15.3. 1978 Specialia 353 at 4~ in an SW 65K rotor in the L3-40 Beckman ultra- centrifuge. A portion of the resultant pellet was fixed for electron microscopy, a portion was negatively stained with 1% uranyl acetate and the remainder electro- phoresed in 5.13% polyacrylamide gel containing 1% sodium dodecyl sulphate 3. Actin and desmin were isolated from chick gizzard smooth muscle as described by Lazarides and Hubbard s, The 8M urea-soluble fraction of gizzard smooth muscle which had been extracted with 0.6 M KC1, and 0.6 M K1 was used for gel analysis. Desmin represents the 50,000 dalton polypeptide that is con- sidered a subunit of the intermediate filaments of chick smooth muscle 5. Microscopic analysis showed that the lens 78,000xg pellet consisted mainly of intermediate-sized filaments (10-12 nm in diameter) (figures 1 and 2). A few membrane profiles were observed in the 78,000 • pellet, and free particles were evident on the negative stain. The electro- phoretic patterns of smooth muscle actin and desmin, and of the lens 8M-USF and 78,000 • g pellet of intermediate filaments are shown in figure 3. The lens 8M-USF (figure 2, B) contains all the crystallin polypeptides (bands 2, 4, 5, 6 and 7) present in the lens water soluble fraction 3, and many noncrystallin components pre- viously identified and characterized by mol. wt a. The most prominent noncrystallin components (bands 1 and 3) correspond in position to that of muscle actin and desmin. Although the 78,000• pellet contains the major polypeptides present in the 8M-USF, it is markedly enriched in the amount of band 1 of mol. wt previously estimated at 49,000 daltons 3. These results strongly suggest that the intermediate filaments of the lens contain a polypeptide of mol. wt identical to that of desmin. Further study is required to determine whether the lens polypeptide and desmin are identical in biochemical structure. Association of IDNA@eplication with 'nuclear.:!membrane in !larvae of ~Chironomus thurnmi ~ C. K. K. Nair and H. N. B. Gopalan 2 Biochemistry and Food Technology Division, Bhabha Atomic Research Centre, Bombay 400 085 India), 11 June 1977 Summary. In the larvae of Chironomus thummi, the newly replicating DNA has been found to be associated with the nuclear membrane, as evidenced by the isolation of DNA nuclear membrane complexes (M-band) of 3H-thymidine labelled larvae. In bacteria it has been shown that chromosomal DNA is attached to the cell membrane at various points, and DNA replication is intimately associated with the cell membrane s, 4. Although there are a few studies indicating absence of any association between nuclear membrane and DNA replication in eukaryotic cells a, ~, several recent reports present compelling biochemical and electron microscopic evidence to suggest a close association be- tween DNA replication and nuclear membrane in both animal and plant cells 7-n. Thus scanning electron microscopic studies of the chromosomes within the nuclei of the salivary glands of the larvae of Chironomus thummi have revealed that the chromosomes are connected to the inner surface of the nuclear membrane ~2. In the ex- periments to be reported here, we have attempted to examine whether, in the larvae of Chironomus thummi, the newty replicated DNA is associated with the nuclear membrane complexes isolated by sedimentation in bi- phasic sucrose gradients using the M-band technique la Chironomus thummi larvae (4th instar) were incubated for 24 h at 28 ~ in distilled water containing 0.1 mCi/ml thymidine-methyl-3H (sp. act. 6 Ci/mM) to label DNA. After incubation the larvae were washed, suspended in I~rebs-Ringer-phosphate buffer (KRP)14 containing 0.25 M sucrose and homogenized at 0-4~ The homogenate was filtered through a cheese cloth (8 layers). The filtrate was centrifuged at 500 • for 10 rain and the pellet was suspended in the same KRP-buffered sucrose solution. This suspension (1 ml) was layered on 10% Ficol in KRP (25 ml) and centrifuged at 3000 • for 30 rain. The pellet 4C o/o 20 ._o M-Band 2 4 6 8 10 12 14 16 18 20 Top Fraction number Bottom Sedimentation profile of labelled DNA of nuclear lysate from Chiro- riotous larvae. Total radioactivity of the nuclear lysate loaded on the gradient was 5.52 • 104 cpm. 1 Acknowledgment. The authors thank Dr D. S. Pradhan for dis- cussions and encouragement. 2 Botany Department, Kenyatta University College, Nairobi, Kenya. 3 A. Klein and F. Bonhoeffer, A. Rev. Biochem. 41, 301 (1972). 4 M.L. Pato, A. Rev. Microbiol. 26, 347 (i972). 5 J.A. Huberman, A. Tsai and R. A. Deich, Nature 241, 32 (1973). 6 D.E. Comings and T. A. Okada, J. molec. Biol. 75, 609 (1973). 7 C.E. Hilderbrand and R. A. Tobey, Biochim. biophys. Acta 331, 165 (1973). 8 P. Binkerd, T. Roach and A. Toliver, Physiol. Chem. Physics 6, 245 (1974). 9 A. Barrieux, L. Long and L. S. Garren, Biochim. biophys. Acta 312, 228 (1973). 10 A.A. Infante, R. Nauta, S. Gilbert, P. Nohart and W. Firshein, Nature New Biol. 242, 5 (1973). 11 W.F. Clay, F. R. H. Katterman and P. G. Bartels, Proc. nat. Acad. Sci. USA 72, 3134 (1975). 12 W. Trosch and B. Lindemann, Micron 3, 370 (1973). 13 G.Y. Tremblay, M. J. Daniels and M. Schaechter, J. molec. Biol. 40, 65 (1969). 14 H. F. DeLuca and P. P. Cohen, in: Manometric techniques, p. 133. Ed. W. M. Umbreit, R. H. Burris and J. F. Stauffer. Burger Publishing Company, Minnesota, 1964.