Scientific Impact of tile Study of Fine Structure of Chromatids
J. Lejeune
Nobel 23 (1973) Chromosome identification.
Sommaire
Since the demonstration of the correct number of chromosomes in man [1] in 1956 and the discovery of the first chromosomal disease in man [2] in 1959, human cytogenetics has developed in an explosive way during the early 'sixties and has since settled down to a steady growth rate.
Since 1965 the number of new syndromes discovered each year has gradually diminished and fears were expressed that cytogenetics had passed direct from infancy to senescence. The reason for this was that whereas the early successes were wan easily and rapidly, the techniques remained quite unchanged except for minor refinements.
Even autoradiography, which registered encouraging advances in the study of the X chromosome and, to a lesser degree, in the field of acrocentrics, failed to break new ground in chromatid analysis.
In 1968 a new era was opened when Caspersson et al. [3] observed a peculiar pattern of fluorescence in the chromosomes of Vicia faba and Trillium erectum following quinacrine mustard staining activated by UV light.
Apparently the importance of the findings was not fully grasped by researchers in human cytogenetics and it was only when Caspersson & Zech [4] revealed the whole human karyotype where every pair was clearly distinguishable that the rush on chromatid fine structure analysis started all over the world.
Although the fluorescence pattern is still the most accurate analytical tool, its technical difficulties prompted research into other procedures more amenable to routine exploitation.
The diversity of tortures inflicted upon chromosomes in order to force them to divulge their inner information is really amazing. Boiling them [5], digesting them [6], burning them with alkali [7] or salts (8), even intoxicating them before fixation [9] - all these maltreatments have been treed in turn.
I do not intend here to review all the available techniques, as I am personally aware of more than 20 variants. I will merely cite some of the more immediate achievements that have resulted from the analysis of the fine structure of chromatids.
Impact on Clinical Cytogenetics
To quote all the improvements in syndrome analysis brought about by the new techniques, whether fluorescence or denaturation, would be an enormous task. I will nevertheless run the risk of missing some of the important improvements by concentrating art only a few of then.
Confirmation of previously established syndromes
New techniques have made refinements in the recognition of chromosomal error in all trisomies and all deletion syndromes.
For instance, in the case of a deletion of the short arm of a group B chromosome, tedious clinical comparisons and painstaking autoradiographic investigation [10] had already led to a distinction between 4p- and 5p- for "cri du chat") syndromes. With chromatid analysis this distinction was now elucidated and definitely established (11).
A curious feature is that the two clinical syndromes, although clearly distinct, are to same extent apparently related, just as chromosomes 4 and 5 exhibit same similarities. Such a consequence could be extremely significant, since it will be encountered repeatedly.
For the medium-sized chromosome, clinical demonstration of a particular C-trisomy has been confirmed by fluorescence identification of pair 8 by de Grouchy et al. [ 12], and now confirmed in at least six cases by our heat denaturation technique [12], Trisomy 8 is the only C-trisomy hitherto individualized but, as we shall see later, partial trisomy 8 can also be recognized now.
In the case of acrocentrics, chromatid analysis has established the syndrome of trisomy 13 beyond doubt and there are good prospects of individualizing the clinical consequences of trisomy 14 and trisomy 15, together with the partial trisomies for these elements.
For the E group, only trisomy 18 and syndromes 18p- and 18q- have been fully investigated and no new syndromes for the F group have yet been recorded [19, 20].
For the small acrocentrics, highly important data have been gathered. Primarily, the Y chromosome can be detected by its remarkable fluorescence, even in the resting nuclei. This technique possesses the same accuracy for Y diagnosis as does the Barr bodies count for the X chromosome.
For pairs 21 and. 22, their first separation by Caspersson & Zech led to unexpected results. The Ph1 chromosome, marker of granulocytic leukemia, was long supposed to be a deleted 21. The fact that this tiny element looks much more like a deleted 22 was a surprise (13), in view of the long-known association between trisomy 21 and acute childhood leukemia.
This again raises the question: Can chromosomes resembling each other control organs or functions of the same "kinship".
However, it is in the analysis of chromosomal rearrangement that fine structure analysis refined most of the old observations.
Improved analysis of chromosomal rearrangement
Rearrangements between acrocentrics:
The detection of each element involved in centric fusion or, more precisely, in reciprocal translocation in the centromeric region of acrocentrics, has confirmed the previously anticipated possibilities.
Hence, in a case of G-G translocation carrier, it is easy to distinguish between a 21-21 and a 2I-22 translocation. In view of the fact that 21-21 translocation leads only to trisomy 21 or to unviable monosomy, this recognition is of the utmost importance for genetic counselling.
The same is true of D-G translocation. A demonstrable 22-15 translocation, for example, would be considered a relatively tolerable burden, since neither possible trisomies nor possible monosomies are compatible with late embryonic development, according to our present knowledge.
A systematic study of the D chromosome involved in D-21 translocation is not yet available, yet it could very well be that the malsegregation risk of a carrier is not always the same, depending on whether the D involved is a 13, a 14, or a 15. If one category had a much more stable meiosis than the others, it might explain the curious fact noted by some workers [14] that some D-G translocations seem to be carried by families without apparent damage, whereas other families exhibit the now-classical one-in-five risk if the mother is the carrier, or one-in-fifty if it is the father.
Here again, if a systematic study could possibly relate each type of translocation to a particular meiotic behaviour, possibly different in the two sexes, this would give a hint as to how to prevent malsegregation.
Besides these classical centric fusions, chromosome 21 can suffer ordinary translocation. We have thus observed a mother carrier of a 21q- 18p+ translocation in which the terminal segment of the 21 could be detected on the top of the short arm of the 18. The 21. trisomic child had a complete trisomy with two no.21 chromosomes, free and normal, and a third present in two pieces: the deleted 21 and its end part translocated to chromosome 18.
There have been other instances showing partial monosomy of chromosome 21, in particular a patient [15] with 45 chromosomes who apparently lacked a whole 21. Half of the long arm of the 21 (terminal part) was translocated on the short arm of chromosome 9. This type of case was thus a partial monosomy for the juxtacentric portion of 21 and the translocation was undetectable with ordinary staining. Thus all alleged cases of monosomy 21 need to be reanalysed with the new techniques.
Rearrangements between other chromosomes
The clearest demonstration of the resolving power afforded by the new techniques is evidenced by the demonstration of trisomy for the short arm of chromosome 9.
The syndrome was isolated at our Institute by M.-O. Rethoré [16], who carefully compared the clinical analyses and chromosomal pictures of four cases of translocation involving the short arm of a C chromosome. Recognition of the donor chromosome as a no. 9 was based on the observation of the secondary constriction which, in some cases, passed over to the carrier chromosome.
As exemplified in various translocations studied by denaturation techniques
9q- 6p+
9p- 22p+
9p- 19p+
9q- 22q+
the short arm of no. 9 remains detectable in each instance and gives definite proof of the clinical entity (fig 1).
In addition to these confirmations, the new techniques have already led to the isolation of new syndromes.
Fig. 1. A case
of trisomy for short arm of chromosome 9 (trisomy 9 p). (Right) the parental
translocation: one of the 9 is amputated of its short arm and one of the 19 has
received this fragment; (left) the child has received two normal 9 and the 19
carrier of the short arm of 9.
Discovery of new syndromes
Among the expected possibilities, trisomy 22 has not yet been detected by the new techniques. On the other hand, cases with an extra G-like chromosome have turned out to be something else, as already seen in a karyotype of the (9p) trisomy syndrome.
Curiously, we have detected, in two apparently unrelated families, a peculiar translocation which would have escaped detection with the old techniques (and had indeed escaped us).
Here the entire long arm of chromosome 22 is transferred to the long arm of no.8 and, reciprocally, the terminal pardon of chromosome 8 is transferred to the centromeric region of no. 22.
The exchanged segments being of equal length, this translocation could not be detected by conventional staining [17].
The children, carriers of an extra G-like chromosome, are trisomics far the distal portion of the long arm of chromosome 8. This type of reciprocal translocation of equal length had been anticipated long ago [18], but awaited new techniques for confirmation.
There is little doubt that ether examples will be discovered, leading eventually to the detection of chromosomal imbalance in abnormal children already classified as "normal karyotype" with the old techniques. This implies that every negative chromosomal examination of the last 10 years must be reinvestigated with the new techniques, not to mention all the positive findings.
Analysis of abnormal cells
Although very impressive studies have been devoted to chromosomal changes in neoplastic cells, further cytogenetic analysis was soon blocked because of the impossibility of defining precisely the identity of rearranged chromosomes.
Application of the new techniques can radically change this state of affairs and I would like to remark in passing that with: Mlle Venuat and Dr Dutrillaux we have been able to observe, in a quasi-diploid malignant strain, a characteristic aberration of chromosome 6, together with a curious streakiness of this element farming pseudo-dicentrics [18].
If the general theory of clonal evolution of the karyotype has any validity, chromatid fine structure analysis should soon provide the evidence by demonstrating "common variants"[19] for some classes of cancer.
Eventually this approach could tale immediate advantage of the enormous wealth of information already gathered by cell hybridization techniques [20], The chromosome maps thus established could give a glimpse of the relationships between gene dosage and metabolic pathways as postulated in the hypothesis of "combinaisons interdites" [21].
No doubt the cytogenetics of tumours will change so rapidly in the next few years that any review of this field is actually impossible.
Impact on Non-human Cytogenetics
Extension of chromatid analyses to animal karyotypes was indeed very attractive and many of us have, fortunately, not resisted that temptation. It is well known that the karyotype of the anthropoid apes orang-utan, gorilla, and chimpanzee, had an "air de famille" and had an obvious resemblance to the human karyotype. A puzzling feature was that although all the elements of the chimpanzee karyotype, for example, shared similarities with the human (apart from the difference in number-48 in the chimpanzee), there was no full metric agreement between recognisable elements of the two species.
As proposed by de Grouchy & Turleau [22], this curious situation might be explained by a number of rearrangements, especially pericentric inversions.
With Dr Rethoré and Dr Dutrillaux, we have studied two closely related chimpanzees: Pan troglodytes (the variety generally seen in zoos) and Pan paniscus (the pygmy chimp), thanks to the courtesy of M. Nouvelle (Paris) and M. Vandenbergh (Liege).
The reason for this choice was the earlier finding by Hamerton [23] and by Chiarelli [24] that P. paniscus differed. from P. troglodytes in that a small acrocentric pair in Paniscus was replaced by a small metacentric pair.
Curiously enough, the expected translocation was not found. All pairs are identical in both species but for the no. 22, acrocentric in Troglodytes and metacentric in Paniscus.
By comparing the results of heat denaturation, fluorescence, and enzymatic denaturation, it appears that the metacentric pair is not an isochromosome. The long arm is similar to that of no. 22, whereas the short arm has a very peculiar chromatid structure, highly fluorescent, extremely faint in denaturation but for its telomeric region, and extremely swollen after enzymatic digestion.
Here, it seems that a particular heterochromatic region constitutes the real distinction, as if Paniscus had amplified enormously a short segment of the short arm of no. 22. Hence the difference in the two species seems to be an increase in material-a de novo synthesis of one part of a chromatid (fig. 2).
If these highly fluorescent regions are related to repetitive DNA and act essentially in the regulation process, what we see at work here is the chromosomal picture of a regulatory system.
Besides chromosomal rearrangements, these comparative studies of chimpanzees have also shown how similar some chromatids can be in man and in apes.
If we count only the very typical structures, and reject the less conspicuous chromatids, it seems difficult to avoid the conclusion that at least the long arm of no. 7 as well as of 10 and 11 are identical in both species. A less precise identity is highly probable for 1, 3, 8, X, 16, 19, 20, 21 and 22, but more speculative for others.
Two remarks are apt here:
(1) Some chromatid structures in primates have remained unchanged for 10 million years at least if we accept morphological similarity as proof of common origin, i.e. if we disregard a possible convergence.
(2) Chromosomal shift by multiple rearrangement is enormous, but reshuffling seems to occur more within a chromosome than between different pairs.
Hence, every heterozygote step must have suffered from a very severe segregational load, a proposition contradictory to the progressive mutational evolution of the neo-Darwinian school. A possible alternative to this paradox is the reduction of the reproductive group, even to the size of a unique couple [25].
It is also possible that the permanence of some chromatids is not fortuitous, but may signify that some combinations are highly preferential, either for positional effect between genes or for regulatory opportunities. For example, it could very well be that an equilibrium exists inside a chromosome, in the sense that a trisomy for one arm could be equivalent to the monosomy of the other, since we have already encountered such a situation in an 18-16 translocation in man [26].
The impact of chromatid analysis on the speciation theory will no doubt prove to be enormous, even if only to exclude some forms from some groups, as is already the case with the Gibbon, which we propose to expel from the primates, for its lack of chromatid structure similarities to man.
Fig. 2. Analysis of the chromosome 22 of Pan paniscus (pigmy
Chimpanzees) and Pan troglodytes (ordinary chimpanzee). The chromosome 22 has a
greatly enlarged short arm in Pan paniscus (three different individuals) A, B
and C compared with the small arm found in Pan troglodytes (individual D). This
enlarged short arm of 22 in Pan paniscus seems rather "euchromatic" by
conventional staining (Aa, Ba and Ca). Controlled denaturation by heat
demonstrates a heterogeneous structure (Ab, Bb and Cb). Quinacrine fluorescence
shows a very brilliant segment (Ac). Enzymatic treatment confirms the
heterogeneous nature of this short arm (Cd).
Impact an Chromosomal Mechanics
The way chromosomes behave in the intimacy of the cell is quite difficult to understand from fixed preparations but fine structure analysis can be used to test hypothetical explanations. Three simple examples can be chosen.
Ring chromosomes
Following the lead of McClintock [37] I have proposed that the aberrations observed in ring chromosomes could be related to chromatid exchanges before the separation of centromeres [27]. Theoretical models showed that, if this were true, double-sized dicentric rings would have a peculiar structure, in the sense that the sequence of the genes in the two segments between centromeres would run in the same order but be opposed by their ends. Secondly, the reduction in size would result essentially from loss of the central part of the ring and not from loss of the juxtacentromeric segments. Although this theoretical model was recently challenged by a double breakage hypothesis [28], chromatid analysis revealed that these previsions are indeed confirmed by observations (fig. 3).
For example, as expected, a ring of chromo-some 13 showed distinctly the dark juxtacentro-meric region and the dark juxtatelomeric region, after rejoining of the short arm and the end of the telomeric region to form a ring. In dicentric rings, the sequence can be observed in both chro-matids which are opposed by their ends. Similarly, in small rings the middle segment is lacking.
Fig. 3. Some examples of a ring 13.
Note that the small ring is restricted to the dark portions of chromosome 13,
the juxtacentromeric one and the juxtatelomeric. On double-sized dicentrics the
dark juxtacentromeric region remains separated from the dark telomeric region
by a a pale region. It can be seen that the sequence of the bands runs in
opposite direction in the two chromatids and are opposed by their ends.
Selective endoreduplication :
This curious chromosomal misdemeanour is exceedingly rare but very interesting. In some cells a distal portion of a chromatid is replicated twice, as we observed nearly 10 years ago. [29]. Curiously, this happens especially often for the terminal segment of chromosomes which exhibits a, structural gap at a fixed point. This point is constant for the affected individual and this chromosomal peculiarity can be inherited as a familial dominant trait [30].
Sometimes the distal endo-reduplicated segment has a moniliform appearance and it can be shown that the sequence of these exceptional structures reproduces, with finer details, the denaturation picture. Hence the possibility that the event involves an abnormal replication which, in turn, may be due to the absence of some chemical required to hold together the chromosomal bands.
Remarkably enough, in some exceptional instances, it is not the distal part which is present twice but the other arm and the adjacent centromeric region, as if the real aberration were at the weak point detected on these chromosomes.
The exact significance of these phenomena regarding the fine mechanics of the chromosome is not yet certain.
"Aneusomie de recombinaison"
Among the possible origins of chromosome imbalance, the eventuality of "anosomie de recombinaison" [31] still lacks definite demonstration in man. The logic of the argue gent was that some rearrangement could, at meiosis, produce unbalanced gametes, even without an apparent change of metrics, if only a chromatid exchange had taken place in a particular exchanged segment. The simplest instance is an insertion, for a crossover inside the inserted segment could produce unbalanced chromosomes in meiosis of a heterozygote carrier.
No such type of chromatid exchange has yet been demonstrated, but the existence of insertion is now certain. Six months ago we found the first case, a piece of short arm of chromosome 3 inserted in the distal third of the long arm of chromosome 7.
With ordinary staining, the appearance was that of a reciprocal translocation, but chromatid analysis reveals the insertion beyond any doubt.
Recently, Grace et al. [32] reported another insertion of part of long amp of 7 inside the long arm of 3, and Gray et al. [33] showed an insertion of the short arm of 1 in the long arm of 4.
In other complex rearrange ments, pericentric inversion can also be recognised and we have recently analysed one in chromosome 2.
At least the topological conditions for "aneusomie de recombinaison" do exist in man and the eventual occurrence or absence of this aberration would increase our knowledge of meiotic mechanisms.
Direct meiotic studies have not yet fully taken advantage of the new techniques, although, from preliminary observation, Dutrillaux thinks that the position of chiasmata could have a curious correlation with the banding pattern.
Finally, the systematic study of break paints in various rearrangements will be statistically feasible with the steady increase of data. In a preliminary study of 28 independent translocations [34], found in different families (with no cases of centric fusion in this sample), we could show that of 56 breaks, 18 were in the centromeric zone, 20 were in the telomeric zone, and. only 18 occurred somewhere in between the two.
Although the definitions of centromeric and telomeric zones are quite arbitrary it can be safely assumed that the "in between" is at least ten times longer than both of then together.
Besides this tendency to break at both ends of chromosome arms, no case of translocation was recorded in which both breaks could be demonstrated to be in the intermediate region in these 28 instances.
Hence it could very well be that breaks themselves are not purely random, but correspond to some peculiarity of chromosomal structure.
This leads us to the last but not the least paint of impact of the new techniques. What kind of structure is revealed by the various pictures we can obtain of a chromosome?
Impact on the Knowledge of Chromosome Structure
This last point is of outstanding importance and is to be discussed more fully later. I shall therefore touch upon this subject with great caution.
To comment bluntly on the present state of observation, we can say that all techniques available are consistent, although they do not all show the same picture.
If we take the four categories now studied, we can compare the so-called Q-bands (after treatment with the quinacrine mustard staining of Caspersson et al.), the R-bands (after treatment with the controlled heat denaturation of Dutrillaux & Lejeune), the E-bands (after treatment with the enzymatic digestion of Dutrillaux, de Grouchy & Lejeune), and the G-bands (produced by various Giemsa techniques).
The correlation is as follows:
| Q-band | R-band | G-band | E-band |
| brilliant | faint | dark | swollen |
| faint | dark | faint | shrunken |
Since the sequences obtained for a given chromosome are perfectly comparable we are positive that each technique acts on the same structural peculiarities.
The first impression from the Q-band observation was that quinacrine could attach specifically at some base-pair of DNA and G-C was considered the most likely [35]. Hence, the banding would result from the preferential reaction of quinacrine mustard with portions of DNA especially rich in G-C.
Such an enormous (at the molecular level) concentration of monotonous formula was disconcerting.
With heat denaturation, this DNA-related banding was less obvious, because with the temperature (87°C) and the time used, preferential denaturation of DNA according to its base sequence is not very likely and only immediate renaturation could occur.
But with the demonstration of the enzymatic bands, the pure DNA hypothesis is even less tenable. Actually, Couturier [36] has systematically used many different enzymes and obtained the same pattern with all the proteolytic enzymes, but not with others (especially DNase and RNase). More precisely, he showed that the best conditions for revealing the banding were the exact pH and temperature conditions optimal far the proteolytic activity of each enzyme studied.
Thus we are forced to accept the fact that the enzymatic banding pattern can be related to protein differences along the chromatids.
How do these differences relate to the DNA-specific sequences? What kind of polypeptide sequence do they represents? Why are some of them more readily "digested" than others? All these questions remain to be answered.
But the DNA hypothesis and the acid protein hypothesis are not necessarily conflicting. Of necessity, both DNA, and protein must be sterically and chemically related to each other so as to build the entity we call a chromosome. With increasing knowledge of the precise mechanism of each staining technique, it could very well appear that all of there reveal a peculiar configuration resulting from DNA/protein interaction and no pure DNA or pure protein properties alone.
In this line of thought, a structural hypothesis for which Dutrillaux is mainly responsible, suggests that chromosomes have special relationships with the nucleoplasm and that banding patterns correspond to these paints of interaction between chromatids and nucleoplasm.
In other words, chromosomes would no longer be noodles floating in a bowl of nuclear juice, but would be organelles having definite and localizable molecular interactions, with a structurally non-homogeneous nuclear content. Although it is difficult to interpret such a picture completely, it would be particularly appealing if some meiotic behaviour were found to be related to banding sequences.
Despite all the satisfaction that cytogeneticists have experienced with chromatid fine structure analysis, one disappointment has been keenly felt. A bending pattern is, for a geneticist, immediately associated with the remarkable "topography" of polytenic chromosomes. Curiously enough, fluorescence studies of giant chromosomes of Dipterae have not yielded any good correlation with the well known maps of these chromosomes, although ordinary mitotic chromosomes of this species have a recognisable fluorescent banding pattern.
Cold it be that the two banding types correspond to two separate physiological functions of chromatid segments? This question really is well worth investigating, for the induction of polyteny in cultured cells would be another milestone in cytogenetics.
To conclude these introductory remarks on the scientific impact of chromatid analysis on the recent development of cytogenetics, it can be stated that the discovery of banding patterns not only gave new impetus to research, but has actually rendered all previous findings obsolete.
In view of what we have learned with these techniques during two years, the old preparations seem like a Palaeolithic research tool. Yet as scientific progress makes rapidly obsolete the keenest refinements of today, it can be ventured and indeed hoped, that at another meeting such as this, ten years hence, the present developments under discussion at this symposium will appear to our successors as neolithic technology.
Fig. 4. A
case of insertion. The middle part of short arm of chromosome 3 is inserted in
the distal portion of long arm of chromsome 7. (a) Actual karyotype of the
individual; (b) cut-out of the chromosomes to show artificially the inserted
piece; (c) the amputated chromosome 3 at left and the long chromosome 7 at
right are the original ones (as shown in a). The amputated chromosome 3 at
right and the long 7 at left are reconstituted by patching together the cut out
fragments seen in (b).
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