* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download localization of histone gene transcripts in newt lampbrush
Transposable element wikipedia , lookup
Gene expression programming wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Molecular cloning wikipedia , lookup
History of RNA biology wikipedia , lookup
DNA vaccination wikipedia , lookup
Metagenomics wikipedia , lookup
Skewed X-inactivation wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Genome (book) wikipedia , lookup
Long non-coding RNA wikipedia , lookup
Non-coding RNA wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
History of genetic engineering wikipedia , lookup
Epigenetics wikipedia , lookup
Genomic library wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Epigenetics in stem-cell differentiation wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Designer baby wikipedia , lookup
Human genome wikipedia , lookup
DNA supercoil wikipedia , lookup
Microsatellite wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Point mutation wikipedia , lookup
Microevolution wikipedia , lookup
Helitron (biology) wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Epigenomics wikipedia , lookup
Y chromosome wikipedia , lookup
Non-coding DNA wikipedia , lookup
Histone acetyltransferase wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Primary transcript wikipedia , lookup
J. Cell Set. 27, 57-79 (1977) Printed in Great Britain © Company of Biologists Limited LOCALIZATION OF HISTONE GENE TRANSCRIPTS IN NEWT LAMPBRUSH CHROMOSOMES BY IN SITU HYBRIDIZATION R. W. OLD Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow, G61 iBD, Scotland, H. G. CALLAN Department of Zoology, University of St. Andrews, Fife, KY16 gTS, Scotland, ANDK. W. GROSS*, MRC Mammalian Genome Unit, Kings Buildings, Mayfield Road, Edinburgh, EH() 3JR, Scotland SUMMARY Denatured sH-labelled DNAs containing histone gene sequences originating from the echinoderms Psammechinus miliaris, Echinus esculentus and Strongylocentrotus purpuratus have been in situ hybridized to RNA transcripts on newt lampbrush chromosomes. Autoradiographs of the hybridized lampbrush preparations show labelling restricted to four or fewer lateral loop pairs all lying within the heteromorphic regions of chromosome I, also one or two loop pairs on chromosome VI, one loop pair on chromosome X and one loop pair on chromosome XI. For oocytes from a single newt, coincident label distribution is found with DNAs of diverse echinoderm origin; however different newts show some specific individual diversity in label distribution, including heterozygosity in the case of loops on bivalents VI and X. The more conspicuously labelled loops, particularly those on chromosome I, show a pattern of labelling which is explicable if the newt histone DNA sequences are confined to short intercepts of lateral loop axis. Transcription is initiated prior to the histone DNA sequences, proceeds through the histone DNA sequences, and beyond, and the histone RNA sequences are cut from the transcripts before the termination of transcription. INTRODUCTION Successful application of the cytological in situ hybridization technique devised by Gall & Pardue (1969) and John, Birnstiel & Jones (1969) depends, inter alia, on the close proximity of many identical nucleic acid sequences in the cytological preparation. This is occasioned both by the inefficiency of molecular hybrid formation in cytological preparations, and by the inefficiency of autoradiography to record radioactive disintegrations from those hybrids which have been produced (see Gall & Pardue, 1971). Thus to date those in situ hybridizations which have allowed the identification of particular nucleic acid sequences in cytological preparations have all concerned reiterated sequences. Two exceptional biological situations have particularly favoured success with the in situ hybridization technique. One of these is the amplification of ribosomal DNA • Present address: Department of Molecular Biology, Roswell Park Memorial Institute, Buffalo, 14263, New York, U.S.A. 58 R. W. Old, H. G. Callan and K. W. Gross sequences during oogenesis in Amphibia and certain other animals. The other favourable situation is provided by polytene chromosomes of larval Diptera, where many identical DNA sequences lie in lateral register with one another. There is yet another favourable situation which has hitherto been little explored. It is provided by the lampbrush chromosomes of Amphibia, where many RNA transcripts of DNA sequences on the lateral loops may lie as closely packed together as the size of RNA polymerase molecules permits (Miller & Beatty, 1969; Angelier & Lacroix, 1975; Scheer, Franke, Trendelenburg & Spring, 1976). The retention of RNA transcripts on the lateral loops produces a situation akin to amplification, in that identical sequences are concentrated in space. Pukkila (1975) has taken advantage of this situation and by hybridizing 126I-labelled 5s DNA to non-denatured lampbrush chromosomes of the newt Notophthalmus viridescens has identified those loops engaged in the transcription of 5s ribosomal RNA. In some preliminary experiments we succeeded in in situ hybridizing 126 I- and H-labelled DNA complementary to total newt ovary polyadenylated messenger RNA with lampbrush chromosomes of the newt Triturus cristatus carnifex. The first attempt resulted in the regular labelling of a few pairs of loops on bivalent X and one pair of loops on bivalent I; the second attempt resulted in a far more complex distribution of label over the chromosome complement, altogether too complex to justify a detailed analysis. The first attempt indicated the potentialities of the in situ hybridization technique, i.e. its specificity, while the second attempt demonstrated that successful identification of particular RNA sequences transcribed on lampbrush chromosomes' lateral loops depends on sequence purity of the labelled probe. The recent availability of short DNA sequences cloned in plasmid or bacteriophage vectors has made possible the production, by nick-translation, of labelled probes of great sequence purity. Such probes, after denaturation, are ideally suited for hybridizing to RNA transcripts on lampbrush chromosomes, and in this paper we describe what happens when echinoderm histone DNA sequences are applied in this manner. Histone coding sequences were chosen because the polypeptide sequences of histones have been stringently conserved in the course of evolution. It has recently been shown by in situ hybridization that echinoderm 9s RNA can be used to identify a small region of Drosophila melanogaster salivary gland chromosomes corresponding to histone DNA sequences in this organism (Birnstiel, Weinberg & Pardue, 1973; Pardue, 1975). On these grounds there was hope that corresponding echinoderm DNA sequences would show a similar hybridization specificity with respect to newt oocyte RNA transcripts. Labelled RNA in solution when hybridized to DNA in a cytological preparation will, if successful, establish the location of a particular sequence in a genome. The same is true of labelled DNA in solution hybridized to DNA in a cytological preparation. However, when labelled DNA in solution is successfully hybridized to RNA transcripts in a cytological preparation, this will establish the location of transcripts, not DNA sequences. One can generally identify the direction of transcription in a lateral loop of a lampbrush chromosome from the way in which loop matrix RNP is distributed, the thin loop insertion being the region of initiation (there may be 3 Localization of histone gene chromosomes 59 more initiation sites in the case of loops with more than one matrix unit). This being so, those silver grains nearest to the thin insertion of a lateral loop in a labelled DNA to RNA in situ hybrid autoradiograph will mark the beginning of the relevant sequence in loop axis DNA, but the end of the relevant sequence will not coincide with the end of the labelled region unless transcription also terminates at this point. As will become apparent, the latter condition manifestly does not apply to the 'histone' loops described in this paper. From the manner in which radioactivity is distributed on the histone loops we can infer that the regions containing histone sequences are considerably shorter than the lengths of the labelled regions. We will return to this matter in the discussion. MATERIALS AND METHODS Preparation of radioactive DNAs Radioactive DNAs containing histone gene sequences were prepared by nick-translation of purified bacteriophage or plasmid DNAs into which echinoderm histone sequences had been inserted by in vitro recombination techniques. Control experiments were performed with nick-translated DNA derived from wild-type bacteriophage lambda. Nick-translation reactions were performed essentially as described by Macgregor & Mizuno (1976). Tritiumlabelled TTP (The Radiochemical Centre, Amersham) was the radioactive precursor. After completion of each nick-translation reaction the solution was extracted with phenol and chromatographed on a column of Sephadex G50. The excluded peak of radioactive DNA was recovered by ethanol precipitation in the presence of Escherichia coli tRNA (sufficient to give afinalcarrier RNA concentration of about 2 mg/ml in the in situ hybridization reaction). The DNA preparations that were nick-translated and the designations given them in this paper were as follows. 1. The complete 6 kilobase pair repeat unit of the histone gene cluster of Psammechinus miliaris cloned in a bacteriophage lambda vector (Clarkson, Smith, Schaffner, Gross & Birnstiel, 1976). Specific radioactivity of the nick-translated product, 2 x io9 cpm//*g. 2. The complete 6-5 kilobase pair repeat unit of the histone gene cluster of Echinus esculentus cloned in a bacteriophage lambda vector (Southern & Murray, unpublished). Specific radioactivity of the nick-translated product, 14 x 10* cpm//*g. 3. A 2 kilobase pair fragment of the histone gene cluster of Strongylocentrotus purpuratus containing the coding regions for histones H2a and H3, cloned in the plasmid pSCioi. This recombinant is the plasmid pSpi7 of Kedes and co-workers (Kedes, Chang, Houseman & Cohen, 1975; Cohn, Lowry & Kedes, 1976). Specific radioactivity of the nick-translated product, 3x10' cpm//ig. 4. A 45 kilobase pair fragment of the histone gene cluster of S. purpuratus containing the coding regions for histones H2b, H4 and Hi cloned in the plasmid pSCioi. This recombinant is the plasmid pSp2 of Kedes and co-workers (Kedes et al. 1975; Cohn et al. 1976). Specific radioactivity of the nick-translated product, 3 x 10' cpm//tg. The specific radioactivity of the nick-translated DNA of wild-type bacteriophage lambda was 3 5 x 1 0 ' cpm//ig. Preparation of lampbrush chromosomes The newts used for the experiments described in this paper were 4 females of Triturus cristatus carnifex purchased from T. Gerrard, Ltd, Beam Brook, Newdigate, Surrey, England. A newt is anaesthetized in 01 % MS222 (Sandoz Ltd, Basle, Switzerland), its ovaries removed, and placed in a sealed container on crushed ice. Ovaries are used fresh, and never stored for more than 6 h. Oocyte nuclei are isolated and their membranes removed using the methods described by Callan & Lloyd (i960). A nucleus is isolated and cleaned in an unbuffered 3: 1 mixture of 60 R. W. Old, H. G. Callan and K. W. Gross o-i M KC1 and 0 1 M NaCl, and transferred by pipette to a modified form of observation chamber (ring cell) containing the 3:1 saline, with in addition 1 x io~ 4 M CaCl s , for removal of the nuclear membrane and dispersal of the chromosomes. Each ring cell is a 24-mm diameter disk of i-mm-thick glass, with a 7-mm diameter central hole. A 22-mm diameter No. 2 coverslip is sealed on one side of the ring cell with 45 °C m.p. paraffin wax, and this forms the base of the chamber. As soon as the nuclear membrane has been removed, the top of the ring cell is covered with an 18-mm diameter coverslip. Preparations made in this manner are each placed in rectangular carriers made from Perspex, the carrier placed in a 90-mm diameter Petri dish covered to restrain evaporation from the saline, and left undisturbed for about 1 h to allow the nuclear sap to disperse and the chromosomes to settle on the floor of the ring cell. Before proceeding further, the rectangular Perspex carrier is placed on the stage of a Zeiss inverted (Plankton) microscope, and the preparation inspected to make sure that complete dispersal of nuclear sap has occurred. The lampbrush chromosomes must be lying flat in one plane on the floor of the ring cell. When sap dispersal is judged to be complete the ring cell is removed from its carrier, excess saline blotted away with filter paper, and the ring cell placed on the flat surface of an Araldite plug made to fit loosely inside a 50-ml polypropylene centrifuge tube of internal diameter 25 mm. The flat surface of the Araldite plug when sitting in the centrifuge tube lies 65 mm from the tube lip. Four such ring cells are placed in 4 plugged centrifuge tubes, the tubes mounted in an HB-4 swinging bucket rotor, and spun for 5 min at 5000 rev/min in a Sorvall superspeed centrifuge. The ring cells when spinning come to lie 10 cm from the centrifuge axis, so the force they experience is about 2800 g. Each ring cell is now removed from the centrifuge tube and submerged in a dish containing 3:1 saline, where its top coverslip is dislodged. The bottom coverslip is now carefully detached from the ring disk by inserting a thin (double edge) razor blade into the wax layer between the 2 adhering surfaces, meantime rotating the ring cell, and the detached coverslip is then placed in a porcelain rack standing in 3:1 saline. When 4 such coverslips have been assembled in a rack, the rack is taken up an ethanol series (30, 50, 70, 90, 95 % ) , 2 changes of absolute ethanol, 4 changes of xylene (long enough to dissolve all traces of wax), 2 changes of acetone, and air dried. The back of each air-dried coverslip, on which the chromosome group can be seen by naked eye, is now cemented to a standard microscope slide using a minimal amount of low viscosity epoxy resin (Spurr, 1969), and baked hard on a hotplate. Once the plastic cement has set (2 to 3 days) the preparations are removed from the hotplate and stored in dust-free conditions. Ideal preparations have the bivalent chromosomes well separated from one another, all their lateral loops should be anchored down on the coverglass surface, and they should be reasonably free from nucleoli. These considerations determine the ideal oocyte size with which to work, as large as possible but below the size where, when the nuclear membrane is removed, it leaves the nucleoli embedded in nuclear sap along with the chromosomes. Below a certain critical size most of the nucleoli remain attached to the nuclear membrane when the membrane is removed; however, if the oocytes are too small the bivalent chromosomes do not separate well. The critical size varies somewhat from one newt to another. For the most part we have worked with oocytes of 0-85 mm diameter. Some control preparations were digested with RNase-A (Sigma), 0 1 mg/ml, in 001 M sodium phosphate buffer, pH 6 0 at 40 °C for 1 h. After digestion the preparations were washed in running tapwater, rinsed in distilled water, taken up an ethanol series, then through 2 changes of acetone, and air dried. Other control preparations were digested with DNase-i(Sigma), o-oi mg/ml, in o-oi M sodium phosphate buffer, pH 7-0 with o-ooi M MgCl,, at 40 °C for 1 h. Thereafter these controls were also washed, dehydrated and air dried from acetone. Hybridization Conditions for hybridization were much like those described by Pukkila (1975). Each preparation of nick-translated ' H - D N A lying in a small capped polypropylene tube is dissolved in 45 fi\ of water, and the tube held in boiling water for 5 min for denaturation. The tube is Localization of histone gene chromosomes 61 then removed, and immediately plunged into a container of crushed ice. Fifteen microlitres of 20 x standard sodium citrate (SSC) pH 70 are added, also 40 fi\ formamide, and the fluids mixed, thus placing the DNA in 3 x SSC, 40 % formamide. Ten microlitres (equivalent to from io6 to 2-5 x 10s cpm) of the above solution of DNA are now pipetted on to a preparation of lampbrush chromosomes, and covered with an acidwashed 13-mm square coverslip. Three such covered preparations are placed in a square plastic dish, propped up on 2 L-shaped glass rods, with a pad of absorbent paper below liberally wetted with 20 x SSC, and the dish covered. The preparations are incubated overnight in a waterbath at 40 °C. In our early in situ hybridizations the pad in each plastic dish was moistened with 2 x SSC as recommended by Gall & Pardue (1971), but we found that in the course of incubation water vapour distils from the pad and condenses, diluting thefluidin contact with the chromosomes under the coverslip. Moistening the pad instead with 20 x SSC, as recommended by Pukkila (197s) overcomes this difficulty. After overnight incubation the coverslip is flushed off each slide with a jet of 2 x SSC from a wash bottle, the slide then taken through 4 changes of 2 x SSC, up an ethanol series, 2 changes of absolute ethanol, 2 changes of acetone, and air dried. Autoradiography, staining and mounting Dilute 'subbing' solution (o-i% gelatin and 0 0 1 % chrome alum in water, Gall & Pardue, 1971) is now carefully painted around, but not over, the preparation of hybridized lampbrush chromosomes, and the subbed preparations dried in a rack over a hotplate. The slides are now taken to a dark room and dipped in Kodak NTB-2 emulsion diluted one part to one part distilled water, dried, and allowed to expose in light-tight boxes at o °C. Slides hybridized with nick-translated preparations 1 and 2 were exposed for about 12 weeks (in the case of preparation 1 this exposure was manifestly excessive) while those hybridized with preparations 3 and 4 were exposed for about 4 weeks. After exposure the slides are developed in Kodak D19 for 5 min at 20 °C, rinsed in water, fixed in Kodak Unifix, thoroughly washed in running tap water and then in distilled water. The slides are now placed in a Coplin jar containing 50 ml of o-oi M sodium phosphate, pH 7, 2 ml of Giemsa stock staining solution (B.D.H.) are added, and the fluids mixed by pipetting in the jar. After staining for 2 h the Giemsa stain is flushed out with a jet of tap water, the slides then briefly rinsed in distilled water, and air dried. This unusual staining procedure was recommended by Gall & Pardue (1971) so as to avoid the preparations passing through, and being contaminated by, the surface scum which quickly forms on top of a Giemsa staining solution. When the stained slide is thoroughly dry the preparation is mounted with a minimal quantity of Canada balsam, using a round coverslip of slightly smaller diameter (18 mm) than that to which the original lampbrush chromosome preparation was attached. RESULTS In well stained preparations examined in white light the majority of the lateral loops appear pale mauve, some of the larger landmark loops bright blue (notably the lumpy structures on chromosome II and the giant fusing loops on chromosomes X, XI and XII), and the chromomeres dense purple. In preparations treated with DNase the chromomeres are less intensely stained than usual, while after digestion with RNase the loops can scarcely be differentiated from the emulsion. Preparations were examined and photographed using a Zeiss Planapo x 63 oilimmersion objective of N.A. 1-4 with a Zeiss broad band green interference filter and Baltzer 'anti-Feulgen' filter in the light path. Photographs were taken on Ilford pan F film. Bivalent chromosomes were drawn with the aid of a camera lucida at a magnification 5 CEL 27 62 R. W. Old, H. G. Callan and K. W. Gross CO IV V 9 9 VI r~P. U Q_ VII VIII . §_ 9 IX . * O no CO gt • g XI i 3i'?. JT XII Fig. i. Working map of the 12 lampbrush chromosomes of Triturus cristatus carnifex (I to XII) showing the major 'landmarks' which serve for chromosome recognition, and giving the locations of loops which hybridize with echinoderm ' histone DNA'. The vertically aligned arrows indicate the positions of the centromeres, with longer chromosome arms directed to the left and shorter to the right. Objects which are bracketed together are frequently fused to one another. The limits of the heteromorphic region of chromosome I are indicated by opposing open arrowheads pointing horizontally. The locations of major histone loops are indicated by large solid arrowheads pointing vertically, those of minor histone loops by small solid arrowheads. The figures associated with solid arrowheads refer to the unit distance from the left end of the chromosome, chromosome V being arbitrarily assigned 100 units total length. The solid arrowheads pointing downwards on chromosome I refer to histone loop locations on the heteromorphic region of that chromosome whose landmark loops are shown on the map. The solid arrowheads pointing upwards refer to the histone loop locations on the partner chromosome, whose landmark loops are not shown, n.o., nucleolar organizer. Localization of histone gene chromosomes 63 of x 1000, and their lengths, and the positions of features of interest, measured by an opisometer to the nearest millimetre. The relative lengths of the 12 chromosomes of T.c. carnifex, and the positions of various 'landmark' structures which assist in chromosome identification, were originally published by Callan & Lloyd in i960. An updated version of the T.c. carnifex 'working map', with additional data derived from the present study, and notably the locations of those loops to which our 3 Hlabelled DNA preparations hybridize, is shown in Fig. 1. In Fig. 1 the positions of labelled loops are given in 'units' measured from the end of the left (longer) chromosome arm. The unit used here was arbitrarily defined by Callan & Lloyd (i960) as one hundredth part of the length of chromosome V. Amongst the chromosomes which concern us in the present work, the longer arm of chromosome I (telomere to centromere) measures 86 units, that of chromosome VI 65 units, while the overall length of chromosome X is 64 units. Of particular concern in the present study are the left-arm pairs of bivalent I. These include a long 'heteromorphic' region extending from 9 to 72 units, within which chiasmata are never formed, and where landmark loops do not show correspondence between the partner arms. It is likely, though not proven, that the heteromorphic regions are concerned with sex-determination in T.c. carnifex, the female being the heterogametic sex. Lacroix (1970) has found a similar heteromorphic region in bivalent IV of Plewodeles poireti and has demonstrated by sex-reversal experiments that this region is indeed concerned with sex determination. In T.c. carnifex there is furthermore a great deal of morphological variation between individuals regarding the heteromorphic regions of bivalent I. The landmarks shown on the working map depict those found on that chromosome I which shows less individual variation than does its partner. Further allusion to this subject will be made later in this paper. The sites of hybridization Successful in situ hybridization was achieved with preparations i, 2 and 4, but not with preparation 3; this latter left the lampbrush chromosomes unlabelled, and apart from postulating a lack of homology between it and the Triturus sequences, we are unable to explain why. The most striking sites of radioactivity are loops on the heteromorphic arms of bivalent I (Fig. 2). It is convenient to begin by considering $$ 9 and 10, whose chromosomes were hybridized with preparation 4. Nine drawn examples of bivalent I from <> j 9, and 6 from $ 10, gave a precisely coincident labelling pattern and, moreover, carried an independent marker loop pair on one chromosome which further served to distinguish it from its partner. These marker loops, which are unlabelled, are large and contorted, stain bright blue rather than mauve, and lie at 24 units. They are evident (bl) in Fig. 2. On that arm of chromosome I which does not carry the ' blue' marker loops, the most prominent labelled loops lie at 24, 29 and 43 units, and we have therefore designated them 1^, I M and L^ respectively. We will use this form of nomenclature throughout. These loops proved to correspond to 3 of the 5 landmark loops shown 5-3 R. W. OU, H. G. Callan and K. W. Gross Localization of histone gene chromosomes 65 on that chromosome I drawn in the working map (Fig. 1), and they are indicated on the map by large, downward pointing arrowheads. I24 is regularly much longer than I29 and 143, and demonstrates particularly clearly that the labelled portion does not extend throughout the loop's length (Figs. 4 and 5). Closer to the centromere, though still within the heteromorphic region of this chromosome, there is a more lightly labelled loop 1^; Fig. 6 shows that this loop too includes a long unlabelled portion. On that arm of chromosome I which does carry the 'blue' marker loops the most prominent labelled loops are 1^, I87 and I M ; they are indicated on the working map (Fig. 1) by large, upward-pointing arrowheads. I M is shown in Figs. 3 and 7; 1^ and I37 are both shown in Fig. 8; in each case it will be noticed that these loops include unlabelled portions. Nearer to the telomere of this chromosome, but again still within its heteromorphic region, there is regularly a sparse patch of silver grains at I13. In none of our preparations is there a clear image of lateral loops associated with this label, but in some preparations the silver grain tracks are sufficiently suggestive of loops (Fig. 9) that we conclude the grains must be associated with lateral loops unusual in that they carry exceedingly little RNP matrix. Bivalent I in $ 7, whether hybridized with preparation 1 or 2, showed great similarity to bivalents I of $$ 9 and 10 hybridized with preparation 4. One arm of chromosome I is virtually identical, with labelled loops 1^, I M , 1^ and L^, 1^ being regularly much longer than the rest and including a long unlabelled portion (Fig. 10). The partner chromosome (Fig. 11) shows the sparsely labelled I13, 1^ and I M are present, but I37 is inconspicuous or absent. Label distribution on bivalent I of $ 8 proved more difficult to homologize with that of $$ 7, 9 and 10, in part because the cytological preparations were of poorer quality and in part because those hybridized with preparation 1 had been overexposed. 1^ and I29 were eventually identified, the former being immensely heavily labelled, the latter remarkable in that the labelled portion is regularly short and far displaced from the chromosome axis, i.e. with long unlabelled portions of loop on both sides of the labelled portion (Figs. 12 and 13). 1^ and 1^ are inconspicuous or absent. On the partner chromosome I of $ 8 1^ is present, sparsely labelled, I M is present, nearly as heavily labelled as 1^ in the partner chromosome, I37 is absent and 1^ present. The sites of in situ hybridization on bivalents I in all 4 newts are summarized in Table 1, which also includes some information about labelled loops on other chromosomes. $ 7 regularly has an intensely labelled loop pair on one chromosome VI, All plates are photographs of autoradiographs of lampbrush chromosomes of Triturus cristatus carnifex following in situ hybridization. Fig. 2. Part of bivalent I of $ 9 hybridized with preparation 4. All 8 labelled loop pairs in the heteromorphic regions of the long arms are evident, bl, 'blue' loops. Arrows indicate the centromeres (c). The 5 round dark objects are nuclebli. Exposure 26 days. Fig. 3. Part of chromosome I of $ 9 hybridized with preparation 4. The labelled loop pair is I H . Arrows indicate the direction of transcription. Exposure 26 days. 66 R. W. Old, H. G. Callan and K. W. Gross 20/mi 6 Localization of histone gene chromosomes 67 about two thirds the way down its longer arm, at VI44, but with no such labelled locus on the partner chromosome (Figs. 14, 16 and 17). This heterozygosity is equally apparent whether the chromosomes be hybridized with preparations 1 or 2. $ 8 has no labelled locus on bivalent VI, but is regularly heterozygous for a labelled loop pair on bivalent X, just to the right of the centromere, at X^ (Figs. 15, 18 and 19). $ 8 is furthermore homozygous for labelled loop pairs on bivalent XI at XI 7 , close to the telomeres of the left arms (Figs. 20, 21). $ 9 has strongly labelled loops at 2 loci on chromosome VI, at VI40 and VI44. The partner chromosome shows traces of labelling at the homologous loci; these would doubtless have been overlooked had the well labelled loops on the other chromosome not been so evident. $ 9 is also heterozygous for labelled loops at X^, just like $ 8. 9 10 is heterozygous for labelling at VI44, just like $ 7, but has no labelled loci on bivalents X or XI or elsewhere. Control lampbrush chromosomes hybridized with 3H-labelled nick-translated lambda DNA, similar to the DNA which carried the sea-urchin histone DNAs used in preparations 1 and 2, were uniformly unlabelled. Lampbrush chromosomes treated with RNase and hybridized with preparations 1, 2 and 4 were also uniformly unlabelled. Lampbrush chromosomes treated with DNase and hybridized with preparations 1, 2 and 4 were as well labelled as untreated chromosomes, and indeed some of them were used, along with untreated chromosomes, for establishing the locations of labelled loops. DISCUSSION In all probability the RNA sequences on the lateral loops of the lampbrush chromosomes to which the applied DNA sequences bind are histone messenger sequences rather than spacer sequences. While histone sequences are generally considered to be stringently conserved in evolution, variable levels of synonymous codon substitution in the histone coding sequences of related organisms has previously been observed (Grunstein, Birnstiel & Schaffner, unpublished). In combination with sequence divergence attributable to small numbers of primary amino-acid sequence changes, third base substitutions could have the effect of lowering the efficiency of hybridization in the heterologous reactions performed here. Previous studies with mRNA, however, have indicated that sufficient homology is generally retained to permit detection of at least a portion of the corresponding sequences in cross-species experiments (Kedes & Birnstiel, 1971). The histone coding sequences are almost certainly more conserved Fig. 4. Part of chromosome I of ? 9 hybridized with preparation 4. The larger labelled loop pair is IM and the smaller I^. Arrows beside I t4 indicate the direction of transcription. Exposure 26 days. Fig. 5. Part of chromosome I of $ 9 hybridized with preparation 4. The larger labelled loop pair is IM, and one of the pair has suffered breakage. Arrows indicate the direction of transcription. The smaller loop pair is IM. Exposure 26 days. Fig. 6. Part of chromosome I of $ 9 hybridized with preparation 4. The labelled loop pair is IM. Exposure 26 days. 68 R. W. Old, H. G. Callan and K. W, Gross Localization of histone gene chromosomes 69 than the non-coding 'spacer' sequences which make up the remainder of the seaurchin histone repeat unit; this at least is true of rDNA and spacer sequences in Xenopus (Forsheit, Davidson & Brown, 1974). Here we show that 3 DNA preparations, derived from Psammechinus miliaris, Echinus esculentus and Strongylocentrotus purpuratus, bind to coincident loci. The high degree of specificity at the sites of labelling is remarkable. A few loops are labelled in any single preparation, but several thousand are not, and absolutely not. This evidently reflects the sequence purity of the radioactive probe DNA, and provides an assurance that the conditions of hybridization do not allow mismatched hybrid sequences to persist. We have noted that many of the labelled loops include at least one, and often two, unlabelled portions. Whenever a long labelled loop happens to lie conveniently displayed, 2 unlabelled portions are regularly present; one, generally the shorter, extends from the thin loop insertion in the chromosome axis and runs out, with increasing quantity of loop matrix, until the labelled region is reached. It is a feature of the labelled region that silver grain density rises steeply, there follows a plateau where grain density is uniform, after that a region where the grains are irregularly clustered and of diminished overall density, and finally an unlabelled portion of loop with, however, plenty of RNP matrix, which returns to the chromosome axis at the thick loop insertion. Before evaluating this pattern of labelling we need to bear in mind that if oocytes of T.c. carnifex are incubated for an hour in the presence of ^HJuridine, loops such as these are found to be labelled throughout their lengths, from which one can infer that all of the loop axis is transcribed. Furthermore when such loops are examined unfixed in phase contrast, each shows a uniformly increasing quantity of matrix from the thin to the thick insertion, without evident interruption, so we can reasonably assume that transcription similarly proceeds without interruption, and that the disposition of thin and thick loop insertions establishes its direction. In the in situ hybrid autoradiographs that portion of the loop where grain density rises steeply from zero must mark the beginning of histone sequences in the DNA, and the fact that there follows a plateau region of uniform grain density suggests very strongly that the DNA histone sequences are confined to a short length of loop axis. If they were not so confined, if indeed they extended throughout the length of the labelled region, one would expect to encounter a continuous gradient of grain density rising up to the point where processing, shedding of histone messenger RNA from loop transcripts, occurs. Fig. 7. Part of chromosome I of $ 10 hybridized with preparation 4. The labelled loop pair is IM. An arrow indicates the direction of transcription. Exposure 26 days. Fig. 8. Part of chromosome I of $ 10 hybridized with preparation 4. The labelled loop pair to the left is I37; that to the right is IM, beside which is an arrow indicating the direction of transcription. Exposure 26 days. Fig. 9. Part of chromosome I of $ 10 hybridized with preparation 4. The grain tracks suggestive of a pair of loops mark the location of IIS. Exposure 26 days. R. W. Old, H. G. Callan and K. W. Gross 20//m • •43 Localization ofhistone gene chromosomes 71 A possible alternative explanation, for the plateau of uniform grain density might be found by proposing that histone messenger RNA transcripts are processed as rapidly as they are transcribed, but the relative abundance of loop matrix in the region where labelling ends argues against such a proposal; there should be no more matrix here than there is where transcription begins, i.e. close to the thin loop insertion. This is manifestly not the case. Fig. 22 is a diagram showing our interpretation of the observations described in this paper, and we think that in principle it applies to all the histone loops of T.c. carnifex except I13, which is a special case to which we will return. Transcription begins at an initiation site near the thin loop insertion, but the RNA transcripts do not include histone messenger sequences until they enter the short (a few microns long) histone sequence zone in the loop axis. Having traversed this zone, the transcripts enter a long region containing other sequences, perhaps repetitious (Macgregor, 1977), where each transcript includes the same length of histone message. It is this uniform concentration of histone messages that is responsible for the plateau region of uniform silver grain density in the autoradiographs. Ultimately the histone messages are processed, cut off from the tips of the continuing transcripts. How far transcription has proceeded before processing occurs would appear to be subject to some variation; this could account for the rather ill-defined ending of the labelled regions of the histone loops. Indeed occasional transcripts may not be processed before they reach the terminus at the thick loop insertion; this would explain the small cluster of silver grains close to the main chromosome axis in the example of I M shown in Fig. 3. The majority of transcripts are evidently processed at an earlier stage, some two thirds to three quarters the way round such loops as I24 (Figs. 4 and 5), I34 (Figs. 8 and 11) and I M (Fig. 7), but the transcript length cannot be greatly reduced by processing, for there is abundant RNP matrix where the loop passes from the labelled region and on to the thick insertion, where transcription terminates. The pattern of labelling over the histone loops has another feature that supports the general scheme of Fig. 22. In accord with the overall thin-to-thick polarity of visible loop matrix, there is a decided tendency for silver grains to be progressively distributed further away from loop axis (e.g. Figs. 3 and 10); this is readily explained by the increase in length in an intact primary transcript condensed into RNP. RNA molecules of high molecular weight have indeed been isolated from nuclear RNP particles of T.c. carnifex oocytes and have been shown to contain sequences found in oocyte polyadenylated mRNA (Sommerville & Malcolm, 1976). Assuming that this scheme of organization applies generally to the histone loops of Fig. 10. Part of chromosome I of $ 7 hybridized with preparation 2, showing labelled loop pairs IM, I N , and \a. Arrows beside loops I24 indicate the direction of transcription. Exposure 87 days. Fig. 11. Part of chromosome I of ? 7 hybridized with preparation 2. The labelled loop pair is IM. One of the pair is broken within the labelled region, the other is intact, and an arrow alongside indicates the direction of transcription. Exposure 87 days. R. W. Old, H. G. Callan and K. W. Gross • u- Localization of histone gene chromosomes 73 T.c. carnifex, different loops show different labelling patterns because the proportionate lengths of the various zones differ. The stubby loops VI40, VI^ (Figs. 16 and 17) and X^ (Figs. 18 and 19) have short zones preceding the histone sequences and processing occurs much closer to the thick loop insertion than is shown in Fig. 22. At the other extreme there are the loops 1^ specifically of $ 8 (Figs. 12 and 13), where the zone preceding the histone sequences is long and the zone succeeding processing longer still. Another arrangement is found in the loops XI 7 , where the zone preceding the histone sequences is short, but the zone succeeding processing long (Figs. 20 and 21). Table 1. Distribution of histone sequences in the chromosome complements of 4 female Triturus cristatus carnifex Lateral loOpS . . . L, IJSI I41 I6] + + + + - + + In I 14 137 I34 + + + + - + + + VI4n VI44 X4, XI, -/+/- -/+/- +/+/- +/ + -/- Animal 98 99 The invariance of these labelling patterns from oocyte to oocyte within individuals leads us to conclude that the histone-coding region of each particular loop occurs at a fixed position around the loop axis. Hence we have proof against the proposition that the loop axis spins out from one half-chromomere and is reeled in at the adjoining half-chromomere (Gall & Callan, 1962; Callan, 1967; Snow & Callan, 1969). The interpretation of labelling at I13 (Fig. 2) is problematic. As mentioned earlier, loops cannot be resolved below the silver grains at this locus in autoradiographs, nevertheless the grains appear to be disposed as though they were tracking along a pair of loops in some preparations (Fig. 9). If one studies freshly isolated lampbrush chromosomes of T.c. carnifex in saline in phase-contrast, there are very occasional loop pairs whose thickness lies close to the resolving limit of the light microscope; such loops are noticeable only because they show much more violent Brownian movement than neighbouring loops which carry normal quantities of matrix RNP. Fig. 12. Part of chromosome I of 9 8 hybridized with preparation 1. An arrow at upper left indicates the direction of transcription along labelled loop I N ; the labelled part of its sister lies across the main axis of the chromosome. The lower and intensely labelled object marks the location of I^. The 'loose' patch of silver grains above IJJ, and just to the left of the chromosome axis, may be over a fragment of loop I54 or more likely over a shed portion of this loop's matrix. Exposure 87 days. Fig. 13. Part of chromosome I of 9 8 hybridized with preparation 1. An arrow at upper left indicates the direction of transcription along labelled loop I,9; the labelled part of its sister lies below, alongside the main axis of the chromosome. The intensely labelled object to the right marks the location of Iu. There are 2 other 'loose' patches of silver grains which probably lie over shed portions of matrix of I.4. Exposure 87 days. R. W, Old, H. G. Callan and K. W. Gross . k/e 14 I' . • 100 •;•>... "»• le Fig. 14. Bivalent VI entire of $ 7 hybridized with preparation 1. It shows heterozygosity for the labelled loop pair VI44 (heavy arrow). Arrows indicate the centromeres (c). re, ends of the shorter (right) arms. The telomeres of the longer (left) arms are fused together. Exposure 87 days. Fig. 15. Bivalent X entire of $ 8 hybridized with preparation 1. It shows heterozygosity for the labelled loop pair XH (heavy arrow), re, ends of the shorter (right) arms, le, ends of the longer (left) arms. Exposure 87 days. Figs. 16, 17. Parts of bivalents VI of $ 7 hybridized with preparation 1, showing 2 examples of the labelled loop pair VIM. Exposure 87 days. Figs. 18, 19. Parts of bivalents X of $ 8 hybridized with preparation 1, showing 2 examples of the labelled loop pair Xj,. Exposure 87 days. Figs. 20, 21. The extremities of the longer (left) arms of bivalents XI of $ 8 hybridized with preparation 1, showing 2 examples of homozygosity for the labelled loop pairs XI 7 . le, ends of the left arms. Exposure 87 days. Localisation of histone gene chromosomes 75 76 R. W. Old, H. G. Callan and K. W. Gross It would seem likely that there are loops similarly devoid of matrix at locus I13, and that this accounts for their invisibility in autoradiographs. If so, their histone sequence organization and transcript processing must be exceptional, for although sparsely labelled the labelling, so far as one can judge, appears to be continuous throughout loop length. The presence of label indicates histone sequences in their transcript, the absence of discernible matrix that transcripts are processed and transcription reinitiated at frequent intervals along the loop. If so, then the histone sequences in DNA at I ]3 must be distributed throughout the length of loop axis. I13 is quite unlike \ Fiv. 22. Diagram to illustrate the general organization of a lateral loop transcribing histone sequences. The DNA duplex in the loop axis is drawn as a single line, thicker in the region containing histone sequences (HS). Projecting from the loop axis are RNA transcripts, arbitrarily drawn contracted to one tenth the length of the transcribed DNA; I, indicates the initiation site and T, termination. Thickened portions of the transcripts represent histone messenger RNA sequences, P indicating where processing of the transcripts occurs, liberating histone messenger RNA from the loop matrix. To the left is shown a portion of the non-transcribed main chromosome axis, including a chromomere. the other histone loops, but it may contribute more to the pool of histone messenger sequences than its sparse labelling might suggest; the other histone loops are much more heavily labelled than I13, not necessarily because they are transcribing more histone sequences, but because those sequences after transcription remain for relatively long periods associated with neighbouring transcribed sequences in loop matrix. How long they remain associated cannot be stated with precision. The rate at which full transcription is re-established following the inhibition of RNA synthesis in oocytes of T.c. cristatus by actinomycin D (Snow & Callan, 1969; inhibition causes the stripping off of loop matrix and the collapse of loop axes on to the main chromosome axis) suggests a period of one or two days. We have surmised that in all but the case of I13 the histone coding sequences transcribed in T.c. carnifex lampbrush chromosomes are confined to lengths of only a few microns within loops which may be upwards of 200/<m long. Further evidence for this proposal should now be sought by hybridizing labelled probe Localization of histone gene chromosomes 77 DNA to lampbrush chromosomes from which the RNA has been removed by prior hydrolysis, and the chromosomal DNA denatured. Such an experiment should also reveal whether there are any histone sequences which are not transcribed in lampbrush chromosomes. If there are such sequences we predict that they would lie in chromomeric rather than loop axis DNA. However the all-or-nothing labelling of the homologues in the heterozygous animals leads us to doubt whether transcriptional control is responsible for the observed heterozygous ' expression' of VI40, VI^ and X^. We have given evidence for the presence of a number of histone loci in T.c. carnifex, distributed on several chromosomes. We do not know how many gene copies are present at each locus, or whether the separate loci are identical in the histone sequences they contain. They may correspond to non-allelic variant forms of histones whose existence has been demonstrated in a variety of animals (Franklin & Zweidler, 1977; Ruderman & Gross, 1974). If so the problem of maintaining sequence identity among such widely separated loci does not arise. Within each locus (with the possible exception of I13) transcription occurs unidirectionally, a single strand acting as template. Such is the case in 3 echinoderms studied so far, all 5 histone genes being transcribed in tandem from the same strand (Cohn et al. 1976; Gross, Schaffner, Telford & Birnstiel, 1976). The organization of histone genes in T.c. carnifex differs in 2 respects from that observed in Drosophila melanogaster where the histone genes appear to be located at a single (complex) locus (Pardue, 1975) and where both strands within a gene cluster serve as template (D. Hogness, personal communication). The experiments described here have allowed us to locate the histone gene transcripts in the lampbrush chromosomes of T.c. carnifex. The autoradiographic exposures are not unduly long and the specific radioactivity of the probe DNA, though high, could be increased. The sensitivity of the method may be due, in part, to the potentially duplex character of the radioactive DNA. Thus, one molecule of the probe may hybridize to the chromosomal transcript leaving a single-stranded DNA tail available for hybridization with additional radioactive DNA. We suggest that the method may be sufficiently sensitive for the localization of transcripts arising from unique gene sequences in newt lampbrush chromosomes. Two extensions of the method come readily to mind. First, the use of separated strands of probe DNA would permit investigation of the asymmetry (or othenvise) of transcription within a small chromosomal region. However, as we have demonstrated, it is possible to draw clear conclusions about the asymmetry of transcription of histone genes without recourse to strand separation. Second, in combination with restriction endonuclease analysis of the cloned DNA, radioactive DNA from noncoding regions could be used to study the extent and nature of transcription around a single gene sequence. These further experiments await the production of appropriate, homologous cloned DNA probes. R.W.O., H.G.C., and K. W.G. wish to thank St Leonards College of the University of St Andrews, the Science Research Council and the Helen Hay Whitney Foundation for financial support. We thank Dr L.H. Kedes and Dr E.M. Southern for the provision of bacteria and bacteriophage containing sea-urchin histone DNA sequences. We gratefully acknowledge the expert assistance of Mrs L. Lloyd. 6 CEL 27 78 R. W. Old, H. G. Callan and K. W. Gross REFERENCES N. & LACROLX, J.-C. (1975). Complexes de transcription d'origines nucle'olaire et chromosomique d'ovocytes de Pleurodeles waltlii et P. poireti (Amphibiens, Urodeles). Chromosonia 51, 323~335BIRNSTEEL, M. L., WEINBERG, E. S. &PAHDUE, M. L. (1973). Evolution of 9SmRNA sequences. In Molecular Cytogenetics (ed. B. A. Hamkalo & J. Papaconstantinou), pp. 75-93. New York & London: Plenum Press. CALLAN, H. G. (1967). The organization of genetic units in chromosomes. J. Cell Sci. a, i-7CALLAN, H. G. & LLOYD, L. (i960). Lampbrush chromosomes of crested newts Triturus cristatm (Laurenti). Phil. Trans. R. Soc. Set. B 243, 134-219. CLARKSON, S. G., SMITH, H. O., SCHAFFNER, W., GROSS, K. W. & BIRNSTLEL, M. L. (1976). Integration of eukaryotic genes for 5s RNA and histone proteins into a phage lambda receptor. Nucleic Acid Res. 3, 2617-2632. COHN, R. H., LOWRY, J.C. & KEDES, L. H. (1976). Histone genes of the sea urchin (Strongylocentrotus purpuratus) cloned in E. coli: order, polarity and strandedness of the five histonecoding and spacer regions. Cell 9, 147-161. FORSHEIT, A. B., DAVIDSON, N. & BROWN, D. D. (1974). Electron microscope heteroduplex study of rDNAs of Xenopus laevis and Xenopus mulleri. jf. molec. Biol. 90, 301-314. FRANKLIN, S. G. & ZWEIDLER, A. (1977). Non-allelic variants of histones 2a, 2b and 3 in mammals. Nature, Lond. 266, 273-275. GALL, J.G. & CALLAN, H.G. (1962). 'H-uridine incorporation in lampbrush chromosomes. Proc. natn. Acad. Sci. U.S.A. 48, 562-570. GALL, J.G. & PARDUE, M.L. (1969). Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. natn. Acad. Sci. U.S.A. 63, 378-383. GALL, J. G. & PARDUE, M. L. (1971). Nucleic acid hybridization in cytological preparations. In Methods in Enzymology, vol. 2iD (ed. L. Grossman & K. Moldave), pp. 470—480. New York: Academic Press. GROSS, K.W., SCHAFFNER, W., TELFORD, J. & BraNSTmL, M.L. (1976). Molecular analysis of the histone gene cluster of Psammechinus miliaris: III. Polarity and asymmetry of the histone-coding sequences. Cell 8, 479-484. JOHN, H.A., BIRNSTIEL, M.L. & JONES, K.W. (1969). RNA-DNA hybrids at the cytological level. Nature, Lond. 223, 582-587. KEDES, L.H. & BIRNSTEEL, M.L. (1971). Reiteration and clustering of sequences related to histones. Nature, New Biol. 230, 165-169. KEDES, L.H., CHANG, A.C.Y., HOUSEMAN, D. & COHEN, S.N. (1975). Isolation of histone genes from unfractionated sea urchin DNA by subculture cloning in E. coli. Nature, Lond. 255, 533-538. LACROIX, J.-C. (1970). Mise en Evidence sur les chromosomes en 6couvillon de Pleurodeles poireti Gervais, Amphibien urodele, d'une structure Ii6e au sexe, identifiant le bivalent sexual et marquant le chromosome W. C.r. hebd. Se'anc. Acad. Set., Paris 271, 102-104. MACGREGOR, H.C. (1977). Possible trends in the evolution of very large chromosomes. Phil. Trans. R. Soc. Ser. B (In Press). 3 MACCREGOR, H.C. & MIZUNO, S. (1976). In situ hybridization of nick-translated H-ribosomal DNA to chromosomes from salamanders. Chromosoma 54, 15-25. MILLER, O.L. & BEATTY, B.R. (1969). Portrait of a gene. J. cell. Physiol. 74, Suppl. 1, 225- ANGELIER, 232. M.L. (1975). Repeated DNA sequences in the chromosomes of higher organisms. Genetics, Princeton 79, Suppl., 150-170. PUKKILA, P. J. (1975). Identification of the lampbrush loops which transcribe 5sribosomalRNA in Notophthalmus (Triturus) viridescens. Chromosoma 53, 71-89. RUDERMAN, J.V. & GROSS, P.R. (1974). Histones and histone synthesis in sea urchin development. Devi Biol. 36, 286-298. PARDUE, SCHEER, U., FRANKE, W.W., TRENDELENBURG, M.F. & SPRING, H. (1976). Classification of loops of lampbrush chromosomes according to the arrangement of transcriptional complexes. J. Cell Sci. 22, 503-520. Localization of histone gene chromosomes 79 M.H.L. & CALLAN, H.G. (1969). Evidence for a polarized movement of the lateral loops of newt lampbrush chromosomes during oogenesis. J. Cell Sci. 5, 1-25. SOMMERVILLE, J. & MALCOLM, D.B. (1976). Transcription of genetic information in amphibian oocytes. Chromosoma 55, 183-208. SPURR, A.R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-34. (Received 20 April 1977) SNOW, 6-3