J. Cell Set. 38, 357-367 (i979)
Printed in Great Britain © Company of Biologists Limited 1979
357
SPECIFIC END-TO-END ATTACHMENT OF
CHROMOSOMES IN ORNITHOGALUM
VIRENS*
TERRY ASHLEYf
Department of Anatomy, Duke University Medical Center, Durham,
North Carolina 27710, U.S.A.
SUMMARY
C-banding of nonhomologous chromosomes in haploid generative nuclei of Ornithogalum
virens (« = 3) reveals a high degree of specificity with respect to end-to-end connexions. The
centromeric end of chromosome 2 preferentially associates with the centromeric end of
chromosome 3 and the telomeric end of chromosome 3 associates preferentially with the
telomeric end of chromosome 1. This same association of nonhomologous chromosomes
persists in prophase nuclei of diploid root tips. In addition, the telomeric ends of the 2 chromosome 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. Centromeric ends lie on one side of the nucleus and telomeric ends on the other. It is proposed that
this specific association of chromosome ends reflects an order which was probably established
at the preceding anaphase ortelophase and which persists throughout interphase.The suggestion
is made that the proximity of homologous ends and consequently homologous alignment may
facilitate initiation of pairing at meiosis.
INTRODUCTION
' Is there a specific arrangement of chromosomes within nuclei ?' is a question
which has often been raised (see Comings, 1968; Ashley & Wagenaar, 1974, for
reviews). The question (and answer) has important implications for one of the central
problems of cytogenetics, initiation of homologous chromosome synapsis in meiotic
prophase; however, conclusive answers have been elusive. Difficulties include the
near impossibility of determining chromosome arrangement in interphase nuclei,
the confusion of large numbers of chromosomes present in most organisms, and the
problem of positive identification of each chromosome in the complement.
Examination of nuclei in which chromosomes are in transition from the interphase
to the prophase state offers one approach to the problem. At this stage the chromosomes are individually visible, but might still be expected to retain their interphase
relationships with each other and with the nuclear membrane. By selecting an
organism with a haploid chromosome number of 3 (Ornithogalum virens), Ashley &
Wagenaar (1974) detected an end-to-end arrangement of prophase chromosomes in
haploid, diploid and tetraploid tissue. They suspected that the chromosomes were
* This paper is dedicated to Professor Sally Hughes-Schrader, who first reported a specific
end-to-end association of chromosomes and whose delight in chromosomes and in life brightens
the lives of all those fortunate enough to know her.
t Present address, to which off-print requests should be sent: Laboratory of Genetic
Research, Delphian Foundation, Sheridan, Oregon 97378, U.S.A.
358
T. Ashley
connected to one another in a specific sequence. However, since all 3 chromosomes of
Ornithogalum virens are acrocentric and approximately the same length, the identity
of each chromosome and consequently the specificity of end associations could not be
positively determined in their material. With the advent of the new banding techniques
(Arrighi & Hsu, 1971), identification of each chromosome and determination of its
prophase relationship with any other chromosome in the nucleus became a possibility.
Stack, Clarke, Cary & Muffley (1974) C-banded root tips of Ornithogalum virens
and determined that the bands were located at the centromeric ends of each acrocentric
chromosome. Therefore, C-banding allows identification not only of each chromosome, but of each end of each chromosome. C-banding of pollen material has proven
more difficult, but by modifying the technique a sufficiently large sample of material
with distinct bands has been obtained to complete a statistical analysis of end-to-end
associations. Analysis of end-to-end associations of the nonhomologous chromosomes
in pollen and of the arrangement of both homologous and nonhomologous chromosomes in prophase diploid root tips is presented below. These observations offer an
explanation for somatic crossing over and somatic association and an insight into how initiation 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 of
Duke University. The bulbs were originally obtained from Dr A. H. Sparrow at the Brookhaven National Laboratory.
Preparation of haploid generative nuclei from pollen
Buds immediately above those which had just opened were collected and placed on moist
filter 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 was
placed 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 were
air-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 °C
for 2 h and rinsed gently in running deionized water, incubated in 6 x SSC (saline-sodium
citrate) at 60 °C for 10 min, rinsed and stained 10 min in 10% Gurr Giemsa (Bio/Medical
Specialties) in 0-12 M sodium-potassium phosphate buffer at pH 6-8. The slides were rinsed
briefly and checked for C-bands. If the stain was too dark, rinsing was continued until C-bands
were distinct. As soon as the bands could be clearly discerned, the slides were removed from
the 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 the
bulbs 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 and
fixed in fresh 3 parts 95 % ethanol : 1 part glacial acetic acid, rinsed in distilled water and
hydrolysed in I N HC1 at 60 °C for 10 to 15 min. The root tips were then rinsed again and
squashed in 45 % acetic acid. The coverslip was removed as described above and the preparations air-dried in a 60 °C oven from 1 to 12 h. Slides were placed directly into 5 % Gurr Giemsa
for 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 for
chromosomes and bands was obtained with phase optics and a Vivitar orange 02 filter. Cells
were 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 2
mitotic 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 Ornithogalum virens the generative nucleus is arrested at prophase until after germination
of the pollen, thus providing a highly synchronized population of cells at precisely
the stage needed for this study.
C-bands of the prophase chromosomes of the generative nuclei (Fig. 1) were
similar to those previously reported for metaphase root tips (Stack et al. 1974). One
chromosome had one C-band (chromosome C of Ashley & Wagenaar, 1974); another
had 2 C-bands (chromosome A), and a third had 3 C-bands (chromosome B). They
are 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, the
banded ends of the chromosomes will be referred to as iC, 2C and 3C, while the
distal (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 probable
associations of ends of nonhomologous chromosomes (Fig. 2). As is readily apparent
from examination of the distribution of associations in Fig. 2, this is not the case
0\!2 = 326; P value for o-ooi confidence level=3i). Clearly, the association of ends is
not random, but highly specific. Most associations are of one of 2 types: 2C-3C and
1T-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 these
2 types of connexions. Of the 16 cells in which there were 2 connexions, 15 exhibited
both the 2C-3C and 1T-3T connexions. It can be concluded that the preferred
arrangement of nonhomologous chromosomes in generative nuclei is the telomeric end
of 1 connected to the telomeric end of 3 and the centromeric end of 3 connected to
the 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 Ornithogalum virens form an open chain, rather than a closed circle. If the associations
resulted 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 in
only 6 out of 116 cases, and these associations were random (Fig. 2). The other end
of 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 nonrandom, although the sample is too small for an adequate analysis.
T. Ashley
Fig. i. End-to-end association of nonhomologous chromosomes in haploid generative
nuclei of the pollen of Ornithogalum virens. Chromosomes are numbered according
to the number of C-bands they possess. Centromeric ends are indicated by solid
arrows. Connexions between chromosome ends are indicated by open arrows.
Nuclei in Fig. IA and G have connexions between the centromeric ends of chromosomes 2 and 3; nuclei in B-F have connexions between the telomeric ends of chromosomes i and 3.
Specific association of chromosome ends
361
The data also support the hypothesis of Ashley & Wagenaar (1974) that centromeric
ends associate preferentially with centromeric ends, and telomeric ends with telomeric
ends. 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-telomere
connexions (Fig. 2).
1T
X
2
0
2T
0
2
X
3C
3
0
41
0
3T
1
62
0
5
X
1C
1T
2C
2T
3C
2C
Fig. 2. The observed frequencies of the 12 possible end-to-end connexions between
the 3 nonhomologous chromosomes of Ornithogalum virens pollen. If associations
were random, all connexions should be equally frequent. C indicates the end of the
chromosome nearest the centromere, T indicates the distal end. The numbers
identify the chromosome by the number of C-bands present. Thus, 1C is the centromeric end of the chromosome with one C-band, etc.
Diploid nuclei of root tips
Ashley & Wagenaar (1974) suggested that there might be an identical specific
sequential arrangement of the chain of nonhomologous chromosomes in both gametes
and that, at fertilization, homologous ends of the two chains joined to form a closed
circle (Fig. 3B). Thus the sequence of nonhomologous chromosomes would be
maintained, but now homologous chromosomes would lie adjacent to one another
within the diploid nucleus (Fig. 3B, c). On this basis, 2C-3C and 1T-3T connexions
should also be found in diploid tissue. In addition, 2T-2T and 1C-1C homologous
connexions would be predicted as a result of the joining of the 2 gametic chains to form
a closed loop.
In squash preparations of pollen the chromosomes are pressed out of the pollen
grain walls along with the cytoplasm resulting in extensive separation and spreading.
Extrusion of the cellular contents is much less frequent in root tip material. The
small 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 arrangement
of the nonhomologous chromosomes in the generative nucleus of the pollen of Ornithogalumvirens. Bent interrupted lines indicate connexions, (B) Proposed arrangement
of chromosomes at time of fertilization in Ornithogahnn virens. The 2 identical chains
of nonhomologous chromosomes come together so that homologous ends of the 2
chains form connexions (broken lines), (c) A schematic diagram of a diploid nucleus
showing B 'folded up' with the 'Rabl orientation' and end-to-end connexion of
chromosomes. Note that homologues lie beside one another.
overlapping chromosome ends, bands, and even whole chromosomes lying on
top of one another. Consequently, nuclei in which each of the 6 chromosomes (and
ends) can be positively identified and followed are rare. However, a total of 67
connexions 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 and
1T-3T) were nonhomologous connexions and of the same type as was observed in the
pollen. The latter 2 (2T-2T and 1C-1C) were between homologues and were of the
type that would be predicted if the 2 ends of the gametic chains join homologously at
fertilization.
Specific association of chromosome ends
0-01 mm
363
MA
1 H
Fig. 4. End-to-end connexions in diploid Ornithogalum virens root tips. Chromosomes
are numbered according to the number of C-bands they have. In the photographs
in the first column, connexions between non-homologous chromosomes are indicated
by hollow arrows; connexions between homologues (ends of the gametic chains),
by black arrows. Column 2 is a tracing of each photograph with bands drawn
in, and column 3 is an interpretative drawing of each photograph. Bands are indicated 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 of
a 1. The centromeric ends of the two l's are connected. The centromeric end of the
second 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. The
centromeric 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. The
centromeric end of one chromosome 3 is connected to the centromeric end of a
chromosome 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 homologous
associations: 1C-1C (Fig. 4.A, G) and 2T-2T (Fig. 4D). It should be noted that
except 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 specific
chromosome association in nuclei. This specificity falls into 3 categories: centromeretelomere 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 a
specific relationship to other nonhomologous chromosomes.
The orientation of chromosomes within the nucleus is one of the most striking
observations reported above. Centromeric ends consistently associate with centromeric ends and telomeric ends with telomeric ends. Only one exception to this was
noted 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 to
lie 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 consequence
of anaphase segregation in the cells studied here. It suggests a rapid reassociation of
ends 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 early
as 1909 by Boveri (cf. Wilson, 1925). Indeed, the high degree of specificity of C-C
and T—T associations observed above suggests that this orientation may be a primary
factor 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 mean
that there is an 11-2% 'error' in non-homologous association of ends within the
generative nuclei. There is a large difference between the frequency of end-to-end
connexions observed in the current sample and the frequency observed earlier (Ashley
& Wagenaar, 1972, 1974). This reduced frequency of end-to-end connexions in the
present material may be due to an undetermined biological factor. Alternatively, the
Giemsa preparative procedure may induce more disruption of end-to-end connexions.
The latter possibility is supported by the observation that there is less association of
telomeres in Allium root tips with the Giemsa technique (Fussell, 1977) than with
3
H-autoradiography and Feulgen staining (Fussell, 1975). For whatever reason, there
appears 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 chromosomes to undergo slight displacements, resulting in an occasional overlap of unconnected ends or 2 ends lying so close that they may be mistaken for an actual physical
connexion, 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 of
the telomeres on the other, the displacement induced by the preparative procedure
might 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 nonhomologous chromosomes. Hughes-Schrader (1946) reported end-to-end association
of the 2 chromosomes in spermatozoa of Steatococcus tuberculatus and noted that
the shorter of the 2 chromosomes always led in the movement of the chromosomes
into the sperm (thus there was a specific order). A specific arrangement of the 8 nonhomologous chromosomes in the spermatozoa of the liverwort, Sphaerocarpus
donnellii has also been noted (Reitberger, 1964). Costello (1970) observed a specific
arrangement of the chromosomes at metaphase of the first cleavage in Polychoerus
(a tubularian with n= 17) that was presumably the consequence of a highly ordered
sequential arrangement of the chromosomes in the 2 gametes.
The molecular basis of these non-homologous end-to-end associations is of course
of interest. It has been suggested (Yunis & Yasmineh, 1971) that nonhomologous
end-to-end associations might be due to heterochromatic attraction and cohesion
(see also the rebuttal by Maguire, 19726). C-bands are generally considered to be
heterochromatic (Arrighi & Hsu, 1971), hence the centromeric connexions might
be explained on this basis. However, the telomeric ends (which accounted for 69 out
of 116 connexions) do not contain visible heterochromatin and therefore cannot be
explained in this manner.
In the haploid nucleus the nonhomologous connexions between chromosomes could
be due to an association of homologous components or to a nonhomologous attraction
(here referred to as complementarity). It should be possible to distinguish between
these 2 possibilities in the diploid material. If the nonhomologous connexions are
due to homology there are now 4 homologous regions for the 2 connexions observed
in the haploid material (e.g., 2 combinations of 2C with 3C, plus 2C with 2C and 3C
with 3C would be expected). However, if the connexion is due to complementarity
then 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 would
mean that any homologous attraction between ends which are also involved in
nonhomologous connexions is achieved by a different mechanism.
As mentioned above, there was no evidence of association between the 2 ends of
the haploid chain in chromosomes in the pollen. If there is an identical sequence of
end-to-end associations in egg cells, it is logical to postulate that homologous ends
of the 2 chains unite at karyogamy (Fig. 3B). The 1C-1C and 2T-2T connexions
observed would be predicted if the homologous ends of the gametic chains from the
egg and sperm unite at karyogamy. These end associations together with the 'Rabl
orientation' result in a ring of chromosomes in which homologues lie adjacent to one
another in the nucleus thereby producing homologous associations (a phenomenon
which has been reported in a wide variety of organisms).
Evidence of association of homologous chromosomes at anaphase, interphase or
24
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 of
homologues in 'pre-meiotic' tissue (Brown & Stack, 1968; Chauman & Abel, 1968;
Maguire, 1972 a). A tendency for homologous chromosomes to lie nearer to one
another on the metaphase plate than might be expected by chance has also been
reported 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 which
existed in the preceding interphase nucleus. Since somatic crossing-over presumably
depends on the physical proximity of homologues at the time of exchange, reports of
occurrence of somatic crossing-over (see Ashley, 1978, for a recent summary of the
literature) are also taken here as evidence of homologous association. Such association
would obviously facilitate initiation of synapsis during meiotic prophase whether
pairing is initiated at the ends or interstitially.
Reports of end-to-end associations in somatic prophase nuclei are rare, although
many published photographs exhibit a conspicuous shortage of 'free ends'. In
actuality, few investigators have closely examined prophase nuclei, presumably
because they are not amenable to simple study as a consequence of the difficulties
mentioned in the introduction. These difficulties have been circumvented in Ornithogahim virens with its low chromosome number and simple C-band pattern, which
together with the natural prophase arrest of haploid generative nuclei made possible
the present analysis. While observations of one species do not allow generalizations
about the universal occurrence of end associations, they do offer an attractive explanation for the somatic association of homologues, a phenomenon which has frequently
been reported in a large variety of organisms (see above reference), and a simple
mechanism for initiation of homologous synapsis at meiosis.
I would like to extend special thanks to Nada Staddon for her patience and initiative in
helping adapt the C-banding technique for use in pollen material. I also wish to thank DannyAndrews for additional technical assistance and Mr L. H. Muhlbaier in the Biostatistics
Section of Community and Family Medicine at Duke University for his assistance with the
statistics.
Thanks are extended to Drs M. J. Moses and M. Y. Menzel for their critical reading of the
manuscript and their helpful comments during the course of this work.
The research was supported by National Institutes of Health Grants GM-23712 to Terry
Ashley 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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