Cytologia 42: 57-63, 1977

Variability in Chromomere Pattern in a Specific Chromosome Region at Pachytenel

Marjorie P. Maguire Zoology Department, Universityof Texas, Austin,Texas, U.S. A. ReceivedJune 24, 1975

A common current assumption holds that the same chromosomal structural subunit is represented by: 1) orthodox chromomeres seen at pachytene of meiosis, 2) lampbush chromosome loops with their accompanying basal structures, and 3) the familiar bands of dipteran polytene chromosomes. It is also widely believed that in each case, any one of these structures consistently includes a specific genetic trait or set of traits. Substantial evidence has accumulated over many years that this is so for the bands of Dipteran polytene chromosomes, and while the total number of these bands may vary somewhat among different tissues of a single organism, this variation is thought to be attributable to differential visibility (Beerman 1956). Individual lampbush chromosome loops are reported to have characteristic and identifiable morphology, although construction of maps as detailed as Dipteran polytene maps has not been possible, and closely related species may have very great differences in loop pattern (White 1973). On the otherhand, pachytene chromomeres are recognizable only if they are exceptionally large or located in the vicinity of landmarks such as telomeres, centromeres or nucleolus organizers. In fact, pachytene chromomeres appear to range in size from the relatively large structures often known as knobs, at least down to apparent particles of a size close to the limit of light microscope resolution. Available observations have suggested, as Rhoades (1961) noted, that although larger pachytene chromomeres seem to be constant in size, position and number, this does not appear to be true for smaller chromomeres. Furthermore, large and small chromomeres seem to be so inter mixed in distribution as to prohibit precise, detailed linear mapping, contrary to apparently common belief. Barry (1967) pointed out that variability in chromo mere pattern at pachytene in Neurospora was too great to permit chromosome identification. Other aspects of chromomere behavior, which are familiar to students of pachytene chromosome cytology, apparently defy popular orthodoxy. Lima-de Faria (1952) found not only rare and sporadic pairing of unequal appearing -chromo meres at pachytene, as well as occasional absence of one member of a pair (in rye), but also differences in paired chromomere number in a specific region among differ ent cells of the same organism. He attributed this variation to differential effect of preparation and to possible small premeiotic deletions or duplications (which were considered potential sources of sterility). Structures at least superficially 1 This work was supported by Grants GM 19582and ResearchCareer DevelopmentAward 5-K03 GM 25988from the National Institutes of Health. 58 Marjorie P. Maguire Cytologia 42 similar to pachytene chromomeres have been observed at other meiotic stages and in other tissues. Lima-de-Faria (1954) reported that the chromomere number at mid-prophase II in Agapanthus was almost twice that at late prophase II and that although chromosome lengths varied with stage and tissue, the number of chro momeres per unit length, as well as the distance between chromomeres, were re markably similar among diverse kinds of cells (pachytene, mid and late prophase II and root tip cells). He reasoned that the accompanying marked differences in total chromomere number among corresponding chromosomes in these differing cell types were probably not due to simple fusion of chromomeres (in those with fewer of them) but rather to more complex differences in chromosome organization. Lima-de-Faria, Sarvella and Morris (1959) drew similar conclusions from obser vation of meiotic and mitotic chromomeres in Ornithogalum. They proposed that chromosomes are differently organized at pachytene and prophase of other divisions. Lima-de-Faria (1974) recently reemphasized that the widespread assumption that the polytene Dipteran chromosome band represents a structure identical to a pachy tene chromomere is inconsistent with observations; he also noted that there tend to be fewer chromomeres per genome, by an order of magnitude, at pachytene than there are bands in the Dipteran polytene chromosome. An obvious inference is that pachytene chromomeres should probably be presumed to have greater genetic complexity than polytene chromosome bands. The observations bear importantly on questions of the relationship of structure to function as well as on the nature of chromosome organization at various stages. If chromosomes consistently contained identical substructures at corresponding positions in prophase cells in diverse tissues, these might be presumed to be genetically constant, and a kind of universal functional significance might reasonably be postu lated for the organization of these substructures. As indicated above this does not appear to be true. A particular functional role seems so far to have been assigned to pachytene chromomeres only in the rather special case of ribosomal DNA carrying, large chromomeres which generally contain this informational DNA repetitiously (Phillips, Kleese and Wang 1971, Lima-de-Faria 1974). Only the larger chromomeres are generally considered consistent morphological features, and even these may vary in size relative to other chromomeres from tissue to tissue. Whether there is generally a fundamental compositional difference between large, consistent chro momeres and smaller chromomeres, which are variable in both presence and size, is of interest. The observable size range appears to be continuous, and no sharp distinction on the basis of size alone seems possible. With these considerations in mind, a specific, readily identifiable region of a maize chromosome at pachytene was examined for chromomere number and pattern in many cells of microsporocyte preparations.

Materials and methods

Acetocarmine smear preparations were examined of maize microsporocyte samples which had been fixed in ethanol-acetic 3:1 mixture and stored in a freezer. Illustrations in Figs. 1-12 are from a sporocyte sample of a single KYS plant. 1977 Variability in Chromomere Pattern in a Specific Chromosome Region 59

Sporocyte samples were also examined from various Maize Coop genetic stocks. A Zeiss Photomicroscope equipped with a 63X oil immerision 1.4 N.A. bright field objective was utilized. Slides were systematically scanned and photomicro graphs were taken of those cells in which the chromomere structure of the satellite region of chromosome 6 could be unequivocally analyzed. Since many cells did not allow such analysis, records were not kept of relative numbers of cells of each type; it is suspected that some types are more readily analyzed than others.

Results and discussion

The satellite region of chromosome 6 was chosen for study because of its relative ease of identification, and it is realized that its behavior may not be representative of the entire genome. Lima-de-Faria and Sarvella (1962) noted that the satellite arm of maize chromosome 6 is variable; many other regions (perhaps all), although less well demarcated, also exhibited marked differences between cells in their illus trations. More intensive studies of additional, well-marked, short pachytene chromosome regions seem desirable. The satellite region of chromosome 6 is une quivocally marked in each cell by the proximally adjacent nucleolus organizer region (a consistent, very large chromomere). The satellite region of chromosome 6 was found to contain: one, two, three, four or five chromomeres, with expression varying from cell to cell within a sample (Figs. 1-12 and 1A-12A). All types of pattern were found among sporocytes of single plants, and no type was extremely rare. The most common type of cell contained three chromomeres in this region. All types of pattern were seen at early, middle and late pachytene and in all stocks studied. Differences between homologues were seen in a few cells of a sample (Figs. 8, 8A). But even in rare cells where homologues were obviously not synapsed in the chromosome 6 satellite regions, they tended to have the same chromomere pattern (Figs. 1, 1A, 9, 9A); on the other hand rare cases of non-matching homologues were seen in the presence of apparent synapsis (Fig. 8, 8A). A tendency for similarity of chro momere pattern in specific regions in homologues within a cell, perhaps in advance of synapsis (in itself a perplexing problem), may result in a high degree of fidelity of matching of chromomeres between homologues, in spite of cell to cell variability in detailed pattern. Such an effect might contribute to a deceptive overall impres sion of constancy of pattern. Observations reported here suggest that an unexpected amount of variability in chromomere pattern may be found among cells of the same plant when a specific chromosome region is examined in detail. It seems unlikely that spontaneous dele tions and duplications in premeiotic tissue could account for the amount of varia bility found or that it was a result of differential chromosome stretching during slide preparation. It is not known whether chromomeres in corresponding positions in the same pattern configuration in different cells represent genetically equivalent components. It is also not clear whether any of the chromomeres of the region studied could be considered to be of the large, constant variety described in the introduction; the 60 Marjorie P. Maguire Cytologia 42 Figs. 1-8: Photomicrographs of microsporocytes showing various patterns of chromomere organization of the chromosome 6 satellite region. 1-3, satellite region with one chromomere. 1, homologues not synapsed in satellite region. 4-6, satellite region with two chromomeres. 7-9, satellite region with three chromomeres. 8, three chromomeres in one homologue, one chromomere in other homologue, homologues synapsed. Figs. 9-12. 9, terminal chromomeres not synapsed. 10, satellite region with 4 chromomeres. 11-12, satellite region with five chromo meres. Arrows mark the nucleolus organizer region. All cells from the same KYS stock plant. Magnification bar represents 5ƒÊ. Figs. 1A-12A: Interpretive drawings of photomicrographs shown in Figs. 1-12. 1977 Variability in Chromomere Pattern in a Specific Chromosome Region 63 terminal chromomere is probably the most consistent, but the possibility cannot be ruled out that variable amounts of material, in some cells represented in other chromomeres, are incorporated into it when fewer than the maximal number are present. It is conceivable that missing chromomeres may reflect failure of conden sation of the regions involved. It is suspected on the basis of the chromomere size variability seen, that alternative chromomere patterns reflect differential aggregation during condensation of subunits. It seems probable that the available array of patterns of chromatin packaging depends ultimately on the distribution of specialized protein-binding sequences such as those postulated by Comings and Riggs (1971). It is improbable that a 1: 1 relationship exists between gene and pachytene chro momere as we know it (whether or not such a relationship exists between gene and polytene chromosome band). It is tempting to speculate that chromomere pattern may be instructive in terms of the distribution of DNA sequences concerned with packaging during chromosome condensation.

Summary The satellite region of maize chromosome 6 was examined for variability of chromomere pattern at pachytene in a large number of microsporocytes. The number of chromomeres visible with the light microscope in this region was found to vary from one to five in different cells from the same plant. The two homologues of the same cell usually, but not always, matched in chromomere pattern. It is emphasized that the common assumption that pachytene chromomeres are genetically constant from cell to cell is not justified and that alternative chromomere patterns may reflect differential aggregation during condensation of subunits.

References

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