J. Cell Set. 38, 357-367 (i979) 357Printed in Great Britain © Company of Biologists Limited 1979

SPECIFIC END-TO-END ATTACHMENT OF

CHROMOSOMES IN ORNITHOGALUM VIRENS*

TERRY ASHLEYfDepartment of Anatomy, Duke University Medical Center, Durham,North Carolina 27710, U.S.A.

SUMMARY

C-banding of nonhomologous chromosomes in haploid generative nuclei of Ornithogalumvirens (« = 3) reveals a high degree of specificity with respect to end-to-end connexions. Thecentromeric end of chromosome 2 preferentially associates with the centromeric end ofchromosome 3 and the telomeric end of chromosome 3 associates preferentially with thetelomeric end of chromosome 1. This same association of nonhomologous chromosomespersists in prophase nuclei of diploid root tips. In addition, the telomeric ends of the 2 chromo-some 2s are connected to one another as are the centromeric ends of the chromosome is.This results in a ring of chromosomes in which homologues lie opposite one another. Centro-meric ends lie on one side of the nucleus and telomeric ends on the other. It is proposed thatthis specific association of chromosome ends reflects an order which was probably establishedat the preceding anaphase ortelophase and which persists throughout interphase.The suggestionis made that the proximity of homologous ends and consequently homologous alignment mayfacilitate initiation of pairing at meiosis.

INTRODUCTION

' Is there a specific arrangement of chromosomes within nuclei ?' is a questionwhich has often been raised (see Comings, 1968; Ashley & Wagenaar, 1974, forreviews). The question (and answer) has important implications for one of the centralproblems of cytogenetics, initiation of homologous chromosome synapsis in meioticprophase; however, conclusive answers have been elusive. Difficulties include thenear impossibility of determining chromosome arrangement in interphase nuclei,the confusion of large numbers of chromosomes present in most organisms, and theproblem of positive identification of each chromosome in the complement.

Examination of nuclei in which chromosomes are in transition from the interphaseto the prophase state offers one approach to the problem. At this stage the chromo-somes are individually visible, but might still be expected to retain their interphaserelationships with each other and with the nuclear membrane. By selecting anorganism with a haploid chromosome number of 3 (Ornithogalum virens), Ashley &Wagenaar (1974) detected an end-to-end arrangement of prophase chromosomes inhaploid, diploid and tetraploid tissue. They suspected that the chromosomes were

* This paper is dedicated to Professor Sally Hughes-Schrader, who first reported a specificend-to-end association of chromosomes and whose delight in chromosomes and in life brightensthe lives of all those fortunate enough to know her.

t Present address, to which off-print requests should be sent: Laboratory of GeneticResearch, Delphian Foundation, Sheridan, Oregon 97378, U.S.A.

358 T. Ashley

connected to one another in a specific sequence. However, since all 3 chromosomes ofOrnithogalum virens are acrocentric and approximately the same length, the identityof each chromosome and consequently the specificity of end associations could not bepositively determined in their material. With the advent of the new banding techniques(Arrighi & Hsu, 1971), identification of each chromosome and determination of itsprophase relationship with any other chromosome in the nucleus became a possibility.Stack, Clarke, Cary & Muffley (1974) C-banded root tips of Ornithogalum virensand determined that the bands were located at the centromeric ends of each acrocentricchromosome. Therefore, C-banding allows identification not only of each chromo-some, but of each end of each chromosome. C-banding of pollen material has provenmore difficult, but by modifying the technique a sufficiently large sample of materialwith distinct bands has been obtained to complete a statistical analysis of end-to-endassociations. Analysis of end-to-end associations of the nonhomologous chromosomesin pollen and of the arrangement of both homologous and nonhomologous chromo-somes in prophase diploid root tips is presented below. These observations offer anexplanation for somatic crossing over and somatic association and an insight into how in-itiation of synapsis of homologous chromosomes may be facilitated at meiotic prophase.

MATERIALS AND METHODS

Ornithogalum virens was grown in a greenhouse belonging to the Botany Department ofDuke University. The bulbs were originally obtained from Dr A. H. Sparrow at the Brook-haven National Laboratory.

Preparation of haploid generative nuclei from pollen

Buds immediately above those which had just opened were collected and placed on moistfilter paper. All 6 anthers from one bud were placed on a slide in 3-4 drops of 45 % acetic acid.The pollen was teased out and the antheridial tissue removed. A 40-50-mm coverslip wasplaced over the pollen and pressure applied. The slide was submerged in liquid nitrogen,removed, and the coverslip immediately flipped off with a razor blade. The preparations wereair-dried in a 60 °C oven for from 1 h to overnight.

Slides were placed in a freshly prepared and filtered saturated solution of Ba(OH)2 at 60 °Cfor 2 h and rinsed gently in running deionized water, incubated in 6 x SSC (saline-sodiumcitrate) at 60 °C for 10 min, rinsed and stained 10 min in 10% Gurr Giemsa (Bio/MedicalSpecialties) in 0-12 M sodium-potassium phosphate buffer at pH 6-8. The slides were rinsedbriefly and checked for C-bands. If the stain was too dark, rinsing was continued until C-bandswere distinct. As soon as the bands could be clearly discerned, the slides were removed fromthe water, air-dried and mounted in Depex (Bio/Medical Specialties).

Preparation of diploid root tips

One to four weeks before use, greenhouse plants were ' transplanted' to fish tanks where thebulbs were supported on test tube racks. Roots were submerged in 0-5 x Hoagland's solution,which was aerated with an aquarium bubbler. To prepare slides, root tips were excised andfixed in fresh 3 parts 95 % ethanol : 1 part glacial acetic acid, rinsed in distilled water andhydrolysed in I N HC1 at 60 °C for 10 to 15 min. The root tips were then rinsed again andsquashed in 45 % acetic acid. The coverslip was removed as described above and the prepara-tions air-dried in a 60 °C oven from 1 to 12 h. Slides were placed directly into 5 % Gurr Giemsafor 2 min, rinsed, air-dried, and mounted in Depex.

Specific association of chromosome ends 359

Observations and photography

Slides were examined with a Biophot (Nikon) research microscope. Maximum contrast forchromosomes and bands was obtained with phase optics and a Vivitar orange 02 filter. Cellswere photographed on Kodak SO 115 film and developed in Kodak HC 110 (dilution D).

RESULTS

Nonhomologous chromosomes in haploid generative nuclei

Following meiosis, the haploid male gametophyte of angiosperms undergoes 2mitotic divisions. The first division gives rise to the vegetative and generative nuclei.The generative nucleus then divides again to produce the 2 sperm nuclei. In Orni-thogalum virens the generative nucleus is arrested at prophase until after germinationof the pollen, thus providing a highly synchronized population of cells at preciselythe stage needed for this study.

C-bands of the prophase chromosomes of the generative nuclei (Fig. 1) weresimilar to those previously reported for metaphase root tips (Stack et al. 1974). Onechromosome had one C-band (chromosome C of Ashley & Wagenaar, 1974); anotherhad 2 C-bands (chromosome A), and a third had 3 C-bands (chromosome B). Theyare here referred to as chromosomes 1, 2 and 3, according to the number of bands.The C-bands are located at the centromeric end of each chromosome. Therefore, thebanded ends of the chromosomes will be referred to as iC, 2C and 3C, while thedistal (telomeric) end of each chromosome will be referred to as iT, 2T and 3T.

If association of ends is random (the null hypothesis) there are 12 equally probableassociations of ends of nonhomologous chromosomes (Fig. 2). As is readily apparentfrom examination of the distribution of associations in Fig. 2, this is not the case0\!2 = 326; P value for o-ooi confidence level=3i). Clearly, the association of ends isnot random, but highly specific. Most associations are of one of 2 types: 2C-3C and1T-3T. In fact, 2C was connected to 3C (as in Fig. IA and G) 41 times out of 41(Fig. 2), and iT was connected to 3T (as in Fig. IB-F) 62 times out of 64 (Fig. 2).Thus, 103 out of 116 connexions (88-8%) observed could be accounted for by these2 types of connexions. Of the 16 cells in which there were 2 connexions, 15 exhibitedboth the 2C-3C and 1T-3T connexions. It can be concluded that the preferredarrangement of nonhomologous chromosomes in generative nuclei is the telomeric endof 1 connected to the telomeric end of 3 and the centromeric end of 3 connected tothe centromeric end of 2 (Fig. 3 A).

The data support the suggestion made previously (Ashley & Wagenaar, 1972, 1974)that the nonhomologous chromosomes in the haploid generative nucleus of Orni-thogalum virens form an open chain, rather than a closed circle. If the associationsresulted in a closed circle, iC and 2T would be connected; this configuration, however,was never observed. The iC end of the chain was associated with another end inonly 6 out of 116 cases, and these associations were random (Fig. 2). The other endof the chain (2T) was observed in association with another chromosome only 5 times.However, here the association was always with 3T and thus appeared to be non-random, although the sample is too small for an adequate analysis.

T. Ashley

Fig. i. End-to-end association of nonhomologous chromosomes in haploid generativenuclei of the pollen of Ornithogalum virens. Chromosomes are numbered accordingto the number of C-bands they possess. Centromeric ends are indicated by solidarrows. Connexions between chromosome ends are indicated by open arrows.Nuclei in Fig. IA and G have connexions between the centromeric ends of chromo-somes 2 and 3; nuclei in B-F have connexions between the telomeric ends of chromo-somes i and 3.

Specific association of chromosome ends 361

The data also support the hypothesis of Ashley & Wagenaar (1974) that centromericends associate preferentially with centromeric ends, and telomeric ends with telomericends. In only one case out of the 116 was a centromere-telomere connexion found(1C-3T); the remaining 115 cases were centromere-centromere or telomere-telomereconnexions (Fig. 2).

1T

2C

2T

3C

3T

X2

0

3

1

1C

0

2

0

62

1T

X41

0

2C

0

5

2T

X3C

Fig. 2. The observed frequencies of the 12 possible end-to-end connexions betweenthe 3 nonhomologous chromosomes of Ornithogalum virens pollen. If associationswere random, all connexions should be equally frequent. C indicates the end of thechromosome nearest the centromere, T indicates the distal end. The numbersidentify the chromosome by the number of C-bands present. Thus, 1C is the centro-meric end of the chromosome with one C-band, etc.

Diploid nuclei of root tips

Ashley & Wagenaar (1974) suggested that there might be an identical specificsequential arrangement of the chain of nonhomologous chromosomes in both gametesand that, at fertilization, homologous ends of the two chains joined to form a closedcircle (Fig. 3B). Thus the sequence of nonhomologous chromosomes would bemaintained, but now homologous chromosomes would lie adjacent to one anotherwithin the diploid nucleus (Fig. 3B, c). On this basis, 2C-3C and 1T-3T connexionsshould also be found in diploid tissue. In addition, 2T-2T and 1C-1C homologousconnexions would be predicted as a result of the joining of the 2 gametic chains to forma closed loop.

In squash preparations of pollen the chromosomes are pressed out of the pollengrain walls along with the cytoplasm resulting in extensive separation and spreading.Extrusion of the cellular contents is much less frequent in root tip material. Thesmall space within the confines of the diploid cell walls results in interstitial chromatin

302 T. Ashley

+e++e-H- -©+

•+<H- +e-hf -eH-

-en-

Fig. 3. Association of chromosomes, (A) A diagrammatic drawing of the arrangementof the nonhomologous chromosomes in the generative nucleus of the pollen of Orni-thogalumvirens. Bent interrupted lines indicate connexions, (B) Proposed arrangementof chromosomes at time of fertilization in Ornithogahnn virens. The 2 identical chainsof nonhomologous chromosomes come together so that homologous ends of the 2chains form connexions (broken lines), (c) A schematic diagram of a diploid nucleusshowing B 'folded up' with the 'Rabl orientation' and end-to-end connexion ofchromosomes. Note that homologues lie beside one another.

overlapping chromosome ends, bands, and even whole chromosomes lying ontop of one another. Consequently, nuclei in which each of the 6 chromosomes (andends) can be positively identified and followed are rare. However, a total of 67connexions were observed in 34 cells. All identified connexions were of 4 types:2C-3C (22); 1T-3T (24); 2T-2T (12); and 1C-1C (9). The first 2 (2C-3C and1T-3T) were nonhomologous connexions and of the same type as was observed in thepollen. The latter 2 (2T-2T and 1C-1C) were between homologues and were of thetype that would be predicted if the 2 ends of the gametic chains join homologously atfertilization.

Specific association of chromosome ends

0-01 mm MA

363

1 H

Fig. 4. End-to-end connexions in diploid Ornithogalum virens root tips. Chromosomesare numbered according to the number of C-bands they have. In the photographsin the first column, connexions between non-homologous chromosomes are indicatedby hollow arrows; connexions between homologues (ends of the gametic chains),by black arrows. Column 2 is a tracing of each photograph with bands drawnin, and column 3 is an interpretative drawing of each photograph. Bands are indi-cated by solid lines and centromeres by open circles.

A-C, the telomeric end of the 3 at the far left is connected to the telomeric end ofa 1. The centromeric ends of the two l's are connected. The centromeric end of thesecond 3 is connected to the centromeric end of a 2.

D-F, a nucleus in which the telomeric ends of chromosome 2's are connected. Thecentromeric end of one chromosome 2 is connected to the centromeric end of a 3.The telomeric end of that 3 is connected to the telomeric end of a chromosome 1.The other 1 is connected by its telomere to the other 3.

G-l, the centromeric ends of the two chromosome l's are connected. Thecentromeric end of one chromosome 3 is connected to the centromeric end of achromosome 2.

364 T. Ashley

Open arrows in Fig. 4 indicate the nonhomologous associations: 2C-3C(Fig. 4A, D, G) and 1T-3T (Fig. 4A, D). Solid arrows in Fig. 4 designate homologousassociations: 1C-1C (Fig. 4.A, G) and 2T-2T (Fig. 4D). It should be noted thatexcept where separated by breaks (presumably induced by the squashing procedure),homologous chromosomes lie adjacent to one another.

DISCUSSION

The data presented above offer definite evidence for a high degree of specificchromosome association in nuclei. This specificity falls into 3 categories: centromere-telomere polarization, specific attachment of ends of nonhomologous chromosomes,and specific attachment of certain homologous ends. With these 3 factors in operation,homologous chromosomes lie opposite one another within the nucleus and in aspecific relationship to other nonhomologous chromosomes.

The orientation of chromosomes within the nucleus is one of the most strikingobservations reported above. Centromeric ends consistently associate with centro-meric ends and telomeric ends with telomeric ends. Only one exception to this wasnoted in the 116 end-to-end associations scored in pollen cells.

The tendency for centromeres to lie on one side of the nucleus and telomeres tolie on the other was first observed by Rabl (cf. Wilson, 1925) and referred to as a'Rabl orientation'. Such an arrangement would appear to be a natural consequenceof anaphase segregation in the cells studied here. It suggests a rapid reassociation ofends at late anaphase or early telophase (as was observed by Ashley & Wagenaar, 1974)and/or little movement of chromosomes during interphase, a fact observed as earlyas 1909 by Boveri (cf. Wilson, 1925). Indeed, the high degree of specificity of C-Cand T—T associations observed above suggests that this orientation may be a primaryfactor in establishing chromosomal order within nuclei.

The 2C-3C and 1T-3T connexions in the haploid material account for 88-8%of the end-to-end associations that were observed. This does not necessarily meanthat there is an 11-2% 'error' in non-homologous association of ends within thegenerative nuclei. There is a large difference between the frequency of end-to-endconnexions observed in the current sample and the frequency observed earlier (Ashley& Wagenaar, 1972, 1974). This reduced frequency of end-to-end connexions in thepresent material may be due to an undetermined biological factor. Alternatively, theGiemsa preparative procedure may induce more disruption of end-to-end connexions.The latter possibility is supported by the observation that there is less association oftelomeres in Allium root tips with the Giemsa technique (Fussell, 1977) than with3H-autoradiography and Feulgen staining (Fussell, 1975). For whatever reason, thereappears to have been more disruption of end-to-end connexions in the current study.When end associations are disrupted there is more opportunity for individual chromo-somes to undergo slight displacements, resulting in an occasional overlap of uncon-nected ends or 2 ends lying so close that they may be mistaken for an actual physicalconnexion, thus accounting for some of the 'random' associations.

If this is the case, more centromere-telomere associations might theoretically be

Specific association of chromosome ends 365

expected. However, if all of the centromeres lie on one side of the nucleus and all ofthe telomeres on the other, the displacement induced by the preparative proceduremight be enough to cause a shift of alignment of 2 centromere ends which had broken,but insufficient to rotate a whole chromosome 180 degrees.

There are several documented examples of specific associations between non-homologous chromosomes. Hughes-Schrader (1946) reported end-to-end associationof the 2 chromosomes in spermatozoa of Steatococcus tuberculatus and noted thatthe shorter of the 2 chromosomes always led in the movement of the chromosomesinto the sperm (thus there was a specific order). A specific arrangement of the 8 non-homologous chromosomes in the spermatozoa of the liverwort, Sphaerocarpusdonnellii has also been noted (Reitberger, 1964). Costello (1970) observed a specificarrangement of the chromosomes at metaphase of the first cleavage in Polychoerus(a tubularian with n= 17) that was presumably the consequence of a highly orderedsequential arrangement of the chromosomes in the 2 gametes.

The molecular basis of these non-homologous end-to-end associations is of courseof interest. It has been suggested (Yunis & Yasmineh, 1971) that nonhomologousend-to-end associations might be due to heterochromatic attraction and cohesion(see also the rebuttal by Maguire, 19726). C-bands are generally considered to beheterochromatic (Arrighi & Hsu, 1971), hence the centromeric connexions mightbe explained on this basis. However, the telomeric ends (which accounted for 69 outof 116 connexions) do not contain visible heterochromatin and therefore cannot beexplained in this manner.

In the haploid nucleus the nonhomologous connexions between chromosomes couldbe due to an association of homologous components or to a nonhomologous attraction(here referred to as complementarity). It should be possible to distinguish betweenthese 2 possibilities in the diploid material. If the nonhomologous connexions aredue to homology there are now 4 homologous regions for the 2 connexions observedin the haploid material (e.g., 2 combinations of 2C with 3C, plus 2C with 2C and 3Cwith 3C would be expected). However, if the connexion is due to complementaritythen 2C and 3C ends will associate only with each other and not with any other end.Since only the 2C-3C and 1T-3T associations have been found in root tip material,the complementarity mechanism of attraction seems to be in operation. This wouldmean that any homologous attraction between ends which are also involved innonhomologous connexions is achieved by a different mechanism.

As mentioned above, there was no evidence of association between the 2 ends ofthe haploid chain in chromosomes in the pollen. If there is an identical sequence ofend-to-end associations in egg cells, it is logical to postulate that homologous endsof the 2 chains unite at karyogamy (Fig. 3B). The 1C-1C and 2T-2T connexionsobserved would be predicted if the homologous ends of the gametic chains from theegg and sperm unite at karyogamy. These end associations together with the 'Rablorientation' result in a ring of chromosomes in which homologues lie adjacent to oneanother in the nucleus thereby producing homologous associations (a phenomenonwhich has been reported in a wide variety of organisms).

Evidence of association of homologous chromosomes at anaphase, interphase or24 CEL 38

366 T. Ashley

prophase has been reported in somatic cells (Boss, 1954, 1955; Hiraoka, 1958;Kitani, 1963), while several additional authors have found somatic association ofhomologues in 'pre-meiotic' tissue (Brown & Stack, 1968; Chauman & Abel, 1968;Maguire, 1972 a). A tendency for homologous chromosomes to lie nearer to oneanother on the metaphase plate than might be expected by chance has also beenreported frequently (Feldman, Mello-Sampayo & Sears, 1966; Galperin-Lamaitre,Hens, Kirsch-Volders & Susanne, 1977; and review by Bahr, 1977). Feldman et al.(1966) have attributed this metaphase association to retention of a relationship whichexisted in the preceding interphase nucleus. Since somatic crossing-over presumablydepends on the physical proximity of homologues at the time of exchange, reports ofoccurrence of somatic crossing-over (see Ashley, 1978, for a recent summary of theliterature) are also taken here as evidence of homologous association. Such associationwould obviously facilitate initiation of synapsis during meiotic prophase whetherpairing is initiated at the ends or interstitially.

Reports of end-to-end associations in somatic prophase nuclei are rare, althoughmany published photographs exhibit a conspicuous shortage of 'free ends'. Inactuality, few investigators have closely examined prophase nuclei, presumablybecause they are not amenable to simple study as a consequence of the difficultiesmentioned in the introduction. These difficulties have been circumvented in Orni-thogahim virens with its low chromosome number and simple C-band pattern, whichtogether with the natural prophase arrest of haploid generative nuclei made possiblethe present analysis. While observations of one species do not allow generalizationsabout the universal occurrence of end associations, they do offer an attractive explana-tion for the somatic association of homologues, a phenomenon which has frequentlybeen reported in a large variety of organisms (see above reference), and a simplemechanism for initiation of homologous synapsis at meiosis.

I would like to extend special thanks to Nada Staddon for her patience and initiative inhelping adapt the C-banding technique for use in pollen material. I also wish to thank Danny-Andrews for additional technical assistance and Mr L. H. Muhlbaier in the BiostatisticsSection of Community and Family Medicine at Duke University for his assistance with thestatistics.

Thanks are extended to Drs M. J. Moses and M. Y. Menzel for their critical reading of themanuscript and their helpful comments during the course of this work.

The research was supported by National Institutes of Health Grants GM-23712 to TerryAshley and HD-12225 and National Science Foundation Grant PCM-76-00440 to Dr M. J.Moses, in whose laboratory the work was performed.

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