Download localization of histone gene transcripts in newt lampbrush

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
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