Jan 3, 1989 - tween rDNA segments located on sister chromatids is apparently ..... intensity midway down the gel i n each lane is a small region of poor.
Copyright 0 1989 by the Genetics Society of America
Premeiotic Changeof Nucleolus Organizer Sizein Neurospora David K. Butler and Robert L. Metzenberg Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin 53706 Manuscript receivedJanuary 3, 1989 Accepted for publication April 15, 1989
ABSTRACT We have investigated the heritability of nucleolus organizer region(NOR)size in Neurospora crassa. By pulsed-field gel electrophoresis, we followed in genetic crosses the size of the normal or “terminal” NORs and the size of a small interstitial NOR. Tetrad analysis revealed that changes in NOR size occur frequently in the sexual phase. Moreover, most size changes occurred in the period between fertilization and meiosis, although some changes occurred during and after meiosis. Unexpectedly, increases and decreases in NOR size were not equally frequent: decreases were more common. The NOR size changes generated during meiosis were not the result of unequal crossing over between NORs on homologous chromosomes.
I
N most eukaryotes, the genes encoding the ribosomal RNAs are arranged in clusters of tandem repeat units called rDNA (LONGand DAWID1980). Generally, the rDNA repeatunits in any one organism are homogeneous in sequence despite the fact that they must be subject to mutation (SMITH1973; LONG and DAWID1980). The rDNA also displays peculiar recombination properties. In many organisms, traditional meiotic recombinationbetweenrDNA segments located on homologous chromosomes is very much depressed compared to that in similar lengths of non-rDNA (PALUMBO, CAIZZIand RITOSSA1973; PETES,SMOLIK-UTLAUT and MCMAHON 1982; CASSIDY et al. 1984; RUSSELL, PETERSEN and WAGNER 1988). However, in yeast, meiotic recombination between rDNA segments located on sister chromatids is apparently frequent. PETES(1 980) constructed strains that contained a single genetically marked rDNA repeat unit within the tandem array. He showed that when diploids heterozygous for the marked repeat unit were induced to undergo meiosis, they usually segregated the marker to two spores of a tetrad, as expected. However, in about 10% of the tetrads examined,onesporecontainedaduplication of the marker while the other three sporescontained no marker. PETESconcluded that unequal crossing over between sister chromatids in the rDNA generated the duplications and deletions. Mitotic unequal sister chromatid recombination in the rDNA is much less frequent in yeast. Using a similarly markedstrain, SZOSTAK and WU (1980) showed that mitotically dividing haploid cells can sometimes form colonies sectored for the marked repeat unit. In such colonies, one sector contained cells with the marker duplicated T h e publication costs of this article were partly defrayed by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. 5 1734 solely to indicate this fact. Genetics 122: 783-791 (August, 1989)
and the other sector contained cells with the marker deleted. The recombination events that gave rise to the duplications and deletions of the marked repeat unit were estimated to occur once in every 5000 cell divisions. T h e suppression of mitotic recombination in the rDNA has recently been shown to depend on the concerted action of topoisomerase I and topoisomerase I1 (CHRISTMAN, DIETRICHand FINK1988). Additionally, the SIR2 gene of yeast has been shown to suppress both mitotic and meiotic recombination in the rDNA (GOTTLIEB and ESPOSITO1989). T h e results of PETES(1980) and SZOSTAK and Wu (1980) suggest that rRNA gene clusters might frequently expand or contract inmeiosis,while being relatively stable in vegetative growth. Consequently, cluster size should usually remainconstant during mitotic growth, though rareunequal sister chromatid recombination within the rDNA duringmitosis would result in the occasional heterogeneity of cluster size in an otherwise clonal population of cells. Sucha process, if reciprocal, would not be detectedusing the traditionalmethods of quantitatingrDNAredundancy in a population of cells, since there would be no net change in the amount of rDNA per culture. In meiosis, however, which exhibits frequent recombination between sister chromatids, one consequence should be agreat deal of heterogeneity of cluster size among the segregants. In most strains of the filamentous fungus Neurosfioru crassu the rDNA is located at a single site in the genome called the nucleolus organizing region(NOR) which formsaterminalsegment on linkage group (LG) V (BARRYand PERKINS 1969). Certain translocation strains, designated QNS for Quasi-Normal Sequence, have been found to harbor a small misplaced block of rDNA, which we will call the “interstitial” NOR todistinguish it from theconventionally located,
standards. interstitial NOR on LG I was confirmed by following DNA preparations: Intact Neurospora chromosomes the segregation of a restriction fragment length polywere prepared using a slight modification of the liquid morphism (RFLP) characteristic of theinterstitial et al. (1988). The spheroplast method described by ORBACH rDNA among the progeny of a genetic cross. Segremodifications were as follows: conidia were germinated at 30" for 3-4 hr; the molten agarose-conidia mixture was gation of the RFLP was Mendelian but, unexpectedly, drawn into 1/16-inch inner diameter Tygon tubing; and the a range of the set of individualprogenydisplayed lysis buffer contained 10% SDS. Neurospora genomic DNA hybridization intensities for the rDNA RFLP of the for rDNA typing was prepared by a modification of the et al. 1986). This could be interstitial NOR (PERKINS method of METZENBERCand STEVENS (1982). Intact S. explained if different progeny containeddifferent cereuisiae chromosomes were prepared by the method of and OLSON(1985) andconcatamers of phage lambda numbers of rDNA repeat units in the interstitial NOR. CARLE DNA by the method of VOLLRATHand DAVIS(1987). We report here an investigation into the heritability Restriction digestion of intact Neurospora DNA: An of rRNA gene cluster size in Neurospora, both in the approximately 7-mm section of the agarose plug extruded interstitial NOR a n d in the terminal NOR. We have from the Tygon tubing was used for restriction analysis. used the pulsed-field gel electrophoresis system CHEF The agarose section was washed three times in 1.5 ml of T E (10 nM Tris, 1 mM EDTA pH 8.0) for 1 hr each at 0 " .The (Contour-clamped Homogeneous Electric Field) second wash solution included 0.1 mM phenylmethylsulfonyl (CHU, VOLLRATHand DAVIS1986) to determine the fluoride (PMSF). The agarose section was then incubated in size of the terminal NOR, and in those progeny that 100 p1 of l x restriction buffer without restriction enzyme contain it, of the interstitial NOR as well. T h e size of for 30 min at 0". After replacement with fresh buffer plus NOR the restriction fragment containing the terminal 30 units of BclI restriction enzyme (New England Biolabs), the mixture was overlaid with 150 pl of paraffin oil and varied from approximately 800 kbp to approximately incubated at50" for 12-15 hr. For NotI digestions, the 1600 kbp in different strains. Both the terminal and agarose sections were handled as above, except that 40 units interstitial NORs changed size with high frequency of NotI (New England Biolabs) were used per reaction at The majority of size changes during the sexual phase. 37" for 14 hr. in both the terminal and interstitial NORs were reCHEF gel electrophoresis: The apparatus used was described by CHU,VOLLRATH and DAVIS(1986). All CHEF ductions. Analysis of tetrads and of cultures from electrophoresis was performed for 24 hr in 0.5X TBE at randomspores from individualperithecia revealed 10-ll", with 1% agarose gelscastin0.5X TBE(CHU, that most of the changes occurred during premeiotic VOLLRATHand DAVIS1986). The pulse times and voltages proliferation of the dikaryotic tissue formed by fertilwere varied from experiment to experiment to optimize ization. This analysis also revealed rare meiotic and resolution of the pertinentfragments (VOLLRATHand DAVIS 1987) and are given in the appropriate figure legends. postmeioticchangesinNOR size. The changes in NOR size estimates: T o determine the numberof repeat terminal NOR size during meiosis are not due to units per terminal NOR, we first estimated the molecular unequal recombination between the NORs onhomolweight in kbp of a given BclI fragment by comparison to a ogous chromosomes. yeast chromosome ladder. We then divided this number by 9.3 kbp, the sizeof the Neurospora rDNA repeat unit (RUSSELLet al. 1984). Two factors make these estimates of the number of repeat units per NOR somewhat uncertain. MATERIALS AND METHODS First, the exact molecular weights of the largest yeast chromosomes are not known. These molecular weightshave Strains, genetic methods and plasmids: The origin and been estimated on the basisof genetic map distance and structure of the translocation strain, QNS-1, were described must be considered approximate (MORTIMERand SCHILD et al. (1986). The normal sequence parent, here by PERKINS 1985). Second, we do not know the distance from the noncalled NS-1 and referred to elsewhere as sat, was obtained rDNA BclI site to the most proximal rDNA repeat unit of from the FungalGeneticsStock Center (FGSC 945). All the terminal NOR,nor do weknow whether there is a crossesweremade on synthetic crossing medium at 25" sizeable telomeric segment free of BclI sites beyond the most (WESTERCAARD and MITCHELL 1947). In one cross, NS-1 distal rDNA repeat unit. However, neither of these considwas inoculated first and allowed to grow for 6 days, during erations seems likely to result in a serious overestimate of which time the female reproductive structures (protoperithe size of the NOR (e.g., >>5 repeat units). The number thecia) were formed. These were then fertilized with a of rDNA repeat units in the most commonly studied wild conidial suspension of QNS-1, which therefore acted as the type strain of Neurospora has been estimated, on the basis male in the cross. In a second cross,the orderof inoculation of saturation hybridization, to be about 185-200 (KRUMwas reversed and, with it, the sexual roles of the two parental LAUF and MARZLUF1980). In our system, the same strain strains. The spores from complete, unordered asci were yields a single rDNA-containing BclI fragment with an apcollected and ripened as described by METZENBERC(1988). parent size of 1630 kbp (data not shown). The maximum The plasmids used in this study were as follows: pKDO18, number of rDNA repeat units that could be contained in a derived from the non-transcribed spacer region of the Neufragment of this sizeis 175.Thus,our assumption that rospora rDNA repeat unit, used to detect the Hind111 RFLP virtually the entire BclI fragment is rDNA probably does et al. (1986) for details of rDNA RFLP analy[see PERKINS not result in a substantial overestimate of the number of sis]; and pRW528, covering all but approximately 0.2 kbp repeat units per terminal NOR, aswouldbe expected if of the 9.3-kbp repeat unit, used to detect intact NOR there were a large amount of non-rDNA in this BclI fragfragments on Southern t lots. Saccharomyces cereuisiae strain ment. In the caseof the interstitial NOR, the smallest 5288C and phage lambda strain cI857S7 (NewEngland
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Nucleolus Organizer Size
observed BclI fragment containing rDNA is about 15 kbp (data not shown). Since we know that this fragment has at least one complete rDNA repeat unit (data not shown), the amount of non-rDNA on this fragment, and other fragments with the interstitial NOR, is at most 6 kbp. RESULTS
Terminal and interstitial NOR sizes are altered We usually before, but occasionally during, meiosis: have exploited three features of Neurospora rDNA in order tostudy the inheritanceof rRNA gene cluster size. First, the rDNAs of laboratorystrains can be distinguished by the presence of either one or two HindIII restriction sites in the nontranscribed spacer of the rDNA repeat unit (RUSSELL et al. 1984). Thus type I repeat units are defined as having one HindIII site in the nontranscribed spacer and type I1 repeat units are those with two HindIII sites in the nontranscribed spacer. Second, the rDNA is inherited as a simple Mendelian trait in Neurospora (RUSSELL, PETERSEN and WAGNER 1988),as in yeast (PETESand BOTSTEIN1977). Within the limits of detection, all repeat units are alike in a given strain (FREE,RICEand METZENBERG 1979).Ina cross heterozygous for rDNA type (e.g., a cross of a type I rDNA strain x a type I1 rDNAstrain) any one segregant normally inherits type I rDNA or type I1 rDNA, but not both. This is interpreted to mean that there is no recombination between rDNA on homologous chromosomes. Therefore, one can follow the inheritance of a NOR in a cross. Third, both type I and type I1 repeat units are devoid of recognition sites forthe restriction enzyme BcZI. Therefore, the tandem array of repeat units in the terminal and/or interstitial NOR will be released intact after BclI digestion of chromosomal DNA (see MATERIALS AND METHODS for a discussion of the proximity of the flanking BclI sites tothe rDNA).These two types of repeat units are also devoid of recognition sites for NotI. Previous results have indicated that the amount of rDNAataninterstitial locus mightchangeduring sexual reproduction (PERKINS et al. 1986). We wanted to determinedefinitively whether terminal and interstitial NOR sizes are altered in the sexual phase, and, if so, at what stage (see Figure1fora schematic representation). The premeiotic stage of the sexual phase begins when an ordinary hypha or conidium of one mating type fuses with a specialized hypha (trichogyne) originating from a protoperithecium of opposite mating type (the protoperithecia and associated trichogynes are the female sexual structures). Normally a single nucleus is transported through the trichogyne into the protoperithecium, which then develops into aperithecium. At this point thereare two paired haploid nuclei of opposite matingtype in the common cytoplasm of a specialized cell called an ascogenous hypha. The two kinds of nuclei in the ascogenous
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IE 0 0 0 0 . 0 . .
FIGURE1.-Schematic representation of the sexual cycle of N . crassa. The small filled and unfilled circles represent haploid nuclei of opposite mating type. (A) Fertilization, to form the dikaryotic ascogenous hypha. (B) Seven to ten premeiotic conjugate mitotic divisions of nuclei (we show only three divisions). The final round of premeiotic DNA synthesis occurs in the haploid nuclei. (C) Karyogamy, to form diploid nuclei with replicated chromosomes. All pairs of nuclei will fuse and undergo the following events, though only one is shown in the diagram. (D) Meiosis I and 11, to generate four haploid nuclei. (E) Postmeiotic mitosis, ascospore delimitation, and ascus formation. The haploid nucleus within the ascospore undergoes several mitoses before germination. All of the asci and ascospores contained within a single perithecium are derived from a single fertilization event.
hypha begin to divide mitoticalIy. Toward the last of seven to ten doublings, each nucleus divides in synchrony with its eventual partner. The haploid nuclei undergo thefinal round of premeiotic DNA synthesis, followed by fusion of nuclei of opposite mating type. This fusion, or karyogamy, is the last premeiotic event. T h e resulting diploid nuclei of the ascogenous hypha promptly undergo the two meiotic divisions. Following meiosis the four haploid products divide once mitotically to generate eight nuclei from which the eight ascospores of an ascus are subsequently delimited (PERKINSand BARRY19’77). During ascospore maturation, there are several more mitotic divisions to generate a multinucleate spore(RAJU1980). If a NOR changes in size during the sexual phase, the stage at which the change occurs will determine the segregation pattern of NOR size inindividual asci. If the sizes of the NORs are fixed prior to the final round of premeiotic DNA synthesis ( i e . , prior to “C”
786
D. K . Butler and R. L. Metzenberg
in Figure l), the segregation pattern for a cross in which the parents have terminal NORs of different size will be 4:4. In contrast, NOR size might change during meiosis (it.,between “C” and “D” in Figure 1); for example, if there were a single unequal sister chromatid exchange in the rDNA at the four strand stage, the segregation pattern would 2:2:4, or if both sets of sister chromatids were to recombine the segregation pattern would be 2:2:2:2. T h e 2:2:2:2 segregationpattern is also consistent with anunequal crossover between NORs on homologouschromosomes (only two strands need be involved). However, if this occurred, then a mixture of rDNA types would be evident in four out of the eight progeny. Finally, if the NORs were to change size postmeiotically, at the mitotic division immediately following meiosis o r in the first few divisions after ascospore delimitation (ie., after “E” in Figure l), then sister spores of an ascus would harbor NORs of different size. Strain NS-1, which had type I rDNA and had no interstitial NOR, was crossed to QNS-1, which had type I1 rDNA in both its terminal and interstitial NORs. Fourteen complete unordered asci were isolated for analysis. Intact chromosomal DNA was prepared from both parents and from the progeny, digested with BcZI, fractionated by CHEF gel electrophoresis, blotted, and hybridized with a radiolabeled r D N A probe (Figure 2). In 1 1 out of 14 asci examined,the sizes of the terminal NORs segregated in a 4:4 pattern. Not surprisingly, therDNA type always cosegregated with terminal NOR size-thatis, the four progeny with the same size NOR also hadthe same rDNAtype (data not shown). Figure 2A shows the results of an experiment in which the DNAs of the two parents, QNS-1 and NS-1, and the eight progeny spores from ascus A, were electrophoresed under conditions that maximally resolve fragments of 1200-1 500 kbp. T h e QNS-I parent yielded a single high molecular weight band corresponding to the terminal NOR. T h e NS-1 parent also yielded a single, but somewhat more diffuse, high molecular weight band. T h e terminal NOR band of NS-1 probably represents a small range of NORs of different size (our unpublished results). Each progenyculture yielded a single high molecular weight band. T h e four type I1 rDNA progeny (A3, A5, A6, and A8) clearly have the same apparent size of terminal NOR, corresponding to 154 repeat units, but their terminal NOR is different in size from that of QNS-1 (the type I1 rDNA parent), which corresponds to 166 repeat units. T h e four type I rDNA progeny ( A I , A2, A4 and A7) also have the same apparent size of terminal NOR, corresponding to14 1 repeat units. However, the size of the terminal NOR of these progeny is only slightly changed, if at all, from that of NS-1 (the type I rDNA parent); the sizes
FIGURE2.- (A) Autoratliogrml showing an example of 4:4 segregation of ternlinal NOR size in ascus A. The DNA samples are from QNS-I. NS-I and cultures from the eight progeny ascospores. The CHEF gel electrophoresis conditions employed were a I 20-sec pulse time at 150 V. The apparent increase i n hybridization intensity midway down the gel i n each lane is a small region of poor resolution (VOLLRATHand DAVIS1987); randonl degradation products of rDNA, which typically form a light background in the wellresolving regions of the gel, become concentrated in this region. Progeny A5 has an interstitial NOR. whichis very diffuse in this gel, b u t is well-defined in other gels (data not shown). The positions of bands for the intact yeast chromosomes used as size standards are indicated by the marks at the left of the gel. Chromosome IV (the second mark from the top) has been estimated to be 1600 kbp (MORTIMER and SCHILD1985). Chromosonles VI1 and X V comigrated a t the position of the third mark from the top and have been estimated to be I 100 kbp. The bracket to the right of the gel marks the terminal NORs. The rDNA type of each progeny culture is indicated in parentheses above the appropriate lanes. (B) Autoradiogram showing a rare example of 2:2:4 segregation of terminal NOR size in ascus B. The DNA samples are from QNS-I, NS-I, and cultures from the eight progeny ascospores. The CHEF gel electrophoresis conditions were as in Figure 2A. The positions of the yeast chromosorne markers are as i n Figure 2.4.
might differ from that of NS-1 by, at most, three or four repeat units (see the last section of MATERIALS AND METHODS for a discussion of the assumptions underlying the estimation of the repeat unit number as a function of NOR size). Nonparental sizes of the terminal NORswere also frequent among the progeny from other asci. One curious observation is that the terminal NORs among the progeny were not equally likely to be increased or decreased relative to those of their parents: decreases were notably more common (see Table 1 forasummary of allof our dataon terminal NORs). A possible trivial explanation for the results pre-
787
Nucleolus Organizer Size r
TABLE I
I
v)
z Number of NOR size classesthat are theresult of independent eventsD and the range of sizes for each t p of NOR
'
a
z
H H"H
E
'
~ " Wu "H
2
P
H"H H '
?igzgzggggg
NOR size classes of progeny KOR
l y e
Type I' Type I I' Interstitial'
-
Parenrd Decreases Increases Rangeb
4
15
. i
1 gd
8 5
2
11
1
112-161 85-1 77 1-12
Progeny from the same ascus with the same size of NOR were consideredtohave arisen from asingleevent,and all random progeny from individual perithecia were considered to have arisen from independent events. ' In repeat units. ' Includes progeny from the 14 asci and eight random progeny from perithecium 1 and five random progeny from perithecium 2. Includes the two 2:2:4 asci. ' Includes progeny from the 14 asci only. a
sented above would be that BclI is not cutting at the same sites relative to the rDNA in the parents and progeny, resulting in different size BclI rDNA fragments without different size NORs. This could be due to incomplete digestion, to some kind of modification of the BclI recognition site, or to addition and elimination of BclI recognition sites duringthe sexual phase. T o determine if any of these possibilities are valid explanations, we digested QNS-1 and four randomly collected type I1 rDNA progeny from the cross of QNS-I x NS-1 with BclI and NotI separately. T h e NotI restriction enzyme does not cut within the Neurospora rDNA repeat unitsof any of the strains tested (data not shown). If the variations in NOR size observed with BclI digests are due toany of these trivial reasons, they should be unrelated to any variations observable with NotI digests. On the other hand, if the differences in BclI rDNA fragment sizes between the parents and the progeny are the result of differences in thenumber of rDNArepeatunits within their respective NORs, then the differences in BclI rDNA fragment sizes between the parents and progeny should be arithmetically related to those seen with NotI. For QNS-1, the BclI rDNAfragmentcorresponding to the terminal NOR is slightly smaller than the NotI rDNA fragment (Figure 3, compare the left two lanes). The same difference in the sizes of the Bcfl and NotI rDNA fragments is maintained for four randomly collected type I1 rDNA progeny (Figure 3). Thus, the differences in NotI rDNAfragment size between the QNS-1 parent andthese progeny are the same as those seen with BcZI. In two out of 14 asci the terminal NOR size segregated in a 2:2:4 pattern. Figure 2B shows one such ascus designated as B. T h e progeny B3, B5, B6, and B7 have only type I1 rDNA (data not shown), yet B3 and B7 have a terminal NOR size different from that ofB5 and B6 (the B3 and B7 terminalNORs are about 157 repeat units, and the B5 and B6 terminal
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4
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FIGURE3.-Autoradiogram showing the separation of the terminal NORs of QNS-I and four randomly collected type 11 rDNA progeny after digesting eitherwith BclI or Notl. CHEF gel electrophoresis conditions employed were a 1.50-sec pulse time at 155 V. Under these conditions yeast chromosome IV migrated as the second marker from the top. ChromosomesVI1 and X V comigrated as the third marker from the top. See Figure 2A for the apparent molecular weights of these bands.
NORs are about 163 repeatunits). Interestingly, both terminal N O R sizes among the progeny with type 11 rDNA are smaller than that of the parent, QNS-1, as if the terminal NORs in the progeny had undergone two roundsof sizechange-atleast one of them during meiosis and the other having occurred either during or before meiosis. Since progeny B3, B5, B6 and B7 have only type I1 rDNA, these data cannot be explained on the basis of unequal recombination between the NORs on homologous chromosomes. In all 14 asci, a low molecular weight rDNA fragment (ie.,
