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J. Cell Set. a, 9-22 (1967)
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A CYCLOID MODEL FOR THE CHROMOSOME
H. L. K. WHITEHOUSE
Botany School, University of Cambridge
SUMMARY
Investigations of lampbrush chromosomes and the nucleolar organizer have suggested that
each gene may be duplicated many times in consecutive linear series within one DNA molecule.
This conclusion is in direct conflict with recombination data which indicate, not only that each
gene is represented only once per chromatid, but that different genes are contiguous.
This paradox is resolved by postulating that the chromosome has the form of a cycloid. Each
loop of the cycloid would correspond to a set of copies of a gene forming a chromomere. It is
suggested that at meiosis the copies of the gene are detached as a result of intrachromatid
crossing-over between the first and last members of the series. The master copy remaining in
the chromatid would then be in a position to undergo crossing-over with a homologous
chromatid, while the duplicate copies in the detached chromomere would all be included in a
single circular DNA molecule. They could subsequently be restored to the chromatid by crossing-over between one of their number and the master copy. This intrachromatid crossing-over
would imply that the chromosome can alternate between two states with each set of duplicate
genes either detached as a circle or integrated with the DNA axis.
Callan's model for matching slave genes against a master copy so that all acquire identical
nucleotide sequences is modified to facilitate coiling and uncoiling of nucleotide chains, by
postulating breakage of the matching chains at one end of the gene. Matching of only one chain
of the slaves against the master is proposed or, if necessary, subsequent matching of the second
slave chain to the first. It is suggested that matching may regularly precede the synthesis of
messenger RNA.
Investigations of dipteran salivary gland chromosomes and amphibian oocyte nucleoli have
established that the chromomere is the unit of replication of the chromosome. On the cycloid
model the replicons would be adjacent to one another, and each would comprise a master gene and
all the copies. It is suggested that the replicator may correspond to the operator of the master
copy of the gene. This hypothesis provides an explanation for several previously unexplained
features of crossing-over, including its occurrence at the four-strand stage.
INTRODUCTION
Ever since the acceptance of the chromosome theory of heredity, studies of mutation and recombination have required that any particular gene should be represented
once only in a chromatid. With the realization, however, that a gene consists essentially
of a segment of a DNA molecule containing a particular sequence of a few hundred
nucleotide-pairs, the problem has arisen of how to reconcile this structure for the gene
with that of the chromosome. Various lines of evidence have suggested that each
gene might be represented many times in a chromosome, either transversely in
duplicate DNA molecules, or longitudinally as duplicate genes within one DNA
molecule. There now appears to be overwhelming evidence that germ-line chromosomes are not multistranded (see Callan, 1967), but the likely existence of duplicate
copies of each gene within one DNA molecule (see later) poses an acute problem.
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H.L. K. Whitehouse
Callan & Lloyd (i960) offered a solution by postulating the existence of a master
gene and, alongside it in the same DNA molecule, a set of slave genes composing a
chromomere, with any changes due to mutation or recombination in the master gene
copied in the slaves. Callan (1967) has now made an ingenious proposal for the
copying mechanism by successive matching of the nucleotide sequence of the slaves
against that of the master. This hypothesis has the added attraction of explaining the
remarkable discovery which he and his associates have made of the polarized extension
of lampbrush loops from chromomeres.
One of the difficulties, however, of this model of chromosome structure is that
neighbouring master genes would be separated by a series of slave genes, and yet there
is evidence from data on recombination suggesting that neighbouring genes may be
contiguous. In particular, the genes ad-g (adenine-9) andpaba-i (pora-aminobenzoic
acid-1) in Aspergillus nidulans, which are thought to be neighbours, appear to be
interrelated in recombination (see Whitehouse & Hastings, 1965). Moreover, the patterns of polarity in recombination within the gene can be accounted for by postulating
that the pattern in any particular gene is partly determined by the behaviour of the
neighbours on each side (Whitehouse, 1966). This would require a gene to be in
contact with its neighbours on both sides. It thus appears that data on recombination
provide no evidence for the existence of slave genes, and not only require each gene to
be represented once only in the chromatid but also suggest that neighbouring genes
may be immediately adjacent to one another. This conclusion is in direct conflict with
the results of studies of lampbrush chromosomes (Callan, 1963) and of the nucleolar
organizer (Beermann, i960; Ritossa & Spiegelman, 1965; Callan, 1966) which point
to serial repetition of each gene (see Callan, 1967).
A CYCLOID MODEL
This dilemma can be resolved, however, by postulating that the duplicate copies of
each gene are removed from the chromatid at the time of crossing-over. Such removal
could be brought about by a crossover between the first and last members of the linear
series of identical genes. All except one would thereby be detached in the form of a
single closed circle. After crossing-over had occurred with one of the homologous
chromatids, the gene copies could be reinserted in the chromatid by the same
mechanism as that which led to their removal from it, namely, a crossover between the
gene in the chromatid and one of the copies of it in the ring. This mechanism for
attaching and detaching the duplicate genes is the same as that proposed by Campbell
(1962) (and now well established—compare Hayes, 1966) for the incorporation of a
circular phage genome (including the sex factor) into that of its host, and for its
removal again. The postulated gene-specific crossing-over between duplicate genes
within one chromatid could be regulated in a similar way to normal crossing-over
between homologous chromosomes, if this is also specific to structural genes, as
evidence suggests (Whitehouse, 1966).
This intrachromatid crossing-over would imply that the chromosome can alternate
between two states, with the duplicate genes detached as rings or integrated with the
Cycloid model for the chromosome
11
DNA axis. A convenient way of representing this situation is to consider the chromosome as having the form of a cycloid (Fig. i). In studies of mutation or recombination,
the loops in the chromosome would appear not to exist and the linear linkage map
would match the linear chromosome axis. This would correspond to the line
A D G J M P S in Fig. i. From other points of view, however, the loops would form
part of a thread which was continuous with the axis, corresponding to A B C D E F . . .
N O P Q R S in Fig. i. Each loop of the cycloid would correspond to a chromomere
(compare Callan, 1966&). According to the present hypothesis, therefore, chromomeres
are detached from the chromosome before crossing-over takes place with the
homologue.
J
Fig. 1. A cycloid, representing the hypothetical structure of a chromosome, with the
loops corresponding to chromomeres. From the point of view of mutation or recombination, the loops would appear not to exist and the chromosome be represented by
the line ADGJMPS. From other points of view the loops would be continuous with
the axis corresponding to the sequence ABCDEF. . .NOPQRS.
This cycloid model of the chromosome bears a superficial resemblance to the chain
model proposed by Stahl (1961), in which the chromosome was supposed to consist
of a linear series of circles. Stahl suggested that the phenomena associated with recombination between closely linked mutants, such as conversion and negative interference, might be due to events within a circle of DNA. On the present model, however, these phenomena are attributed to crossing-over by hybrid DNA formation in the
linear part of the cycloid, and the loops contain duplicate copies of genes and do not
take part in recombination. Thus, the cycloid model does not really resemble the chain
model, and has been proposed for quite different reasons.
Keyl (1965 a, b) has proposed a chromosome model involving loops of DNA for the
chromomeres, but differing from mine in having the loops joined by non-DNA
material. Basic features of the cycloid model are the continuity of DNA throughout
the chromosome and the ability to detach the loops by crossing-over.
Chromomere detachment
The hypothetical steps in the process of detaching a chromomere are shown in
Fig. 2 (a)-(d). A gene M, and 16 linearly arranged copies of it, are shown in diagram (i),
and the succeeding diagrams illustrate stages in crossing-over between the first and
last members of the series. It is assumed that the mechanism of crossing-over is the
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H. L. K. WHtehouse
same as the normal process between homologous chromatids, and that this follows
the operator model (Whitehouse, 1966). The detached chromomere resulting from
the crossover and consisting of 16 copies of the gene serially arranged in a ring is
shown in diagram (v). (The number of copies of a gene in a chromomere is likely to
differ from one gene to another but to be a power of 2—see discussion of Keyl's work
below.) The master gene remaining in the chromatid would then be in a position
Fig. 2. Diagram to show the postulated behaviour at meiosis of a gene, M, and 16
copies of it arranged in a consecutive linear sequence.
(a)—(d) Detachment of the 16 copies as a result of intrachromatid crossing-over between the first and last members of the series, leaving the master gene M in the
chromatid in a position to undergo crossing-over with a homologous chromatid from
the other parent.
(e)-(A) Reincorporation of the copies into the chromatid by crossing-over. The
lines represent nucleotide chains, broken lines indicating newly synthesized chains.
Wavy lines show the position of the operator of the gene. A, annealing; B, breakdown of unpaired chains; D, dissociation; L, N, neighbouring genes to M and its
copies; M, master copy of gene; O, breakage at operator; S, synthesis; 1-16 = 16
copies of gene M.
Cycloid model for the chromosome
13
to undergo normal crossing-over with the corresponding gene in a homologous
chromatid. This gene would have shed its chromomere in the same way. One of the
functions of the synaptinemal complex might be to hold detached chromomeres in
place at pachytene while crossing-over occurred between homologous chromatids.
The reinsertion of the chromomere into the chromatid is shown in Fig. 2 (e)-(h) and,
as indicated, could occur by precisely the same mechanism as its previous release.
Any of the copies of the gene in the chromomere might be involved in the crossover
which restores it to the chromatid or, alternatively, the crossover might regularly
involve the same copy as took part in the releasing crossover.
Another open question, on the basis of the present hypothesis, is whether detachment of chromomeres occurs at meiosis for all the genes in both chromatids of each
chromosome, or whether it is restricted to the genes which participate in crossingover with the homologous chromosome. I have suggested that the pattern of polarity
in recombination within a gene is partly due to the behaviour of the neighbouring
genes (Whitehouse, 1966). If this is true, it would be necessary to suppose that when
crossing-over took place at a particular locus, the chromomere not only of that
gene but also of at least one neighbouring gene on each side had been detached
from the chromatid. This suggests that release of all the chromomeres may occur at
meiosis.
Chromomere activity
Callan (1967) has suggested how slave genes might be matched against a master
gene and how in the process the DNA thread of the chromomere would be fed out as a
loop which extended from one side in just the way he has observed lampbrush loops
to develop. The chief features of this hypothesis are that, for each copy of the gene
in turn, the two nucleotide chains of the copy would dissociate and pair with the
complementary chains of the master gene, to be followed by correction of mispairing. This correction would always occur in the same direction, that is, from master
to slave and not the converse. After correction, the slave chains would dissociate
from the master chains and reanneal with one another.
A modified form of this model is given in Fig. 3 with matching of only one of the
chains of the slaves against the master, and with breakage of the matching chains at
one end of the gene. This breakage would facilitate the coiling and uncoiling of
nucleotide chains. Matching of only one chain might suffice, since it is known that
messenger RNA is the complement of one specific DNA chain. Since it is also known
(see Guest & Yanofsky, 1966) that messenger synthesis begins at the operator and
proceeds in the 5' to 3' direction (that is, grows from its 5' end by adding nucleoside
5'-phosphates to the 3'-hydroxyl end), the complementary DNA chain would be
polarized 3' to 5' with respect to the operator. It is this chain in the slaves which
would need to agree with the master, and so the master chain which showed complementary pairing with successive slave chains would need to be that which was
polarized 5' to 3' with respect to the operator, or in other words had the same nucleotide sequence (apart from thymine in place of uracil) as the messenger. This argument
is based on the assumption that the messenger is synthesized with one DNA chain as
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H. L. K. WMtehouse
the template. If the duplex DNA acts as template, modification might be needed,
depending on how the nucleotide sequence in the DNA was recognized.
Fig. 3. Diagram to show the postulated behaviour of a gene M and 16 copies of it
when messenger RNA is to be synthesized. The copies are matched against the
master and then extend as a lampbrush loop. The lines and symbols have the same
meaning as in Fig. 1. C, correction of mispairing to correspond with the chain with
descending arrow; T, breakage at terminus (non-operator) end of gene.
It is suggested that the initial step in the process of copy matching might be breakage of one of the nucleotide chains of the master copy of the gene at the operator. This
breakage would be in the chain which had the 5' to 3' polarity, that is, the sequence
-O-P-O-5'-4'-3'-O-P-O- in the atoms of its backbone reading from the operator end.
Cycloid model for the chromosome
15
Following this breakage, it is assumed that dissociation of the chains would occur
over the length of the gene. A cycle of 6 steps is then postulated for the matching of
each slave in turn against the master, namely: (1) breakage of the complementary
chain of the slave at the terminus (non-operator) end of the gene; (2) dissociation of
the chains of the slave over the length of the gene; (3) annealing of complementary
broken chains of master and slave; (4) correction of mispairing in the direction from
master to slave; (5) dissociation of master and slave chains; and (6) annealing of the
two chains of the slave. These steps are shown diagrammatically in Fig. 3 (a)—(c) and
again in Fig. 3 (c)-(e) for another cycle of matching. As in Callan's model, the lampbrush loop would represent that part of the series of copies which had been corrected.
The motive force for formation of the loop would be the attraction of complementary nucleotide chains, one from the master and one from each slave in succession.
In Fig. 3 the copy labelled M of the gene is taken to be the master, with breakage of
the 5' to 3' chain at the operator. Copy number 1 is then the first to emerge as a lampbrush loop after correction, followed by number 2 and so on. Alternatively, if copy
number 1 were the master and showed the 5' to 3' breakage at the operator, then copy
M would be the first to be matched, followed by number 16. The first alternative
(illustrated in Fig. 3) leads to the terminus (non-operator) end of each copy of the
gene emerging first into the lampbrush loop. The direction of messenger synthesis
would then coincide with that of loop movement. The second alternative leads to the
operator end emerging first and to m-RNA being synthesized in the opposite direction
to that of the movement of the loop. The alternative shown in Fig. 3 is considered to
be the more likely, for reasons discussed later.
The mechanism of correction of mispairing would need to be able to distinguish
a nucleotide chain from the master copy and one from a slave. This would require the
correcting enzyme to recognize some distinguishing feature such as the operator
region of the gene. In Fig. 3, at the times when correction would take place (at (iii),
(v) and (vii)), the slave chain has the operator at the 3' end (that is, the chain is
attached to the operator and is polarized 3' to 5' with respect to it), while the master
chain does not have the operator attached (or, if it were attached, it would necessarily
be at the 5' end). In addition to correction in a specific direction determined by a
distinction of this kind, there must subsequently either be no correction between the
chains of the slaves, or correction in a predetermined direction, namely, from the
slave chain which had paired with the master to that which had not. The first alternative would mean that mispairing would persist in the slaves. The second would
require a different recognition mechanism from that for correction from master to
slave. This is because the slave chain which received corrections from the master
would now have its role reversed and would have to pass the corrections on to the
complementary slave chain. A model of the kind proposed by Callan (1967) in which
both chains of each slave were corrected directly from the master would pose an
acute recognition problem for the correcting enzymes, because correction from
master to slave in one pair would correspond to correction from slave to master in the
other. The question of whether both slave chains are corrected or not is discussed
further later.
16
H.L. K. Whitehouse
During the correction of the slaves it would be possible for the matching process to
be broken off. This correcting of some but not all of the slaves might occur regularly,
or only as an abnormality. These alternatives are related to the question of whether
the process of matching slaves against master takes place once only per generation
(after crossing-over has occurred with the homologous chromosome at meiosis), or
whether it regularly precedes messenger synthesis. The number of lampbrush loops
at diplotene far exceeds the number of chiasmata, so clearly the matching of copies is
not directly related to the occurrence of crossing-over with the homologous chromosome. On the other hand, all the loops are associated with RNA and protein synthesis,
which indicates that the genes concerned are functioning. It seems, however, most
unlikely that all the genes are active in the oocytes. Those that are inactive at this
stage might undergo matching of slaves to master in whatever tissue they first became
active. The simplest hypothesis appears to be that the matching of slaves against
master does not occur only once per life-cycle, but always precedes messenger
synthesis at whatever stage of development the gene is functioning. This would
explain the similarity of puffing in salivary gland chromosomes to lampbrush loop
formation (compare Pelling, 1966). The peculiarity of the lampbrush phase of oocytes
would then be merely that a large number of genes were active over a prolonged
period. The hypothesis of matching whenever m-RNA was to be synthesized would
also accommodate the data on somatic mutation and recombination, because the
functional genes—the slaves—would always correspond to the master gene, at whatever stage they functioned.
If matching occurs regularly before m-RNA synthesis, there will be two categories
of genes: those that function in the germ-line and those that do not. For genes which
do not function in the germ-line, there is the possibility that, under special circumstances, selection might favour slight differences between the copies of a gene in the
chromomere. Recombination between such non-identical copies might explain antibody variability (see Whitehouse, 1967).
There seem to be no exceptions to the rule that protein synthesis occurs throughout
the length of lampbrush loops, although in giant granular loops RNA synthesis is
confined to the region of the loop that has recently emerged from the chromomere
(Callan, 1963). Presumably in these loops the messenger persists in contact with the
loop and functions repeatedly for protein synthesis, whereas in the majority of loops
the messenger breaks down or leaves the loop, and in either case is continually resynthesized. It might, therefore, be anticipated that the size of a loop was some indication of the scale of activity of the corresponding gene. It would then be expected (on
the assumption that matching always occurs before m-RNA synthesis) that when the
activity of a particular gene was low or of short duration, only a few of the copies
would be corrected against the master, while for a high rate or prolonged activity, all
the copies might be matched and act as templates for m-RNA synthesis. If an average
gene codes for a polypeptide containing 500 amino-acid residues, it would occupy
approximately 0-5 fi of DNA (1500 nucleotide pairs 0-34 m/i apart). If an average
lampbrush loop in Tritiums cnstatus is 30 /i in circumference, it might contain 26 copies
of a gene. On the other hand, allowing for the loop being fed back into the chromo-
Cycloid model for the chromosome
17
mere, Callan (1963) has estimated that a giant granular loop and its chromomere
might contain 1-2 mm of DNA, which would be equivalent to perhaps 2 U or 212
copies of the gene. If a gene with so many copies were active at some stage of development for only a short period of time, it is unlikely that all these copies would be
matched against the master. Interrupted slave matching thus seems probable, if it is
accepted that correction of slaves always precedes m-RNA synthesis.
Callan (1963) has described the occurrence in T. cristatus of a small number of
lampbrush loops which are symmetrical. In these loops, each half is the mirror image
of the other and resembles a normal asymmetrical loop. The basal part of each half of
the symmetrical loop resembles the end of a normal loop that appears to be re-entering
the chromomere, and the part of each half which corresponds in appearance to the
emerging end of a normal loop is that which is farthest from the chromosome axis.
Callan has interpreted symmetrical loops as due to reversed repeats. This explanation
is in keeping with the hypothesis of chromosome structure proposed here. A symmetrical loop would be accounted for if half the copies of the gene were reversed
relative to the other half, and if the matching occurred separately in each half. The
situation could be represented diagrammatically by including the master gene and the
16 copies of it twice in Fig. 3, so that the sequence in the DNA was L, 1-16, M,
M, 16-1, N. According to this interpretation, the matching of copies against the
master takes place at the tip of each hah0 of the symmetrical loop. That the DNA
thread emerges linearly from each half, and not as a loop, is in keeping with
the model proposed here, in which the first slave to be matched is the one which
is farthest from the master. According to Callan's model (1967), the first slave to
be matched adjoins the master, but the structure of symmetrical loops contradicts
this.
Chromomere replication
Keyl (1965 a, b) and Pelling (1966) have found evidence that the transverse band of
dipteran salivary gland chromosomes, or its counterpart in normal chromosomes, the
chromomere, is a unit of replication of DNA. By thymine autoradiography they had
found (Keyl & Pelling, 1963) that DNA synthesis in the transverse bands of the
salivary gland chromosomes of Chironomus thummi begins simultaneously in all the
bands, but that those with larger DNA content take longer to replicate. Keyl (1964,
1965 a, b, 1966) has found, by microspectrophotometry after Feulgen staining, that
particular bands in two subspecies of C. thummi differ in DNA content by a power of 2
(either 1, 2, 4, 8 or 16 times as much DNA in a band in one subspecies as in the
corresponding band in the other). Similar variation was found between different
individuals within one of the subspecies. These findings have led Keyl (1965 a, b) and
Pelling (1966) to infer that each band is a unit of replication, and that the doubling in
evolution of the DNA content of a particular band is due to doubling the length of the
DNA molecules it contains. They have suggested that this duplication of particular
chromomeres could occur most readily if the DNA of the chromomere were arranged
in a ring, so that the end of the replicating segment lay close to its beginning. Keyl
(1965 a, b) has accordingly proposed a chromosome model with this feature, and has
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Cell Sci. 2
18
H.L. K. WHtehouse
suggested that the doubling arises through an error of replication. On the cycloid
model, however, the doubling would be explained if the crossover between the first
and last copies of the gene to detach the chromomere (see Fig. 2) occasionally
took place between the first copy in one chromatid and the last copy in the sister
chromatid.
Evidence for circularity in the DNA of the chromosome has been obtained by Hotta
& Bassel (1965). By sedimentation analysis and electron microscopy, they have found
that at least part of the DNA of the sperm of the boar (Sus domesticus) is in the form
of circles with circumferences ranging from 0-5 to 16-8 /i.
Direct support for the idea that the unit of replication of the DNA of the chromosome is circular and corresponds to the chromomere has been obtained from study of
the hundreds of free nucleoli which occur in the nuclei of amphibian oocytes. Miller
(1964) found from enzymic treatment and electron microscopy that each nucleolus of
Triturus pyrrhogaster contains a circular DNA molecule surrounded by matrix. This
construction closely resembles that of lampbrush loops and led him to suggest that a
single chromosomal locus may replicate independently of the rest of the genome.
Callan (1966) has made discoveries with Ambystoma mexicanum which lead to the
same conclusion. He has found that the free nucleoli show the same range of form as
those remaining attached to the chromosome in the region of the nucleolar organizer,
and that whether free or attached they may be ring-shaped. Moreover, he has found
that a typical lampbrush loop may occur at the nucleolar locus, and that this loop
closely resembles the ring-shaped nucleoli. It is inferred, first, that the nucleolar locus
is constructed in the same way as other gene loci, and secondly, that it is capable of
generating several hundred DNA threads, each of which resembles its own lampbrush
loop in appearance, but has the form of a closed circle. These conclusions are of outstanding importance because they imply that the lampbrush loop is the unit of
replication of the chromosome, in precise agreement with Keyl and Pelling's comparable discovery for the salivary band.
Jacob & Brenner (1963) proposed the term 'replicon' for a unit of replication of
DNA which was capable of controlling its own replication, as in bacteria and viruses.
They suggested that this control would take place by means of a specific 'initiator',
which would act on a recognition site, or 'replicator', and initiate DNA replication in
a specific direction from that point. It is not yet known how the replication of chromomeres is controlled, but it is convenient to apply the term 'replicon' to these units of
replication, even though control of their replication from elsewhere in the cell would
imply a modification of the original definition of the term 'replicon'. The circular
configuration of the units of replication in bacteria and viruses on the one hand, and in
the nucleoli of amphibian oocytes on the other, has been remarked upon by Miller
(1964), and suggests a similarity in the control mechanism.
On the cycloid model, the unit of replication would be expected to correspond to all
the copies of a particular gene, such as numbers 1-16 and M in Figs. 2, 3. One
possibility is that the replicator is at the operator of the master copy (M) of the gene,
and the replication terminus at the non-operator end of copy number 1 (see Figs. 2, 3).
This would mean that each replicon consists of a chromomere and parts of the
Cycloid model for the chromosome
19
neighbouring interchromomeric segments, and that successive replicons along the
chromosome would be contiguous. At the replicon junctions, one of the nucleotide
chains of the DNA would need to be free to rotate about the other (compare Whitehouse, 1965, p. 201). If, as suggested above, the replicator coincided with the operator
of the master gene, this would imply that the master gene always had the position
shown in Figs. 2 and 3 relative to the copies, because if (as discussed earlier) the master
were at the position of copy number 1 in the diagrams, its operator would not be at
either end of the series of copies of the gene. The ring of DNA in each nucleolus of
amphibian oocytes is evidently synthesized from the nucleolar lampbrush loop as
template, and its synthesis is presumably controlled in the same way through the
replicator. One possibility is that the rings are synthesized successively along the
DNA of the chromomere and each in turn detached from the chromosome by the
mechanism already proposed as a normal feature of meiosis, namely, crossing-over
between the first and last copies of the gene as in Fig. 2 (i)-(v). Another possibility is that
the crossover occurs first and successive replications of the ring follow.
Since the DNA of a lampbrush loop at the nucleolar locus can replicate many times
to give rings of DNA in the nucleoli, and these rings (as well as the loop) can then
function as RNA templates, both chains of the nucleolar slave genes presumably
correspond with those of the master. For this locus, therefore, it would appear
necessary for both chains of each slave to match against the master (as suggested by
Callan, 1967), or for one to do so and then to correct the other slave chain. It is
arguable, however, that matching may be abnormal at the nucleolar locus because it is
not a normal structural gene specifying a polypeptide through m-RNA.
The mechanism of crossing-over
There are several aspects of the hypothesis of chromosome replication postulated
here which appear to be relevant to the mechanism of crossing-over. I have suggested
(Whitehouse, 1966) that crossing-over is organized on a gene-specific basis with a
similar control mechanism to transcription. It now appears that the replication of the
chromosome is also gene-specific. There is the possibility, therefore, of an interrelationship between the control of replication and of crossing-over. Thus, the model
of crossing-over which I have proposed requires DNA synthesis to occur in both
chromatids from the operator of the gene. If, as was suggested above, the replicator
coincided with (or was adjacent to) the operator of the master copy of the gene, the
synthesis necessary for crossing-over could be initiated in the same way as normal
replication.
The operator model of crossing-over requires breakage at the operator of nucleotide chains of opposite polarity in the two recombining chromatids. The elaborate
enzymic breakage organization that this would need can be dispensed with, if it is
supposed that the breakage is a failure of joining of the phosphodiester backbones
after the preceding replication. Such failure might be required in order to allow the
chains of the next replicon to rotate about one another, if it was later in replicating.
The failure to join would necessarily leave one chain of each polarity unjoined in sister
chromatids, and similarly in the homologous chromosome. Moreover, if the primary
20
H. L. K. Whitehouse
breakage of nucleotide chains in crossing-over is really a failure to join after the previous replication, this would account for crossing-over, whether meiotic or mitotic,
always occurring at the 4-strand stage. Evidence for a relationship between crossingover and events at the preceding DNA replication has been obtained from studies of
recombination in organisms as diverse as Liliitm (Lawrence, 1961), Chlamydomonas
(Hastings, 1964; Lawrence, 1965) and Drosophila (Grell & Chandley, 1965). These
authors have shown that a change of temperature or treatment with chemicals or
radiations at the time of the premeiotic DNA replication can affect recombination. This
suggests, as Hastings (1964) has proposed, that the initial steps of the process of
crossing-over may take place at this time, before the homologous chromosomes
associate.
The chains remaining unjoined at the end of the replicon would necessarily be the
newly synthesized ones. It follows that, in the intrachromatid crossover to detach a
chromomere, the breakage of a nucleotide chain at the operator of copy number 1 of
the gene (Fig. 2 (a)) would always be in the old chain. This is on the assumption that
the intrachromatid crossover follows the mechanism proposed for normal crossing-over.
DISCUSSION
The model of chromosome structure and organization proposed here has direct
relevance to all the basic aspects of genetics—replication, recombination and transcription, and their regulation—and so could be tested by studies in all these fields.
Investigation of the mechanism of synthesis of nucleolar DNA, and its control,
would be particularly instructive. Information about the correction of slave genes
against a master gene might be obtained from electron microscopy of the tip of symmetrical lampbrush loops at different stages of development of amphibian oocytes,
and perhaps also by isotope labelling, if mutation or recombination had occurred in
the master gene. The idea that the replicator of a chromomere coincides with the
operator of its master gene could be tested if it were possible to determine the direction
of messenger synthesis in a lampbrush loop, since synthesis coinciding in direction
with that of loop movement is predicted.
It is known that heterochromatic segments of chromosomes replicate late, and it
would be interesting to know more about their detailed replication pattern. It is
possible that the cycloid structure might apply only to the euchromatic regions,
since chromomeres are largely restricted to euchromatin and may, therefore, be a
specific feature of structural genes.
Apart from the circularity of the replicon, the organization of a chromosome appears to be quite different from that of its counterpart in bacteria and viruses, for which
I have proposed the term 'chromoneme' (Whitehouse, 1965). The chromosomal
organization, with its duplicate genes in chromomeres, is not known in bacteria. It
appears significant that no example of an operon, in the sense of a group of structural
genes with a common messenger, has yet been conclusively demonstrated in any
chromosomal organism. It may be that the operon is not appropriate to the chromosomal (as distinct from the chromonemal) organization.
Cycloid model for the chromosome
21
The term 'operator' has been used in this paper for the recognition site at one end
of the gene. Jacob, Ullman & Monod (1964) have shown, however, that in the lactose
operon of Escherichia colt, the operator, defined as a site of repressor action, is distinct from the site of initiation of transcription, for which they have proposed the
term 'promoter'. It is possible that for each chromomere in a chromosome there is
only one operator, situated at one end of the master gene, and that for each slave what
I have called an operator is a promotor.
I am most grateful to Professor H. G. Callan, F.R.S., for showing me the manuscript of
his paper on the organization of genetic units in chromosomes. The ideas in the present paper
are a development of his and owe much to discussion with him. I thank Mr G. J. Clark for
preparing the diagrams.
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{Received 15 September 1966)