Preliminary Account of an Electron Microscope Study
of Chromosomes from Newt Oocytes
By S. G. TOMLIN AND H. G. CALLAN
(From the Wheatstone Physics Laboratory, King's College, London, and the Animal
Breeding and Genetics Research Organisation, Agricultural Research Council, Institute
of Animal Genetics, Edinburgh)
With one Plate (fig. i)
SUMMARY
Chromosomes from oocytes of the newt Triturus cristatus carnifex have been photographed by means of the electron microscope. These chromosomes are shown to con. sist of single axial filaments, approximately 200 A wide, with 'lamp-brush' loops
attached at intervals along their lengths. Good fixation of the lamp-brush loops has not
yet been obtained. These chromosomes are attached in pairs to their homologues at
points which, in the past, have been considered to be chiasmata: it has been assumed
that the stage is diplotene of the first meiotic division. The singleness of the chromosome strands which we have observed is at variance with observations of diplotene in
other material by means of the light microscope.
S
INCE the electron microscope was introduced as a tool for the study of
the fine structure of biological specimens there have been a number of
attempts to photograph chromosomes (Buchholz, 1947; Clark, Barnes, and
Baylor, 1942; Clark, Quaife, and Baylor, 1943; Elvers, 1941 and 1943;
Guyenot, Danon, Kellenberger, and Weigle, 1950; Hovanitz, 1947; Hovanitz,
Denues, arid Sturrock, 1949; Palay and Claude, 1949; and Pease and Baker,
1949). On reading the accounts of this work it is evident that little new information on chromosome structure has so far resulted. In many cases there
would seem to be grave doubts as to whether the objects photographed were,
in fact, chromosomes. This criticism does not apply to the research of Palay
and Claude (shadowed replicas of salivary gland chromosomes of Drosophila)
or to the work of Pease and Baker (thin sections of fixed salivary gland
chromosomes). These two studies confirm earlier observations on salivary
gland chromosomes made by means of the light microscope and indicate
that these chromosomes consist of many thin chromonema-strands running
parallel to one another and connecting large and dense chromomeres like
beads on a string. Hovanitz and collaborators studied 'chromosomes' recovered from bird red blood-cells treated en masse by the drastic extraction
procedures introduced in 1943 by Claude and Potter and by Pollister and
Mirsky. The criticisms of Lamb (1949) as to the identity of the 'chromosomes' of Claude and Potter and of Pollister and Mirsky point to the need for
caution when assessing the origin of filamentous material recovered from
[Quarterly Journal of Microscopical Science, Vol. 92, part 2, June 1951.]
222
Tomlin and Callan—Preliminary Account of an
cells broken down by gross mechanical means. Elvers studied sections of
fixed pollen mother-cells of Lilium; Buchholz studied fragments from teased
pollen mother-cells of maize; Clark and collaborators studied the desiccated
contents of whole nuclei, presumably of some amphibian species since they
claim to have photographed 'lamp-brush' chromosomes. In all this work it is
difficult to assess the degree to which the chromosomes studied were contaminated with nuclear sap; this is a serious defect and makes the observations of little value. Finally there is the work of Guyenot and collaborators:
the material which they investigated consisted of chromosomes from newt
oocytes. The nuclei were isolated in amphibian Ringer solution, then placed
in distilled water where tBey generally burst; a drop containing the nuclear
material was pipetted on to collodion stretched over a metal grid. The
preparations were then dried, floated on distilled water to dissolve the inorganic salts and some of the nuclear sap proteins, dried once more, goldshadowed, and then examined. In an alternative method the nuclei were
isolated in Ringer solution, fixed in 45 per cent, acetic acid, centrifuged direct
on to collodion and the material dried, gold-shadowed and examined. Guyenot
and his collaborators observed long thin filaments which they describe as
chromosomes. This interpretation is probably correct but in the absence of
direct visual identification throughout the isolation procedures some doubt
must still remain. There are more serious criticisms. First, Ringer solution
contains calcium and this ion has very undesirable actions on the nuclear sap
and on the chromosomes. The sap undergoes some degree of precipitation
and the chromosomes are rendered brittle (Duryee, 1937, 1938; Callan,
unpublished). Secondly, washing in distilled water before desiccation or
fixation results in the lateral loops ('lamp-brushes') of amphibian chromosomes passing into solution. Chromosomes treated in this manner are thus
incomplete.
We have been able to photograph chromosomes whose identity was never
in doubt throughout the isolation and mounting procedure. The chromosomes chosen for study were obtained from almost mature oocytes of the
newt Triturus cristatus carnifex. The stage is somewhat more advanced than
that illustrated by Duryee (1941), and the lateral loops or 'lamp-brushes' have
passed their maximum development. The nuclei were isolated and cleaned in
0-2 M potassium chloride solution. The chromosome group is visible as a
rounded mass lying in the middle of the nucleus (fig. IA) : the diameter of this
mass is about one-third that of the nucleus itself and is approximately 150 fi.
Individual chromosomes are not easily observed at this stage since they form
a tightly packed mass embedded in nuclear sap whose refractive index is
very close to that of the chromosomes themselves.
The nuclear membrane is now broken and removed by means of finepointed tungsten needles and the jelly-like sap exterior to the chromosome
group is chopped away. The chromosome group is now pipetted into a fresh
drop of 0-2 M potassium chloride solution lying on a coverslip coated with a
thin film of metallized formvar. Under a binocular microscope at a magnifica-
FIG. I
Electron Microscope Study of Chromosomes from Newt Oocytes
223
tion of X 87, a tungsten needle is now agitated free-hand near the chromosome group, causing the sap to disperse. As the sap disperses the chromosomes become clearly visible individually: this is because of the lowering of
the refractive index of the medium in which they are lying and not, as stated
by Guyenot and his collaborators, due to the deposition of some material
from the sap on to the chromosomes. Loose chromosome arms swing free
from the mass: the end of an arm is now touched by the tungsten needle, to
which it adheres, and carried to the formvar film. A slight tear is made in the
film and the attachment of the chromosome transferred from the needle to
the film. By briskly rotating the needle near to the attached end of the chromosome arm, the chromosome is first disengaged from the mass of chromosomes
and, as it is freed from sap, adheres to the formvar film.
The chromosomes of the newt oocyte at this stage consist of 12 pairs, the
members of a pair being joined to one another at a few points which have
formerly been considered to be chiasmata (cf. Duryee, 1941; Dodson, 1948).
The lateral loops projecting from the chromosome are visible under the
binocular and serve to indicate where the chromosome is lying. The axial
filament is not visible, except by inference from the linear arrangement of the
loops.
After being washed in 0-2 M potassium chloride solution, the chromosomes
are fixed in o-i per cent, phosphotungstic acid solution, washed in distilled
water after fixation and dried in a desiccator. The films bearing chromosomes
are now mounted on copper grids by the method described by Martin and
Tomlin (1950). The first preparation was examined without shadowing, the
remainder after shadowing with palladium at an angle of 150.
Photographs were taken in a Siemens electron microscope at 52 kV. and at a
magnification of X 7500 or 10,000. The plates used were Ilford special process,
experimental; they were developed in undiluted D.8 or I.D. 2 developer of
normal strength. Typical composite prints are shown in fig. ic and a single
print at higher magnification in fig. IB. It will be seen that the chromosome
consists of a very thin axial filament of relatively even diameter, about 200 A,
on which, attached at intervals, are larger objects which represent the remains
of the lateral loops. The larger objects should probably be considered comparable with 'hypertrophied' chromomeres of leptotene chromosomes as seen
by ordinary microscopy, the thin axial filament being the chromonema of
classical cytology. On a very rough estimate there are about 25,000 of the
lateral loops in the entire chromosome complement of the newt.
We have not yet obtained clear evidence of chromosome structure at the
points where the chromosome pairs are joined to one another: the only parts
which we have been able to photograph satisfactorily are free arms distal to
the points of junction. If the points of junction between homologous chromosomes in newt oocytes are to be considered as comparable with chiasmata, it is
significant that the axial filaments which we have photographed are single
undivided structures. However, further work is needed on this matter and it
will be necessary to examine other stages. We have not yet found a suitable
224
Tomlin and Callan—Chromosomes from Newt Oocytes
method for the fixation of the lateral loops: it is evident that the structure
which they show in the photographs is a product of the technique of mounting,
fixation, and drying, and bears little relation to what exists in life. However,
the relative solidity of the lateral loops gives reason for the belief that future
work should provide evidence as to the structure of the lateral loops and therefore of the genes.
We wish to thank Professors C. H. Waddington, F.R.S., and J. T. Randall,
F.R.S., for the encouragement and help which they have given us in this
research. The study is to be continued.
REFERENCES
BUCHHOLZ, J. T., 1947. Science, 105, 607.
CLARK, G. L., BARNES, M. R., and BAYLOR, E. R., 1942. Ibid., 95, 250.
— — QUAIFE, M. L., and BAYLOR, M. R. B., 1943. Biodynamica, 4, 153.
CLAUDE, A., and POTTER, J. S., 1943. J. exp. Med., 77, 345.
DODSON, E. O., 1948. Univ. Calif. Publ. Zool., 53, 281.
DUHYEE, W. R., 1937. Arch. exp. Zellforsch., 19, 171.
1938. Collecting Net, Woods Hole, 13, 161.
1941- Cytology, Genetics and Evolution. Philadelphia (Univ. Pennsylvania Bicentennial
Conference).
ELVERS, I., 1941. Ark. Bot., 30, 1.
I943- Acta Hort. berg., 13, 150.
GUYENOT, E., DANON, M., KELLENBERGER, E., and WEIGLE, J., 1950. Arch. Klaus-Stift.
Vererb-Forsch., 25, 47.
HOVANITZ, W., 1947. Genetics, 32, 500.
DENUES, A. R. T., and STURROCK, R. M., 1949. Wasmann Collector, 7, 233.
LAMB, W. G. P., 1949. Nature, 164, 109.
MARTIN, A., and TOMLIN, S. G., 1950. Biochem. et Biophys. Acta, 5, 154.
PALAY, S. L., and CLAUDE, A., 1949. J. exp. Med., 89, 431.
PEASE, D. C , and BAKER, R. F., 1949. Science, 109, 8.
POLLISTER, A. W., and MIRSKY, A. E., 1943. Genetics, 28, 86.
EXPLANATION O F FIG, 1.
A. Optical section through nucleus of Triturus cristatus carnifex oocyte (diameter 1 -6 mm.).
The nucleus was isolated in a mixture of 19 volumes of o z M potassium chloride with 1 volume
of 0-2 M magnesium chloride. The chromosomes are grouped in the small inner mass
which is rendered the more visible by the presence of magnesium ions in the medium. A few
nucleoli lie near to the chromosome group and large numbers lie adjacent to the nuclear
membrane. Light microscope, magn. X 65.
B. Photograph of fragment of axial filament of a chromosome from a nucleus such as that
shown in A. The filament runs from top right to bottom left, the loop to the right of
the filament being
a 'lamp-brush'. Chromosome mounted on formvar and shadowed with
palladium at 150. Printed as a negative in order to show shadows dark. Electron microscope,
magn. x 13,000.
c. Composite photograph of segment of chromosome running between two supports of a
copper mounting-grid. The chromosome runs through the three blocks from top right to
bottom left and shows the continuous axial filament and several attached 'lamp-brush'
loops.
Chromosome mounted on formvar film and shadowed with palladium at 150; the pits and
folds are irregularities in the film. Printed as a negative in order to show shadows dark. Electron
microscope, magn. X 4,800.