Metaphase position of an interchange quadrivalent of Allium triquetrum

Transcription

1 Metaphase psitin f an interchange quadrivalent f Allium triquetrum III. The expected distributin based n randmness GEOFFREY K. RICKARDS and WENDY A. BAKER* Btany Department, Victria University f Wellingtn, Private Bag, Wellingtn, Xete Zealand Present address: Ppulatin Genetics Grup, Australian Natinal University, Canberra, Australia Summary The psitin f an interchange quadrivalent at metaphase I f Allium triquetrum was mdelled s as t simulate the riginal placement f the quadrivalent in the spindle and the preparatin f linear spreads thrugh squashing. In this way an expected distributin fr the quadrivalent in linear spreads was generated. The prcedure used plar views f metaphase I t which the quadrivalent was assigned pairs f psitins nrmally ccupied by bivalents. The psitins f the bivalents and assigned quadrivalent were then prjected int a linear array and analysed as 'real' data. Cmparisns with bserved distributins shwed that a general bias in favur f marginal placement f the quadrivalent in the linear array is expected; and als shwed that the quadrivalent is psitined nn-randmly in the metaphase plate prir t slide preparatin. Key wrds: chrmsme psitin, metaphase I, expected distributin. Intrductin Whilst the study f nn-randm chrmsme psitining cntinues t attract attentin, it is still nt clear hw widespread the phenmenn is and what its functinal significance might be (Libbus, 1985; Hubert & Burgeis, 1986; Schwarzacher-Rbinsn et al. 1987, and references therein fr brief reviews and recent wrk). Interpretatin f data in the field has been hampered by the unavailability in many cases f theretically expected randm distributins fr chrmsme psitining specific t the particular rganism under study and specific t the particular investigative technique emplyed. Data n the psitin f interchange quadrivalents in metaphase I linear spreads f chrmsmes are available fr fur plant species (Rickards, 1984, 1985, in Allium triquetrum; Rickards, 1986, in hybrid rye; Murray, 1986, in tw species f Briza). Similarities within these fur data sets are apparent in the tendency fr the interchange quadrivalent t lie in marginal rather than central psitins in the linear spread. Differences in quadrivalent psitining, hwever, d exist in respect f the species cncerned, the rientatin adpted by the quadrivalent (alternate versus adjacent), the verall percentage f alternate rien- Jurnal f Cell Science 90, (1988) Printed in Great Britain The Cmpany f Bilgists Limited 1988 tatin, the quadrivalent type (chain r ring) and the presence r absence f B chrmsmes. T date, interpretatin f these data has prved difficult, precisely fr the reasn utlined abve: n expected distributins have been established with which t cmpare the bserved distributins. Therefre, the pssible relatinship f these bserved data t the general phenmenn f chrmsme psitin in the nucleus prir t meisis and t rientatin/rerientatin mechanics f multivalents has remained unclear. Interpretatin is a special issue in the cases cited abve because the psitinal data have been btained after flattening cells frm the side f the metaphase plate, s mechanically transfrming chrmsmes frm their riginal psitins in the spindle t a linear array (Fig. 1). We reprt here, therefre, the use f a prcedure that mdels the riginal quadrivalent psitins n the metaphase plate f A. triquetntm and simulates the events f the preparatin f metaphase linear arrays. In this way an expected psitinal distributin fr the quadrivalent is established. The prcedure makes use f plar views f metaphase I upn which the psitin f quadrivalents can be assigned cnceptually t psitins nrmally held by bivalents. The psitins f the bivalents and assigned quadrivalents are then prjected int a linear array, t 493

2 Fig. 1. Metaphase I f pllen mther cells f interchange/!, triquetnnit. A. Plar view in an unsquashed cell, shwing nine chrmsme 'bdies', cmprising seven bivalents and the tw halves (centrmere pairs) f the quadrivalent. B. Linear spread in side view in a squashed cell. The arrw lcates the interchange quadrivalent, which is in adjacent (adj.) rientatin and ccupies psitin 4/6. 4/6 adj. simulate flattening during slide preparatin, and analysed as 'real' data. Because each plar view is mdelled nce thrugh a single line f prjectin we use the phrase 'single angle prjectin squash' (SAPS) mdel, t distinguish it frm multiple-angle cmputer-assisted mdels currently under develpment. After cmparisns between the bserved and expected distributins, it is cncluded, in supprt f Rickards (1986), that a general bias in favur f marginal placement f the quadrivalent is t be expected. Evidence is als prvided t indicate that the interchange quadrivalent f A. triquetrum is psitined in a nn-randm manner n the metaphase plate, perhaps reflecting its psitin in the preceding interphase. Materials and methds Materials Metaphase I plar views in unsquashed pllen mther cells (Fig. 1A) were btained frm anthers f nrmal A. triquetnim that had been fixed in acetic ethanl and stained in Snw's ethanlic carmine (Snw, 1963). T btain these plar views in sufficient number, anther cntents were liberated int 45 % acetic acid in a cavity slide, where abut 10% f metaphase I cells are fund in plar view. Recrding plar views (Fig. 2A) Fr each plar view the utlines f the bivalents were recrded by camera lucida drawing under X 1000 magnificatin. The centre pint f each bivalent was taken as the mid-pint f the line that bisected the area enclsed by the bivalent utline. Assigning quadrivalents t the mdelled metaphase plate In plar views f metaphase I f bth nrmal and interchange pllen mther cells, nine chrmsme bdies can be distinguished (Fig. 1A). In nrmal cells, these nine bdies represent nine bivalents. In interchange cells, they represent seven bivalents and the tw 'halves' f the quadrivalent, given by its tw c-riented centrmere pairs, which align vertically in the spindle. In the mdelling prcedure the quadrivalent was assigned the equivalent f tw psitins held by bivalents in nrmal cells. This assignment is referred t as quadrivalent placement. With nine psitins available there 494 G. K. Rickards and W. A. Baker Dual psitin N. Placement type Prjected psitin Transfrmed psitin (i) (ii) (iii) Peripheral Central Radial 1/2 5/6 6/8 1/2 4/5 2/4 Fig. 2. Illustratin f the mdelling prcedure. A. Outlines f nine bivalents f an unsquashed metaphase I cell in plar view, frm nrmal material. The area midpint f each bivalent is marked. One each f three main quadrivalent placement types (peripheral, central and radial) are shwn as lines jining apprpriate bivalent midpints and labelled (i) (iii), respectively. B. Randmly chsen prjectin line; and the linear array f numbers (1-9) derived by parallel prjectin f the bivalent midpints. C. Dual psitins f quadrivalent placements (i)-(iii), shwing fr each its prjected psitin and transfrmed psitin. are 36 theretical quadrivalent placements, irrespective f the number f psitins in the centre f the plar view (Fig. 3, left). Of the 36 pssible placements, nly thse between neighburing bivalent pairs were accepted as being realistic. This restrictin n the randmness mdelled is justified because in

3 Plar view type c 8 c D c c PQ Ttal number f theretical quadrivalent placements (36) Central Subset Accepted quadrivalent placements (0) (3) Peripheral Subset linear spreads f metaphase I there is a sharp decline in the frequency f cells in which the quadrivalent verlies successively mre bivalents (Rickards, 1984, and see belw). Quadrivalents verlying mre than three bivalents are rare, because the tw c-riented centrmere pairs f the quadrivalent are linked by their hrizntal r diagnal cmpnent (in adjacent r alternate rientatin, respectively) and thus are physically restrained frm ccupying psitins widely spread acrss the metaphase plate (Rickards, 1984). The impsed restrictin in the mdels eliminates nearly all f the unrealistic, high verlay assignments that ccur if quadrivalent placements are made in a cmpletely randm manner. The number f neighbur placements available fr the quadrivalent varies accrding t the number f central bivalents in the plar view being mdelled, i.e. whether the mdelled metaphase plate is unicentric (having a single bivalent in the centre), bicentric r tricentric (Fig. 3). The ttal array f placements divides int three subsets, as fllws: (i) Central subset: placements assigned t tw psitins that are in the centre f the plar view, (ii) Peripheral subset: placements assigned t tw psitins that are immediate neighburs in the periphery f the plar view, (iii) Radial subset: placements assigned tw psitins that link each peripheral psitin t its nearest central psitin. Simulating the squash prcedure Plar views f metaphase I with vid distributins f chrmsmes are rarely seen in cnventinally prepared slides. The cell type analysed fr quadrivalent psitin in the studies f Rickards and Murray (lc. at.) is seen in mre than 95 % f cases: it has chrmsmes in a linear array, which is the result f the chrmsmes being flattened in a plane parallel t the spindle axis (Fig. IB). T mdel this situatin in plar views, a prjectin line was chsen at randm t Radial Subset \ / A A V (6) Fig. 3. Illustratin f quadrivalent placements in stylized plar views f metaphase I f unicentric, bicentric and tricentric types, based n ttal randmness (left) and n 'realistic' randmness. In the latter nly placements between neighburing bivalent pairs arc accepted. The ttal array f accepted placements is shwn divided int their three subsets. The numbers f quadrivalent placements in each categry are shwn in parenthesis. Fr the tw lwer cmpartments under 'Radial Subset', the brken lines indicate either/r placements because, being a stylized situatin, the distances invlved are equal. simulate the (presumed randm) plane thrugh which the cell is nrmally squashed. T simulate the mvement f chrmsmes int their linear array psitins, the area midpints f the bivalents were translated at right angles nt the prjectin line and numbered frm the left (Fig. 2B). Within the resulting array f numbers each quadrivalent placement was nw represented by a dual psitin. During the squash prcedure chrmsmes are spread laterally t a certain extent (Fig. IB). Hwever, different degrees f spreading f chrmsmes during slide preparatin d nt significantly affect the relative psitins f bivalents and the quadrivalent (Rickards, 1984). Fr this reasn the parallel (as ppsed t angled) prjectin f bivalent psitins is justified. A prjectin grid transparency was used ver each plar-view drawing t ensure crrect translatin f each mid-pint psitin int the linear array. Transfrmatin f dual psitins (fig- 2C) The dual psitins f the linear array were then transfrmed accrding t the ntatin riginally emplyed by Rickards (1984). This ntatinal methd recrds dual psitins by numbering frm the margin nearest the quadrivalent. Each transfrmed dual psitin indicates thepsilinalgmup f the quadrivalent placement (i.e. the psitin f its mst marginal half), and als indicates the number f bivalents that lie between the tw halves f the placement (the degree f verlay). Results Plar views f 100 cells frm three pllen sacs were used. Of the 100 cells, 88 were bicentric, while six were unicentric and six were tricentric. Cntingency chisquared tests indicate that the psitinal data frm the Metaphase psitin f quadrivalent /Allium 495

4 A U T3 a 3 T J > JT 40- t Percent, n- Md. / / / _ ri ' ' 1 ' ' ' 1 ' r. ' 1 ' Psitinal grups ; g n n [In 1/2 1/3 1/4 1/5 1/6 1/7 2/3 2/4 2/5 2/6 2/7 2/8 3/4 3/5 3/6 3/7 4/5 4/6 Dual psitins Fig. 4. Graphical presentatin f mdel data (brad, pen bars) and bserved data fr adjacent (ADJ) and alternate (ALT) quadrivalents, accrding t individual dual psitins (A) and psitinal grups (B). Fr clarity the data fr the alternate medium (ALT-M) inflrescence type nly are shwn in A; but fr alternate high (ALT-H) and alternate lw (ALT-L) inflrescent types as well in B. The data fr adjacent quadrivalents (ADJ-Ttal) are thse f the three inflrescence types pled. three pllen sacs d nt differ significantly frm each ther (data n request) and therefre were pled and treated tgether in the fllwing analysis. In ttal, 1506 quadrivalent placements were assigned, 106 t the central subset and 700 t each f the peripheral and radial subsets. The psitinal data are presented in Fig. 4. Fr cmparisn, this figure includes representative data, cnverted t histgram frm, frm Rickards (1984, fig. 2; and 1985, figs lb and 2b). These are the psitinal data fr alternate and adjacent quadrivalents frm first, secnd and third inflrescences f A. triquetrunt. These three inflrescence types are characterized by having 75%, 65% and 80% alternate rientatin and will be referred t as 'alternate medium 1, 'alternate lw' and 'alternate high', respectively. Adjacently riented quadrivalents are psitined similarly in the three inflrescence types and have a much strnger tendency twards marginal lcatin in linear spreads than d alternately riented quadrivalents. As the verall percentage f alternate rientatin in the three inflrescence types decreases, the tendency fr marginal psitining f alternate quadrivalents increases systematically (Rickards, 1984, 1985). The main pints t emerge frm the mdelling are as fllws. (1) Degree f verlay (Fig. 4A). Within psitinal gwup I (1/2-1/7) f the mdel data there is a decline in the prprtin f assignments in which the quadrivalent verlies successively mre bivalents. In brad terms this is the same as the decline fr the bserved data. The rate f decline in the mdel data is less than fr the bserved data. Mrever, fr the mdel data there are assignments with extreme verlay (l/6 and 1/7) nt fund in the bserved data. Thus fr the mdel data in psitinal grup 1 there is a higher degree f verlay than is fund in the real cell situatin. This is als the case, thugh t a lesser extent, fr the data f psitinal grups 2 and 3. (2) Psitin in the linear array (Fig. 4B). Fr the mdel data there is a decrease in the frequency f quadrivalent placements acrss the fur psitinal grups. A similar pattern f decrease ccurs in the bserved data (Rickards, 1984, 1985). N significant difference exists between the psitinal grup distributin f the mdel and that f the alternate medium quadrivalents (Table 1). Fr the ther tw alternate distributins, hwever, the fit is pr, and is especially pr fr the adjacent quadrivalents. Thus the distri- 496 G. K. Rickards and \Y. A. Baker

5 r n _ ttal p & 20 i Percent 3 O Table 1. Cntingency chi-squared values fr mdel psitinal grup data (ttal set) versus bserved data Mdel data cmpared with: Adjacent Alternate Alternate Alternate (ttal) lw medium high Chi-squared value Prbability (P) <0-0O01 < Fr each cmparisn the degrees f freedm arc 3. B Central Radial L C ' i * u 4 1 Psitinal grups Peripheral ADJ-Ttal w I2 n 3 4 rloo 1 1 npheral placeme luadnvalents -40 Q < g CJ -0 a- Fig. 5. Mdel psitinal grup data fr central (A), radial (B) and peripheral (C) subsets, expressed as percentages f the ttal mdel placements. In C the mdel data and thse fr the bserved adjacent quadrivalents are als shwn expressed as percentages f the ttal peripheral placements and ttal adjacent quadrivalent, respectively. butin f the alternate quadrivalents in first inflrescences is mdelled by quadrivalent placement in all neighburing pairs f psitins in the spindle. Such randmness, hwever, des nt explain the ther alternate distributins, r the distributin f the adjacent quadrivalents. (3) Central, peripheral and radial subsets (Fig. 5). Placements in the periphery f the plar views cmprised 46% f the ttal mdel set. These peripheral placements prduce an especially sharp decline in the number f assignments acrss psitinal grups 1-4 (Fig. 5C), with nearly 60% being in psitinal grup 1 and nly 1-3 % being in psitinal grup 4. As indicated in Fig. 5C, the distributin f the peripheral subset mst clsely mdels that f the bserved adjacent quadrivalents. Hwever, significant differences exist between these tw distributins (X Z = 17-9, P = ) and s, even with the restrictin f placements t the periphery f the metaphase plate, the highly marginal distributin f the adjacent quadrivalents cannt be mdelled. The radial and central placements, cntributing 46% and 8%, respectively, t the mdel's ttal data set, give psitinal grup distributins that are very different frm all bserved distributins (statistics nt shwn, but cmpare Fig. 5A,B with the mdel data f Fig. 4). Discussin General The mdel data f Fig. 4 prvide a clear indicatin f the linear array distributin expected when the quadrivalent halves are randmly lcated in neighburing pairs f psitins in the three-dimensinal spindle. Overlay f bivalents by the quadrivalent in the mdel data (Fig. 4A) is the prduct f the prjectin prcedure, and des nt represent real verlay n the metaphase plate. The decline in the frequency f cells within a psitinal grup (ver 1/2 t 1/7, e.g.) is then primarily the result f the restrictin f quadrivalent assignments t neighburing pairs f psitins. Fr the psitinal grup data f the mdel (Fig. 4B) there is a significant tendency twards marginal placement f the quadrivalent in the linear array after prjectin. There are mre marginal than central placements after prjectin, primarily because the scring prcedure assigns the quadrivalent t a psitinal grup accrding t the lcatin f its mst marginal half, and nt because f any preferential psitining in the riginal distributin prir t prjectin int the linear array. The marginal tendency als results frm the facts that there are mre peripheral than central psitins in the spindle and peripheral psitins are mre likely t becme marginal than therwise in their lcatin after prjectin; central psitins, indeed, never becme marginally lcated after prjectin. While ther scring methds can be applied t the data t vercme these prblems (Baker, 1987), it has been fund that the psitinal grup ntatin is the mst apprpriate ne t use, thugh it must be understd that it prduces a tendency fr quadrivalents apparently t be 'psitined in the margin f the linear array'- Cmparisns by dual psitins: accunting fr the degree f verlay The mdel data indicate that in the real cell situatin it is likely that the quadrivalent verlies n bivalents at all: the bserved verlay is cnsidered t be induced by the flattening prcedure that cnverts a circular distributin f chrmsmes int a linear array. This cnclusin is entirely cmpatible with the fact that the tw 'halves' f the quadrivalent are linked and thus their independence (wide separatin) restricted (Rickards, 1984). If all 36 pairwise cmbinatins f psitins (Fig. 3) had been included in the mdelling prcedure a much Metaphase psitin f quadrivalent /Allium 497

6 greater and quite unrealistic degree f verlay in the mdelled linear arrays wuld have resulted. As it is, the mdel data shw a greater degree f verlay than bserved, and include wide placements nt seen in the real cell situatin (Fig. 4A). This may mean that even restricting placements t neighburing pairs f psitins separates the quadrivalent halves mre than is physically pssible in the real situatin. The degree f verlay (but nt the psitinal grup) is als affected by the degree f spread during slide preparatin (Rickards, 1984). By analg)', the degree f verlay seen in the mdel data will be affected by the methd f prjectin during the mdelling prcedure. In the mdelling prcedure adpted here, all riginal psitins were prjected in parallel (withut spreading). It is therefre t be expected that the mdel prduces a higher degree f verlay than in the real cell situatin, where chrmsmes are always spread t sme degree. The prjectin line allcated fr any plar view plays a part in determining the degree f verlay fr a given quadrivalent placement within that plar view. A prjectin line parallel t a quadrivalent placement will result in higher verlay than ne that is perpendicular t that placement. Linked with this, the distributin f nearby bivalents influences the recrded verlay fr any placement. These factrs are presumed t have similar effects in bth the real and mdelled situatins. It is apparent, therefre, that differences between the bserved and mdel data in respect f the degree f verlay (Fig. 4A) are explained by: (1) the different restrictins put n the separatin f the tw halves f the quadrivalent in the real versus mdelled situatins; and (2) the differing effects f spreading brught abut by the squash prcedure, n the ne hand, and the parallel prjectin prcedure used in the mdel, n the ther hand. Cmparisns by psitinal grups: accunting fr the bserved distributins The distributin f the alternate quadrivalents in first inflrescences (alternate medium) is explained adequately by the mdelling prcedure. On the surface this implies their randm lcatin in all neighburing pairs f psitins in the three-dimensinal spindle befre squashing (see, hwever, later discussin). As indicated abve, the bserved tendency twards marginal placement in the linear array after squashing is t be expected and des nt in itself indicate nnrandmness in the selectin f metaphase psitins. The very strng marginal psitining f adjacent quadrivalents, hwever, is nt explained by the mdel, indicating that there is real nn-randm placement f these quadrivalents in the three-dimensinal spindle. Even the theretical cnfinement f the adjacent quadrivalents t peripheral placements (as in the peripheral subset f the mdel; Fig. 5C) des nt prduce a psitinal grup distributin with a sufficiently high marginal frequency in the linear array t mdel the bserved adjacent distributin. The highly marginal distributin f adjacent quadrivalents implies that special relatinships exist between chrmsme psitin, spindle rientatin and cell shape. It is necessary t suppse that either (1) cell shape r spindle rientatin is adjusted accrding t the psitin an adjacent quadrivalent adpts in the periphery f the spindle, s that sme planes f squashing relative t the quadrivalent psitin are mre likely than thers, r (2) quadrivalent psitin in the periphery f the spindle is fixed in relatin t a certain gemetry/structure within the cell r spindle, thereby revealing a nn-randm distributin f adjacent quadrivalents in linear spreads after squashing. This intriguing prblem was addressed by Murray (1986), wh cited ther evidence in the literature in supprt f the case. The alternate high and the alternate lw distributins are als nt adequately explained by the randm assignment f quadrivalent placements in neighburing pairs f psitins thrughut the spindle. T accunt fr these distributins it is necessary t invke sme frm f preferential placement fr these quadrivalents n the metaphase plate. The alternate lw distributin can be explained if sme alternate quadrivalents are preferentially lcated in peripheral psitins in the spindle; and the alternate high distributin can be explained if sme alternate quadrivalents preferentially ccupy radial and/r central placements. One pssible explanatin fr the psitining f alternates is that early rienting quadrivalents are placed preferentially in the periphery f the metaphase plate (as with adjacent quadrivalent). Late rienting alternates wuld, by relegatin, be psitined mre twards the centre f the plate. Rickards (1986) and Baker (1987) give specific prpsals as t hw differences in the distributins fr alternate quadrivalents might be accunted fr, in relatin t rientatin and re-rientatin phenmena. Under their prpsals, the 'randmness' f alternate medium quadrivalents seemingly revealed by the mdel data is viewed as cincidental, being derived frm the particular relative frequency f early rienting (peripheral) and late rienting (central) quadrivalents that cmprise the ttal alternates in first (alternate medium) inflrescences. Under such a view the prcess f psitining f alternate medium quadrivalents is thus n different frm that f the ther tw alternate quadrivalent types. Of curse, the abve level f preferential psitining f alternate quadrivalents is likely t have superimpsed n it thse same factrs f spindle rientatin and cell shape that are f imprtance in explaining the distributin f adjacent quadrivalents. 498 G. K. Rickards and W. A. Baker

7 The mdelling prcedure given abve has prvided expected distributins fr quadrivalent psitining that are specific t A. triquetrum. Hwever, the prcedure can easily be applied t metaphase spreads f ther species with differing chrmsme numbers, s allwing fr mre infrmed cmparisns between species t be made. In additin, the mdelling prcedure can be perfrmed using trignmetric rather than diagrammatic/mechanical prjectin, s enabling the mdelling t be cmputer based (wrk in prgress). References BAKER, W. A. (1987). Mdels f metaphase I quadrivalent psitining in AlHum triquetrum. Unpublished M.Sc. thesis, Victria University f Wellingtn. HUBERT, J. & BOURGEOIS, C. A. (1986). The nuclear skeletn and the spatial arrangement f chrmsmes in the interphase nucleus f vertebrate smatic cells. Hum. Genet. 74, LIBBUS, B. I. (1985). The rdered arrangement f chrmsmes in the Chinese hamster spermatcyte nucleus. Hum. Genet. 70, MURRAY, B. G. (1986). Interchange quadrivalents and chrmsme rder at meitic metaphase in Bnza L. (Gramineae). Chrmsma 94, RAPPOLD, G. A., CREMER, T., HAGER, H. D., DAVIES, K. E., MILLER, C. R. & YANG, T. (1984). Sex chrmsme psitins in human interphase nuclei as studied by in situ hybridizatin with chrmsme specific DNA prbes. Hum. Genet. 67, RICKARDS, G. K. (1984). Psitin and rientatin in the metaphase equatr f an interchange quadrivalent f AlHum triquetrum. Genet. Res. 43, RICKARDS, G. K. (1985). Metaphase psitin and rientatin f an interchange quadrivalent f Allium triquetrum. II. Changes in psitin with changes in percentage alternate rientatin. Genet. Res. 45, RICKARDS, G. K. (1986). Psitin and rientatin in the metaphase equatr f an interchange quadrivalent f hybrid rye. Chrmsma 94, SCHWARZACHER-ROBINSON, T., FlNCH, R. A., SMITH, J. B. & BENNETT, M. D. (1987). Gentypic cntrl f centrmere psitins f parental genmes in Hrdeum X Secale hybrid metaphases.j. Cell Sci. 87, SNOW, R. (1963). Alchlic HCl-carmine as a stain f chrmsmes in squash preparatins. Stain Tecltnl. 38, (Received 5 January Accepted 8 March 1988) Metaphase psitin f quadrivalent f Allium 499

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