MEIOTIC CHROMOSOME BEHAVIOR OF AN INVERTED INSERTIONAL TRANSLOCATION IN NEUROSPORA1

Transcription

1 MEIOTIC CHROMOSOME BEHAVIOR OF AN INVERTED INSERTIONAL TRANSLOCATION IN NEUROSPORA1 EDWARD G. BARRY Department of Botany, Uniuersity of North Carolina Chapel Hill, N. C Manuscript received October 22, 1971 Revised copy received January 17, 1972 ABSTRACT Cytological study of meiotic chromosomes heterozygous for the T(I+ll) translocation confirm genetic evidence (PERKINS 1972) that a section of linkage group I including the mating type locus has been inserted into linkage group 11. Pachytene chromosomes when fully paired show that a segment from chromosome 1 has been inserted into chromosome 6. When pairing fails between the translocated segment in 6 and its homologous region in chromosome 1, buckles or loops are formed at pachynema in the deletion or insertion areas of the bivalents.-acentric fragments and anaphase bridges occur at both meiotic divisions and in the subsequent two mitotic divisions in the ascus. These provide supporting evidence that the translocated segment is inverted with respect to centromere in its new location.-unexpectedly the acenhic fragment, formed by crossing over in the inverted translocated segment, persists without degradation in a micronucleus, and it replicates and divides in synchrony with the centric chromosomes in adjacent nuclei HE insertional translocation T(Z+ZZ)39311 is analyzed genetically in the accompanying paper by PERKINS (1972). The break points are identified and evidence is given that a long interstitial segment of the left arm of linkage group I extending between un(b39) and un(46006t) is inserted into the right arm of linkage group I1 between 2-3 and pe. This paper presents cytological observations of T(Z-+ZZ)39311 which confirm the genetic analysis. In addition, a description is given of the behavior of the acentric fragment which is produced in translocation heterozygotes when meiotic exchange occurs between the transposed segment and its homologous region in the standard chromosome sequence. The occurrence of such an acentric fragment confirms PERKINS genetic finding that the translocated chromosome segment has been inserted into the second chromosome in reverse order in its orientation to the centromere. MATERIALS AND METHODS Neurospora crassa strains containing T(1+11)39311 were obtained from Dr. DAVID D. PERKINS. 74-OR23-1A and 74-OR8-la wild types were used as the standard chromosome com- Supported by Public Health Service Research Grants GM and AI Genetics 71 : 53-GP May, 1978.

2 54 E. G. BARRY plement. The procedures for preparing meiotic chromosomes for examination have been described (BARRY 1966). The quality of chromosome staining and spreading in Neurospora cytological preparations varies among different isolates of the same strain (MCCLINTOCK 1945). As a general procedure, five to ten isolates of each aberration strain are crossed to wild type to obtain meiotic chromosome stages. After a preliminary inspection of the cytological quality of each separate cross, the one or two crosses giving chromosome preparations of the best quality are then used for intensive subsequent study. Such routine selection has substantially aided the cytological study of chromosome aberrations of Neurospora. Observations were made with a Spencer microscope equipped with Leitz 12x periplan oculars and a 1.3 na, 9Ox objective. Photographs were taken with a Leica M2 35" camera on Kodak High Contrast Copy film. RESULTS T(I+ II)39311 possesses some of the cytological characteristics of both paracentric inversions and translocations, plus some novel features. Meiotic chromosome pairing shows a complex association of four chromosomes when pairing is complete between the translocated segment and its homologous sequence in the standard chromosome. Pachytene chromosomes, when almost or completely synapsed in translocation heterozygotes, show the extent of the translocation, and that chromosomes 1 and 6 are involved (Figure 1 ). However, when synapsis does not occur between the translocated segment and its homologous region in the standard sequence, there is bivalent pairing with chromosome bulges or loops showing the position and extent of the deletion from chromosome 1 and insertion into 6 (Figures 2, 3, 4, 5). At diakinesis or metaphase I, appropriate complexes of rings or chains of four chromosomes may be observed as expected when synapsis is complete, but in other figures an apparently normal complement of bivalent chromosomes may be present, as expected in the second situation where pairing is incomplete. Dicentric bridges, or residual evidence of bridges, are frequently observed at the first meiotic anaphase (Figure 6). For technical reasons it is difficult to determine the actual frequency of bridges because sometimes lagging chromosomes of homologs separating asynchronously mimic bridge appearance. The bridges are presumably a consequence of crossing over within the inverted, translocated segment. Acentric fragments are also observed at anaphase I (Figures 7, 8). Ordinarily dicentric bridges and fragments are diagnostic of paracentric inversions, as originally shown by MCCLINTOCK (1938). Dicentric anaphase bridges at the second meiotic divisions (AII) are also frequently observed in translocation heterozygotes, and these are characteristic for the insertional translocation. Other examples of situations where AI1 bridges may be expected are: (1) when certain classes of exchanges occur between ring and rod or ring chromosomes (MORGAN 1933); (2) when a 3-strand-double crossover has occurred in a paracentric inversion heterozygote, with the position of one exchacge in the inversion loop and the second between the centromere and the proximal break point (MCCLINTOCK 1938); (3) when exchange has occurred between sister chromatids of internally paired tandem

3 CYTOLOGY OF AN INSERTIONAL TRANSLOCATION 55 All figures are from crosses of T(I-,II)3YJII by wild type. Magnification is approximately 3000x. FIGURE 1.-Pachynema. A-F. Five focal levels and an interpretive drawing showing pairing between the standard chromosomes 1 and 6 and their rearranged homologues with the segment from 1 inserted into 6 in T(I+11) One end of 6 is unpaired; the translocated section is paired throughout its length with the homologous region in normal sequence in 1. reverse repeats or duplications (MCCLINTOCK 1939) ; (4) when (illegitimate) exchanges occur between nonhomologous chromosomes forming dicentric chromatids (JONES 1968); and (5) when a breakage-fusion-bridge cycle has been initiated by a first anaphase double bridge (from a 4-strand-double exchange) followed by breakage and sister chromatid fusion (this has probably never been documented). The second-anaphase bridges of the inverted translocation, however, are explained by normal chromosome behavior. The nonhomologous centromeres of the dicentric chromosome can segregate together at the first division, but go to opposite poles at the second division. Not only are second anaphase bridges frequent, but dicentric bridges are found with a substantial frequency at the third and fourth divisions in the ascus in crosses of by normal (Figure 9). Third and fourth division (mitotic)

4 56 E. G. BARRY bl., // +: vj \.\- -~. Ii~tiwit!; 2,- -P~IC~I~IIC~I:I~I. A- I). 1"our rac::l Irwls. E. 11iterp.etive clrawi~ig of chroniosoincs 1 and 6 showing loops as a consrquencc of the translocation when the translocated segment has not paired with its normal homologue. F. The remaining normal chromosome complement. FIGURE 3.-A-D. Three focal levels and drawing of chromosomes 6 where the translocated segment has not paired with its normal homologue. FIGURE 4.-A-D. Three focal levels and drawing of chromosome 1 with the loop showing the approximate position and extent of the deletion.

5 ~~ - CYTOLOGY OF AN INSERTIONAL TRANSLOCATION 57 FIGURE 5.-LOop in chromosome 6 resulting from the translocation in linkage group 11 where the translocated segment has not paired with its normal homologue in chromosome 1. FIGURE 6.-Meiotic anaphase I. Dicentric bridge. FIGURE 7.-Telophase I1 nucleus. Centric chromosomes clustered at the pole with an acentric fragment passively included in the nucleus. FIGURE 8.---Meiotic anaphase I. A-D. Three focal levels and an interpretive drawing. TWO acentric fragments at arrows, and a dicentric bridge (not in focus in the photographs).

6 58 E. G. BARRY FIGURE 9.-Mitotic anaphase, division I11 in the ascus. Dicentric bridge. FIGURE 10.-Mitotic anaphase, division IV in the ascus. A-B. Two focal levels. M indicates the micronucleus with two chromosomes at anaphase. N indicates the chromosome groups of the normal nuclei. FIGURE 11.-Telophase, division IV. A-B. Two focal levels. M indicates the four micronuclei, N the two normal nuclei.

7 CYTOLOGY OF AN INSERTIONAL TRANSLOCATION 59 anaphase bridges would ordinarily only be expected for an inversion or an inverted insertional translocation where a breakage-fusion-bridge cycle has commenced, but for the inverted insertional translocation they will also occur without the initiation of a breakage-fusion-bridge cycle. That is, where a dicentric tie has formed following crossing over, there is equal probability that both centromeres will go to opposite poles (bridging) as to the same anaphase pole (no bridge), first for the successive meiotic divisions and then for each subsequent mitotic division. If, however, the chromosomes are twisted more than one-half gyre about each other, bridges will be formed whether or not the centromeres of the dicentric chromosomes go to the same or opposite poles. In Neurospora the paired homologous chromosomes have little or no relational coiling during stages preceding diplonema, in contrast to maize, for example, where synapsed homologs are twisted about one another. As a consequence, bridges observed in the second and later ascus divisions of heterozygotes may result from the centromeres of the dicentric chromosome failing to disjoin at anaphase I, only to do so later. No further acentric fragment is expected to be generated after the first division, regardless of when dicentric bridges appear. However, rupture of the dicentric bridges produces chromosome fragments with broken ends. It is my interpretation that such broken fragments are digested in the cytoplasm and do not persist for long. Fragment Behavior: The acentric fragments formed by a meiotic crossover in the transposed segment of inverted insertional translocation heterozygotes have very interesting properties and potentialities in Neurospora. The acentric fragment from T(Z-+ZZ)39311 is enclosed in a nuclear envelope at telophase and forms a micronucleus, as described for a paracentric inversion in maize by Mc- CLINTOCK (1938) and for another inverted insertional translocation in Neurospora by J. R. SINGLETON (personal communication 1961; ST. LAWRENCE and SINGLETON 1963). In subsequent stages of division, the chromosome fragment appears to be closely coordinated in its activities with the chromosomes of adjacent macronuclei. The fragments elongate or contract and even replicate and divide in concordance with the chromosome cycle of other nuclei in the same ascus. Thus, by second metaphase of the meiotic division, the fragment is very condensed and the nuclear membrane of the micronucleus has disappeared. However, the fragment does not divide at this point, in accordance with the notion that it consists of one chromatid and has not yet doubled. In the subsequent interphase the fragment, again in a micronucleus, elongates and has an appearance which matches the chromosomes of adjacent nuclei. It must also replicate at this time because in the following mitotic divisions two fragments may be found in appropriate configurations (Figure 10). In fact, as many as four fragments have been observed within one ascus (Figure 11 ); this would only be possible if there were a 4-strand-double crossover producing two acentric fragments, which each subsequently duplicated and divided; or alternatively, if one fragment went through two mitotic cycles. Throughout the mitotic divisions in the ascus, the chromosome fragment proceeds through a cycle in syn-

8 60 E. G. BARRY chrony with the adjacent nucleus or nuclei. Apparently the chromosome cycle in adjacent nuclei is coordinated through the cytoplasm, and this is supported by observations of nuclei in vegetative cells of the paraphyses and ascogenous hyphae where nuclei within the same cell all have the same appearance and are thus presumably in synchronous division. DISCUSSION Insertional translocations have been identified genetically by BRIDGES first in Drosophila (BRIDGES 1923, 1936; see MULLER 1967), by RHOADES (1968) in maize, by DE SERRES (1957), ST. LAWXENCE (1959), and BARRY (1960) in Neurospora, and by OHNO and CATTANACH (1962) and EICHER (1967) in the mouse. However, in the above cases there is no genetic indication of whether the inserted segment is in inverted or noninverted order with respect to the centromere. Cytological confirmation of the chromosome rearrangement has been found for many Drosophila insertionals (LINDSLEY and GRELL 1967), for maize (RHOADES 1968), and for the T(X;Z)Ct insertional translocation in the mouse (OHNO and CATTANACH 1962), which has also been reported to be a reciprocal translocation (SLIZYNSKI 1967). Cytological identification of inverted insertional translocations is not easily accomplished without genetic analysis. They may be misclassified cytologically as inversions unless pachytene chromosome analysis is possible. This apparently occurred in the case of the aberration in strain S1325 of Neurospora crassa which was originally misidentified cytologically as a paracentric inversion by SINGLE- TON ( ST. LAWRENCE and SINGLETON 1963), but was subsequently shown to be an inverted insertional translocation (MURRAY 1968; BARRY and PERKINS 1969). The associations of four chromosomes which would indicate an interchromosomal translocation may be infrequent if the translocated segment is short or rarely paired. Only if loops or buckles can be seen in one member of each of two pairs of pachytene chromosomes will the aberration be recognized when pairing of the interchanged segment has not occurred. Acentric fragments: The behavior of the acentric fragment obtained from crossovers within the translocated segment of T(Z+ZZ)39311 is in some respects like that of acentric fragments in maize as described by MCCLINTOCK (1938). She reports that the maize acentric fragment acts as does a normal chromosome in its cytological behavior and metabolic stage, and possibly its genetic influence, when the fragment was included in a telophase nucleus. There was, further, some cytological evidence for the continued inclusion of the maize fragment in subsequent nuclear cycles. Fragments left in micronuclei in the cytoplasm at the end of the meiotic divisions became pycnotic, however, and there was evidence of their degenerating during the subsequent mitotic division cycle in the microsporocyte. In contrast, when the fragment in Neurospora is left behind in the cytoplasm and is enclosed in a micronucleus, it behaves as does a chromosome included in the macronucleus. (Observations of acentric fragments derived from

9 CYTOLOGY OF AN INSERTIONAL TRANSLOCATION 61 other Neurospora aberrations suggest that not all fragments behave as do fragments.) It is of special interest in Neurospora that the fragment may persist in the cytoplasm of the cell. Since the hyphae are multinucleate and cell wall formation does not separate the fragment from other nuclei, the acentric fragment may persist and replicate indefinitely if it continues to behave vegetatively as observed in the two mitotic divisions following the meiotic division where it is generated. If the fragment were included in an ascospore with a nucleus containing a complete complement of chromosomes, the genes on the fragment would be accessory to the normal function of the nucleus. However, if the fragment were included in an ascospore where there is a complementary genetic deficiency, the continued presence, replication, and functioning of the fragment would be necessary for the survival of the deficient nucleus. This provides a possible model for the evolution of episomes and other nonchromosomal hereditary and cytoplasmically transmissible particles. This cytological investigation was conducted at Stanford University, and I am grateful to Dr. DAVID D. PERKINS for providing the facilities, the collaboration, and the instigation which brought it about. LITERATURE CITED BARRY, E. G., I960 Genetic analysis of an insertional translocation in Neurospora crassa. (Abstr.) Genetics 45: , 1966 Cytological techniques for meiotic chromosomes in Neurospora. Neurospora Newsl. 10: BARRY, E. G. and D. D. PERKINS, 1969 Position of linkage group V markers in chromosome 2 of Neurospora crassa. J. Heredity 60 : BRIDGES, C. B., 1923 The translocation of a section of chromosome-i1 upon chromosome-i11 in Drosophila. (Abstr.) Anat. Record 24: , 1936 Demonstrations (1) of the first translocation in Drosophila melanogaster and (2) of normal repeats in chromosomes. (Abstr.) Am. Naturalist 70: 41. DE SERRES, F. J., 1957 A genetic analysis of an insertional translocation involving the ad-3 region in Neurospora crassa. (Abstr.) Genetics 42: EICHER, E. M., 1967 The genetic extent of the insertion involved in the flecked translocation in the mouse. Genetics 55: JONES, G. H., 1968 Meiotic errors in rye related to chiasma formation. Mutation Res. 5: LINDSLEY, D. L. and E. H. GRELL, 1967 negie Inst. Wash. Publ Genetic variations of Drosophila melanogaster. Car- MCCLINTOCR, B., 1938 The fusion of broken ends of sister half-chromatids following chromatid breakage at meiotic anaphases. Missouri Agric. Exp. Sta. Res. Bull. 290: , 1939 The behavior in successive nuclear divisions of a chromosome broken at meiosis. Proc. Natl. Acad. Sci. U.S. 25: , 1945 Neurospora. I. Preliminary observations of the chromosomes of Neurospora crassa. Am. J. Botany 32: MORGAN, L. V., A closed X chromosome in Drosophila mehnogaster. Genetics 18: 250- MULLER, H. J., 1967 Translocational Pale Drosophila and Snaker mice, a semicentennial parallel. Mutation Res. 4:

10 62 E. G. BARRY MURRAY, N. E., 1968 Polarized intragenic recombination in chromosome rearrangements of Neurospora. Genetics 58: OHNO, S. and B. M. CATTANACH, 1962 Cytological study of an X-autosomal translocation in Mus musculus. Cytogenetics 1: PERKINS, D. D., 1972 An insertional translocation in Neurospora that generates duplications heterozygous for mating type. Genetics 71 : RHOADES, M. M., 1968 Studies on the cytological basis of crossing over. pp In: Replication and Recombinaiion in Genetic Material. Edited by W. J. PEACOCK and R. D. BROCK. Australian Acad. Sci., Canberra. ST. LAWRENCE, P., 1959 Gene conversion at the nic-2 locus of Neuospora crassa in crosses between strains with normal chromosomes and a strain carrying a translocation at the locus. (Abstr.) Genetics 4.4:532. ST. LAWRENCE, P. and J. R. SINGLETON, 1963 The cytogenetic effects of a paracentric inversion in Neurospora crassa. (Abstr.) Proc. 11th Intern. Congr. Genet. 1: 119. SLIZYNSKI, B. N., : Oocyte pachytene analysis of Cattanach s fd translocation. Genet. Res.