Meiotic Dysgenesis Associated with Behavioral Mutants of Phycomyces. Isabel L6pez-Diaz I and Edward D. Lipson. Department of Physics, Syracuse University, ...
© Springer-Verlag 1983
Meiotic Dysgenesis Associated with Behavioral Mutants of Phycomyces Isabel L6pez-Diaz I and Edward
D.
Lipson
Department of Physics, Syracuse University, Syracuse, NY 13210, USA
Phycomyces mutants, recently isolated for enhanced bending responses (hypertropic phenotype), have unusual genetic properties. In sexual crosses between hypertropic mutants and other strains, the progeny showed the following features: a) many incomplete tetrads, b) distortion of segregation ratios, c) progeny with nonparental phenotypes when hypertropic strains carrying mutations in the same gene or even the same allele were crossed, and d) morphologically abnormal progeny with phenotypes unrelated to those of the parents. In particular, the mesophorogenic colonies, which produced short sporangiophores, were genetically unstable; their mycelia produced sectors with normal morphology and segregated several alleles for different markers. Most of the phenomena (mutation, segregation distortion, and sterility) described in this paper resemble the "hybrid dysgenesis" syndrome in Drosophila. The results suggest that all seven hypertropic mutations affect the process of meiosis and thereby lead to unstable aneuploid progeny. Abstract.
Key words: Phycornyces - Phototropism - Hypertropic - Dysgenesis
Introduction
Behavioral mutants of Phycomyces were isolated recently for their enhanced tropic responses, in particular at high light intensity (Lipson et al. 1983). Genetic analysis of the "hypertropic" mutations in heterokaryons Offprint requests to: E. D. Lipson 1 Present address: Microbiology Unit, Department of Bio-
chemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England
with wild type (L6pez-Diaz and Lipson 1983) showed that four of them were strongly dominant while the other three were strongly recessive. The recessive mutations were found to comprise a single complementation group, madH, which was shown further by recombination analysis to represent a new gene. This paper deals with the abnormal genetic properties that arise when hypertropic mutants are crossed with other strains. In such crosses, the proportions of phenotypes among the progeny become distorted, and many progeny have phenotypes very different from the expected parental and recombinant phenotypes. These abnormal phenotypes include various forms of abnormal growth and morphology, as well as a propensity for sectoring that leads to further segregation. A striking feature is the high proportion of progeny with nonparental phenotypes from crosses between allelic hypertropic strains. In Phycomyces genetics, recombination analysis employs the sexual cycle. Like other Mucorales, Phycomyces produces haploid mycelia of two different mating types, (+) and (-), which are morphologically indistinguishable. When hyphae of opposite mating type come together, they differentiate into multinucleate gametangia which fuse to form the zygospore (Cerd~Olmedo 1974). In its early stages, the zygospore contains a large number of haploid nuclei derived from both parents. Occasionally, pairs of nuclei may fuse to form diploids, but normally only one meiosis takes place. After a dormant period of a few months, the zygospore germinates to produce a germsporangium containing several thousand germspoms. Each germsporangium is the product of a single meiosis followed by mitotic divisions and therefore may be considered as an enlarged tetrad. In the early work on sexual genetics of Phycomyces, irregular germination and frequent loss of genotypes in
314 the p r o g e n y c o m p l i c a t e d the analyses o f crosses. These irregularities were a t t r i b u t e d to differences in genetic b a c k g r o u n d s (nonisogenicity). The recent availability o f highly isogenic strains, as well as the establishment o f conditions for efficient and reproducible g e r m i n a t i o n o f zygospores h a v e m a d e r e c o m b i n a t i o n suitable for standard genetic analyses in Phycomyces (Eslava et al. 1975a, b; Cerd~-Olmedo 1975). The e x p e r i m e n t s r e p o r t e d h e r e e m p l o y e d isogenic strains. The a b n o r m a l genetic properties f o u n d in this s t u d y are p h e n o m e n o l o g i c a l l y similar to the " h y b r i d dysgenesis" s y n d r o m e in Drosophila (distorted p r o p o r t i o n s o f segregants, c h r o m o s o m a l aberrations, high p r o p o r t i o n o f sterile progeny, and appearance o f p r o g e n y w i t h m u t a n t p h e n o t y p e s ; Kidwell et al. 1977). In the c o n t e x t o f fungal genetics, we relate our results t o those asso, elated w i t h m e i o t i c aberrations in Neurospora and Aspergillus. We have chosen to denote the s y n d r o m e i n d u c e d b y crosses with the h y p e r t r o p i c m u t a n t s as " m e i o t i c dysgenesis". This s y n d r o m e in P h y c o m y c e s can be explained b y the h y p o t h e s i s that crosses w i t h h y p e r t r o p i c strains result in unstable aneuploid progeny.
I. L6pez-Dlaz and E. D. Lipson: Meiotic Dysgenesis in Phycomyees Table 1. Strains of Phycomyces used in this work Strain
Genotype
NRRL1555 A56 A125 A269 C288 C305 L1 L2 L82 L83 L84 L85 L86 L87 L88 L89 L92 L94 L96 L98 L123
(-) (+)
ribA4(+) madB103(+) madG131(+) madE102(+) madCl l 9(- ) madC119( +) mad- 702(-) madH703(-) mad- 704(-) madH705(-) mad- 706(-) madH707(-) mad- 708(-) earA5(+) mad- 702 (+) mad- 706 (+) mad- 703(+) mad- 704 (+)
a
Materials and Methods
NRRL1555 and A56 are standard wild types; L82-L88 are the hypertropic strains studied in this paper; L92-L98 are (+) hypertropic strains obtained by crosses; L123 is a mesophorogenic strain (see text) b rib, mad, and car genotypes indicate abnormal riboflavin synthesis, phototropism, and carotene synthesis respectively
Strains. The strains used are listed in Table 1. Media. Minimal medium (MM), complete medium (CM) and
Analysis of the Progeny. Germspore samples were inoculated
potato dextrose agar medium (PDA), as described by Lipson et al., (1980) were used in plates (8.5 cm diameter) or shell vials (diameter 1 cm and height 3 cm). For colonial growth, the media were acidified with HC1 to pH 3.3. For selection ofdar mutants, a concentration of 1 gg/ml of the toxic riboflavin analog, 5deazariboflavin (provided by the late Prof. P. Hemmerich), was used in MM acid plates.
from a concentrated suspension were spread on a slide (by means of another slide or a cover slip) and air dried. Cells were then fixed by immersion in methanol/glacial acetic acid (3:1 v/v) for about 10 min and hydrolyzed in 1 N HC1 at 60 °C for 30 to 60 s. Nuclei were visualized with Giemsa stain in Sorensen's phosphate buffer pH 6.9 (Robinow 1957a, 1957b; Brody and Williams 1974). Germinating spores required a treatment of only a few minutes, while dormant spores required several hours (overnight treatment).
on CM acid plates. After two or three days, colonies were transferred to PDA vials or PDA plates for further analysis of phenotypes. To determine the phototropic behavior of the progeny colonies, vials containing one colony each, were incubated two or three days under diffuse illumination and then transferred to test boxes with bilateral alternating illumination (Lipson et al. 1983; Lopez-Diaz and Lipson 1983). At low intensity (10 - 6 W/m 2) with an alternation period of 8 h, sporangiophores from wild type and hypertropic colonies showed a conspicuous sinusoidal pattern, because of their phototropism back and forth. Mutants with any mad mutation, other than hypertropic, grew straight at this intensity. At high intensity (10 W/m 2) only hypertropic sporangiophores showed a sinusoidal pattern. To determine mating type, colonies were inoculated on PDA plates together with (+) and ( - ) standard strains. Mycelia of opposite mating type formed zygospores in the area of contact. Mycelia that failed to form zygospores with either mating type were classified as sterile.
Sexual Crosses. Our procedures for sexual crosses followed
Segregation of Mesophorogenic Colonies. Many colonies from
Eslava et al. (1975a). Strainsofoppositematingtype were crossed by alternately placing two pieces of mycelium from each strain, around the edge of the plate: After 6 weeks of incubation in the dark at room temperature, the zygospores were transferred to wet filter paper on plates kept under inverted glass beakers. The zygospores were exposed to overhead white fluorescent illumination until germination. For "mass spore analysis", over a hundred ripe germsporangia from each cross were pooled into 2 ml of water. For "tetrad analysis", a separate germspore suspension was kept for each of several germsporangia.
these crosses produce sporangiophores limited in height to only 1 to 2 cm. Colonies that produce these short sporangiophores, which we call "mesophores", are termed mesophorogenic. Mesophorogenic colonies were transferred to Petri dishes containing PDA or CM. These colonies often produced sectors with different phenotypes. From sectors that produced normal sporangiophores (macrophores), pieces of mycelium were transferred to new plates to test phototropic behavior and mating type. This procedure was repeated several times, and several sectors from the same colony were studied. When spores
Nuclear Staining Procedure. Germspores or vegetative spores
315
I. L6pez-Dtazand E. D. Lipson: MeioticDysgenesisin Phycomyces Table 2. Abnormalities in the progeny of individualgermsporangiafrom crossesinvolvinghypertropic strains Number of germsporangia
Cross
L89
x
L85
carA5(+)
madH705(-)
L89
L82
x
carA5(+)
Total
Producing viable progeny
Producing incomplete tetrads
P r o d u c i n g Producing mesophoro- abortiveand genie pilobologenic progenya progeny
12
9
4
3
4
12
8
6
3
1
mad-702(-)
a Tetradsproducing mesophorogenicprogeny alone or a mixture of mesophorogenicand normal progeny
from mesophorogenic strains were tested, samples were plated on acid CM. After three days, when the sporangiophoresbegan to appear, the covers were removed to allow maerophores to grow unobstructed. Results
Abnormalities in Crosses Involving Hypertropic Strains When any hypertropic mutants were crossed with other strains, the progeny showed several features not expected for a normal meiosis. The main features were distortion in the segregation of phenotypes and production of colonies with abnormal mycelial growth and/or abnormal formation of sporangiophores (phorogenesis). The abnormal progeny included (a) abortive colonies, (b) pilobologenic colonies, producing piloboloid sporangiophores (see below), and (c) mesophorogenic colonies, producing short sporangiophores (mesophores). These phenotypes were absent from the parents. The abortive colonies constituted an inhomogenous population. The colonies reached diameters of only a few millimeters. Some produced no sporangiophores at all. In those that did, the sporangiophores grew very irregularly; specifically, they fell over during elongation. In some cases, even after these colonies were transferred to nonacidified medium, they still would not grow. Pilobologenic colonies, which produced piloboloid sporangiophores, appeared similar to existing pil mutants (so-called because their sporangiophores bulge in the growing zone just below the sporangium, like those of the related fungus, Pilobolus). Mutants of this type, obtained by mutagenesis, were previously assigned to four complementation groups (Yoshida et al. 1980; Ootaki, personal communication). Pilobologenic colonies obtained in the progeny of these crosses still produced piloboloid sporangiophores when the mycelium was
retested in non-acid medium. Spores from these progeny also produced pilobologenic colonies. Mesophorogenic colonies produced short sporangiophores, which stopped elongating after reaching heights of 1 or 2 cm. They were also sterile, in that they failed to produce zygospores in crosses with the standard ( - ) and (+) wild-type strains, NRRL1555 and A56. They did, however, show partial sexual reactions with either of the two mating types. These abnormalities were found either when the progeny of individual germsporangia were analyzed (tetrad analysis) or when a pool of germspores from many germsporangia was analyzed (mass spore analysis). The progeny of an individual germsporangium normally should contain either two or four genotypes depending on the type of tetrad (ditype o r tetratype) produced by meiosis. However, crosses involving hypertropic strains yielded many incomplete tetrads with missing genotypes. Table 2 shows two crosses between the albino mutant L89 [carA5(+)] and two different hypertropic mutants L82 [mad-702(-)] and L85 [madH705(-)]: Besides the large number of incomplete tetrads, some germsporangia did not produce any viable progeny while others produced, only or in part, abnormal progeny (see above). From mass spore analysis (Table 3), we found considerable distortion of the segregation ratios. The two alleles of each marker were expected to appear in the progeny in the ratio 1:1. However, in the 22 crosses involving hypertropic mutants (of which 10 examples are shown in Table 3), the ratio of nonhypertropic parentals to hypertropic parentals varied from 0.2 to over 85. With a few exceptions, the ratios were highly unbalanced in favor of the nonhypertropic parental. These crosses also produced mesophorogenic colonies, and (in smaller number) abortive and pilobologenic colonies. All of the crosses involving a hypertropic
I. L6pez-Dtaz and E. D. Lipson: Meiotic Dysgenesisin Phycomyces
316
Table 3. Abnormalities in mixed progeny of crosses involving hypertropic strains Cross
L2x
L84 L85
C288 x L82
Genotypes
Germsporangia pooled
madC119(+) x mad-704(-) madH705(-)
80 100
madG131(+)x mad-702(-) madH703(- ) mad. 704(-) madH705(-) mad-706(-) madH707(-) mad- 708(-)
L83 L84 L85 L86 L87 L88
ribA4(+)
A125 x L85
Parental ratios a
0.2 1
166
39
318
x madH705(-)
Mesophorogenic colonies (%) 38.5 52.5 28
38
446 422 103 318
>55 6 >85 >77
518
65
80
72 48 31 32
53 42
a Ratio between the non-hypertropic parental and the hypertropic parental
Table 4. Frequency of sectoring in mesophorogenic mycelia Cross L92
Plates or vialsa x
mad- 702(+) L89
x
carA5( +) C288
x
madG131 (+) C305
x x
madB103( +)
30
L85
4
3
madH705(- )
P
L88
V
123
10
V
85
7
V
67
14
V
92
5
V
104
17
L82 L84
mad- 704(-) x
madC119( +) A269
70
P
mad- 702(- )
madE102(+) L2
Colonies sectoringb
mad- 708(-)
madE102(+) C305
L84
mad- 704(-)
Colonies tested
L84
mad: 704(-) x
L85
madH705(- )
a mycelia were transferred and grown in plates (P) of diameter 8.5 em or in vials (V) of diameter 1 cm b colonies producing sectors with normal sporangiophores
strain produced a high proportion of mesophorogenic colonies in the progeny. We also found a considerable n u m b e r of mating-type heterokaryons among the progeny. In the cross L2 x L85, for instance, 8% of the progeny were mating-type heterokaryons.
and generally exhibited normal sexual reactions. In order to understand the genotype of these colonies, we studied their instability and the markers segregated by these sectors, as follows.
Instability o f the Mesophorogenic Progeny. Table 4 Segregation o f Mesophorogenic Mycelia The mesophorogenic colonies were unstable. While growing on petri dishes from an initial mycelial transfer, they produced sectors with normal morphology. These sectors developed macrophores (normal sporangiophores)
shows the frequency of segregant sectors with normal macrophores. More than 40% of the mesophorogenic colonies produced such sectors when they were transferred to PDA or GAYC plates. When the colonies were transferred to vials, a smaller proportion segregated normal sectors. However, the opportunity for sectoring was severely limited b y the 1 cm diameter of the vials.
317
I. L6pez-Diaz and E. D. Lipson: Meiotic Dysgenesisin Phycomyces Table 5. Segregation of markers in sectors from mesophorogenic mycelia Cross
Mesophorogenic mycelia
Phenotypes of sectors
Color
Mating typea
Sporangiophores
Color
Mating type
Phototropismb
L89 x carA5(+)
L85 madH705(-)
white
sterile
normal normal normal normal piloboloid piloboloid
white white yellow yellow white yellow
(+) (-) (+) (-) n.t. (+)
wild type wild type wild type wild type n.t. n.t.
L92 x mad-702(+)
L84 mad-704(-)
yellow
sterile
normal normal normal normal normal
yellow yellow yellow yellow yellow
(+) (-) (-) (+) (-)
blind blind wild type hypertropic hypertropic
"sterile" indicates that the mycelia did not mate with either mating type The phototropism phenotypes were hypertropic (phototropic response at high and low intensity), wild-type (response at low but not high intensity) and blind (no phototropism at high or low intensity). "n.t." means not tested (piloboloid sporangiophores could not be tested for phototropism under our conditions)
Although mesophorogenic mycelia produced segregant sectors with normal appearance and macrophore development, spores from mesophorogenic strains failed t o produce normal colonies. When samples of several thousand spores from different mesophorogenic strains were plated in acid medium, they produced only mesophorogenic colonies. When we plated spores from segregant maerophorogenic (normal) sectors, only normal colonies were produced.
Segregation of Markers in Normal Sectors. Sectors with normal morphology, that arose from mesophorogenic mycelia, usually showed normal macrophores and sexual reactions. We could study, therefore, which alleles for mating type and behavior were expressed. We tested mating type and phototropic responses in several sectors from the same and from different mesophorogenic colonies. The results are shown in Table 5. We examined mesophorogenic progeny from the cross between the albino mutant L89 and the hypertropic strain L85. From these progeny, we observed segregation of both mating types and of both color alleles. However, in phototropism tests, we found no hypertropic behavior in any of the sectors studied. We also found sectors with piloboloid sporangiophores. This marker (pi/) was not present in any of the parental strains. Usually, several markers segregated simultaneously. Most of the sectors that segregated macrophores also recovered sexual potency. Color segregation often occurred together with segregation of macrophorogenesis and mating type. For instance, a white mesophorogenic
sterile mycelium developed a sector that was yellow, had mating type ( - ) , and produced macrophores. On several occasions, however, a mesophorogenic colony segregated markers sequentially. For instance, a white mesophorogenic colony produced a sector with macrophores and (+) mating type, but still with white color; later, a new yellow sector appeared. In some cases, one colony produced homokaryotic sectors of both mating types, as well as mating-type heterokaryons. In a cross between two dominant hypertropic strains, L92 and L84, the mesophorogenic progeny mycelia produced sectors with macrophores of either mating type, showing three different phenotypes for phototropism: hypertropic, wild type, and blind (Table 5). Sectors heterokaryotic for mating type appeared frequently. Mating-type homokaryons were obtained by spore segregation from these heterokaryotic sectors.
Nuclei in Germspores and Vegetative Spores In the crosses involving hypertropic strains, germspores frequently produced mycelia that segregated several genotypes (see above). The presence of multiple genotypes might be explained by heterokaryotic germspores arising from multinucleate protospores. If so, then one would expect that the alteration in the process of germspore formation might also change the final number of nuclei per germspore. An alternative explanation is altered ploidy of the nuclei, leading to heterozygous germspores. In this case, nuclei of different sizes might be expected in the germspores. With these hypotheses
318
I. L6pez-Dlaz and E. D. Lipson: Meiotic Dysgenesis in Phycomyces i
0 1 2 3 4
i
,
,
,
i
56 ,
t
4(1
2(1 0
6(1 (n
4O
o
2(1
.E
o
®
611
0 1 2 3 4 5 6 i
j
j
r
t
i
i
type of progeny they would produce. These cases cannot account for the production of mesophorogenic colonies because the frequencies of occurrence do not correspond. In some of the crosses with many mesophorogenic colonies (more than 50%) we did not observe any germspores with large nuclei among several hundred examined.
J
¢k 2t
6(1
Nuclei in Vegetative Spores. In vegetative spores, the nuclei of the mesophorogenic strain L123 were not noticeably different in size from those of wild type, whether examined before germination or after several hours of growth. Because of the small and variable size (1 to 2 ~m) o f the nuclei, small differences in size could not be resolved under the microscope. A nucleus with twice the normal volume would have only a 30% difference in diameter and this would be hardly recognizable.
40 4)
20 0
o ®
50 40 20 C 5( 4(
20i
Frequency of dar Mutations 0 1 2 3 4 5 6 number
of
0
1 2 3 4 5 6
nuc nuclei per germspore
Fig. 1. Distributions of nuclei in germspores for crosses involving hypertropic strains, a) L2 x L84, b) C288 x L83, c) C288 x L84, d) C288 x L85, e) C288 x L86, f) C288 x L87, g) C288 x L88, h) L92 x L84, i) A125 x LI,j) vegetative spores of NRRL1555. The crosses in a to g involve one hypertropic strain; in h, both strains carry hypertropic mutations; in i, neither strain has a hypertropic mutation, the data in j are from Heisenberg and Cerd~t-Olmedo (1968)
Table 6. Frequency of dar mutations in hypertropic and mesophotogenic spores StrNn
Phenotype
NRRL1555 L83 L85 L87 L123
wild type hypertropic hypertropic hypertropic mesophorogenic
Number of spores tested x 10 - 6 4.5 0.53 0.87 3.6 25
Mutation frequency x 10 - 6 47 150
59
21
80
1
mad-706(-)
×
L88 mad- 708(-)
a For description of phototropism phenotypes, see footnote to Table 7
320
I. L6pez-Diazand E. D. Lipson: MeioticDysgenesisin Phycornyees
to wild-type phototropic behavior, showing no response at high intensity (where hypertropic strains do bend) and normal response at low intensity. However, in a few cases, some of the colonies with recombinant phenotype (no response at high intensity) did not show the wild phenotype b u t were unresponsive at low intensity as well. A striking result is the large number of progeny with recombinant (nonparental) phenotypes from a cross between two strains, L98 and L84, with the same mutant allele. The hypertropic strain, L98, was obtained from the cross A56 [(+)] x L84 [mad-704(-)] and therefore supposedly carries the same mutant allele as L84. About half of the progeny showed wild-type behavior for phototropism while the rest were hypertropic. In crosses between L96 and L85, and between L96 and L87, the proportion of progeny with nonparental phenotypes was also remarkably high. L96 came from the cross A56 [(+)] x L83 [madHT03(-)] and supposedly carries the same allele as L83. Complementation analyses (Ldpez-Diaz and Lipson 1983) showed that the hypertropic alleles of strains L96, L85, and L87 were apparently in the same gene, madH. The proportion of progeny colonies with nonparental phenotypes is inconsistent with intragenic recombination. Therefore, these results are incompatible with the occurrence of a normal meiotic process. Explanations in terms of meiotic aberrations will be discussed below.
genicity. The isolation of (+) strains, isogenic to the standard wild-type NRRL1555(-), has alleviated these problems. Crosses with these isogenic strains, which were obtained by repeated backcrosses, generally produce regular meioses (Eslava et al. 1975b). However, in our present study, lack of isogenicity cannot account for the abnormalities found in the crosses with hypertropic mutants. Some of the characteristic features of these crosses were observed early in crosses between nonisogenic strains. Others, like the production of mesophorogenic colonies and the high frequency of nonparental phenotypes in progeny between allelic hypertropic mutants, have not been reported before. All crosses described in this paper involved strains with a degree of isogenicity equivalent to at least four backcrosses and in many cases ten backcrosses. According to Eslava (personal communication), the number of incomplete tetrads decreases from 85% in crosses between two nonisogenic strains, to 30% between two strains with four successive backcrosses, and to 10% for ten backcrosses. For the degree of isogenicity between the strains crossed, the number of incomplete tetrads we obtained is abnormally high. So are the irregularities in the segregation of phenotypes in the progeny. The possibility of extranuclear inheritance of the hypertropic mutations has been ruled out by heterokaryosis tests (L6pez-Diaz and Lipson 1983), and therefore cannot be invoked to explain their irregular segregation in the progeny.
Discussion
Hypertropic Mutations Affect the Meiotic Process
Dysgenic Properties of Hypertropic Mutants
The genetic properties of these mutants and their progeny clearly indicate an effect of the hypertropic mutations upon the meiotic process. Mutants affecting meiosis are known in many organisms, including Neurospora (Smith 1975). Since many of these mutants affect chromosomal disjunction, the occurrence of aneuploidy in the meiotic products increases (Smith 1975). Various types of aberrant meiotic products could cause the appearance of mesophorogenic and abortive colonies. In Aspergillus, aneuploids have characteristic morphology (Kafer and Upshall 1973) and can be produced in two ways: (a) by mitotic nondisjunction from diploids, and (b) by meiotic nondisjunction among ascospores. The various types of disomic (n + 1) and trisomic (2n + 1) aneuploids, affecting each of the eight linkage groups, differ in frequency and morphology. In Aspergillus, diploids can be distinguished from haploids by the size of their conidia as well as by genetic properties. In Neurospora, diploids and aneuploids are formed during the sexual cycle (Smith 1974). Diploid and aneuploid strains in fungi tend to be unstable and undergo spontaneous haploidization to produce euploid
In crosses involving hypertropic strains, we have found the following abnormal properties: (a) high proportion of incomplete tetrads, (b) distortion of segregation ratios, (c) appearance among the progeny of abortive colonies, pilobologenic colonies, and mating-type heterokaryons, (d) high proportion of mesophorogenic colonies with special morphology, sexual properties, and segregation, and (e) high proportion of progeny with nonparental phenotype, when hypertropic strains with mutations in the same gene or even with the same allele were crossed.
Previous Irregularities in Phycomyces Genetics The occurrence both of incomplete tetrads and of abortive colonies in the progeny has been observed in previous genetic crosses of Phycomyces (Eslava et al. 1975a; Cerd~-Olmedo 1975). These abnormalities were attributed to irregular meioses caused by lack of iso-
I. Ldpez-Dlazand E. D. Lipson: MeioticDysgenesisin Phycomyces sectors (Fincham et al. 1979). In the crosses involving hypertropic strains, occurrence of chromosomal nondisjunction during meiosis could generate diploid or aneuploid nuclei. Abortive and mesophorogenic progeny would then result from these aberrant meiotic products. The instability of mesophorogenic colonies, which leads to segregation of markers, is one of the main arguments in favor of this hypothesis. Spontaneous haploidization of a heterozygous nucleus by chromosome loss (eventually yielding euploid sectors) could explain the occurrence of sectors with normal morphology and the segregation of different markers. Disomy for more than one chromosome is indicated by the segregation pattern. Different markers for behavior, mating type, and color segregated independently. These markers are in different chromosomes according to the recombination data of Eslava (personal communication). Since the relatively high frequency of spontaneous dar mutations (Table 6) indicates that mesophorogenic colonies are not diploid, we assume that they are aneuploid. Meiotic aberrations can also explain the segregation distortion that we have observed (Table 2). Disproportion among the segregants is due to loss of progeny. The loss of genotypes could be due to chromosomal nondisjunction during meiosis. This explanation would account too for the large number of incomplete tetrads we observed. High mutability also can be associated with meiotic aberrations. Breakage of chromosomes could explain the appearance of morphological markers, such as pilobologenic colonies, that were not present in any of the parental strains. Translocations and loss of chromosome fragments could produce lethal and nonlethal mutants among the progeny. Hot spots for breakage would yield certain types of mutants preferentially. The crosses among hypertropic strains gave some puzzling results (Table 8). Crosses between allelic hypertropic strains gave progeny with nonparental phenotype, mostly wild-type. Our results are consistent with the hypothesis that some hypertropic (+) strains are disomic heterozygous for the hypertropic marker. The (+) hypertropic strains used in these crosses were derived from crosses between the original hypertropic mutants and an isogenic wild-type strain of (+) mating type. Chromosomal nondisjunction during meiosis could produce aneuploids heterozygous for the hypertropic marker. These dlsomic strains would then segregate the wild-type allele when crossed with other hypertropic strains, thereby producing pseudorecombinants, similar to the case of "pseudowild-types" in Neurospora (Pittenger 1954). These pseudorecombinants of wild phenotype (disomic heterozygous for two complementing mutations) segregate both mutations when crossed with standard wild-type strains.
321 The few blind pseudorecombinants obtained in some of the crosses, lacking responses at any intensity, could perhaps be due to the presence of two hypertropic mutations in a disomic strain. Another possibility is a new mutation, produced in the same manner as the pilobologenic colonies (see above), leading to blind behavior.
Similarities with Hybrid Dysgenesis Syndrome in Drosophila The set of properties associated with the sexual cycle in the hypertropic strains resemble in many ways the "hybrid dysgenesis syndrome" in Drosophila (Kidwell et al. 1977). There, dysgenic traits such as mutation, chromosomal aberration, segregation distortion, and sterility appear to be associated with the germline of specific hybrids. This syndrome has been attributed to a family of transposable DNA sequences, activated in the germline of hybrids between certain interactive strains (Rubin et al. 1982; Bingham et al. 1982). Insertions of these DNA sequences, called P elements, are responsible for mutations arising in dysgenic hybrids. Furthermore, these P elements are located in breakpoint hot spots for chromosome rearrangements. Further studies on the genetic properties of hypertropic mutants in Phycomyces are needed to determine whether phenomena similar to those in Drosophila could be causing these dysgenic traits in the crosses with hypertropic strains. If so, then the new markers and the abnormal progeny could be explained by insertion of DNA elements. To help us understand the molecular basis of these phenomena, special attention should be given to the abnormal progeny from the crosses, specifically the mutant progeny with pilobologenic or blind traits.
Relation Between Sensory Behavior and Dysgenic Properties of Hypertropic Mutants The connection between the enhanced sensory behavior of the hypertropic mutants and the meiotic dysgenesis syndrome remains to be determined. Although for phototropic behavior, three of the seven hypertropic mutations are expressed in a strongly recessive way, they are nevertheless strongly dominant in their meiotic abnormalities (Ldpez-Diaz and Lipson 1983). Furthermore, the recessive and dominant hypertropic mutants have similar effects on meiosis. All of them have a dominant expression since they produce the genetic abnormalities in crosses with any other strain tested. Besides their enhanced behavioral properties, the hypertropic mutants are unexpectedly offering new opportunities for Phycomyces genetics. If the aneuploidy
322 hypothesis is correct, these crosses may well provide tools analogous to those o f the parasexual cycle in other fungi (Fincham et al. 1979), for example new methods for linkage and complementation analyses. Alternatively, i f processes like those underlying hybrid dysgenesis in Drosophila are in operation, here that t o o would offer valuable tools (viz. transposons) for genetics research on Phycomyces. These two alternatives are n o t mutually exclusive: it m a y be that transposons provide the molecular basis for the meiotic alterations leading to aneuploidy and the other effects. Finally, the question remains as to h o w these mutations induce b o t h the hypertropic p h e n o t y p e and the meiotic dysgenesis syndrome. It is an intriguing problem for future research to fred the underlying molecular processes affected b y these mutations.
Acknowledgements. We are indebted to Drs. Paul Galland and Darrel Falk for helpful discussions and for critical reviews of the manuscript. We thank Craig Chmielewicz for technical assistance. This work was supported by a grant (PCM-8003915) from the National Science Foundation. ILD held a postdoctoral fellowship from the Spanish government. EDL held a research fellowship from the Alfred P. Sloan Foundation.
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Received February 10, 1983
