The absence of labeled RNA on metaphase chromosomes of Taricha and Rana embryos

640

M. L. Freedman,

P. J. Sfambrook

and R. A. Flickinger

the typical collagen staining. Hence, of collagen is present in plant nuclei. to that of the animal sources, in so Summary.-The results presented detectable amount of collagen which

it may be concluded that a detectable amount Moreover, the collagen in plant nuclei is similar far as the stainability is concerned. indicate that nuclei of plant cells contain a can be demonstrated by specific staining procedure, only when nucleic acids and some proteins are removed by TCA hydrolysis and pepsin and trypsin digestion in succession. The authors are grateful to Dr S. R. Sen Gupta (Director) and Prof. A. C. Pandya of the Indian Institute of Technology, Kharagpur, for providing necessary facilities and encouragement for the present investigation.

REFERENCES 1. HUMASON, G. L., Animal Tissue Techniques, pp. 165. W. H. Freeman and Co. London, 1962. 2. PEARSE, A. G. E., Histochemistry Theoretical and Applied. pp. 816 and 818. Little, Brown and Co., Boston, 1960. 3. POLLISTER, A. W., Expfl Cell RES. Suppl. 2, 59 (1952). 1. STEDMAK, E. and STEDMAN, E., Cold Spring Harbor Symp. Quanf. Biol. 12, 224 (1947). 5. STEELE, W. J. and BUSCH, H., Expfl Cell Res. 33, 68 (1964).

THE

ABSENCE

OF LABELED OF TARICHA

M. L. FREEDMAN, Department

RNA ON METAPHASE AND

RANA

P. J. STAMBROOK

of Biology,

State University

Received

February

CHROMOSOMES

EMBRYOS1 and

of New

R. A.

FLICKINGER

York, Buffalo,

N.Y.

13, 1967

IF

recently synthesized radioactive RNA were retained on metaphase chromosomes during mitosis, it might offer a means of examining RNA synthesis at specific regions of the chromatin in different cells. Thus, one could look at a specific chromosome, or region of that chromosome, in embryonic cells that will specialize into different cell-types and assume that the retention of labeled RNA at a specific region denoted RNA synthesis by the expanded chromatin of that region during interphase. Autoradiographic evidence, however, indicates that labeled RNA is not retained on the metaphase chromosomes of cultured Chinese hamster or HeLa cells 131, although biochemical studies have demonstrated RNA associated with chromosomes isolated from these cells [l, 51. Most of this RNA sedimented at 28s and 16S, suggesting that it is ribosomal RNA. However, one group attributes the presence of this RNA to adventitious binding during the preparation [5]. Our work utilized gastrulae (stages 10 and 12) and neurulae (stage 16) of Taricha forosa [4], and 1 This research Experimenfal

was sponsored

Cell Research 47

by a grant from the National

Science Foundation.

Absence of labeled RNA on metaphase chromosomes

641

Fig. l.-Metaphasc chromosomes from a cell of the animal hemisphere of an early gastrula (stage 10) of Taricha torosn cultured with 3H-5-uridine (25 ,x/ml) for 21 hr. Colcemidc was present during the final 12 hr.

blastulae, neurulae and tailbuds of Kuna pipiens (stages 8, 15, and 19) [6]. The early stages were cut from their jelly and vitelline membranes and all stages were washed several times in sterile Niu-Twitty solution [a]. The embryos were cut into smaller groups of dorsal ectoderm plus mesoderm or endoderm cells for incubation in NiuTwitty solution containing 3H-5-uridine (25 PC/ml) at 20°C. After the explants of Taricha had been cultured for 12 hr, colcemide was added to a final concentration of 0.01 per cent and the explants were incubated for another 12 hr period. The explants of Rana pipiens were cultured for 6 or 24 hr with colcemide (0.01 per cent) present throughout the incubation period. At the conclusion of the incubations the explants were washed extensively in Niu-Twitty saline, then cut into smaller pieces and washed again. These clumps of Experimental

Cell Research 47

642

M. L. Freedman,

P. J. Stambrook

and R. A. Flickinger

Fig. 2.PChromosomes from a cell of the dorsal axial region of a neurula (stage 14) of Rana pipiens cultured with SH-5-uridine (25 PC/ml) for 6 hr. Colcemide was present throughout the incubation.

cells were placed in 2 x glass distilled water for 15 min to spread the chromosomes in situ. The explants were then fixed in aceto-orcein (2 per cent aceto-orcein in 45 per cent acetic acid) for 2 hr. The fixed cells were squashed on slides on which albumin had been heat-dried. After squashing, the coverslips were removed by freezing on dry ice and the tissue was dehydrated in ice cold 50, 75, 95 per cent and absolute alcohol. The slides were coated with liquid emulsion (NTB-2) at 45°C and exposed in light-tight boxes in the cold for up to six weeks. Inspection of the radioautographs revealed that label was not associated with the metaphase chromosomes of cells of any region of any of the developmental stages used of Taricha (Fig. 1) or Rana (Fig. 2) embryos. In some of the metaphase preparations of Rana cells there were silver grains around the chromosomes, but the label was not directly on the chromosomes. It is likely that this is cytoplasmic or nuclear Experimental

Cell Research 47

Acrosomd

reaction

in A. lumbricoides

vnr. suum

643

RNA which was trapped in clumped metaphase figures. The nuclei showed a very heterogeneous labeling pattern. In any region of any of the stages there were nuclei which showed all variations between light and heavy labeling. Incubation of the slides of Rana with ribonuclease (0.02 per cent RNase in 0.2 n/l acetate buffer, pH 5.5 for 2 hr) before coating with the liquid emulsion virtually abolished the subsequent appearance of silver grains in nuclei. This indicates that the 3H-5-uridine was not incorporated into DNA. In conclusion, results of our autoradiographic study show that labeled RNA is not retained by metaphase chromosomes, but the data do not indicate at what time before metaphase the recently synthesized RNA is lost from the nuclei. REFERENCES 1. MAIO, J. Jr,, and SCHILDRANT, C., L., J. Mol. Biol. 24, 29 (1967). 2. NW, M C. and TWITTY, V. C., Proc. Nafl Acad. Sci. Wash. 34, 985 (1953). 3. PRESCOTT, D. M. and BENDER, M. A., Expff Cell Res. 26, 260 (1962). 4. RUGH, R., Experimental Embryology, p. 90. Burgess Publishing Co., Minneapolis, 1962. 5. SALZMAK, N. P., MOORE, D. E. and R~ESDELSOHN, J., Proc. I\‘afl Acad. Sci. r.S. 56, 1449 (1966). 6. SHUMWAY, W., Anat. Rec. 78, 139 (1940).

ELECTRON THE PRESENCE ASCARIS

MICROSCOPIC

EVIDENCE

OF AN ACROSOMAL LUMBRICOIDES

W. H. CLARK, JR., R. L. MORETTI

FOR

REACTION

IN

VAR. SUUM. and W. W. THOMSON

Department of Life Sciences and the Air Pollution Research Center, University of California, Riverside, Calif., U.S.A.

Received February 14, 1967l

A LTHOUGH an acrosome has been found in most sperm, its presence in the nonflagellated sperm of nematodes has been a controversial subject for many years. According to light microscope studies [5-81, the mature nematode sperm is slightly elongated with one end narrower than the other. There is a centrally located nucleus surrounded by mitochondria. Occupying most of the narrow end is a refractile, gram positive cone which is composed of a protein, ascaridine [3]. Bowen [l] suggested that the refringent cone might correspond to the acrosome. Sturdivant [8], using Ascaris megalocephala, also considered the refringent cone to be acrosomal in nature. Recently Favard [4], in an electron microscope study of A. megalocephala, showed that the refringent cone did not originate from the Golgi complex as is true of acrosome formation described to date. It has also been shown that the refringent cone does not exhibit a positive histochemical test for mucopolysaccharides which is a generally accepted criterion for acrosomal identification [2]. 1 Revised

version

received

April

24, 196i.

Experimental

Cell Research 47