Preliminary Account of an Electron Microscope Study of Chromosomes from Newt Oocytes

With one Plate (fig. 1)

Since 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, 194i and 1943; Guyénot, Danon, Kellenberger, and Weiglé, 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 x943 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 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 Guyénot 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 they 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. Guyénot 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 crrstatus 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. 1A) : the diameter of this mass is about one-third that of the nucleus itself and is approximately 150 μ. 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 fine-pointed 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 magnification of × 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 Guyénot 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 0·1 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 15°.

Photographs were taken in a Siemens electron microscope at 52 kV. and at a magnification of × 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. 1B. It will be seen that the chromosome consists of a very thin axial filament of relatively even diameter, about 200 Å, 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 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 futute 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.

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 0·2 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. × 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 15°. Printed as a negative in order to show shadows dark. Electron microscope, magn. × 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 15 °; the pits and folds are irregularities in the film. Printed as a negative in order to show shadows dark. Electron microscope, magn. × 4,800.