Chromosoma (1992) 101 : 527-537

CHROMOSOMA 9 Springer-Verlag1992

Chromosoma Focus

A survey of the first hundred volumes of Chromosoma H.G. Callan

In this survey I have attempted to evaluate the importance for cell biology of some of the work published in Chromosoma over the years from 1939 to 1991, albeit in a partisan and selective manner. For simplicity I have referred to authors of such work by volume number only. Work published in other journals that I have included to put topics in perspective, or to fill gaps in a commentary, is referred to by year of publication, and included in the list of references as usual. It is a measure of the importance that scientists attach to publishing in a journal of repute that one can read the pages of Chromosoma alone and yet obtain a pretty fair idea of what has been happening in nuclear cytology over the past 50 years. Publication of Chromosoma could hardly have begun at a less auspicious time, spanning the outbreak of the second World War. In the first two volumes there was a distinctive mix of papers, for along with rather few of what might be termed run-of-the-mill papers on chromosome numbers, idiosyncratic meiotic behaviour and the like, there were six important contributions by Caspersson, two by Schmidt, and one by Frey-Wyssling, including an argument as to the relative sensitivities of birefringence measurements (Schmidt) and UV absorption and dichroism measurements (Caspersson). Inter alia, these observations established beyond reasonable doubt that the division spindle contains protein fibres Gatty Marine Laboratory, Universityof St. Andrews, Fife KY16 8LB, Scotland, and CarnegieInstitution of Washington,Department of Embryology,Baltimore,MD, USA

aligned along its length, and that the elongated sperm heads of grasshoppers contain nucleic acid fibres aligned along their lengths. These papers concerned me directly, for along with H.N. Barber during 1942/44 I had been in spasmodic controversy with the Drs. Stedman, when occasion permitted, for they held precisely the contrary view (Callan ] 943; Barber and Callan 1944). I am afraid that here, as elsewhere, my survey of Chromosoma cannot but be partisan! The 50 years since Chromosoma first appeared have seen astonishing progress in our understanding of the material basis for heredity, and how it works. If one looks back to 1939, it was then widely assumed that only proteins are endowed with the specificity of structure, in their amino-acid sequences, that are required of substances capable of passing on instructions as to how a zygote should develop. Although the chromosomal basis for Mendelian genetics had become generally accepted during the 1920s, and although by the 1930s thymonucleic acid (later DNA) was known, at least by the majority of cytologists, to be universally a constituent of chromosomes, DNA was still thought to be a monotonous polymer having a "protective" function for the protein with which it was associated. That DNA rather than protein might be the carrier of genetic information did not seem plausible until the dramatic discovery of pneumococcal transformation was published by Avery et al. (1944). It was scarcely surprising that two papers on the structure of the salivary gland (SG) chromosomes of Chironomus should appear in the first two volumes, with

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Hans Bauer as Managing Editor of the new journal, for along with Heitz, Bauer in 1933 had been responsible for rediscovering giant chromosomes in the SGs of the larva of Bibio. Bauer himself contributed a paper to the first volume, using the SG chromosomes of Drosophila for analysing structural mutations induced by X-rays. Chromosoma quickly became a recognized haven for work on polytene chromosomes, and Fig. 1 shows the percentage of such papers published in five-volume aggregates spanning the years from 1939 to 1991. The steep rise to about 15% by 1970 reflects the prestige of Bauer and his student Beermann's school. 1948 saw the publication of Sengiin and Kosswig (vol 3) where the banding patterns of giant chromosomes from different tissues of the "bloodworm" larvae of Chironomus were claimed to be diverse, and which if true would have undermined the prevailing claim that this banding pattern is in some manner determined by boundaries between genes, and that gene order does not vary from one tissue to another. In 1952 came papers by Bauer and Beermann (vol 4) and Beermann (vol 5) giving direct evidence for the polytene nature of giant chromosomes in the Diptera, largely based on the observed structure of Balbiani rings (BRs) in the SG cells of Chironomus. Beermann (vol 5) also supplied a satisfying alternative explanation for the supposedly diverse banding patterns in cells of different tissues, namely that the patterns are substantially the same in all, but are overlaid by the "puffing" of particular bands that are distributed in a tissue-specific manner, the BRs being outsize puffs restricted to SG cells and therefore a conspicuous example. In my opinion this marked a turning point in studies on polytene chromosomes, for it was soon apparent that as well as offering their by now traditional scope for refined cytogenetic analysis, SGs (and other) cells of larval Diptera provide remarkably favourable opportunities for probing the intracellular mechanisms responsible for gene "action". In order to understand why there was such a cascade of seminal papers on polytene chromosomes in Chromo-

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soma during the 1950s and 1960s, one needs first to consider the then prevailing perspective. The nature of D N A was revealed by Watson and Crick (1953a, b), including the proposal as to how the molecule is replicated. A few years later came the physico-chemical proof of the semi-conservative replication of bacterial D N A by Meselson and Stahl (1958), and the cytological proof by Taylor et al. (1957) that such a mechanism also operates in eukaryotes. Proof using enzymatic digestion that DNA, not protein, holds a eukaryotic chromatid together lengthwise, was supplied by Callan and Macgregor (1958). The demonstration that cell nuclei do not become irreversibly differentiated from one another, at least during early embryogenesis, came from the results of nuclear implantation experiments with amphibian eggs, initiated by Briggs and King (1952) on Rana and continued by Gurdon et al. (1958) on Xenopus. The role of mRNAs in information transfer from D N A to protein became apparent as the result of several independent discoveries published round about 1960, and the crucial involvement of nucleoli in ribosome synthesis in eukaryotes was demonstrated independently by Perry (1962), by Brown and Gurdon (1964), and by Ritossa and Spiegelman (1965). A connection between BRs and SG secretions appeared likely in the early 1950s. It began with Beermann's (1952) description of two BRs that are present in six cells with particularly granular cytoplasm that lie alongside the duct in the SGs of Trichocladius larvae but are absent from the "normal" cells, which possess a single BR, in another location, that is absent from the six special cells. A similar and particularly striking association was found by Mechelke (vol 5) in Acricotopus. The next important paper on polytene chromosomes to appear in Chromosoma was that of Breuer and Pavan (vol 7) on Rhynchosciara. They studied, in relation to stage of larval development, the sequence of formation

529 and regression of various "bulbs" (=giant puffs) on the SG chromosomes; during the expansion of some though not all of these giant puffs there occurs a local increase in DNA content of the bands involved relative to neighbouring loci. This observation to some extent called in question the theory of the constancy of DNA per cell per species (Boivin et al. 1948) and gave origin to the term " D N A puff"; such puffs occur in other sciarid flies (see Crouse and Keyl, vol 25), though not in Chironomus. During the early 1950s Beermann made an exhaustive cytogenetic analysis (vol 7) of the SG chromosomes of two species of Chironomus, C. tentans and C. pallidivittatus, which he succeeded in hybridizing. This work led to several important discoveries. The first of these was a demonstration that at least one, or part of one, nucleolus is essential for the development and survival of a Chironomus egg (vol 11). The two Chironomus species differ in the number and locations of nucleolus organizers (NOs) on their chromosomes. Recombinant genotypes in F 2 hybrids may have any combination from three homozygous NOs to none at all; all but the last are viable and fertile. Moreover, normal NOs when broken into fragments by irradiation can join up with other broken chromosomes and still form nucleoli. Any such partial NO when introduced into an otherwise nucleolusless genome can sustain normal development. Beermann's next paper (vol 12), and in my opinion his finest, described a study of an extra BR on chromosome 4 that is associated with an extra granular component of the SG secretion produced by four special cells that lie alongside the duct exit in C. pallidivittatus. This extra BR is not present in the normal cells of the SG, though there are three other BRs, also on chromosome 4, that are present in both the normal and special cells. In C. tentans these same three BRs are present, but there are no extra BRs in SG cells lying adjacent to the duct exit, nor is there a granular component in the saliva. In all F1 hybrids this granular component is present, and so is the extra BR on their C. pallidivittatus chromosome 4, though this is absent from their C. tentans chromosome 4. In backcrosses of such F1 hybrids to C. tentans, whichever way round, half of the offspring show the extra BR and the granular secretion, the other half show neither. Moreover if recombination in the F1 parent happens to have transferred the region of the C. pallidivittatus chromosome 4 bearing the extra BR to an otherwise C. tentans chromosome 4, the outcome is the same. A clear correlation was thereby established, including the further likelihood that all the BRs " . . . supply the genetic information for non-enzyme proteins, e.g. the proteins of the saliva." This set the stage for a variety of attacks on the problem of cell-specific gene activation, and the transcriptional response. Before continuing with this topic, I wish to return for a while to the matter of polyteny. Bier (vols 8, 10 and 11) showed that in the developing nurse cell nuclei of Calliphora (and some other higher Diptera), the early four or five rounds of endomitosis result in narrow, cross-banded chromosomes. Further rounds of endomitosis produce either bulky and compact cross-banded

chromosomes just like the typical SG chromosomes of Chironomus, or loose bundles of narrow cross-banded threads, which because of the breakdown of lateral association between these threads directly demonstrate the polytene nature of the alternative, tightly associated state. Experimental proof of polyteny was provided by Beermann and Pelling in 1965 (vol 16). A single C. tentans fertilized female was constrained to lay its eggs in a drop of water containing tritiated thymidine; the membranes of the eggs are only permeable to water for half a minute or so after oviposition. The eggs so treated were thereafter raised to 4th stage larvae in the usual way, squash preparations of their SGs were made, the slides were made up as autoradiographs and left to expose. After 10 weeks exposure some slides were developed, but these were not informative. After 2 years exposure the rest of the slides were examined and these gave decisive proof for polyteny. Various patterns of chromosome labelling were found, as anticipated. Of these patterns, the most illuminating showed one or two chromosome pairs per nucleus with a single narrow overlying track of silver grains over all or part of their lengths, the rest of their bulk, and other chromosomes, being unlabelled. Returning to the topic of gene activation and transcription, Clever (vol 12) showed that when the insect prothoracic gland hormone ecdysone is injected into larvae of C. tentans that are approaching the prepupal moult, a puff is induced within minutes at a specific locus on one SG chromosome, its size related to the dose of hormone injected; thereafter other loci puff in a characteristic sequence, suggestive of a cascade reaction system where t h e " product" of the first puff induces another to follow suit, and so on. Further analysis by Clever followed in vols 13, 14 and 17. In the meantime Pelling (vol 15) had shown by autoradiography that after injecting tritiated uridine into C. tentans larvae, the nucleoli and all puffed loci come to contain labelled RNA, the nucleoli being especially heavily labelled, and likewise the BRs. This focussed attention on the accurate description of puffing patterns, and Ashburner (vol 21) published the first of several papers on this topic, studying the SG chromosomes of Drosophila to take advantage of the sophisticated knowledge of this animal's genetics. His lead was to bear particular fruit after the discovery, or rather re-discovery, o f " heatshock" puffs. I will return to this topic towards the end of my survey. Now I wish to consider another topic that was centre stage in the early 1960s: the question of whether single chromatids are unineme or multineme in regard to their DNA. Polytene chromosomes are by definition and on experimental evidence multineme, and they are built up by successive rounds of DNA replication. They occur in many unexpected places, including suspensor cells in the ovules of several Angiosperm plants (Hasitschka, vol 8; Tschermak-Woess, vol 8; Nagl, vol 16), SGs of Collembola (Cassagnau, vol 24) and the macronuclear Anlage of heterotrich Ciliates (Ammermann, vol 25). The problem, which came to be known as the C-value

530 paradox in the early 1960s, concerned how to account for considerable differences of genome size between evolutionarily related organisms. Some of these differences are of close to an order of magnitude, and over the years several examples, in both higher plants as well as animals, have been recorded in Chromosoma. Polyploidy can be invoked in some instances, but not when chromosome numbers are substantially the same in the species being compared, as is for example the case in two turbellarian flatworms, Mesostoma ehrenbergi (2n = 10) and M. lingua (2n = 8). The genome of M. ehrenbergi, its C-value, contains ten times as much D N A as that of M. lingua (Goltenb6th, vol 44). The authors of several papers in Chromosoma, as well as elsewhere, have argued that such differences are only explicable if the chromosomes of eukaryotes are multineme; the higher the C-value the greater the degree of multinemy. However, this notion ran directly counter to the autoradiographic information coming from tritiated thymidine incorporation followed through successive rounds of mitosis, as first demonstrated by Taylor et al. (1957) and many times substantiated thereafter. A chromosome is labelled in both of its chromatids after one S-phase in the presence of a labelled D N A precursor but after a following S-phase in the absence of a labelled precursor only one chromatid shows labelling at any one level along each chromosome, the other chromatid being unlabelled. This is precisely what would be expected if each chromatid contains only a single D N A duplex, i.e. is unineme. Significant evidence against the multineme "doctrine" came, surprisingly enough, from another line of work on the polytene chromosomes of Chironomus. In vol 8 of Chromosoma, Keyl published a paper comparing the banding patterns of the SG chromosomes of two subspecies of C. thurnmi, C. thummi thummi and C. t. piger, and ofF1 hybrids between them. The patterns in the two subspecies are essentially the same, but there are many bands in the C. t. thummi chromosomes that are larger and stain more intensely than their counterparts in C. t. piger. This is clearly demonstrated in F1 hybrids, for the partner chromosomes include unpaired regions in which most of the single b a n d " heterozygosities" lie. Keyl and Pelling (vol 14) used autoradiography to follow the time course of D N A synthesis in SG chromosomes of C. t. piger/thummi hybrids that had been injected, either with [3H]thymidine alone, or first with [14C]thymidine and then later with [3H]thymidine. Apart from showing that D N A synthesis begins in all bands at about the same time, i.e. that there are a multiplicity of initiation sites for replication per chromosome, the bands in C. t. thummi that are conspicuously larger than their counterparts in C. t. piger continue to label with [aH]thymidine after the corresponding C. t. piger bands have completed their replication. Keyl (vol 17) extended this study, using microspectrophotometry of Feulgen-stained SG preparations of C. t. piger/thummi hybrids, to determine the relative D N A contents of 30 bands amongst those that differentiate the two subspecies. Fourth instar larvae that had been raised at 4~ C until just before pupation were utilised

in order to reduce the size of puffs and to avoid fixing SG glands during rounds of D N A replication, for both of these factors would have confused the issue being addressed. The outcome was clear-cut: C. t. thummi bands that are larger than their C. t. piger counterparts contain 2, 4, 8 or 16 times as much DNA. Keyl demonstrated that these C. t. thummi bands to not contain more D N A because they have engaged in one or more extra rounds of replication during development of the SGs; rather they are integrated as longer replicating units in the gametic D N A of C. t, thummi, for overall the 4C D N A content of C. t. thummi primary spermatocytes is 27% more than that of C. t. piger. Keyl considered the possibility that the duplications might have arisen as a result of unequal crossing over, but rejected this because of the regularity of the doubling series that he had observed. Instead, he proposed that the duplications occur as evolutionary accidents, that the replicating units are looped domains where, during replication, irregular union at the base of a loop leads to one D N A duplex having two lengths of the loop joined together in series, the sister duplex suffering a corresponding deletion. Such errors of replication, if they were to occur on a sufficient scale, could explain the origin of major C-value differences without the need to invoke multinemy (which was finally and decisively shot down by Kavenhoff and Zimm as described in vol 41), but the paradox itself remains. Bearing in mind Keyl's and other evidence for repetitous DNAs, I attempted (1967) to account for the paradox by proposing a generalised Master/Slave organization for the genetic units of eukaryotic chromosomes, but this came to grief when more became known about coding sequences in DNA; the Master/Slave theory presupposed that coding sequences would be highly reiterated in organisms with high C-values; some are, but most are not. I return now to the BRs of Chironomus. By the early 1960s, and stimulated by Beermann's (vol 12) genetical analysis of the relationship between a limited number of BRs and SG secretion, it occurred to several cell biologists that this topic might yield to a thorough-going molecular approach. Edstr6m and Beermann (1962) began by showing that it is possible to microdissect various fixed components of SG cells of C. tentans, extract R N A (and in some cases DNA) from these separate components, and subject their nucleic acids to base ratio analysis on a micro-scale. In vol 28, three papers by Daneholt et al. described physico-chemical and kinetic properties of chromosomal R N A from the SGs of C. tentans, in the third of these papers concentrating on the R N A synthesized on BR 2, which is generally the largest of the BRs. In the same volume, Grossbach described a micro-electrophoretic analysis of the polypeptide units, separated from one another by reduction of the disulphide bonds that hold them together, in the SG secretions of C. tentans, C. pallidivittatus, and hybrids between them. In C. tentans, there are five polypeptides, in C. pallidivittatus there are these same five plus two more, and Grossbach showed that one of these latter is synthesized by the special cells that lie adjacent to the salivary duct and whose chromo-

531 somes carry the extra BR. Few of the later and more molecular developments in this field were published in Chromosoma; the situation as it stood 4 years later was summarized for the polypeptides by Grossbach (1973), and for the RNAs by Daneholt and Hosick (1973). However, in a tour de force published in 1980 (vol 81) Derksen et al. were able to identify the precise band, and it is one band only, that gives rise to BR 2 in C. tentans. This was achieved by in situ hybridization of 3H-labelled BR 2 R N A to denatured squash preparations of Malpighian tubules, for in this tissue the banding pattern of Chromosome 4 is not distorted by the presence of BRs ! BR 2 harbours an exceptionally long transcription unit of 37 kb, and the translated product of its m R N A is a correspondingly large polypeptide of more than 500,000 daltons (Rydlander and Edstr6m; Rydlander et al. vol 81). Both the m R N A and the polypeptide consist of serially repeated subunits, a finding that for a little while appeared to bolster the Master/Slave theory. I have not finished with polytene chromosomes, but it is convenient at this point to turn elsewhere. In 1968, Gall, and Brown and Dawid independently, demonstrated that during pachytene in Xenopus oocytes there occurs an amplification of 18S+28S rDNA sequences, which become dissociated from the chromosomes of the oocytes and engage on their own in rRNA synthesis in multiple "free" nucleoli during later stages of oogenesis. The amplified rDNA forms a conspicous Feulgenpositive " c a p " in pachytene. Using this material, the technique of in situ hybridization was invented. 3H-labelled Xenopus 18S + 28S rRNA was purified by density gradient separation and applied to denatured squash preparations of young Xenopus ovaries under conditions allowing renaturation and hybridization to occur, and the preparations were subsequently autoradiographed. The slides showed radioactivity in the pachytene caps but not in the chromosome" bouquet" (Gall and Pardue 1969). Complementary preparations where sheared, denatured 3H-labelled Xenopus DNA, from which the heavier 18S + 28S rRNA sequences had been removed before hybridization, was applied instead, showed radioactivity in the chromosomes but not in the caps (Pardue and Gall 1969). The development of in situ hybridization, which has had a tremendous impact on cell, biology, was soon followed by the discovery, announced by Caspersson et al. (vol 30), that the application of acridine derivatives, quinacrine and quinacrine mustard, to cytological preparations induces metaphase chromosomes to display reproducible fluorescent banding patterns. The uniformity along the lengths of metaphase chromosomes had for long tantalized cytogeneticists, who knew that both polytene and lampbrush chromosomes are anything but uniform, and that their lengthwise heterogeneity is concealed in the compact chromosomes of dividing cells. Thus of the fluorescence techniques, and they quickly multiplied, were a blessing to cytogeneticists, especially those studying human karyotypes where unambiguous chromosome recognition can be of grave import. Moreover, Arrhigi and Hsu (1971), utilizing an incidental outcome of Pardue and Gall's (1970) in situ

hybridization of mouse satellite D N A to denatured mouse metaphase chromosomes, published a C-banding technique for detecting "constitutive heterochromatin" (see also Hsu and Arrhigi, vol 34), where the crucial step is denaturation of the material on the slide, just as though preparatory to in situ hybridization, but instead followed directly by Giemsa staining. Banding techniques, which have since proliferated, have revolutionised cytogenetics; they have not made the identification of small portions of metaphase chromosomes as precise as the natural banding patterns of polytene chromosomes, but they have gone some considerable way in that direction. I will now turn to another topic in which I have for long had a particular interest. Scarcely any volume of Chromosoma has appeared without some reference to meiotic chiasmata, which is hardly surprising in view of the universality of genetic recombination. However, I will refer only to a few critical experiments that have been performed during the last 15 years. The idea that a chiasma represents, to a cytologist, the outward and visible sign of a genetical crossover having occurred, and that this has involved the reciprocal breakage and rejoining of non-sister chromatids during meiosis, was based for the most part on evidence already available prior to the publication of the first volume of Chromosoma, i.e. by 1939. However, with the advent of autoradiography came the opportunity to put the idea to decisive test. Taylor (1965) was able to show that prior to male meiosis in the grasshopper Romalea the D N A of chromosomes replicates in the standard semi-conservative way, and that if chromosomes are labelled with [3H]thymidine in the last spermatogonial S-phase, and not during the following premeiotic S-phase, they enter meiosis with one labelled and one unlabelled chromatid. Half of the recombinational events should then occur between labelled and unlabelled chromatids, and Taylor showed that such exchanges can be recognized, as anticipated, but only with confidence when the chromatids are sufficiently spread apart from one another, i.e. at first anaphase. However, the analysis was complicated by two factors: most of the bivalents of Romalea form several chiasmata, so there is the potential for multiple label switch points per chromatid; and some of the label switch points are, just as in mitosis, a consequence of sister chromatid exchange. Therefore, proof of breakage and reunion at chiasmata was still not absolute. Proof absolute was provided by Jones (vol 34), who carried out a similar autoradiographic study, but using the grasshopper Stethophyrna; all the chromosomes of Stethophyrna are acrocentric, its three largest bivalents form only a single chiasma during the meiosis of males, and this is close to the centromeres. These features make for ease of analysis. The labelling patterns visible at first meiotic anaphase were just as predicted: chiasmata generate proximal label exchanges in half of the chromosome pairs, while sister chromatid exchanges are fewer in number and are distributed at random over the length of the chromosomes. A fascinating outcome of Latt's work (vol 49) with fluorescent dyes bound to metaphase chromosomes, was

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his discovery (1973) that if chromosomes incorporate bromodeoxyuridine (BrdU) in place of thymidine during two rounds of replication, those chromatids in which only a single D N A polynucleotide chain contains BrdU fluoresce more intensely than do their sister chromatids where both D N A chains contain BrdU. The observations are in complete accord with autoradiographic resuits in showing that D N A replicates semi-conservatively, including the detectability of sister chromosome exchanges, but the cytological resolution is higher than is attainable with tritium autoradiography. To use this technique, a fluorescence microscope is required, and the preparations cannot be preserved indefinitely because the fluorescence fades with time. However, both these drawbacks were quickly overcome when Perry and Wolff (1974; see also Wolff and Perry, vol 48) showed that such preparations can be made permanent if they are stained thereafter with Giemsa, when the differential fluorescence is mirrored by differential staining and can thus be observed with an ordinary light microscope. This technique has permitted a further investigation of chiasmata on account of its higher resolving power. Tease and Jones (vols 69, 73) were able to demonstrate in Locusta that visible crossover exchanges coincide precisely with chiasmata, that there is no such phenomenon as "terminalization of chiasmata," and that "terminal associations" in bivalents result from conventional, near-terminal chiasmata (see also Jones and Tease, vols 73, 89). In 1956 Moses, when studying thin sections through spermatocytes of the crayfish Cambarus, discovered dense structures that he termed axial cores associated with the chromosomes during pachytene. Similar structures were observed by Fawcett (1956) in the spermatocytes of vertebrates, and they have since been found in a wide variety of eukaryotes, both plant and animal. Single linear cores first appear during zygotene, and as pairing progresses they contribute to tripartite structures, synaptinemal (later synaptonemal) complexes (SCs), with the cores to the outside, with most of the chromatin lying still more lateral, and a central "element" in between; Moses (1958) proposed that SCs play a crucial role in genetic recombination, and thus chiasma formation. The distance between the lateral elements of SCs is about the same in all those organisms that have been investigated, some 100 to 200 nm. A large number of papers on SCs have appeared in Chromosoma following their discovery. The connection of SCs with recombination seemed to be assured when they were found to be absent from Drosophila spermatocytes, which show no recombination; nevertheless, SCs were found to be present in the achiasmate male meiosis of scorpion flies and the mantid Bolbe (Gassner, vol 26), and also in the achiasmate female meiosis of the silkworm Bombyx (Rasmussen, vol 54). Moreover lamellate "polycomplexes," the separate components of which are similar in appearance (except for the absence of chromatin) and in dimensions to SCs, have been found in spermatid nuclei of Gryllus (Sehin, vol I6) and in other insects. It is now generally believed that the polycomplexes

in post-meiotic cells are the polymerized relics of SCs discarded from meiotic bivalents, and that in organisms with achiasmate meiosis SCs hold bivalent chromosomes together so as to ensure correct disjunction; in Bombyx oocytes they are left on the spindle equator at first ariaphase (Rasmussen, vol 60). The connection between SCs and genetic recombination began to gain a firmer footing with the discovery of discrete nodules, about 100 nm diameter, associated laterally with the SCs of Lilium spermatocytes (Moens, vol 23). Similar "nodes" were found by Gillies (vol 36) in Neurospora and by Zickler (vol 40) in several other fungi. They were recognized by Carpenter (1975) to be transient structures present during pachytene in Drosophila oocytes, and because their numbers and locations along bivalents correspond, to a first approximation, with those of genetic crossovers, they were given the name "recombination nodules." Determination of the transient nature and the number of recombination nodules required painstaking and laborious serial sectioning (see Carpenter, vol 51) for transmission electron microscopy of Drosophila ovarioles, convenient in only two respects, the relatively small size of their pachytene nuclei, and the wealth of available genetic data on recombination. Carpenter's proposal has been corroborated in several later studies, including those of Zickler (vol 61) on the fungus Sordaria, Carpenter herself (vol 75) on recombination-deficient mutants of Drosophila, and perhaps most strikingly by Rasmussen and Holm (1979) on the silkworm Bombyx. Here SCs are present at pachytene in both sexes, but females of Bombyx have achiasmate meiosis and lack recombination nodules. Furthermore, Carpenter (vol 83) has demonstrated by autoradiography that [3H]thymidine is incorporated in the vicinity of SCs, though not elsewhere, in the pachytene nuclei of Drosophila oocytes; a limited amount of D N A repair synthesis is known to be required at sites of genetic recombination. The labour involved in analysing synapsis by serial sectioning has prompted attempts to reach the same objective by simpler surface spreading techniques, much as squash techniques largely came to replace paraffin sectioning for karyologists using light microscopy in the 1930s. Several papers appeared in Chromosoma with this aim in view, beginning with Comings and Okada (vol 30). Progress was made by Counce and Meyer (vol 44) who devised a method whereby the axial cores and SCs of meiotic prophase stages are well preserved, and, because they are virtually devoid of chromatin, these axial structures have well defined outlines. This has led to '~SC karyotyping" (e.g. Moses, vol 60; Dresser and Moses, vol 76), and, because many spreads of complete nuclei at various stages of meiotic prophase can be examined by light as well as by electron microscopy, the analysis of synapsis in exceptional as well as normal individuals has been greatly facilitated. A large number of studies using the technique have been reported in the more recent volumes of Chromosoma. However, I now propose to tackle the subject of lampbrush chromosomes (LBCs). Studies on LBCs began in unobtrusive fashion in

533 Chromosoma with a paper by Meyer, Hess and Beermann (vol 12) concerning "Phasenspezifische Funktionsstrukturen" in spermatocytes of Drosophila melanogaster, and their connection with the Y chromosome. Working with thin sections and electron microscopy they observed various unusual structures in prophase nuclei, all of which were absent from spermatocytes lacking Y chromosomes. Meyer followed (vol 14) with a study carried out on several other species of Drosophila, including in particular D. hydei and D. neohydei, where the unusual structures are large enough to be clearly seen in living spermatocytes by phase contrast microscopy. He concluded that they are precisely comparable to the lateral loops of amphibian LBCs, each loop pair having its own distinctive morphology. Hess (vol 16) then described the results of a sophisticated genetical analysis, using various translocation stocks of D. hydei; he established the order of the loop loci on the Y chromosome and in 1967 demonstrated by complementation tests that each and every one of the Y loops must be present if functional spermatozoa are to develop. In the same volume, Meyer and Hess showed that the Y loops of D. hydei disappear immediately following the injection of the R N A synthesis inhibitor actinomycin into larvae, and that they reappear during recovery from this poison. This precisely parallels what occurs in amphibian oocytes when similarly treated. Direct evidence that R N A is present and is synthesized on the Y loops of D. hydei was supplied by Hennig (vol 22). In view of the remarkable lengths of the Yloops, several of which are clearly single transcription units, this prompted much speculation and experimentation as to whether they are themselves the sources of mRNAs coding for specific proteins, or whether they serve some other function. Papers on "main-line" amphibian LBCs began to appear from Mancino and his associates in vols 26, 3~ and later. In vol 36 Lacroix and Loones demonstrated that X-ray induced chromosomal aberrations in male Pleurodeles can be readily recognized in LBC preparations of their female progeny, and in particular they were able to show that, if a break happens to have occurred at a sphere-organizing locus, spheres are formed at both fragments of such an organizer. Spheres are present in most amphibian oocyte nuclei (though see Nardi et al., vol. 37), free as well as attached to organizer loci; I will make further reference to these enigmatic structures later. Kezer and Macgregor (vol 42) described the astonishingly large ring nucleoli that are attached to the NOs of Plethodon LBCs. It is worth pointing out that this condition is by no means universal amongst Amphibia. Although rDNA amplification is widespread in the group, and leads to the formation of a multiplicity of free nucleoli that are active in the transcription of 18S + 28S rRNA in oocytes, the NO itself may be completely inert, as in species such as Xenopus (Callan et al., vol 97). One sees here a remarkable morphological parallel with free spheres and their relationship to sphere-organizing loci, as yet still obscure. At about this time (the early 1970s), three techniques, in situ hybridization, "Miller-spreads" (Miller and

Beatty 1969), and the use of immunofluorescence (Desai et al. 1972) began to have an impact on studies on LBCs. Angelier and Lacroix (voI51) produced handsome spreads from Pleurodeles showing transcription units entire from origin to terminus, with neighbouring units of similar or opposed polarity, confirming impressions gained from light microscopy. Turning to in situ hybridization, Pukkila (vol 53) succeeded in hybridizing labelled 5S rDNA to 5S transcripts on LBC preparations of the newt Notophthalmus, and demonstrated that their synthesis takes place on a few loops, and more particularly on some of the compact centromere bars in this organism. Her study pointed towards the practicability of identifying the nature of other transcripts on the many long loops of amphibian LBCs because of the "amplification" of sequences provided by transcripts accumulated in close juxtaposition to one another. Macgregor and Andrews (vol 63) in situ hybridized labelled middle repetitive D N A from Triturus carnifex to R N A transcripts on the loops of its LBCs. They found that most loops were labelled to some degree, but that loops on the heteromorphic arms of bivalent I were especially heavily labelled, some of these being labelled only in restricted regions. Mixed probes such as those used in this work have limited powers of discrimination, but the study clearly demonstrated the promise of in situ to transcript hybridization (see the follow-up by Varley et al., and by Morgan et al. in vol 80). A new line was struck by Sommerville et al. (vol 66) who used immunofluorescence to study the binding distribution of antisera to various size classes of nuclear RNPs on LBC preparations of T. carnifex. Some of these proteins were demonstrated to be common to transcripts on all loops, while others were restricted to transcripts on a few only. Loones (vol 73) showed that if female Pleurodeles are exposed to 7-irradiation, LBC preparations made from large oocytes when taken a few hours later show virtually complete retraction of the lateral loops, and condensation of the chromosomes to the size they would reach just after ovulation (i.e. first meiotic metaphase). However full recovery follows, so that by 3 days after irradiation the lateral loops are back to their normal size. The astounding reversibility of this process parallels recovery from actinomycin poisoning (Snow and Callan 1969, on T. cristatus) and recovery of the Y loops after X-irradiation of D. hydei (Hennig, vol 22). Although the shutdown of transcriptional activity is evidently the cause of loop retraction, and recovery of this activity the cause of loop extension, a full explanation of this phenomenon remains for the future. Schultz et al. (vol 82) broke new ground by devising an incubation medium in which germinal vesicles isolated from newt (Notophthalmus) oocytes could be cultured in vitro and tested for their capacity to synthesize R N A in the presence of ~-amanitin. In the absence of this toxin, synthesis proceeds normally for a few hours in the LBCs and extrachromosomal nucleoli. RNA polymerase II is most sensitive to ~-amanitin, and at 0.5 gg/ ml R N A synthesis on all the lateral loops is inhibited. There are, however, sites of continuing R N A synthesis

534 at a few, not all, of the centromere bars (compact regions without lateral loops, including the centromeres), at a further 15-20 small discrete axial sites that are widely distributed over the LBC complement, and additionally in the many extrachromosomal nucleoli. In the presence of 200 gg/ml c~-amanitin, R N A synthesis is confined to the nucleoli. R N A polymerase I is the enzyme least sensitive to e-amanitin, and evidently this is responsible for the continuing rRNA synthesis. By deduction, the chromosomal sites where R N A synthesis proceeds in the presence of ~-amanitin at 0.5 ~g/ml should therefore involve R N A polymerase III, i.e. the synthesis of 5S rRNA at a few centromere bars (so confirming some of the observations of Pukkila, vol 53) and tRNAs. A connection between a regularly identifiable "landmark" structure in an LBC complement and a defined transcriptional product was established by Gall et al. (vol 84) who showed that in the newt Notophthalmus viridescens (and indirectly in T. cristatus and T. alpestris as well) histone genes are located at the sphere organizer loci. The historic gene cluster, along with a repeated sequence "satellite 1 ", are transcribed together on loops that project from the sphere organizer loci, closely associated with the spheres themselves. The nature of the relationship between spheres and histone loops is problematical, but it has since been established that the same connection exists in the anm'an Xenopus, so its antiquity suggests a functional relationship. Another outcome of this study of histone gene transcription was the demonstration by Diaz and Gall (vol 92) that the transcription of satellite sequences occurs by readthrough from adjacent histone clusters, histone sequences remaining attached to transcripts of satellite sequences on the loop matrix. Moreover Diaz and Gall also proved that reversals in the polarity of transcription on these histone loops occur as a consequence of the switch of transcription from one polynucleotide strand to its complementary partner. Sommerville and Scheer (vol 84) demonstrated that complementary repeat sequences are abundant in the heterogeneous R N A of Xenopus, Triturus and Necturus oocyte nuclei; this was assessed both by hybridization kinetics and by direct visualisation of duplex structures in preparations spread for electron microscopy. Such sequences, and indeed repeated sequences in general, must therefore be correspondingly abundant in the transcription units of the lateral loops. This presumably accounts for the complexity in secondary or "super structure" of the various Y loop transcripts of D. hydei demonstrated by Hennig and his co-workers (see Grond et al., vols 88, 89), and equally so of the landmark loop transcripts on Pleurodeles LBCs (Angelier et al., vol 89; Penrad-Mobayed et al., vol 94). We now have the evidence of molecular sequence organization of Y loop D N A in three papers by Vogt and Hennig (vol 94), and also papers by Wlaschek et al. (vol 96) and Trapitz et al. (vol 96), which indicates that no protein-coding sequences are present, and a general consensus seems to be emerging that the Y loop transcripts, and similar transcripts on landmark amphibian LBC loops, function as traps for the sequestration of particular RNPs. As early

as 1977, Yamasaki (vol 60) had shown that the various Y loops can be stained differentially and in a highly specific manner by Giemsa at pH 10 (see also Yamasaki, vol 83). She suggested that her observations implied a site specificity of loop proteins, though this deduction might be questioned because pretreatment with RNase removed the differential staining, which indicates that R N P specificity is responsible. Kloetzel et al. (vol 84) reached a similar conclusion, on the evidence that some RNPs present in the spermatocyte nuclei of both X Y and J20 males of D. hydei are markedly under-represented in the latter. In the search for protein specificity resident in the Drosophila Yloops, indirect immunofluorescence studies have yielded valuable information. Gl~tzer (1984) has found three monoclonal antibodies raised against D. melanogaster non-histone nuclear proteins that bind specifically and differentially to Y loops of D. hydei. In XO males the corresponding antigens are evenly distributed in the spermatocyte nuclei. Furthermore the antigens that are assembled on the Y loops remain as stable "organelles" detectable throughout meiosis and during the transformation of spermatids into spermatozoa. In another immunofluorescence study, Tischendorf et al. (vol 98) have demonstrated that four different spermatocyte nuclear glycoproteins present in XO D. hydei, and thus not Y encoded, possess epitopes that require for their detection by microscopy the presence of two particular Y loops in spermatocyte nuclei that possess them. Studies on amphibian LBCs using monoclonal antibodies have proceeded along similar lines. Lacroix et al. (vol 92) raised such antibodies against germinal vesicles of Pleurodeles waltl, and ten of these reacted with antigens on the lateral loops of this urodele. A group of five reacted with all the transcription units on all the loops, and likewise labelled cell nuclei of embryos. Two monoclonal antibodies also reacted with all loops, but not with the nuclei of embryos. Most of the antibodies raised against Pleurodeles cross-react with Triturus LBCs (Ragghianti et al., vol 97). One antibody labelled certain specific loops in Pleurodeles LBCs, these being otherwise unremarkable in morphology, including several that lie, though differently distributed, in the heteromorphic regions of the ZW sex bivalent (Lacroix et al., vol 99); this antibody was notable in labelling a single transcription unit in a loop comprising several such units. Another antibody reacted with chromomeres only. Finally yet another antibody reacted with spheres, both free and attached, and with another structure that, like the spheres, does not have a fundamental loop organization. Another twist to the LBC story was exposed by DiMario et al. in vol 97. If preparations of newt LBCs are probed with 3H-labelled single-stranded newt D N A in 0.1 x SSC, nothing labels. However, if such preparations are first treated with RNase and then exposed to the same probe under identical conditions, the great majority of loops bind the probe. If the probe is applied in 1 x SSC, only a small subset of loops become labelled, and these same loops are recognized by a polyclonal serum raised against, of all substances, histone H1 ! This is about how matters stood on the LBC "front" when

535 the 100th volume of Chromosoma appeared in 1991. There is more to come, but it would not be appropriate to discuss it at this juncture. I think it appropriate to conclude this survey by returning yet again to polytene chromosomes, which have figured so prominently throughout Chromosoma's history. I take up the story where I left it earlier and the year is 1970 or thereabouts. The technique of in situ hybridization had just recently been devised, technical progress in molecular biology was advancing at a phenomenal rate and it was clear that molecular methodologies would have a tremendous impact on all aspects of cell biology, not least on cytogenetics. The first focus of this impact would be on the SG chromosomes of D. melanogaster. Such indeed was the stated aim when Pardue et al. (vol 63) successfully used sea urchin historic m R N A probes to locate the Drosophila historic genes. Drosophila would be the target because more was known about its genetics than that of any other eukaryote, manipulation of its stocks had long since reached a high level of sophistication, and its SG chromosomes, with their invaluable cross-banded patterns and "lateral sequence amplification" provided by polyteny, had been mapped for mutations from " A to Z." With the power to generate and clone specific probe sequences isolated from Drosophila the full potential of in situ hybridization could be realised. This was made apparent in the paper by Wensink et al. (1974) describing the preparation, isolation and cloning of three D. melanogaster D N A segments, and in situ hybridization of RNAs copied from them to its SG chromosomes. One of these cloned sequences proved to be frequently repeated in the Drosophila genome, and hybridized to the chromocentre and 15 other sites, but the two others (later four more out of a total of six) hybridized to single sites only. The frequency of papers appearing in Chromosoma in which the technique of in situ hybridization has been employed is shown in Fig. 1. The first such papers came in 1970, and rose to about 10% 5 years later. There followed a plateau at this level for a further 5 years, and thereafter the frequency rose rapidly, to reach about 26% by 1990. The first 10 years, including the plateau, corresponds roughly to that when satellite and other repeated sequences were used as probes; the following period, with rapid expansion, marks the introduction and use of cloned sequences as probes. One example will have to suffice, and I will consider the heat-shock story, mentioned earlier. Ritossa (1962) discovered that when Drosophila larvae are transferred from normal culture temperature (25 ~ C) to about 37~ C, three major puffs quickly appear in their SG chromosomes; in 1964 Ritossa further showed that similar changes occur in other tissues containing polytene chromosomes, and 6 years later Ashburner (vol 31) added to the list of such heat-shock puffing loci. A most significant observation in this field was that the pattern of protein synthesis in several different tissues of Drosophila changes dramatically as a result of a similar increase in culture temperature (Tissi6res et al. 1974); synthesis of most of the polypeptides that are produced at 25 ~ C

ceases, while the synthesis of several new polypeptides is induced. Spradling et al. (1975) then demonstrated that there are corresponding changes in the populations of mRNAs extractable from Drosophila tissue culture cells; such mRNAs, when labelled and extracted from cells grown at 25 ~ C, in situ hybridize to a great many loci on SG chromosomes, whereas mRNAs extracted from cells grown at 37~ bind to far fewer loci, and these coincide with the heat-shock puffs that were identified by Ashburner (vol 31). The isolation and cloning of heat-shock genes proceeded independently in several laboratories towards the end of the seventies. For example, Holmgren et al. (1979) demonstrated in D. melanogaster that two heat-shock loci contain unique sequences that code for two proteins, and that tandemly repeated coding sequences are present at two other heat-shock loci. This paper presented a very sophisticated molecular analysis, and the clones when used as probes hybridized in situ to the appropriate regions of the SG chromosomes. Several papers where cloned heat-shock genes were used as probes for in situ hybridization to SG chromosomes appeared in Chromosoma from vol 80 onwards. In situ hybridization was given improved resolution by the introduction of biotinylated probes in 1975 (Manning et al., vol 53). This was taken a step further by Langer et al. (1981), who used biotinylated nucleotide triphosphates as substrates for R N A or D N A polymerases to generate biotinylated polynucleotide probes. Manuelidis et al. (1982) used such probes for in situ hybridization to mammalian cells, and determined the sites of hybridization by immunological methods, using as a first antibody rabbit anti-biotin, followed by goat antirabbit IG conjugated to horseradish peroxidase for bright field microscopy, or followed by fluorescein isothiocyanate-labelled goat anti-rabbit antibody for fluorescence microscopy. The high resolution potential of this technique was demonstrated on Drosophila polytene chromosomes by Langer-Safer et al. (1982). However, biotinylated probes are unlikely entirely to supplant radioactive ones because of the possibility of artefacts being generated during antigen-antibody reactions and the desire for permanent preparations. Papers where either biotinylated or radioactively labelled probes or both have been used continued to appear in Chromosoma right up to vol 100, some particularly arresting examples being those of Pardue and Dawid (vol 83) on mobile genetic elements (=transposable elements=jumping genes); Izquierdo et al. (vol 83) on an ecdysone-inducible structural gene; Bauman et al. (vol 84) on the use of diverse fluorochrome-labelled RNAs; and Carmona et al. (vol 92) on telomeric sequences of Chironomus. Running in parallel with studies whose main concern has been to determine the sequence organization of Drosophila SG chromosomes, latterly effort has also gone into analysing the kind and the distribution of proteins associated with these chromosomes. Here the ultimate aim is to lay bare the molecular mechanisms that control the transcription of D N A sequences using by the technique of indirect immunofluorescence. In an early paper,

536

Silver and Elgin (1976, also vol 68) raised antisera in rabbits against fractionated nonhistone proteins extracted from Drosophila chromatin, and applied these antisera to squash preparations of SG chromosomes; the binding of antisera was visualised by reacting the preparations with fluorescein-conjugated goat anti-rabbit 7globulin, and the preparations were then examined using a fluorescence microscope. Antisera to three different nonhistone protein fractions gave three different labelling patterns. Silver and Elgin paid critical attention to the technique used for making squash preparations, because the traditional cytological method, squashing in 45% acetic acid, extracts most of the histones and does not stabilize the nonhistone proteins. The problem was largely overcome by treating SGs first with a neutral detergent to permeabilize membranes, then fixation for 30 min in 2% neutralised formaldehyde, bathing in 45% acetic acid, 10raM MgC12 for 15rain, and finally squashing in this same solution. Much the same technique was used by Plagens et al. (vol 59) and Greenleaf et al. (vol 65). An antiserum against Drosophila RNA polymerase II was shown to bind to many sites throughout the chromosome complement, and in SG preparations made from heat-shocked Drosophila the binding was especially intense at the heatshock puffed loci. Later papers on this topic by Sass and Bautz (vols 85, 86), using electron microscopy, demonstrated that all binding sites for anti-RNA polymerase II lie in decondensed regions of the SGs of Chironomus, that is in interbands as well as puffs. At about this time, Hill and Watt (vol 63) introduced a technique for preparing SG chromosomes that avoids all use of acetic acid, and exposure to pH" outside the range 7 to 8. Chromosomes prepared in this manner showed good retention of their antigenicity when reacted with antisera against Drosophila chromatin and chromosomal proteins raised in mice. In 1975, K6hler and Millstein published an account of their hybridoma technique for producing monoclonal antibodies. This great advance was put to good use by Saumweber et al. (vol 80), who used total nuclei or crude nuclear proteins to immunize mice. Saumweber et al. had no hesitation in squashing SGs in 45% acetic acid, despite the reservations made by Hill and Watt about its use, because treatment given before and during squashing guarded against protein loss. The fact is that good quality squash preparations cannot be made without using acetic acid. Hill and Watt's technique serves for the ultrastructural study of relatively few incomplete SG preparations (as in Hill et al., vol 94; Mott and Hill., vol 94) but not for the repetitive preparation of complete SG complements required for screening purposes. Saumweber et al. found that six out of seven monoclonal antibodies bound to puffs and interbands, and one to bands alone; only three of these monoclonals bound to SG chromosomes that were squashed in 45% acetic acid without formaldehyde to guard against protein loss. The continuing and outstanding studies of Saumweber and his colleagues rightly terminate this final part of my survey. Having by now swum right out of my depth, I will only list them: Hofmann and Korge

(vol 96), Hofman et al. (vol 96), Frasch and Saumweber (vol 97), Saumweber et al. (vol 99), yon Besser et al. (vol 100). Together these studies demonstrate how essential it is nowadays to harness together the powers of molecular biology with the old-fashioned genetical and cytological and embryological skills if seemingly intractable biological problems are to be resolved. The days of the lone ranger are over. In conclusion, I would say that the coverage in 100 volumes of Chromosoma of those topics that I have discussed in outline here, most of which are tolerably familiar to me, could certainly be equalled had other topics been surveyed by a different cell biologist. The quality of plate reproduction has remained exemplary throughout, as was Bauer's clear intention from the start. Bauer died in January 1988 at the ripe age of 83, and a tribute to the founder of the journal, written by Beermann, appeared in vol 96. I remember long ago reading a paper by Ruch (vol 3) in which he demolished the then widely held view that chromosome condensation from interphase through prophase to metaphase is accomplished by hierarchies of spiralization. This view had been a pet theory of Darlington and indeed, as if to emphasize the point, a paper on this topic by Darlington and Upcott was the second to be published in the new journal (vol 1). No direct evidence was provided in Darlington and Upcott's paper, whereas Ruch's study was accompanied by actual (and expensive) photographic prints of Tradescantia meiotic chromosomes in which the "major" coils were beautifully displayed, but there was no evidence whatsoever for the so-called "minor" coil. I perceive the hand of Bauer in this extravagance, and it is a tribute to his wisdom. References Arrighi FE, Hsu TC (1971) Localization of heterochromatin in human chromosomes. Cytogenetics 10:81-86 Avery OT, MacLeod CM, McCarty M (1944) Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. J Exp Med 79:137-158 Barber HN, Callan HG (1944) Distribution of nucleic acid in the cell. Nature 153:109 Beermann W (1952) Chromosomenstruktur und Zelldifferenzierung in der Speicheldriise von Trichocladius vitripennis. Z Naturforsch 7b: 237-242 Boivin A, Vendrely R, Vendrely C (1948) L'acide d6soxyribonucl6ique du noyau cellulaire, d~positaire des charact6res h~r~ditaires; arguments d'ordre analytique. CR Acad Sci [III] 226:1061-1063 Briggs R, King TJ (1952) Transplantation of living nuclei from blastula cells into enucleated frog eggs. Proc Natl Acad Sci USA 38:455--463 Brown DD, Dawid IB (1968) Specific gene amplification in oocytes. Science 160: 272-280 Brown DD, Gurdon JB (1964) Absence of ribosomal RNA synthesis in the anucleolate mutant of Xenopus laevis. Proc Natl Acad Sci USA 51 : 139-146 Callan HG (1943) Distribution of nucleic acid in the cell. Nature 152: 503 Callan HG (1967) The organization of genetic units in chromosomes. J Cell Sci 2:1-7

537 Callan HG, Macgregor HC (1958) Action of deoxyribonuclease on lampbrush chromosomes. Nature 181:1479-1480 Carpenter ATC (1975) Electron microscopy of meiosis in Drosophila melanogaster females. II. the recombination nodule - a recombination associated structure at pachytene? Proc Natl Acad Sci USA 72:3186-3189 Daneholt B, Hosick H (1973) Evidence for transport of 75S RNA from a discrete chromosome region via nuclear sap to cytoplasm in Chironomus tentans. Proc Natl Acad Sci USA 70:442446 Desai LS, Potier L, Foley GE, Adams RA (1972) Immunofluorescent labelling of chromosomes with antisera to histones and histone fractions. Exp Cell Res 70:468-471 Edstr6m J-E, Beermann W (1962) The base composition of nucleic acids in chromosomes, puffs, nucleoli and cytoplasm of Chironomus salivary gland cells. J Cell Biol 14:371-380 Fawcett DW (1956) The fine structure of chromosomes in the meiotic prophase of vertebrate spermatocytes. J Biophys Biochem Cytol 2: 403-406 Gall JG (1968) Differential synthesis of the genes for ribosomal RNA during amphibian oogenesis. Proc Natl Acad Sci USA 60:553-560 Gall JG, Pardue ML (1969) Formation and detection of RNADNA hybrid molecules in cytological preparations. Proc Natl Acad Sci USA 63 : 378-383 G1/itzer KH (1984) Preservation of nuclear RNP antigens in male germ cell development of Drosophila hydei. Mol Gen Genet 196: 236-243 Grossbach U (1973) Chromosome puffs and gene expression in polytene cells. Cold Spring Harbor Syrup Quant Biol 38:619627 Gurdon JB, Elsdale TR, Fischberg M (1958) Sexually mature individuals of Xenopus laevis from the transplantation of single somatic nuclei. Nature 182: 64-65 Heitz E, Bauer H (1933) Beweise f/Jr die Chromosomennatur der Kernschleifen in den Knfiuelkernen von Bibio hortulanus L. Z Zellforsch 17 : 67-82 Hess O (1967) Complementation of genetic activity in translocated fragments of the Y chromosome in Drosophila hydei. Genetics 56: 283-295 Holmgren R, Livak K, Morimoto R, Freund R, Meselson M (1979) Studies of cloned sequences from four Drosophila heat shock loci. Cell 18:1359-1370 K6hler G, Millstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495497 Langer PR, Waldrop AA, Ward DC (1981) Enzymatic synthesis of biotin-labelled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci USA 78 : 6633-6637 Langer-Safer PR, Levine M, Ward DC (1982) Immunological method for mapping genes on Drosophila polytene chromosomes. Proc Natl Acad Sci USA 79:4381-4385 Latt SA (1973) Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc Natl Acad Sci USA 70:3395-3399

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