/. Embryol. exp. Morph. 83, Supplement, 51-73 (1984)
Printed in Great Britain The Company of Biologists Limited 1984
Chromosome order — possible implications for
development
By J. S. HESLOP-HARRISON AND M. D. BENNETT
Plant Breeding Institute, Maris Lane, Trumpington, Cambridge CB2 2LQ,
U.K.
TABLE OF CONTENTS
Summary
The recognition of order
The demonstration of order
The developmental implications of order
Molecular effects of order
The nucleotype and order
Effects of order on gene expression
Order and chromosome behaviour
Chromosome elimination
Chromosome transmission frequencies
The central bivalent and meiosis
The 'end' chromosome
Developmental changes in order
Changes between somatic and meiotic cells
Karyotype evolution
Multiple translocations
Evolution of Hordeum species
Conclusions
References
SUMMARY
Chromosomes are arranged in ordered haploid sets around the centre of the metaphase
plate at mitosis in several grass species and hybrids. Each chromosome is in a fixed mean
position relative to other, heterologous chromosomes, this order can be predicted using
Bennett's model, and is clearly demonstrated from reconstructions of electron micrographs
of serial sections (see Heslop-Harrison & Bennett, 19S3a,b,c).
The nucleus contains spatial domains of genes with similar functions. Chromosomes with
major effects on nuclear behaviour - division or meiotic pairing - may be at special positions
in the order. Changing spatial relationships of chromosomes with respect both to each other
and the nuclear envelope (during the cell cycle and during development) may affect cell
differentiation and gene activity.
Chromosome order may have implications for the control of development within the
nucleus and the organism. Order may constrain karyotype and hence species evolution.
52
J. S. HESLOP-HARRISON AND M. D. BENNETT
THE RECOGNITION OF ORDER
The possibility that chromosomes may have fixed relative dispositions at
mitosis, meiosis and interphase has been considered for many years (e.g. Rabl,
1885; see Comings, 1968, 1980). However, it has generally been assumed that
chromosomes in vivo are as they 'appear' in most metaphase spread preparations, that is, more or less randomly scattered, and with no fixed relationships in
their positions relative to either cell structures or to any other chromosomes.
Nevertheless, there are a few well known cases where chromosomes lie in fixed
relative positions, such as the association of homologues in bivalents at meiosis
(see Darlington, 1937). This is a very tight association, which remains during,
and in spite of, the randomizing influences of fixation and spreading. Indeed, the
chromatin of each bivalent cannot be separated into chromosomes at metaphase
I of meiosis. Another example of the fixed relative positioning of chromosomes
in nuclei is the association of homologous chromosomes seen in Diptera (e.g.
Stevens, 1908).
The subject of this paper is another form of order, less distinct than those just
mentioned - namely order involving each chromosome or bivalent lying in a
fixed mean position relative to one or more other chromosomes or bivalents. The
first section of this paper will review some of our recent findings about this latter
type of order before discussing the evidence that chromosome order might have
implications for the control of development.
THE DEMONSTRATION OF ORDER
Most of the original work discussed here has been carried out using complete
reconstructions of serially sectioned nuclei, or metaphases, made from electron
micrographs of untreated (or ice-water treated) cells from various intergeneric
or interspecific hybrids and species of grasses. The results discussed were collected from analyses of over 300 reconstructions of complete metaphases and interphase nuclei which were made from prints of electron micrographs of 0-ljUm
thick, serial sections examined at 11800 times magnification (Fig. 1). Details of
the techniques used have been published elsewhere and will be referenced in the
discussion below.
The serial section reconstruction technique for looking at relative metaphase
chromosome disposition has the major advantage that the chromosomes in the
reconstructions are not greatly distorted from their in vivo positions (Bennett,
1984c). All the tissue, including the cell walls and cellular organelles, as well as
Fig. 1. Two consecutive sections from a serially sectioned root-tip metaphase of the
hybrid Hordeum vulgare (cv. Tuleen 346) x H. bulbosum (clone L6), showing parts
of the chromosomes (dark areas), centromeres (arrowed) and a satellite region (S).
Bar,
Chromosome order - possible implications for development 53
Fig. 1
54
J. S. HESLOP-HARRISON AND M. D. BENNETT
the nuclear components, are completely intact and clearly seen in the sections.
In contrast to all chromosome-spreading techniques, the electron-micrograph
serial section reconstruction technique causes no deliberate physical or chemical
distortion of either the chromosomes themselves or their positioning within the
nucleus. Each chromosome appears in its in vivo position, with its normal shape
and size. Chromosomes are not the highly contracted, widely spaced, rod-shaped
objects which are seen in many two-dimensional spreads made for the light
microscope.
To show order conclusively, one must know the identity of each chromosome
and the position of that chromosome as it was in vivo in real space (i.e. relative
to all other chromosomes or to an arbitrary origin). Both position and identification data can be accurately obtained from serial section reconstructions of
metaphase nuclei in cells with suitable karyotypes. However, the widely applied
cytological spreading techniques are generally unsuitable for examination of
relative chromosome disposition at any level other than the most gross because
the in vivo positions of chromosomes are largely unknown and certainly change
during preparation for examination.
Using the serial section reconstruction technique, chromosomes were identified by their morphology - their relative and absolute volumes, arm volume
ratio and the presence or absence of secondary constrictions at the nucleolar
organizing region and of constitutive heterochromatin (C-bands). Chromosome
arm volumes were obtained by summing the areas of chromatin from each arm
on all the sections where the arm occurred and then multiplying by the section
thickness to give the absolute arm volume (Bennett, Smith, Ward & Finch, 1982;
Heslop-Harrison & Bennett, 19836). The volume of each arm of each
chromosome was thus calculated separately, and arm volume ratios were calculated. Fig. 2 A shows an idiogram of the relative volumes and arm volume
ratios of all the 14 chromosomes in the nucleus seen in Fig. 1. Comparison of the
idiogram with that of the mean karyotype in these two species (Fig. 2B) allowed
almost unequivocal identification of each chromosome. Each chromosome type
in a cell must have a distinctive and unique morphology for this technique of
identification to be applied with success. The species and hybrids we have worked with have been chosen partly because all, or almost all, of their chromosomes
can be identified with certainty in most cells.
To obtain the position of each chromosome, the coordinates of the centromere
were obtained in three dimensions from the serial section reconstructions - in the
X and Y axes relative to an arbitrary origin near the outside and in the Z axis from
the section number and thicknesses, as described in Heslop-Harrison & Bennett
(1983c).
This three-dimensional centromere position in real space was used in the
subsequent analyses of chromosome order and disposition.
Three major results concerning chromosome disposition have been obtained
from the reconstruction of the serially sectioned nuclei:
Chromosome order - possible implications for development 55
T2
Tl
T3
T4
Bl
B2
17
B3
B4
B7
B5
B6
T6
Fig. 2. Idiograms showing the relative volumes of the chromosome arms from serially sectioned root-tip metaphases of the hybrid shown in Fig. 1. Numbers refer to
the gene linkage group assigned to each centromere; T indicates the Tuleen 346
chromosomes and B indicates the H. bulbosum chromosomes. The length of each
arm is proportional to its volume relative to the other chromosomes in the cell. (A)
The individual chromosomes of the cell shown in Fig. 1. (B) The mean relative
chromosome sizes in six cells of the hybrid. (Data from Schwarzacher, Finch &
Bennett, unpublished.)
1. All centromeres are non-randomly positioned on the metaphase plate;
2. Complete haploid genomes tend to be separated in diploid and hybrid
cells; and,
3. Heterologous chromosomes are in a predictable mean fixed order within
each of the haploid genomes.
The results on the disposition of the centromeres show that centromeres at
mitotic metaphase were aligned on a flat plane as expected. However, they were
not randomly distributed over the whole area of the plate (Heslop-Harrison &
Bennett, 1983c), but showed a normal distribution around a circle about the
centre of the metaphase plate, with the distribution of all distances between pairs
of centromeres differing significantly from the expectation assuming centromeres were randomly distributed over the whole area of the plate.
T5
56
J. S. HESLOP-HARRISON AND M. D. BENNETT
Using reconstructions of electron micrographs of serial sections for the
identification and positioning of chromosomes, we showed that the centromeres
of homologous chromosomes were not associated in either the root meristem
cells at mitotic metaphase or pollen mother cells at the last mitosis before meiosis
(premeiotic mitosis or PMM). Indeed, on average, homologues were significantly further apart than heterologues. Thus, there was no evidence for the somatic
association of homologues.
In the reconstructed metaphases, the reverse situation - the spatial separation
of complete haploid genomes of chromosomes - occurred. In interspecific and
intergeneric hybrids, the metaphases tended to show concentric genome separation. Complete haploid chromosome sets could be separated by drawing a circle
on a view of the metaphase centromeres seen from a spindle pole. Fig. 3 shows
polar views of a sample of cells from a hybrid. In pure species, there was a
significant tendency for cells to show genome separation with the two haploid
chromosome sets lying side by side (Finch, Smith & Bennett, 1981; Bennett,
1983).
From such results, we concluded that the disposition of whole genomes of
chromosomes and their centromeres is non-random. Further analyses have
shown that chromosome positions in reconstructed metaphases were predictable
and that each chromosome tended to be in afixedmean position relative to other
heterologous chromosomes. The chromosome order is predictable using a
o
o
o
o
o
o
o
o
o
0
0
o o
o
•00
00
o
o
o
o
%
0
0
0
o
o
0
o
• o
o
0
o
o
Fig. 3. Polar views (as they would appear when viewed from a spindle pole) of six
root tip metaphase cells of the hybrid shown in Fig. 1. Positions of the centromeres
of the H. bulbosum chromosomes are represented by open circles and the positions
of the H. vulgare centromeres by solid circles.
o
Chromosome order - possible implications for development 57
simple model which associates each chromosome in a simple haploid set with two
constant neighbours. The model was developed by Bennett (1983) and mainly
uses the relative sizes of chromosome arms in the complement as the basis for
making the prediction of order.
Fig. 4 shows the mean karyotype of H. bulbosum and the prediction of the
mean order of chromosomes made using the Bennett model. Most-similarlysized pairs of long and pairs of short arms within a simple haploid genome were
associated to complete a sequence of chromosomes, which is shown in Fig. 4B.
In species with an odd number of chromosomes, there is a discontinuity where
a long and short chromosome arm come together. The chromosome or chromosome arms at this point in the order may have a special significance which will
be discussed below.
An average order of chromosomes in haploid sets of chromosomes using their
positions in reconstructed cells was computed using techniques described
elsewhere (e.g. Heslop-Harrison & Bennett, 1983a,fr; Heslop-Harrison, 1983a).
Comparison of the mean order of chromosomes, in the reconstructed cells, with
the prediction of the order made using Bennett's model (see Bennett, 1983)
showed that the predicted order of chromosomes was significantly expressed.
Thus, in samples of seven to twenty reconstructed serially sectioned
metaphases of the species Aegilops umbellulata and Hordeum vulgare and hybrid
nuclei including haploid genomes of Secale africanum and H. bulbosum, we have
shown that the order of chromosomes predicted before the start of the experiment was that actually present as a mean when all the cells were summarized. For
example, in H. bulbosum the predicted order of chromosomes,
-4-1-2-3-6-5-7-,
(using the numbering of chromosomes shown in Fig. 4) ranked first of all the 360
possible orders of chromosomes in complete haploid sets in this species.
From these results, we can conclude that the chromosomes are in afixedmean
spatial order within the nucleus at metaphase. The same - or perhaps only
slightly different - order is probably also present at interphase, and may even be
more strongly expressed at this stage in the cell cycle. Varied evidence has
suggested that metaphase chromosome positions reflect the interphase positions
(e.g. Tanaka, 1981). For example, Cremer etal. (1982) examined the relationship between interphase and metaphase chromosome positions using u.v.
microbeam irradiation techniques and concluded that their experiments supported the assumption that interphase and metaphase chromosome positions were
related.
Mathog et al. (1984) found that the giant chromosomes in Drosophila had 'an
underlying order [which was] present beneath the considerable flexibility in the
overall structure', which supports our findings at metaphase, and suggests that
a similar degree of order may be present both at interphase and metaphase.
Because interphase is the time when genes are actively transcribed, it is the
58
J. S. HESLOP-HARRISON AND M. D. BENNETT
period when the ordering of chromatin is most likely to affect cellular, as well as
nuclear, functioning. Therefore, it is important to know if chromosome ordering
is less, equally, or more strongly expressed at interphase than at metaphase.
Volume. Difference
d
Long arms
7 <
1
10-18
3 <
1 -i
9-33
2 <
J
8-98
discontinuity
0-35
4 <
8-14
015
1 ^
7-99
5 <
7-53
6 <
6-39
1-14
Short arms
1 <
1
2 <
]
3 <
-7-68
0-78
6-90
6-34
0-32
6 <
0 1
4 <
5-69
1
5 <
7 <
6-02
I
ID
]
4-67
discontinuity
0-50
4-17
Sum d = 3-24
Fig. 4. The prediction of the chromosome order in the haploid complement of H.
bulbosum. (A) The chromosome arms ranked by decreasing volume (as a percentage
of a haploid complement) for long and short arms. Those predicted to be adjacent
by the model are linked and the difference in percentage volume between each pair
is shown and differences are summed. The two arms (one long and one short) at the
discontinuity of the order are not linked. (B) The haploid complement ordered as
predicted by Bennett's model. The numbers beside each long arm (L) and each short
arm (S) refer to their volumes as a percentage of the total haploid set, while numbers
in the circles refer to the chromosome linkage group.
Chromosome order - possible implications for development 59
THE DEVELOPMENTAL IMPLICATIONS OF ORDER
This section summarizes some aspects of chromosome order which may affect
the growth, development and differentiation of organisms. The potential implications of an order of chromosomes may extend through all levels from the
molecular to species evolution. All the features to be discussed eventually affect
which genes are transcribed or active, and so can be considered as developmental
effects of order.
Molecular effects of order
In the plant species so far analysed, we have shown how chromosome arms on
heterologues which are adjacent in the order are matched closely in size as
predicted by the Bennett model. One feature which can be examined in the
resulting order is the distribution of highly repeated DNA sequences, which may
9-33
jSji-90
s
7-68
7-53
L7-99
L8-14
1
—' discontinuity
10-18
Fig. 4B
60
J. S. HESLOP-HARRISON AND M. D. BENNETT
be visualized by heterochromatin banding with Giemsa or other stains. When the
chromosomes' C-banding patterns are superimposed on the pairs of arms which
are adjacent in the order, particular patterns and features of their distribution are
seen. For example, in Secale africanum, Bennett (1982) noted that chromosome
arms which were adjacent in the order usually had similar telomeric C-bands
(Fig. 5), while one of the arms at the discontinuity had a unique intercalary
C-band. Further results supporting this work were presented by Greilhuber &
Loidl (1983), who found that the positions of heterochromatin blocks along
chromosome arms of Scilla species matched when the chromosomes were
ordered according to the Bennett model.
A second aspect of repeated sequence positioning, size and type which
chromosome order may affect is the 'concerted evolution' of families of repeated
DNA sequences. This has been shown to occur on two or more non-homologous
chromosomes by Dover and colleagues (e.g. Strachan, Coen, Webb & Dover,
1982). It is unclear how the process of homogenization of interspersed repeated
DNA sequences between chromosomes occurs over a long timescale as proposed
by the molecular drive theory of Dover (e.g. 1982). However, Coen, Strachan,
Brown & Dover (1983) have suggested the interesting possibility that a fixed
spatial order of chromosomes might 'facilitate and perhaps determine the pattern of inter-chromosome transfer of these sequences' between spatially adjacent arms.
Both the fact of order and the occurrence of similar DNA families on
heterologous chromosomes, tend to limit the concept of the independence of the
chromosomes, since each one is dependent and related to other chromosomes in
the complement. The ordering of chromosomes provides a rationale for the
positioning of certain repeated DNA families.
The nucleotype and order
Bennett (1982) defined the nucleotype as those non-genic characters of the
nuclear DNA that affect or control the phenotype, independent of its encoded
informational content. The ordering of chromosomes is, at least partly, a nucleotypic phenomenon because the disposition of chromosomes is determined by
their relative arm sizes and hence the relative DNA contents of the arms.
The results described above showing that chromosomes are in a fixed position
implies that not only are all the genes on one chromosome linked, but also that
genes on chromosomes adjacent in the order tend to be associated. Genes which
have the same name, and thus, often, the same function, are known as
paralogous genes. Evidence from a species with an extensive gene map in which
we have investigated order using the serial section technique has now shown that
paralogous genes are also often mapped to adjacent chromosomes in the order.
The test made in Zea mays (Bennett, 1983) showed that significantly more
paralogous genes were on adjacent heterologous chromosomes than would be
expected by chance alone. In the other species where we have investigated order
Chromosome order - possible implications for development 61
1
2
3
4
5
7
X
X X
1
6
1-12
1-85
1-54
2-34
1-05
1-38
5-87 L
\
5-43
5-17
6-30
Fig. 5. The haploid complement of Secale africanum showing the major C-bands (A)
arranged in order of decreasing chromosome size (as a conventional idiogram; arm
ratios are shown beneath each chromosome) and (B) ordered according to Bennett's
model and shown when position data were averaged from nine reconstructed
metaphases (numbers by arms refer to the mean chromosome volume as a percentage of the total haploid complement). (After Bennett, 1982.)
EMB 83S
62
J. S. HESLOP-HARRISON AND M. D. BENNETT
using reconstructions of serial sections, there are not enough mapped genes to
allow any such tests of gene locations.
Tests for gene associations have been carried out by Vogel & Kriiger (1983)
using human chromosome positions estimated from chromosome reunion figure
frequencies found in chromosome translocation syndromes. However, they
found that 'there was no general tendency for a closer location of chromosomes
containing genes with related function. A few such chromosomes do show below
average distances but this could easily be a chance result.' They cite a number
of reasons why their results may not have shown any gene association even if it
were there. These included possible errors in the gene map, errors in determining or in analysing chromosome positions, and the use of genes which although
close in function were active at different times or developmental stages.
If chromosomes are in an order, then all the genes in the nucleus could be
arranged in a systematic sequence which has relevance to development. This
could be similar to the genes which are on the lambda phage described by
Brammer & Hadfield (this volume), where there is a logical sequence of different
domains of coding and regulatory DNA sequences throughout the genome. E.
coli gene distributions and their possible relationship to any ordering of genes in
eukaryotes are discussed by Vogel & Kriiger (1983). In E. coli (which has a very
well known gene map), some of the functionally related genes are clustered (e.g.
chemotaxis, flagellar synthesis and motility are all 41-43 % along the genome
from an arbitrary starting point, the gene thrA) while other genes are dispersed
throughout the genome (e.g. the Aromatic genes, aroA to aroT; see Bachman,
1982). However, more complex genomes may need a more systematic arrangement of genes with respect to the relative locations of functionally related genes
and the replication schedule (since transcription and translation are mutually
exclusive).
In future, knowledge of the relationships of genes in space, as well as in
function, could be used to place genes (by genetic engineering) in positions
suitable for transcription, and perhaps to interrelate the functioning of particular
groups of genes active at one developmental period.
Effects of order on gene expression
Evidence from interspecific hybrids studied in serial section reconstructions
indicates that chromosome positions are important for the control of gene expression and of DNA transcription. All of the five hybrids examined had stable
karyotypes with the complete chromosome complement present in most cells,
even although some hybrids were infertile or apparently failed to reach meiosis.
As discussed above, there was a highly significant tendency for concentric
parental genome separation (Fig. 3). However, in four of these five cases, the
appearance of the hybrid plant was similar to that of the parent providing the
outermost centromeres in the ring (Bennett, 1984/?). Because the appearance of
the plant is under genetic control, this evidence strongly implies that the outermost
Chromosome order - possible implications for development 63
genes in the nucleus are the ones most likely to be expressed in the phenotype.
Conversely, Hsu (1975) proposed a theory that the expressed genes were inside
the nucleus with a 'bodyguard' of non-transcribed, C-heterochromatin rich,
chromosomes or chromatin around the nuclear periphery. He suggested that
these heterochromatic bodyguard chromosomes 'absorb the assault' of
'mutagens, clastogens or even viruses attacking the nucleus.' However, more
recently, Hens, Kirsche-Volders & Susanne (1983) discussed the spatial
positions of chromosomes within human nuclei with relationship to this
hypothesis, but found only very poor evidence that the most gene-dense
chromosomes were at the centre of the nucleus while the others were more
peripheral.
There is one major exception to the, perhaps quite general, rule that the
outermost chromosomes or chromosome segments carry the genes which are
most likely to be expressed. The exception is the nucleolar organizing (NO)
genes which, when active, form a nucleolus which is usually approximately
central within the nucleus. Our results from hybrid cells showed that the NO
region(s) of the inner chromosome complement was usually expressed but the
NO of the outer chromosome complement was not. This indicated that the
spatial position within the nucleus of a gene was related to its expression.
Nicoloff, Anastassova-Kristeva, Rieger & Kiinzel (1979) found that the
relative positions of rDNA genes in the nucleus can have a major effect on their
expression. Barley has two pairs of nucleolar organizing chromosomes in the
diploid species. Using a genotype including a translocation, so that both nucleolus organizing regions were translocated onto one chromosome, they found a
maximum of two normally sized nucleoli in the cells, because only one pair of
nucleolus-organizing regions were fully active (rather than both pairs as is usual).
The suppression of the second pair of nucleolar genes was apparently caused by
interference between the two sets of nucleolus-organizing regions due to their
proximity, giving a clear position effect with respect to the control of DNA
transcription.
The work of Jack & Judd (1979) is important in highlighting a locus, the Zeste
white, in Drosophila which may be affected by the physical position of the genes
in the nucleus, and the spatial relationship of the genes and controlling loci. They
proposed 'a model (which) necessarily imposes a specific architecture on the
chromatin of the interphase nucleus.' Using insertional translocations and
various alleles of two loci, they concluded that there was communication which
was strongly dependent on the close proximity of gene alleles, demanding a
precise nuclear architecture. They then predicted that chromosomes in interphase nuclei 'occupy precise positions relative to one another and that an important aspect is the close association of homologues.' While we have found no
evidence for the somatic association of homologues in the nuclei of several higher
plants (unlike Drosophila which shows close somatic association of homologous
chromosomes), our data support their conclusion that chromosomes could be in
64
J. S. HESLOP-HARRISON AND M. D. BENNETT
fixed relative positions. The work of Mathog et al. (1984) also supports this
theory.
Order and chromosome behaviour
Chromosome elimination
Effects of concentric separation of haploid genomes of chromosomes on gene
expression in stable hybrids were mentioned above. However, crosses between
different genotypes of the same species can also be made which are not stable,
and eliminate single or all chromosomes from one parental set, often giving rise
to haploid plants. In all cases where we have examined chromosome disposition
in potentially unstable hybrids, the chromosome set on the outside of the
metaphase plate at mitosis is the one which was eliminated (Finch & Bennett,
1981, 1983; Bennett, 1984a).
In addition to the peripheral positioning of chromosomes, the centromere size
and number of microtubules attached to each centromere also varied between
the genomes. The centromeres of the genome most likely to be eliminated were
much more difficult to identify (because of their weaker expression) than those
of the stable genome, and also tended to have fewer spindle microtubules
attached (in cells where the microtubules could be easily counted).
Chromosome transmission frequencies
In most plants, many aneuploid lines can be stably maintained and the plants
may breed freely. However, the ease with which individual chromosomes can be
obtained as aneuploids - and thus the behaviour of the individual chromosomes
- varies widely. While the genetic effects from imbalances caused by aneuploidy
are obvious, there might also be a basically mechanical effect on chromosome
and nuclear behaviour which is caused by the positions of chromosomes. In
barley, the aneuploid behaviour is particularly well documented; if a trisomic
(2n+l) plant is self pollinated, the progeny would be expected to contain a
proportion of tetrasomic plants (2n+2). The progeny of each of the seven different types of parental trisome might be expected each to contain approximately
similar frequencies of tetrasomics. However, one of the end chromosomes,
number 7, occurs as a tetrasomic in about 6 % of the number of seeds set, while
the two chromosomes next to it in the order, 4 and 5, are found in less than 1 %
as tetrasomics (Bennett, 1981). No other chromosomes have been found as
tetrasomes in these crosses. It seems likely that the chromosome order has some
effect on the behaviour of chromosomes in these crosses. Other examples of
transmission frequencies following the chromosome order in barley include
trisomic transmission frequencies (Bennett, 1982).
The central bivalent at meiosis
At mitosis, it is unusual to find a chromosome at or near the centre of the
Chromosome order - possible implications for development 65
metaphase plate. However, at meiosis, one or more bivalents or unpaired
chromosomes regularly occupy this position (Heslop-Harrison, 19836).
Preliminary evidence shows that this central bivalent often has special properties. In hexaploid wheat chromosome 5B contains the gene locus Phi, which
controls bivalent rather than multivalent occurrence seen at metaphase I of
meiosis (Riley & Chapman, 1958). In serial section reconstructions, evidence
from size indicated that the 5B bivalent occupies a position near the centre of the
metaphase plate at metaphase I of meiosis (Heslop-Harrison, 19836).
In several animal species, the sex chromosome is centrally located at meiosis
with the other, autosomal, bivalents located around it. For example, Darlington
& LaCour (1960) noted the central co-orientation of the sex chromosomes at
metaphase II in Cimex rotundatus. More recently, in serial section reconstructions of cells of the nematode, Caenorhabditis elegans, Albertson & Thomson
(1982) showed that the sex univalent was in the central position at metaphase I.
It seems likely that the central position on the meiotic metaphase plate is important in the development in these species as well as wheat.
While the significance in developmental terms of a bivalent (or chromosome
or group of particular chromosomes or bivalents) occupying the central position
on the plate at metaphase I is still unknown, a bivalent at this position is potentially unique in having an equal spatial relationship to other bivalents within the
cell. The data reported above imply that this central location on the plate might
have a special influence on other chromosomes or bivalents.
The 'end' chromosome
In the description of the model for chromosome order above, the discontinuity
in the order, where arms were not matched was shown (Fig. 4A). The end arms
of the order, at this discontinuity, are usually exceptional in the karyotype either
in the genes they contain or in their morphology.
The min gene in Barley is located on the end arm of the order of chromosomes
(Bennett, 1981) and provides an example of a 'gene' with an unusual effect. Cells
with this gene become polyploid - up to 60X - and the plant's roots appear as
though they had been treated with colchicine. It may be significant that the locus
is at the end position in the chain, where it might be at a controlling location
where the events of cell division could be determined.
In most of the species where we have examined order, the chromosome arms
at the discontinuity tend to have morphological features unique in the karyotype.
These include intercalary C-bands (5. africanum, Fig. 5), the longest or shortest
arms in the chromosome complement (A. umbellulata) or extreme arm ratios (H.
bulbosum).
In species which other researchers have worked with, there are particular
chromosome arms with special features which make them unique and perhaps
'end' arms of an ordered set of chromosomes. In two species of Triturus, the
longest arm of the chromosome complement has unusual properties. It is much
66
J. S. HESLOP-HARRISON AND M. D. BENNETT
larger and more variable in its appearance and banding properties than any other
arm in the complement, and it is also always heteromorphic in the adults of some
species (Callan & Lloyd, 1960). In T. cristatus carnifex the two homologous
chromosomes are always different and the 50 % of progeny with homomorphic
chromosomes die during development (Macgregor & Horner, 1980). Bennett's
model would predict this particular chromosome to be an end arm of a
chromosome chain, where firstly, it is free from the size constraints the order
model imposes on other chromosome arms (and so can vary freely), and secondly
might have a controlling influence on cell division and gene expression.
Interspecific variation of one large chromosome arm has been reported by
Rumpler & Dutrillaux (1980) in Lemur species. In their extensive work on
karyotype evolution in this genus, they have shown that differences between
species largely involve fusion, fission and translocations between chromosomes.
However, in the long arm of the longest chromosome large blocks of heterochromatin vary widely between species.
In these cases, the end or predicted end chromosome may be particularly
significant in control of development or of features of chromosome behaviour
which can be considered as mechanical. In other cases, the constraints on
karyotype variation to match similarly sized long and short chromosomes arms,
which apply to all chromosome arms except those at the discontinuity, allow this
particular arm to show greater variation than all the other arms in the complement.
Developmental changes in order
We have strong reservations about the use of spreading techniques to analyse
order (Bennett, 1984c). Nevertheless, comparisons of different cells prepared
using identical spreading techniques may indicate some real differences in
chromosome order between cells from different tissues or cultures from one
organism. Comparison of results of genome separation in spread metaphases
with those from reconstructed serially sectioned metaphases of intergeneric
hybrids showed a basically similar distribution of whole genomes (haploid
chromosome sets; Finch & Bennett, 1981). Thus, this type of analysis, if interpreted with care, is valid on spread preparations, although clearly the resolution
is much lower than that of the reconstruction technique since in vivo
chromosome positions are not known and identification may be less certain.
Several examples where different cells have been found to have different
orders of chromosomes in spread analyses have been claimed. For example,
Avivi & Kariv (1984) analysed lymphocyte cells from young and old adult
humans, and adults with Fanconi's anaemia, finding significant differences in the
association of A group chromosomes between some groups. Wollenberg, Closse
& Zang (1983) have examined chromosome order in a child with a de novo
translocation 21 and its parents, finding that the mother and child were more
similar to each other in this respect, than to the father.
Chromosome order - possible implications for development 67
There are indications of differences in chromosome order in different studies.
Comparison of Juricek (1975) and Hens (1976) 'reveals that there is a fundamental difference between the chromosome position in uncultured corneal
metaphase cells and a23 Chinese hamster fibroblast' (Hens, 1976) cell culture
lines - cells of the former had small chromosomes nearer the centre of the
metaphase plate, and showed somatic association of many pairs of homologous
chromosomes, while the latter had association for only one chromosome and a
tendency for the larger chromosomes to be centrally located.
However, different laboratories have often reported conflicting results about
order even when they are using the same techniques (e.g. Darvey & Driscoll,
1972 and Feldman, Mello-Sampayo & Sears, 1966). Hence comparisons of work
from different laboratories - or even one laboratory at different times - must be
made with caution.
We have analysed reconstructions from electron micrographs of metaphases
at three different developmental stages:
1. Seedling root tip meristems,
2. Male pollen mother cells at the last division before meiosis (the premeiotic mitosis) and
3. Metaphase I of meiosis in the pollen mother cells (also examined in the
light microscope).
In the types of analysis of order used so far (Heslop-Harrison, 1983a; HeslopHarrison & Bennett, 1983a, c) we found no significant difference in order between the two types of mitotic cell. For instance, in an analysis of order in barley
cells (genotype Tuleen 346) at both mitoses (root tip and pre-meiotic), the same
mean order of chromosomes,
-4-1-2-3-5-6-7(which was predicted) ranked first out of all the possible 360 orders (Bennett,
19846). This evidence indicates that the mean order of chromosomes is unchanged at the two stages of development. However, there is a possibility for a more
subtle modulation of order which these analyses have not yet clearly revealed.
The change might be in the absolute mean chromosome order or else in other
features of relative chromosome positioning such as the spatial separation of
particular groups of chromosomes.
Changes between somatic and meiotic cells
The clearest difference in chromosome order between mitosis and meiosis is
the change from a tendency to genome separation to the intimate association
found in the bivalents. Relatively little is known about this process of pairing in
mechanistic and control terms. However, the existence of a pre-existing order
of chromosomes may facilitate the pairing of complete haploid sets of
chromosomes at meiosis without the involvement of long-range interchromosome forces or structures (Wagenaar, 1969; Bennett, 19846), since
68
J. S. HESLOP-HARRISON AND M. D. BENNETT
pairing could occur merely by superimposing pre-existing, ordered chains onto
each other before synaptonemal complex formation. Ashley & Pocock (1981)
also proposed the existence of an arrangement of non-homologues (in a specific
sequence, with involvement of certain homologues) which would simplify the
processes of homologous recognition, and hence, the initiation of pairing at
meiosis. They suggested that this type of ordering of chromosomes is general, but
that it has not been widely recognized because of the selection of organisms for
study, the preparative techniques used, and the objectives of the cytologists
examining the material.
The Bennett model imposes limitations upon chromosome variation within
karyotypes because of the requirement for similarly sized arms to match in the
order, both so that existing pairs of arms continue to lie together and so that new
pairs do not form. With the exception of those chromosome arms at the discontinuity, pairs of arms must change together, while those at the discontinuity are
uniquely free to alter in size.
Multiple translocations
The ordering of chromosomes may put a limitation on which multiple translocations between chromosomes are viable. For example, according to the
literature, single, reciprocal translocations are known between all pairs of
chromosomes in barley (Ramage, Burnham & Hagberg, 1961). Many fewer
multiple translocations have been characterized. One translocation stock is
Tuleen 346 (Finch & Bennett, 1982), which includes three reciprocal translocations involving six of the seven chromosomes in the haploid complement. When
the order of chromosomes in this species is compared to that in normal barley,
the same pairs of telomeres are found to be together at all points around the
order. The centromere order only varies in one position (from
-4-1-2-3-6-5-7in normal barley to
-4-1-2-3-5-6-7in Tuleen 346). The ordering of chromosomes might limit which multiple translocation stocks of barley are viable, and hence place constraints on changes
Fig. 6. A comparison of the karyotypes oiH. vulgare and H. bulbosum ordered as
found in serial section reconstructions. To enable comparison, the order of
chromosomes is shown in a straight line rather than a circle as is seen in Fig. 4A. The
numbers in circles are the linkage groups in H. vulgare and their presumed homoeologies in H. bulbosum. Numbers beneath each long (L) and short (S) arm give their
volumes as a percentage of the mean total for the haploid set in each species, and the
sums and arm ratios are also given. The largest differences between the two species
are in the arms at the discontinuity (*), while the other arms which are adjacent in
the order tend to change in length as pairs. (See also Bennett, 1984a.)
Chromosome order - possible implications for development 69
_J
oo Il oi o
J
70
J. S. HESLOP-HARRISON AND M. D. BENNETT
within the genome. At the same time, where chromosomal rearrangements,
mutations or translocations change an order spontaneously, breeding barriers
may be set up between individuals (because of their different chromosome
orders), and eventually lead to speciation through reproductive isolation.
Evolution of Hordeum species
Fig. 6 shows a comparison of the chromosome order in H. vulgare and H.
bulbosum (shown in Fig. 4) where the order of chromosomes has been predicted
and found in reconstructed metaphases. These species are closely related, with
karyotypes which differ as shown in the figure. The two greatest differences in
chromosome lengths and arm ratios involve the two chromosomes at the discontinuity. Other pairs of chromosome arms have tended to change together, with
pairs becoming longer or shorter.
Other possible evidence for constraints on chromosome size evolution comes
from the work of Macgregor and colleagues. Before the use of chromosome
banding techniques, newt species were considered to have very similar
karyotypes on the basis of chromosome morphology. However, when compared
with Giemsa C-banding techniques, the chromosomes appear very different,
leading to the suggestion that there is much more intrakaryotypic variation than
previously considered (Macgregor, Horner & Sims, 1983). These authors pointed out that the Bennett model for chromosome order may exert 'strong pressure
to conserve the overall morphology of a karyotype without excluding interchromosome rearrangement.' In another genus of newt, Plethodon, Mizuno &
Macgregor (1974) have shown that 'the karyotypes of all (three) species are
identical with respect to relative lengths, centromere indices and arm ratios of
corresponding chromosomes' (Macgregor, 1982) despite a nuclear DNA range
from 20 to 70pg. The constancy in shape of the chromosomes, despite the
changes in DNA content, involving amplification and diversification of the
middle repetitive sequences, possibly could be caused by the constraints of
matching arms sizes which force the karyotype to evolve as a whole and not as
independent chromosomes.
While examples of different species having similarities in karyotypes are common, it is impossible to determine the effect an order of chromosomes has on
speciation without knowledge of the actual chromosome order. However, as
more data accumulate about the cereals and related species and genera, it will
be interesting to see if there is a single basic design for the karyotype in a large
group of grasses, within which variation is limited because of the maintenance
of an order of chromosomes.
CONCLUSIONS
When taken together, the results and features of the karyotype discussed
above demonstrate that the order of chromosomes has many implications for the
Chromosome order - possible implications for development 71
control of the development of cells. Perhaps the order limits the independent
behaviour of individual chromosomes by linking paralogous genes on adjacent
heterologous chromosomes, controlling and limiting the association of
homologues, and perhaps controlling the position of active chromosome segments. Chromosome position in the order is apparently important for control of
chromosome behaviour in aneuploids and perhaps for the activity of loci affecting cell division and chromosome pairing.
There are a number of step-like changes during the development of organisms
where the main cause of the change is unknown; stability and reversibility of the
differentiation are often important factors. Examples of these 'determinations'
include the apical changes at flowering, root/shoot differentiation and juvenile/
mature stages of development. Perhaps such changes are modulated by changes
in chromosome order. Experimental modulation of the order might be possible
(by either mechanical or chemical means), which changes the developmental
pathway of a particular cell by manipulation of chromosome disposition alone.
The title of the session where this paper was presented, 'Changes in Nuclear
Hardware and their Consequences for Development', makes a comparison between the 'architecture' of computers and of cells. A differentiated cell can be
considered as a microcomputer, since both react to wide ranges of inputs in
preprogrammed ways. Output conditions reflect the data inputs, the resources
available and time, although cells (unlike microcomputers) can carry out many
processes simultaneously, without interrupting operation. The cell's structure
and synthetic or degrading capabilities can be regarded as part of the hardware
of the installation which also includes the data store, processing unit and
peripheral input/output devices. The chromosomes are the memory which
stores the data and programs for development of the organisms - the software.
In between the hardware and the software there is firmware - the operating
system and features of memory and input/output device access which are transparent (and invisible) to the software. However, the firmware allows the
programs to work and controls which portion of the program is active at any time.
The firmware may also control all access to hardware components.
Perhaps order within the nucleus can be considered as firmware, which can
modulate the expression of genes and oversee the behaviour of chromosomes
during development, thus giving the cell a new level of control over the genes.
J.S. H.-H. thanks Peterhouse, Cambridge, for the award of a William Stone Research
Fellowship. The authors thank Mr J. B. Smith for immaculate preparation of the serially
sectioned nuclei discussed in this paper.
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