
a63
z0gs;zg
N
0
2.2
N
;I ::
p5
Eo3*'C g
* m a
D m
h
a?
E(DN
N
col?
4 9 h
*-m
? J =? p ? ?
x;
m m m
h ( D ( D
Y C C " C
W N
.e
8 g@+
' D & m " m
1z",.;
"m
:se3
55
m
00
;.
2
I
w-n
I
2
$ s$qg
g s ?e"!
L
z$?*
.e
3
2o
23
2m
n
cCasm:
z+t
0
y1
i
l
gm$j
p?
a
&gz.aa,
2 ;ggs,,
2,zmU
¶ U
0
pa= a
2 -2 -c
5':c h25
.E
g*;lE
z
?
z
? ? ?
3
c"
G2J
ym 5m 10 2
3
222 ; 2 2 2 2
2gc:33m
Pe'-,2,eg
.-
g q $
Ez'gav.M
t.N
t-
h
3
3
33%- I 3
11 2 .e .E 3 s
D
$74212
MhEZ
22;
222
m - 3
m 3 v
t
(ON?.
.
.-
u
aZ--Yo.
gg2 z ' s ;
22:
&
"
3
N
4
3
23
ha??
w23
22:
L?
ri
N
t.-
h
c!
n
?ai?
X"r;
3
a
,cx
a
a2
2
$ $gzlz
6
E =ti I +
%
x
z
0
s
*
r
$?$!Cqg
2 22:
3
c???
22%
O N 3
C O
m
-
-
+
2
h
+I I
+
U
-HI
I
$ 8- 5
2
-
;y z
W
N
h
(D
ti I
+
U
(D
tl I
+
U
U
..
* I
I
.,
.,
:
s
2
x
c 9a;.ca'
c!
i
l
e
;gas:$
+E 3.E c:
D a
ah g O $
.3
4
0
- -ng&$3
&zz:
(D
+
h
'0 z g c $ q 2 . a
-Za%J,,:o
0 Bm
, -.-2- i
e ~"
, mY m
s
m
9
j.I
v
* I ' I
3
e?Ja+&c
+
p\
0
* I
I
..
+
-
W ' Z G 3 0 5.2
a
$Zsz:3gs 5
~ 2 $ ; U a D ~0
&:$ 9 2
a
$;z
4
E
+z
PI
g +;
.5+
0
?
E
+z
v-
F;'
Z
0
a
zz:
F;'
Z
2
a
z;z
--
--
aE
2+
4
6
.t
+;
4+
T
E
+e
vv
:+
(0
h
i+
-s +
0
T
E
+z
U-
,.l
v
,-..-
U
m
'.E
U3A623
v
6%
z
v - E
mi+*
.
U
'CzCm. 2c "e-g &
" Cz" c
2
N -3 I L3
n
% z s / z $z51$
a%?ls
v
:
U
$,sWmQC
g g $c !$mso 32
0"
h
2.-3
:go$::?
2E3;.$;
szglg
~r,
2
:
a
G
3:;ig.g
N
sm-,cw
<2a E2 G6 z g 2 1 2
$
5x
$
:7%3zg
v?z
L?N?
m - o
w
a
SgEm
-c:g;2
3
3
a
???
n
x
U
'3
n
o . 2 w z g ;
;2 2 ; : gs
U
h.
k.*IZ
g551g
e
p ' m5 m +m m
s e
2 .-s
h??
ggW
m
g
* .z
2
L?
%
?
I
3
gsz
:U
x
-
o
qilh
a m 5 02
2
5
2 - i :
.E
3
2
'1°C
$ a
:
V
> R m
3
-3 ;
$
q p ? ?
;g
!?
6%
-2 j % ; C " Z @ $
h
&
a
0 $ z g
(D
h
-aE
+z
h+
(0
h
z
?+
?
E
+E
v-
0
.C-zELk%5k
-
(D
E
p wmo ,. -,-ac ,4 z
Ia
2 2 uC Zx.s0 g e 3
x g g ::y;t-& .:
I
&o
$:sgii"$zps
:2 2 2 m aS
0
mmsw-*
2
2;
m w -
k %
b
g.;X;$
c c 1 3
5s g % < s & . g
3 a- c z = 3 c
4s 8 w z $ 4
mAm-4
a
o.>
S.Z+E
gz0x,.
c
m m U"
P Zccc E
419
MITOCHONDRIAL CROSSES IN ASPERGILLUS
this had happened in all of the crosses except one: yA2 pyroA4 cnxC3 (camAZ22
CS-67sumDZ6) to biAZ luAZ cnxH5 (oliAl).
The variation in the ratio of the (cs-67) alleles was analyzed statistically in
those crosses in which (cs-67) and (oliA1) were in coupling. The nuclear
background in which the cross was analyzed was treated as an independent
variable. A
analysis (with seven degrees of freedom) showed no overall
heterogeneity at a 0.05 level of significance (x2= 10.44). However, a analysis
of a 2 X 2 contingency table, designed to test the interaction of the nuclear
background in which the (cs-67) alleles originated and the nuclear background
in which their ratio was scored, was significant at the 0.01 level (2= 8.65, with
one degree of freedom. We believe the higher than expected frequency of
appearance of the (cs-67) marker in the background in which it originated
reflects a genuine feature of the genetic system. However, further experimentation is required to substantiate this view.
As the major source of heterogeneity, identified before, could be partially
overcome by averaging the relative frequencies over the two nuclear backgrounds in which they were obtained, we considered that it was valid to analyze
the crosses as a unit.
In Table 5, we have considered six possible orders for the four markers.
(oliAZ) is always shown as an outside marker since heterokaryon sector analysis
showed that the other three were loosely linked. The first four orders show (cs67) and (sumDZ6) as neighbours, whereas the last two show (camAZZ2) in
between them. As the genotype (cs-67+ sumD16) has the wild-type phenotype,
only the relative frequencies of the (cs-67 sumD+)and (cs-67 sumDZ6) genotypes
could be ascertained in any cross. The crossover events producing these genotypes will depend on the cross involved and the order of the markers. Table 5
shows schematically the types of crossover events that would have been
required to produce these two genotypes in the four main mitochondrial crosses
for each order postulated and the mean relative frequency with which they
would have occurred in practice as derived from the data in Table 4. Each mean
relative frequency figure was the average relative frequency of two crosses set
with interchanged parental backgrounds, each already averaged over the two
nuclear backgrounds in which it was scored (see Table 4).
On the assumption that a double crossover event occurs less frequently than
a single crossover event, the marker order that fits the experimental data best
can be identified. Order 5, (oliA1 cs-67 camAZZ2 sumD16) is probably wrong
because a double crossover event would have occurred more frequently than a
single crossover event, Order 6 is probably wrong because the two single
crossover events indicate that (oliAZ) and (sumD16) recombine much more
frequently than (sumDZ6) and (camAllZ), whereas the two double crossover
events, which both include a crossover between (camAZZ2) and (cs-67),indicate
that the converse is true. Order 1, (oliAl cs-67 sumDZ6 camAZ12) appears to be
wrong because a triple crossover event would have occurred more frequently
than a single. Order 3 (oliAl camAI12 sumDZ6 cs-67) appears to be wrong
because a triple crossover event would have occurred more frequently than a
double. Orders 2 and 4 which both have the order (camAZ12 cs-67 sumDZ6) in
2
2
420
R. B. WARING AND C. SCAZZOCCHIO
TABLE 5
Determination of map order by correlation of the observed relative frequencies or
recombinants with types of crossover event
Type of cross-over event
Map order with four mitochondrial crosses
1.oliA
cs-67 sumD camA
+
+
oli
+ cs + cam
+
oli cs sum
+ + + cam
+ +
oli cs
+ + sum cam
oli
+
. ! L . X X ! a ! J X K
+
+
cs
+
sum
sum
53.8
10.5
42.0
17.8
cam
7.7
23.5
5.9
22.5
2. oliA sumD cs-67 camA
oli
+
+
sum
oli sum
+
+
oli sum
+
+
+
+
+
cs
cam
cs
cs
cam
+
+
+
+
10.5
42.0
17.8
+
+
23.5
7.7
22.5
5.9
cam
cs
sum + cam
3. oliA camA sumD cs-67
+ + +
oli
+ cam sum cs
+ sum +
oli
+ cam + cs
+ sum cs
oli
+ cam + +
oli
+ + cs
+ cam sum +
4. oliA camA cs-67 sumD
+ + +
oli
+ cam cs sum
+ + sum
oli
+ cam cs +
oli
+ cs sum
+ cam + +
oli
+ cs +
+ cam + sum
5. oliA cs-67 camA sum D
+ + +
oli
+ cs cam sum
+ + sum
oli
+ cs cam +
oli cs
+ sum
+ + cam +
oli
cs
+ +
+ + cam sum
oli
53.8
53.8
10.5
42.0
17.8
7.7
23.5
5.9
22.5
53.8
10.5
42.0
17.8
7.7
23.5
5.9
22.5
53.8
10.5
42.0
17.8
7.7
23.5
5.9
22.5
42 1
MITOCHONDRIAL CROSSES IN ASPERGILLUS
TABLE 5-continued
Type of cross-over event
Map order with four mitochondrial crosses
s
l
X
~
!
X
s
%
X
K
6. oliA sumD camA cs-67
ob
+
+
+
sum cam
oli sum +
+ + cam
oli sum +
+ + cam
oli
+ +
+ sum cam
+
+
53.8
10.5
42.0
17.8
cs
cs
cs
7.7
23.5
cs
5.9
22.5
+
+
The data of Table 4 are considered under six possible map orders. The four mitochondrial crosses
(each was repeated with interchanged parental nuclear background) are considered under each map
order. The figures given are mean relative frequencies from the crosses in Table 4 of either the
(oliAI camAI12 cs-67 sumDI6) genotype having the suppressed phenotype or the (oliAl camAI12
CS-67 sumD+) genotype having the cold-sensitive phenotype. The figures are the mean relative
frequencies of two crosses, having interchanged parental nuclear backgrounds, each averaged over
the two nuclear backgrounds in which the cross was scored (these figures are found in the farthest
right column of Table 4). The mean relative frequencies are placed under the type of crossover
event, schematically represented, that would be required to produce the genotype that occurs with
that mean relative frequency in the appropriate cross. The four types of mitochondrial cross are
listed in the same order as Table 4.
common fit the data. However, do they do this significantly better than orders
1and 3 which have the order (camA112 sumD16 cs-67) in common?
Eight independent crosses have been performed. The mean frequencies
(derived from the average of the two nuclear backgrounds) of the triple crossover
event under orders 2 or 4 were 10.3, 5.1, 5.6 and 6.4% (see Table 4). The mean
frequencies of the single crossover event under order 2 or the double crossover
event under order 4 were 14.2, 6.8, 19.7 and 16.0%(see in Table 4). We made the
hypothesis that the frequencies of the triple crossover events were significantly
less than those of the single (order 2) or double (order 4) crossover events. The
data were then tested against the null hypothesis that there was no difference
between the two classes of frequencies. A one-tailed Mann-Whitney U test
(MANNand WHITNEY
1947) was conducted with n l = n2 = 4, which gives U =
1. The probability of U being this low was 0.029. This indicated that the order
camA112 cs-67 sumD16 (orders 2 and 4) was considerably more likely than the
order camA112 sumD26 cs-67 (orders 1and 3).
Two qualifying points must be made concerning the frequencies of any two
types of crossover event that we compared. First, they were never obtained
from the same cross, and second, the frequency of one crossover event was also
derived from crosses in which (oliAl) and (cs-67) were in coupling, whereas
the frequency of the other crossover event was always derived in crosses with
these two markers in repulsion. Despite this we consider that the most likely
map order is (camA112 cs-67 sumDl6).
It was not possible to distinguish between order 2, oliA1 sumD16 CS-67
camA112, and order 4, oliA1 camAI22 cs-67 sumD16. This essentially indicated
422
R. B. WARING AND
C.
SCAZZOCCHIO
that (oIiA1) showed little linkage to any of the other three markers. When order
2 (or order 4 by a similar argument) was used the frequencies of recombination
between (camAZ12) and (cs-67) and between (cs-67) and (sumD16) could be
compared using the data in Table 5. The relative frequency of the former was
estimated as the average of 23.5 and 22.5% which equals 23.0% and of the latter
as the average of 10.5 and 17.8% which equals 14.2%.This indicated that (cs-67)
recombined with (camA112) about 1.5 times more frequently than with
(sumD16). This compared favorably with the results obtained in heterokaryon
sector analysis.
DISCUSSION
To clarify the analysis of our results, the methods used to investigate mitochondrial recombination in A. nidulans will be discussed, with comparisons
being made with S. cerevisiae. A mitochondrial cross conducted using heterokaryons can be envisaged as producing three classes of “segregants” on the
basis of the degree of cytoplasmic mixing undergone by the segregant’s markers.
The first involves segregants whose markers never underwent cytoplasmic
mixing but that were derived from homokaryotic sections of the heterokaryon.
This is not strictly a true segregant class. A close but not complete analogy to
this class in S. cerevisiae is found in the occasional end bud segregant that buds
off distal to the point of cell fusion shortly after fusion and appears to contain
exclusively the mitochondria of the parent that formed that end (STRAUSBERG
and PERLMAN
1978). The second class consists of segregants whose markers
were located in mitochondria that underwent cytoplasmic mixing but did not
fuse with mitochondria of a different genotype. The third class contains segregants whose markers were located in mitochondria that fused with mitochondria of different genotype. The ratio of the three classes is not the same in
different heterokaryons of A. nidulans, and recombination frequencies expressed as a ratio of the total conidia in the heterokaryon are very variable
(ROWLANDS
and TURNER
1974, 1975; WARING1978). ROWLANDS
and TURNER
(1974,1975) attempted to circumvent this by calculating recombination frequencies as the ratio of the number of recombinants to the number of colonies in
which either of the extranuclear markers have reassorted with respect to their
nuclear genotype. The basis of this approach was to exclude the contribution of
class one segregants from the cross. However, it also meant excluding the
segregants from classes 2 and 3 in which the two parental mitochondrial
genotypes had segregated in their original parental nuclear background. This
modification did not sufficiently reduce the variation in recombination frequencies (ROWLANDSand TURNER
1975; WARING
1978).
Recombination frequencies calculated as the ratio of reassorted types are
often dissimilar in the two nuclear backgrounds in which the cross is scored. If
the input of the two parental mitochondrial genomes is not equal, the two
conidia types may not receive the same ratio of recombinant to reassorted types,
the critical factor probably being the relative proportion of mitochondria that
exchange nuclear backgrounds without undergoing fusion with mitochondria
of a different genotype.
MITOCHONDRIAL CROSSES IN ASPERGILLUS
423
DUJON,SLONIMSKI
and WEILL(1974) have shown that in S. cerevisiae the
frequency of recombination of mitochondrial markers depends upon the relative
input of the two parental mitochondrial genomes. In A. nidulans each hyphal
fusion event is likely to have a different mitochondrial genome input ratio
resulting in a different frequency of recombination. The input ratios might take
any value from 0:lto 1:0,the recombination frequency being maximal when the
input ratio is 1:l (DUJON, SLONIMSKI
and WEILL
1974). An extreme input ratio of
mitochondrial genome may often be correlated with an extreme input ratio of
the two nuclei and a corresponding extreme output ratio of the two types of
nuclei in conidia. In such individual hyphal fusion events, the mitochondrial
segregants would be more frequently distributed with the more frequent nuclear
type. Summed over the entire heterokaryon the frequency of mitochondrial
recombinant classes would not be the same in the two types of conidia except
when the overall mean mitochondrial genome input ratio was 1:1.
In seven of the eight crosses conducted using double marker selection, the
(cs-67) allele segregated more frequently in the nuclear background from which
it originated. We suggest that multiple rounds of recombination may be responsible for the following reason. If there is a bias in the input ratio of mitochondrial
genomes, multiple rounds of recombination will result in a bias in the output
ratio of the two (cs-67) alleles among the selected (camA112 oliA1) recombinants. As the number of rounds of recombination increase, the segregation of
the (cs-67) marker will depend less on linkage and more on the actual ratio of
the (cs-67) alleles in the panmictic pool. The core of a heterokaryon is probably
made up of many hyphal fusion events having a range of mitochondrial genome
input ratios. The input ratio of the two nuclei into the heterokaryotic hyphae
probably correlates with the input ratio of the mitochondrial genomes. Thus, a
bias toward one (cs-67) allele as a result of a biased input ratio will be more
frequently expressed in the conidia of the nuclear type from which that
particular (cs-67) allele originated.
In a heterokaryon one can never identify those conidia whose cytoplasms
have been derived from a mixture of the parental cytoplasms from those conidia
which have not. The formation of a heterokaryotic sector by necessity involves
cytoplasmic mixing. Heterokaryon sector analysis, therefore, enables one to
study exclusively segregants that have derived from a mixed cytoplasm, that is,
it excludes class one segregants as defined earlier. As one is analyzing sectors
rather than conidia, the mitochondrial cross does not have to be analyzed in
both nuclear backgrounds. Heterokaryon sector analysis is comparable to random diploid analysis in S . cerevisiae.
In double marker selection analysis, cytoplasmic mixing and recombination
of markers will have taken place in all segregants analyzed. This selects against
both class 1and 2 segregants as defined earlier. As one selects against segregants
in which cytoplasmic mixing has occurred without recombination taking place,
absolute recombination frequencies cannot be obtained. However, in principle
it provides a powerful method for establishing the relative order of markers,
particularly as many segregants can be analyzed for each cross. The cross does,
however, have to be analyzed in both nuclear backgrounds.
424
R. B.
WARING AND
C.
SCAZZOCCHIO
Four-point crosses were performed using heterokaryon sector analysis and
double marker selection analysis with a view to mapping the four markers,
especially (sumD16). Certain assumptions were made based on what is known
about organelle genetics in S. cerevisiae. These included the assumption that in
nonpolar crosses, reciprocal recombinant classes occurred with equal frequency
at the population level. Although this is generally true in S. cerevisiae, exceptions have been noted for chloroplast loci in Chlamydomonas (HARRIS
et al.
1977) whose organelle genetics share common features with S. cerevisiae
(reviewed in BIRKY
1978).We were not able to test the validity of this assumption
in our work, first, because (sumD16) could not be scored in a (cs-67+) genetic
background and, second, because (oliAZ) had a low transmission frequency.
LAZARUS
and TURNER
(1977) have identified another oligomycin-resistant marker
that does not result in slow growth. G. TURNER
(personal communication) has
also identified a mitochondrial mucidin resistance marker. These two markers
will allow some of the assumptions made in this work to be tested.
(camAI12), (cs-67) and (sumD16) all recombined with (oliA1) at a frequency
of about 30%. As recombination frequencies in nonpolar mitochondrial crosses
in yeast have an upper limit of 20-25’70 (DUJON,
SLONIMSKI
and WEILL1974), it is
likely that 30-35% is the upper limit in A. nidulans and that (oliAZ) is unlinked
to the other loci. The upper limit of 25% in yeast probably results from two
factors. First, the absence of any obvious mechanism to maximize mixing of
unlike mitochondria and unlike DNA strands will decrease recombination
frequencies. Second, the presence of multiple rounds of recombination as
observed in bacteriophage crosses (VISCONTI
and DELBRUCK
1953; DUJON,SLONIMSKI and WEILL1974) will increase recombination frequencies. The two
achieve a balance at about 25%. We suggest that the same factors are operating
in A. nidulans but achieve a balance at the higher level of 30-35%, possibly
because well-balanced heterokaryotic sectors only appear in regions where
good cytoplasmic mixing has occurred, thus increasing opportunity for recombination.
Heterokaryon sector analysis showed that (camA112) recombined at a frequency of 14% with (cs-67) and (sumDZ6) and that (cs-67) and (sumD16)
recombined at an approximate frequency of 8%. Double marker selection
analysis indicated that (cs-67) and (sumD16) were more closely linked to each
other than (camAIZZ), and a similar picture was seen in preliminary random
spore analysis work done to identify suppressors of (cs-67) (WARING1978;
WARING
and SCAZZOCCHIO
1980). LAZARUSand TURNER
(1977) showed that the
frequency of the wild-type recombinant class in crosses involving either
(camAZZ2) and (camB52) or (oliA1) and (oliB322) was about 0.2-0.7% of the
reassorted colonies, using random spore analysis. Although it is very likely that
intragenic recombination was occurring in their case, the considerably higher
frequency of recombination between (cs-67) and (sumD16) (probably a third
that of two unlinked mitochondrial markers) might suggest that these two
mutations map in different genes.
However, in yeast, mutations in the same gene can be loosely linked or even
unlinked due to the presence of introns (reviewed in BORSTand GRIVELL
1978;
MITOCHONDRIAL CROSSES IN ASPERGILLUS
425
SLONIMSKI
et al. 1978). Indeed, in S . cerevisiae it has been estimated using the
chloramphenicol- and erythromycin-resistant markers (KNIGHT1980), oIi2 and
oIi4 (MACINOand TZAGOLOFF
1980) and olil and oli3 (SEBALD, WACHTER
and
TZAGOLOFF
1979), that 1% recombination frequency represents 50-300 base
pairs. If recombination is as active in the mitochondrial genome of A. nidulans
(cs-67) and (sumDZ6)and even (camAZl2) could map in the same or neighboring
genes.
The map order of (camAlZ2) (cs-67) and (sumDZ6) could not be determined
by heterokaryon sector analysis. This was partly due to the small number of
segregants that could be analyzed in a single cross. In principle the use of
markers to select only for segregants that have undergone DNA exchange, as in
the double marker selection analysis, should have solved this problem. However,
the approach depended on the assumption that reciprocal recombinant classes
occur with equal frequency at the population level. In these particular crosses
the analysis was confused by the fact that a strain carrying (sumDl6) has a
wild-type phenotype. Despite this, on the basis of a statistical analysis, we
consider the most likely order of the three markers to be (camAll2 cs-67
sumD26).
It is known that the physical map of the mitochondrial DNA of A. nidulans
is circular (STEPIEN
et al. 1978; LAZARUSet al. 1980). However, this does not
imply that the genetic map is circular. Our data are consistent with both a linear
and circular map (Figure 2).
Recombination of the mitochondrial genome has been demonstrated in the
fungi P. anserina, where it has been used to test loci for allelism (BELCOUR
and
BEGEL1977) and N. crassa by analyzing restriction enzyme patterns (MANNELLA
oli A1
=30
\
sumD16
-cs-67
8
camA112
14
FIGURE2.-Mapping of four mitochondrial loci in A. nidulons. The two maps show the two
possible map orders of the four loci-data presented here suggest that the top one is the more likely
order. The figures given are recombination frequencies expressed as a percentage derived from
heterokaryon sector analysis. The parenthesis are included to show (oIiAl) as probably not being
linked in the genetic sense to the other three loci, 30% being the probable maximum value of
recombination (see text). Given that the physical map is circular (LAZARUSet 01. 1980) the genetic
map may also be circular.
426
R.
B.
WARING AND
C.
SCAZZOCCHIO
and LAMBOWITZ1978, 1979). Recombination has also been shown in the petitenegative yeasts K. lactis (BRUNNER,
DE COBOSand GRIFFITHS
1977) and S. pombe
(SEITZ-MAYR,
WOLFand KAUDEWITZ
1978; WOLF,SEITZ-MAYR
and KAUDEWITZ
1978). Extensive genetic mapping is possible in S. cerevisiae because petite
mutants can be isolated having large deletions of the mitochondrial genome. In
A. nidulans, N. crassa, P. anserina, K. lactis and S. pombe this is not possible.
This coupled with the possible high frequency of recombination may make it
difficult to map loci in these five organisms. However, their genomes are smaller
than S. cerevisiae and certainly in the case of A. nidulans there is much less
intergene spacer region and there are more genes (DAVIES
et al. 1982).
In both N. crassa and S. cerevisiae a portion of the mitochondrial genome
(about 30%) appears to be concerned primarily with mitochondrial protein
synthesis (KROONet al. 1976; MARTIN,RABINOWITZ
and FUKUHARA
1977; reviewed in BORSTand GRIVELL
1978). As (camAZZZ), (cs-67) and (sumD16)
probably all affect mitochondrial protein synthesis, specifically the mitochondrial ribosome (LAZARUS
and TURNER
1977; WARINGand SCAZZOCCHIO
1980),
we suggest that these mutations might map in part of a similar region in A.
nidulans. The region encompassing the rRNA genes, which are close together in
A. nidulans (LAZARUSet al. 1980), is a particularly likely site since mutations
conferring either resistance to chloramphenicol or cold sensitivity through
impairment of the mitochondrial ribosome generally map in the larger rRNA
gene in yeast (SINGH,
MASONand ZIMMERMAN
1978; SPITHILL
et al. 1978; DUJON
1979).
1980; KNIGHT1980; BOLOTIN-FUKUHARA
During this work R. B. WARING
was in receipt of a Science Research Council Studentship.
LITERATURE CITED
BELCOUR,
L. and 0. BEGEL,1977 Mitochondrial genes in Podospora anserina: recombination and
linkage. Mol. Gen. Genet. 153: 11-21.
C. W., JR., 1978 Transmission genetics of mitochondria and chloroplasts. Ann. Rev. Genet.
BIRKY,
12: 471-512.
BOLOTIN-FUKUHARA,
M., 1979 Mitochondrial and nuclear mutations that affect the biogenesis of
the mitochondrial ribosome of yeast. I. Genetics. Mol. Gen. Genet. 177:39-46.
BORST,P. and L. A. GRIVELL,
1978 The mitochondrial genome of yeast. Cell 15: 705-723.
BRUNNER,A., A. T. DE COBOSand D. E. GRIFFITHS,
1977 The isolation and genetic characterization
of extrachromosomal chloroamphenicol and oligomycin-resistant mutants from the petitenegative yeast Kluyveromyces lactis. Mol. Gen. Genet. 152: 183-191.
COVE,
D. I., 1966 The induction and repression of nitrate reductase in the fungus Aspergillus
nidulons. Biochem. Biophys. Acta 113: 51-56.
DAVIES,R. W., C. S~AZZOCCHIO,
R. B. WARING, S . LEE,E. GRISI,M. MCPHAILBERKSand T. BROWN,
1982 Mosaic genes and unidentified reading frames with homology to human mitochondrial
sequences are found in the mitochondrial genome of Aspergillus nidulans. In: Mitochondrial
Genes. Cold Spring Harbor Monographs, New York. In press.
DUJON,B., 1980 Sequence of the intron and flanking exons of the mitochondrial 215 rRNA gene
of yeast strains having different alleles at the w and rib-1 loci. Cell 20: 185-197.
DUJON,
B., P. P. SLONIMSKI
and L. WEILL,1974 Mitochondrial genetics. IX.A model for recombi-
MITOCHONDRIAL CROSSES IN ASPERGILLUS
427
nation and segregation of mitochondrial genomes in Saccharomyces cerevisiae. Genetics 78:
415-437.
GUNATILLEKE,
1. A. U. N., 1974 Biochemistry and genetics of mitochondrial function in Aspergillus
nidulans. Ph.D. Thesis, University of Cambridge.
GUNATILLEKE,
I. A. U. N., C. SCAZZOCCHIO
and H. N. ARST,JR., 1975 Cytoplasmic and nuclear
mutations to chloramphenicol resistance in Aspergillus nidulans. Mol. Gen. Genet. 137: 269276.
HARRIS,E. H., J. E. BOYNTON,
N. W. GILLHAM,
C . L. TINGLEand S . B. Fox, 1977 Mapping of
chloroplast genes involved in chloroplast ribosome biogenesis in Chlamydomonas reinhardtii.
Mol. Gen. Genet. 155:249-265.
KNIGHT,J., 1980 New antibiotic resistance loci in the ribosomal region of yeast mitochondrial
DNA. Genetics 94:69-92.
KROON,A. M., P. TERPSTRA,
M. HOLTROP,
H. DE VRIESand C. VAN DEN BOGERT,1976 The
mitochondrial RNAs of Neurospora crassa: their function in translation and their relation to
the mitochondrial genome. pp. 685-698. In: Genetics and Biogenesis of Chloroplasts and
W. SEBALD
and S . WERNER.
North Holland,
Mitochondria, Edited by T. BUCHER,W. NEUPERT,
Amsterdam.
LAZARUS,C. M., H. LUNSDORF,
U. HAHN,P. P. STEPIEN
and H. KUNTZEL,
1980 Physical map of
Aspergillus nidulans mitochondrial genes coding for ribosomal RNA: An intervening sequence
in the large rRNA cistron. Mol. Gen. Genet. 177: 389-397.
LAZARUS,
C. M. and G. TURNER,
1977 Extranuclear recombination in Aspergillus nidulans: closely
linked multiple chloramphenicol and oligomycin resistance loci. Mol. Gen. Genet. 156 303311.
MACINO,
G. and A. TZAGOLOFF,
1980 Assembly of the mitochondrial membrane system: sequence
analysis of a yeast mitochondrial ATPase gene containing the oli-2 and oli-4 loci. Cell 2 0 507515.
MA", H. B. and D. R. WHITNEY,
1947 On a test of whether one or two random variables is
stochastically larger than the other. Ann. Math. Statist. 18 50-60.
MANNELLA,
C. A. and A. LAMBOWITZ,
1978 Interactions of wild-type and poky mitochondrial DNA
in heterokaryons of Neurospora. Biochem. Biophys Res. Commun. 8 0 673-679.
MANNELLA,
C. A. and A. LAMBOWITZ,1979 Unidirectional gene conversion associated with two
insertions in Neurospora crassa mitochondrial DNA. Genetics 9 3 645-654.
MARTIN,
N. C., M. RABINOWTIZ
and H. FUKUHARA,
1977 Yeast mitochondrial DNA specifies tRNA
for 19 amino acids: deletion mapping of the tRNA genes. Biochemistry 16:4672-4677.
MASON,1. R. and G. TURNER,
1975 Transmission and recombination of extranuclear genes during
sexual crosses in Aspergillus nidulans. Mol. Gen. Genet. 143 93-99.
PONTECORVO,
G . , J. A. ROPER,L. H. HEMMONS,
K. D. MACDONALD
and A. J. BUPTON,
1953 The
genetics of Aspergillus nidulans. Adv. Genet. 5: 141-238.
REVER,
B. M., 1965 Biochemical and genetical studies of inorganic nitrogen metabolism in Aspergillus nidulans. Ph.D. Thesis, University of Cambridge.
ROWLANDS,
R. T. and G. TURNER,
1973 Nuclear and extranuclear inheritance of oligomycin
resistance in Aspergillus nidulans. Mol. Gen. Genet. 126:201-216.
ROWLANDS,R. T. and G. TURNER,
1974 Recombination between the extranuclear genes conferring
oligomycin resistance and cold-sensitivity in Aspergillus nidulans. Mol. Gen. Genet. 133:151161.
ROWLANDS,
R. T. and G. TURNER,
1975 Three-marker extranuclear mitochondrial crosses in
Aspergillus nidulans. Mol. Gen. Genet. 141: 69-79.
SEBALD,
W., E. WACHTER
and A. TZAGOLOFF,
1979 Identification of amino acid substitutions in the
428
R. B. WARING AND C. SCAZZOCCHIO
dicyclohexylcarbodiimide-bindingsubunit of the mitochondrial ATPase complex from oligomycin-resistant mutants of Sacchoromyces cerevisiae. Eur. J. Biochem. 100: 599-607.
SEITZ-MAYR,
G., K. WOLFand F. KAUDEWITZ,1978 Extrachromosomal inheritance in Schizosaccharomyces pombe. VII. Studies by zygote clone analysis on transmission, segregation, recombination and uniparental inheritance of mitochondrial markers conferring resistance to antimycin, chloramphenicol and erythromycin. Mol. Gen. Gent. 164: 309-320.
SINGH,A., T. L. MASONand R. A. ZIMMERMAN,
1978 A cold-sensitive cytoplasmic mutation of
Saccharomyces cerevisiae affecting assembly of the mitochondrial 50s ribosomal subunit. Mol.
Gen. Genet. 161: 143-151.
C. JACQ, A. KOCHKO,
A. LAMOUROUX,P. PAJOT,G.
SLONIMSKI,
P. P., M. L. CLAISSE,M. FOUCHER,
1978 Mosaic organization and expresPERRODIN,
A. SPYRIDAXIS
and M. L. WAMBIER-KLUPPEL,
sion of the mitochondrial DNA region controlling cytochrome c reductase and oxidase. 111. A
model of structure and function. pp. 391-402. In: Biochemistry and Genetics of Yeast, Edited
and A. 0. M. STOPPANI.
Academic Press, New York.
by M. BACILLA,B. L. HORECKER
SPITHILL,
T., K. J. ENGLISH,
P. NAGLEY
and A. W. LINANNE,
1978 Altered mitochondrial ribosomes
in a cold-sensitive mutant of Socchoromyces cerevisioe. Mol. Biol. Rep. 4: 83-86.
STEPIEN,
P. P., U. BERNARD,H. J. COOKE
and H. KUNTZEL,1978 Restriction endonuclease cleavage
map of mitochondrial DNA from Aspergillus nidulons. Nucl. Acids Res. 5: 317-330.
R. L. and P. S. PERLMAN,
1978 The effect of zygotic bud position on the transmission
STRAUSBERG,
of mitochondrial genes in Sacchoromyces cerevisiae. Mol. Gen. Gent. 163: 131-144.
STRAUSBERG,
R. L. and R. A. BUTOW,1981 Gene conversion at the varl locus on yeast mitochondrial
DNA. Proc. Natl. Acad. Sci. USA 78: 494-498.
TURNER,
G. and R. T. ROWLANDS,
1977 Mitochondrial genetics of Aspergillus nidulons. pp. 319337. In: Genetics and Physiology of Aspergillus, Edited by J. E. SMITHand J. A. PATEMAN.
Academic Press, New York.
G. and C. M. J. WATSON,1976 A new extranuclear oligomycin-resistance locus in
TURNER,
Aspergillus nidulans. Heredity 3 7 151.
K. P. and C. W. BIRKY,JR., 1978 The non-reciprocality of organelle gene
VAN WINKLESWIFT,
recombination in Chlamydomonos reinhordtii and Saccharomyces cerevisiae. Mol. Gen. Genet.
166: 193-209.
VISCONTI,
N. and M. DELBRUCK,
1953 The mechanism of genetic recombination in phage. Genetics
38: 5-33.
WALDRON,
C. and C. F. ROBERTS,1973 Cytoplasmic inheritance of a cold-sensitive mutant in
Aspergillus nidulans. J. Gen. Microbiol. 78: 379-381.
WARING,
R. B., 1978 Suppression of a mitochonrially inherited mutation in Aspergillis nidulans.
Ph.D. Thesis, University of Essex.
WARING,
R. B. and C. SCAZZOCCHIO,
1980 Nuclear and mitochondrial suppression of a mitochondrially inherited cold-sensitive mutation in Aspergillus nidulans. J. Gen. Microbiol. 139: 297311.
WOLF,K., G. SEITZ-MAYR
and F. KAUDEWITZ,1978 Extrachromosomal inheritance in Schizosacchoromyces pombe. VIII. Extent of cytoplasmic mixing in zygotes estimated by tetrad analysis
of crosses involving mitochondrial markers conferring resistance of ontimycin, chloramphenicol
and erythromycin. Mol. Gen. Genet. 164: 321-329.
Corresponding editor: J. E. BOYNTON