COMMENTARY Lampbrush chromosomes

H. C. MACGREGOR

Department of'/.oology, Univeisity of Leicester, Leicester LEI 7RH, UK

1986 was a celebration year for lampbrush chromo- problems of the fate and significance of the massive somes (LBCs), marked by the publication of Callan's amounts of non-coding transcripts, and the manner in comprehensive and authoritative book on these struc- which certain initiation sites are 'selected' for high-level tures. My commentary begins where Callan's book transcription on lateral loops, whereas others remain ends, standing on tiptoe beside a large and rather relatively inactive on or within the chromomeres. Of neat assembly of well-established facts, principles and particular interest are the recent studies of Epstein et hypotheses, and trying hard to catch a glimpse of what al. (1986) and Epstein & Gall (1987). These investi- lies ahead. gators have focussed their attention on the nature and Several major principles can now be accepted with transcription of the 330 base-pair (bp) satellite DNA of confidence. LBCs are a feature of the growing oocytes Notophthalmus viridescens (Nv2). Nv2 occurs in tan- of most animals, except mammals and certain insects. demly repeated clusters throughout the genome and its Callan (1986) has reviewed this principle in depth, and sequence is highly conserved among the salamandrid has discussed the significance of exceptional cases. The species so far examined (Epstein et al. 1986). It is main defining feature of LBCs, their lateral loops, are transcribed on lampbrush loops by read-through from regions of intense RNA synthesis, and together these adjacent structural gene promoters, and homologous loops produce a large variety of transcription products, strand-specific cytoplasmic transcripts are found in the though not all coding sequences in the genome are cytoplasm of a variety of tissues. The transcripts transcribed. Much of the transcribed polyadenylated correspond in size to the Nv2 repeat unit or to simple RNA is passed to the cytoplasm and its quantity is multiples of this unit. The transcripts seem to be maintained at a steady-state level by continuing syn- encoded by a specific subset of the genomic Nv2 thesis and turnover. This RNA is stored in the sequences, and they undergo site-specific self-catalysed cytoplasm and probably functions in the regulation and cleavage in vitro (Epstein & Gall, 1987), a reaction that maintenance of oocyte maturation and early embryonic resembles the self-cleavage of certain small infectious development. RNAs found in plants. The cleavage of Nv2 occurs at a The relationship between a transcription unit (TU) site that is homologous to the conserved cleavage site of and a lampbrush loop (Fig. 1) has been well defined by these infectious RNAs. The significance of this remark- Gall et al. (1983). Transcription initiates at structural able discovery remains a mystery, but perhaps will not gene promoters at the ends of loops or TUs, fails to do so for long! terminate and 'reads through' into adjacent non-coding Future directions for the study of LBC RNAs and sequences. The 'read through' hypothesis (Varley et al. transcription processes are at present unclear. An 1980; Gall et al. 1983) probably applies to all lamp- unequivocal demonstration of the location and pattern brush loops. The very large size of lampbrush TUs of transcription of any well-defined low-copy-number suggests that they must include transcripts of inter- 'structural' gene would be welcome. The question of spersed repetitive elements of the genome. Structural co-selection of initiation sites on sister loops, especially genes in large genomes are more widely spaced, inter- with regard to multigene families, is challenging, and is spersed with non-coding repetitive DNA, than in small related to the important questions of whether sister genomes. One might therefore expect LBCs from large loops, arising from the same chromomere, are indeed genomes to have longer loops (TUs) than those from molecularly identical, and if so, how this remarkable small genomes, and this is precisely what has been feat is accomplished? observed (see Callan, 1986; Macgregor, 1980). With regard to the proteins of LBCs, there is On the molecular front, with the main questions exciting progress, mainly through the application regarding RNA synthesis resolved, there remain the of monoclonal antibody technology. Antibodies have Journal of Cell Science 88, 7-9 (1987) Printed in Great Britain © The Company of Biologists Limited 1987 chromosomes, but present in the cytoplasm surround- ing the chromosomes (Roth & Gall, 1987). Callan (1986) remarks that the use of monoclonal antibodies for investigation of the cytogenetics of LBCs and their loops is potentially "on a par with in-situ hybridiz- ation". We shall see! In-situ hybridization has taken us a long way in a short time, the answers it has given have been highly and immediately significant, and there is surely more to come. In this connection I wish to offer a comment on the objects known as 'spheres' on LBCs (see Callan & Lloyd, 1960; Callan, 1986). In his book, Callan remarks that "intensive study of the relationship be- tween spheres and histone transcripts could be reward- ing". His view is based on the close proximity in several amphibia of the spheres, whose antigenic specificity has been established, and the loops that transcribe histone mRNA. The inference is that the spheres and histone loops are in some way functionally linked. This may be so, and there is no doubt that the spheres should continue to be investigated vigorously; but it should be borne in mind that urodele karyotypes are conserva- tive, and the fact that two 'genes' have remained next to one another for more than 50 million years does not necessarily mean that they are functionally related. On the cytological front, there has been one major breakthrough in the past year. Callan et al. (1987) have shown that it is possible to obtain excellent prep- arations of LBCs from Xenopus laevis and this paper gives a detailed technical protocol and an excellent LBC map. With the considerable arsenal of molecular probes from A', laevis and a wide knowledge of the genetics and molecular biology of this species, the way is now open to combine cytological and molecular approaches on a scale that hitherto seemed impossible. Fig. 1. Phase-contrast micrograph of a long lampbrush loop that consists of three tandemly arranged TUs each Persistent looking at LBCs continues to confirm with the same direction of polarization. The arrows existing ideas and open up new questions. The giant numbered 2 and 3 indicate the starts of the second and loops on chromosome 2 of A', vtridescens, reported by third TUs. X 1000. (Reproduced with the kind permission Gould et al. (1976) as uniquely unbreakable by the of Van Nostrand Reinhold Company, New York, from: restriction enzyme Haelll, have been shown to be Macgregor, H. C. (1986). The lampbrush chromosomes of breakable with this enzyme, but only on loops that animal oocytes. In Chromosome Structure and Function consist of tandem TUs and only at points where there is (ed. M. S. Risley), pp. 152—186. New York: Van Nostrand an abrupt change from the end of one TU to the start Reinhold Company.) of another: good confirmation of the principle behind the read-through hypothesis (Macgregor & Fairchild, unpublished observation). Scanning and transmission been prepared that bind specifically to chromomeres electron microscopy have confirmed the doubleness of (histones), hnRNP (heterogeneous nuclear RNP) core the interchrom6meric fibril (Bakken & Graves, 1975), proteins on loops, particular loops or sets of loops, and shown that all loop matrices are composed of standard morphologically distinct regions of certain loops. Most 30 nm RNP particles (N'Da et al. 1986) and helped to of the loop-specific antibodies are almost certainly explain the specific morphologies of the matrices of binding to proteins that are associated with nascent certain loops and other parts of the chromosomes RNA transcripts, and some of them are present in the (Bonnanfant-JaiseJa/. 1985; Macgregor, 1986). One of nuclei of somatic cells and oocytes (Sommerville et our own (unpublished) observations is that the inci- al. 1978; Lacroix et al. 1985; Roth & Gall, 1987). dence of sister loops that are of distinctly unequal Predictably, such proteins are absent from metaphase length is much higher than might be supposed from a

8 //. V. Macgregor reading of the early descriptive literature. There is no identification, and distribution of 5S DNA sequences. easy answer to this point, and the observations are Chwmosoma (in press). suggestive of real molecular differences between sister EPSTEIN, L. M. & GALL, J. G. (1987). Self-cleaving chromatids or discordant transcriptional activity on transcripts of satellite DNA from the newt. Cell 45, sister loops. Accordingly, I believe it may now be 535-543. misleading to say that sister loops are 'identical', and EPSTEIN, L. M., MAHON, K. A. & GALL, J. G. (1986). the significance of non-identity needs attention. Transcription of a satellite DNA in the newt. J. Cell Biol. 103, 1137-1144. Finally, let us not be so intensely occupied with the GALL, J. G., DIAZ, M. O., STEPHENSON, E. C. & MAHON, functioning of LBCs that we forget that they are K. A. (1983). The transcription unit of lampbrush meiotic chromosomes, and their organization may chromosomes. In Gene Stnicture and Regulation in include features that are of long-term adaptive signifi- Development, pp. 137-146. New York: Alan R. Liss. cance in relation to evolution and speciation. LBCs are GOULD, D. C, CALLAN, H. G. & THOMAS, C. A. (1976). unique in so far as they allow us to look at half bivalents The actions of restriction endonucleases on lampbrush and at sister chromatids, and to examine DNA se- chromosomes, jf. Cell Sci. 21, 303-313. quence organization in a germ cell that has undergone LACROIX, J. C, AZZOUZ, R., BOUCHER, D., ABBADIE, C, many rounds of mitosis with ample opportunities for PYNE, C. K. & CHARLEMAGNE, J. (1985). Monoclonal sister chromatid exchanges, followed by all the events antibodies to lampbrush chromosome antigens of of early meiotic prophase. After all, oocytes are cells Pleurodeles ivaltlii. Chwmosoma 92, 69-80. that work for evolution, and in their differentiation MACGREGOR, H. C. (1980). Recent developments in the their chromosomes are subject to some of the main study of lampbrush chromosomes. Heredity 44, 3-35. molecular driving forces of that process. MACGREGOR, H. C. (1986). The lampbrush chromosomes of animal oocytes. In Chromosome Structure and Function (ed. M. S. Risley), pp. 152-186. New York: References Van Rostrand Reinhold. N'DA, E., BONNANFANT-JAIS, M. L., PENRAD-MOBAVED, BAKKEN, A. H. & GRAVES, B. (1975). Visualization of the M. & ANGELIER, N. (1986). Size uniformity of tertiary structure of lampbrush chromosomes with nbonucleoprotein matrix particles in loops of Pleurodeles scanning electron microscopy. J. Cell Biol. 67, 17a. zvaltlu lampbrush chromosomes visualised by electron BONNANFANT-JAIS, M. L., N'DA, E., PENRAD-MODAVED, M. & ANGELIER, N. (1985). Amphibian lampbrush microscopy, j'. Cell Sci. 81, 17—27. chromosome loops: correlative light microscopy, ROTH, M. B. & GALL, J. G. (1987). Monoclonal antibodies transmission electron microscopy and scanning electron that recognize transcription unit proteins on newt microscopy observations. Scanning Electron Microscopv lampbrush chromosomes. J. Cell Biol. (In press.) 2, 889-896. SOMMERVILLE, J. (1978). Immunofluorescent localisation of CALLAN, H. G. (1986). Lampbrush Chromosomes. Berlin, transcriptional activity on lampbrush chromosomes. Heidelberg, New York, Tokyo: Springer-Verlag. Chwmosoma 66, 99-114. CALLAN, H. G. & LLOYD, L. (1960). Lampbrush VARLEY, J. M., MACGREGOR, H. C, NARDI, I., ANDREWS, chromosomes of crested newts Triturus cristatus C. & ERBA, H. P. (1980). Cytological evidence of (Laurcnti). Phil. Trans. R. Soc. bond. B 243, 135-219. transcription of highly repeated DNA sequences during CALLAN, H. G., GALL, J. G. & BERG, C. A. (1987). The the lampbrush stage in Tnturus cristatus canufex. lampbrush chromosomes of Xeuopus laevis: preparation, Chwmosoma 80, 289-307.

Lampbrush clnvinosonies