David R. Wolstenholme

Ultracentrifuge and electron microscope studies on the structure of mitochondrial DNA

The structure of mitochondrial DNA from oocytes of Xenopus laevis and Rana pipiens has been studied by band centrifugation and electron microscopy. The molecular weight as determined by length measurements is 10·6×106. The population of molecules contained two classes which can be distinguished by electron microscopy as twisted and open circles. These two types of circles have physical properties similar to those described by Vinograd, Lebowitz, Radloff, Watson & Laipis (1965a) for polyoma virus DNA. Fresh preparations of mitochondrial DNA contained two components, sedimenting at 39·3 and 27·4 s; the proportion of these components was the same as that of twisted and open circular molecules, respectively. A fraction of the material corresponding to the proportion of twisted circles was reversibly denatured, i.e. it returned to native density after exposure to alkali and neutralization. Molecules indistinguishable from native twisted circles were abundant in material thus treated. Circles and linear forms of single-stranded DNA with contour lengths in the range of those found for native molecules were also seen in such preparations as well as in DNA denatured in the presence of formaldehyde. Conversion of component I (39 s) to component II (27 s) was achieved by treatment with small amounts of DNase I and with hydroquinone. Two components were also observed in centrifugation in alkaline solutions, sedimenting at 24 s and 87 s. The fast material is regarded as the cyclic coil form comprising two interlocked single strands of mitochondrial DNA. In sedimentation velocity titration a reduction in the sedimentation rate of component I was observed in the pH range around 11·4, followed by a rapid increase. The sedimentation coefficients of different forms of mitochondrial DNA were combined with the values from the literature for viral DNAs to provide calibrations for S versus molecular weight for circular forms of DNA.

Replicating DNA molecules from eggs ofDrosophila melanogaster

Eggs ofDrosophila melanogaster were lysed with sodium dodecyl sulphate within 110 minutes after laying and the lysate prepared for electron microscopy by the protein monolayer technique. Long, non-circular DNA molecules were found with a form suggesting they contained either a single replicated region, or two, three or four replicated regions arranged in tandem. Each replicated region was delimited by two forks. The two segments of DNA spanning the region between the forks were approximately equal in length and appeared to be totally or almost totally double-stranded. The appearance of replicating molecules was not altered by digestion with pronase or treatment with phenol or chloroform. The lengths of replicated regions varied from 0.2 μ to 22.1 μ with a mean value of 2.97 μ. The distances between midpoints of adjacent tandemly arranged replicated regions ranged from 1.2 μ to 9.7 μ with a mean value of 3.87 μ. Circular molecules found in these preparations, and presumed to be of mitochondrial origin, were estimated from comparative length measurements with circular double-stranded DNA molecules from the bacteriophages lambda, φX174 and fd to have a molecular weight of 12.36 X 106 daltons.

The distribution of DNA and RNA in salivary gland chromosomes of Chironomus tentans as revealed by fluorescence microscopy

SummaryIn acridine orange stained chromosomes of Chironomus tentans, ribonuclease-sensitive reddish-orange fluorescence is found in all bands and in all interband regions as well as in nucleoli and Balbiani rings.Following ribonuclease digestion, deoxyribonuclease-sensitive yellowish-green fluorescence is found in all bands and in all interband regions. Banded fibres, apparent in Balbiani rings and in nucleoli, and formed by the splitting of the chromosome axis, also show no evidence of discontinuities in their yellowish-green fluorescence. From these results it is concluded that DNA is present in interbands and (at least at the level of the light microscope), is continuous through these regions of polytene chromosomes.

Some facts concerning the nature and formation of axial core structures in spermatids of Gryllus domesticus

Examination of living spermatid nuclei of Gryllus domesticus has revealed the presence of the same structures, the X chromosome, the round body and the axial core structures, which have been described from electron microscopic observations. The outer ribbons of the axial core structures and the round body are composed of 100 Å fibres indiscernible from and often continuous with the fibres composing the X chromosome. That the outer ribbons of the axial core structures and the round body are chromosomal is further substantiated by the results of cytochemical examinations of formaldehyde fixed material which show that the axial core structures and the round body contain RNA, DNA and basic protein. Neither acetic acid-ethanol nor cold ethanol fixation preserve the round body and the axial core structures suggesting that a protein may be responsible for maintenance of the central core structure. The central core structures are always found in close association with condensed chromatin in regions where the chromosome elements are about 1000 Å apart, suggesting that the relative state of condensation of the chromatin and the spacial relationship between condensed regions may be two of the chief factors concerned in central core formation. Maintainance of the condensed state of the chromatin, however, may in turn depend upon central core integrity.

Renaturation and Hybridization Studies of Mitochondrial DNA

The products of the renaturation reaction of mitochondrial DNA from oocytes of Xenopus laevis have been studied by electron microscopy and CsCl equilibrium density gradient centrifugation. The reaction leads to the formation of intermediates containing single-stranded and double-stranded regions. Further reactions of these intermediates result in large complexes of interlinking double-stranded filaments. The formation of circular molecules of the same length as native circles of mitochondrial DNA was also observed. The formation of common high molecular weight complexes during joint reannealing of two DNAs with complementary sequences was used as a method to detect sequence homology in different DNA samples. Although this method does not produce quantitative data it offers several advantages in the present study. No homologies could be detected between the nuclear DNA and the mitochondrial DNA of X. laevis or of Rana pipiens. In interspecies comparisons homologies were found between the nuclear DNAs of X. laevis and the mouse and between the mitochondrial DNAs of X. laevis and the chick, but none between the mitochondrial DNAs of X. laevis and yeast. These results are interpreted as indicating the continuity of mitochondrial DNA during evolution.

Circular mitochondrial DNA from Xenopus laevis and Rana pipiens

Mitochondrial DNA from oocytes of Xenopus laevis and Rana pipiens and from the liver of Gallus domesticus was studied by electron microscopy using the Kleinschmidt technique. A high percentage of circular molecules, either highly twisted or open, was observed in all preparations. The mean contour length of circles from X. laevis was 5.40 μ, from R. pipiens 5.56 μ and from G. domesticus 5.26 μ. Highly twisted circles were found in greater abundance in a fresh preparation than in preparations left standing for 3 months. These molecules are considered to be the native form of mitochondrial DNA.

Electron microscopy of double-stranded RNA induced by turnip yellow mosaic virus and tobacco mosaic virus

SummaryDouble-stranded RNA isolated by phenol extraction from turnip yellow mosaic virus-infected chinese cabbage leaves and from tobacco mosaic virus-9nfected tobacco leaves was rotary shadowed and examined in the electron microscope. The TYMV and TMV molecules are similar in appearance, having uniform width and a linear configuration similar to that previously described for double-stranded RNA and double-stranded DNA molecules. More than 99.5% of the molecules of each virus fall within the range 0.1 μ to 2.2 μ, there being a predominance of smaller molecules in both cases (TYMV mean=0.24 μ, TMV mean 0.42 μ). The mode of the larger molecules of TYMV ≃ 1.92 μ and of TMV ≃ 1.8 μ. These values are close to the expected lengths of whole molecules, calculated from biophysical data. Apparently branched molecules were observed in preparations of both TYMV and TMV double-stranded RNA. It was found, however, that the number of such branches per unit length of RNA decreases with a decrease in density of the RNA in the fields examined.

Electron microscopic identification of the interphase chromosomes of Amoeba proteus and Amoeba discoides using autoradiography; with some notes on helices and other nuclear components

Data obtained from light and electron microscope autoradiographs of cells of Amoeba proteus and Amoeba discoides previously incubated in medium supplemented with H3 thymidine, indicate that fibrous material, the basic unit of which is about 150 Å in diameter, represents the interphase chromosomes of these amoebae. The helices of interphase nuclei do not appear to incorporate H3 thymidine, which is in opposition to the hypothesis of Taylor (1963) that they are G2 chromosomes, and makes it unlikely that they represent any form of the DNA-containing component of the amoebas interphase nucleus. Stereo-electron microscopy reveals that the direction of spiralization of helices may be either left or right handed and that the direction of spiralization of a single helix can reverse. The specific location of helices and of 850 Å–1150 Å electron dense bodies suggests that they are either primary chromosome products which subdivide before entering the cytoplasm, or units for the intranuclear transportation of primary chromosome products. In each nuclear membrane pore complex one central and eight peripheral regions of dense material are found. At each of the nine points, the dense material appears to traverse the nuclear membrane.