Chromosoma (Berl.) 52, 49--58 (1975) �9 by Springer-Verlag 1975

The Volume and DNA Content of Extrachromosomal Inclusions in the Dorsal Foot-pad Nuclei

of Tricholioproctia impatiens (Sarcophagidae, Diptera)

Brian Roberts

Depar tment of Zoology, Monash Universi ty, Clayton, Victoria 3168, Austral ia

Abstract, The chromosomes of the giant dorsal foot-pad cells of Tricholioproctia impatiens produce numerous extrachromosomal bodies which consist of a central spherical core of DNA surrounded by a ribonucleoprotein layer. The bodies are ei ther free within the nucleoplasm or a t tached by th in threads of chromat in to chromosomal bands. The DNA content and the volume of DNA of unattached spherules fell into distinct classes in a regular geometric pro- gression.

Introduction

The occurrence of small inclusions lying free in the nucleoplasm or attached to specific chromosomal bands or nucleolar organizer regions in poly%ene nuclei of various Diptera is now fairly well documented. These inclusions appear to have similar morphological characteristics independent of their synthetic origins or their location within the nucleus. Such inclusions include the multiple micro- nueleoli of the salivary gland nuclei of Hyboseiara/ragilis (da Cunha et al., 1969) and Sciara eoprophiIa (Oabrusewycz-Garcia and Kleinfeld, 1966; Gabrusewycz- Garcia, 1972), the proliferation of the heterochromatic kinetechore regions into "H-bodies" in the salivary gland nuclei of Chironomus melanotus (Keyl and Hiigele, 1966), and the DNA granules of the dorsal foot-pad nuclei of Sareophaga bullata (Whitten, 1965; Roberts, 1968). Similar multiple mieronucleoli occur in the oocytes of the amphibian Triturus (Miller, 1964; 1966; Miller and Beatty, 1969).

All of these bodies, regardIess of their location or synthetic origins, appear to consist of a central core or matrix of material rich in DNA and surrounded by a nueleolonema layer comprised of ribonucleoprotein (Miller and Beatty, 1969; Gabrusewyez-Gareia, 1972). These nuclear inclusions may be attached to specific chromosomal bands to form beadlike arrangements or detached to lie free in the nucleoplasm.

t~or the present study it was found that the dorsal footpad nuclei of the Australian sareophagid, Tricholioproetia impatiens, possesses DNA bodies similar to those described for S. bullata. The present investigation examines the mor- phology and characteristics of the DNA of bodies which had been extruded free into the nucleoplasm.

Present Address: The Biological Laboratories, 16 Divinity Avenue, Harvard University, Cambridge, Massachusetts 02138, U.S.A.

4 Chrolnosoma (Berl.), Bd. 52

50 B. Roberts

M a t e r i a l s a n d M e t h o d s

1. Materials and Tissue Preparation. Larvae of the flesh-fly, Trieholioproctia impatiens (Walker), were reared in uncrowded conditions on a surplus of beef liver at 25~ and 50-70 % R.H. Foot-pads were amputa ted from 168 hour male pharate adults which had been staged from the onset of pupar ium formation. For observation of living whole nuclei and their in- clusions, foot-pads were placed on a slide in a drop of insect Ringer solution. The pupal cuticle was removed and the nuclei dissected free from the dorsal aspect of the developing pulvillus. Wi th in 10 minutes of the operation the preparat ions were photographed wittl Leitz inter- ference contrast optics.

For Feulgen-DNA investigations the pupal cuticle was removed and the tissues fixed in alcohol acetic acid (3: 1) for 10 min, hydra ted through a series el ethanol solutions to distilled water, hydrolyzed in 5 hi HCl a t 25~ for 1 hour (Decosse and Aiello, 1966), and stained for 1 hour in freshly prepared Schiff's Feulgen reagent made according to de Thomasi (Pearse, 1968). The pads were placed in drops of 45% acetic acid on gelatinized slides; the nuclei were then dissected free and squashed under siliconized coverslips. The slides were frozen on dry ice and the coverslips removed. After dehydrat ion and clearing in xylol, the preparat ions were mounted in an oil of matching refractive index (n D 1.528-1.566, Cargille Laboratories, Inc., Cedar Grove, N.J . ) . Some squashes from the same stages of development were stained with Azure B a t pH 4 (Flax and Himes, 1952). The squashes were viewed wi th brightfield illu- mination, darkfield i l lumination, and Nomarski interference contrast optics.

2. Calculation o/the Volume o/DNA o/Spherical Inclusions. The diameter of the DNA of the inclusions was calculated from projected photographic negatives obtained with darkfield i l lumination (Loveland, 1970). The preparat ions were mounted in non-matching refractive index oil (R.I., 1.42). A Leitz oil immersion condenser (D- -1 .20 A) and a 90 • 1.32 NA apochromatie oil objective with a 1.0 NA funnel stop were employed. Polystyrene latex spheres (Dow Chemical Co., Midland, Mich.) with a specified diameter of 0.557 ~ and a cal- culated size deviat ion of 0.011 ix were used as s tandard objects. The polystyrene spheres, the DNA of the inclusions and a Leitz graduated micrometer were photographed and processed under identical conditions and the diameter of the objects calculated. The location of each inclusion was noted by the use of an England finder (Graticules Ltd., London) so t h a t their Feulgen-DNA content could be determined.

8. Microspectrophotometry. A Leitz MPV-1 microscope photometer was used for all ab- sorption measurements. Light from a stabilized 150 W Xenon high-pressure lamp was directed into a Leitz monochromator. The monochromatic l ight beam of selected wavelength was re- flected by a non-absorbing first surfaced mirror through a centerable oil immersion eondensor lens (50 • 0.60 NA), equipped with a variable field diaphragm to reduce light flare, to the object plane. A Leitz 90 • 1.32 NA eenterable oil immersion apochromatic objective and a Leitz periplanatic 25 • large field (G.F.) eyepiece with set d iaphragm were selected for all measurements. The photometer head consisted of a 1.25 X tube lens, measuring diaphragms, a pilot lamp for viewing the dimensions of the diaphragm and a photomultiplier . Leitz variable (0.5 to 6.0 mm) and set d iaphragms of 0.8 and 1.25 m m in diameter were chosen. The dia- meters of the set d iaphragm image a t the object plane were calculated to be 1.13 tz and 1.77 ix respectively when used in conjunction with the optical system specified above. The use of these diaphragms enabled the images of the inclusions to be accurately centered within the boundaries of the measuring field and pass a known area of l ight to the photocathode. The photomult ipl ier chosen was an EMI 1Vfultiplier No. 6904A (Electric and Musical Industries, Ltd., England), eormected to an ul trastabil ized high-voltage supply (Knott-Electronic, Mfinchen, Germany). The un i t was of high sensi t ivi ty and shows low dark current of the multiplier (Thaer, 1966). A digital vol tmeter was used as the reading instrument .

The elements of the MPV-1 were carefully aligned in order to reduce ins t rument error. To check the homogeneity of l ight and the uniformity of the photocathode responses, the five positions tes t of Garcia and Iorio (1969) and the l ineari ty tes t of Pollister et al. (1969) were used.

After Feulgen staining the D N A content of the larger spherical inclusions was measured by the two-wavelength method of cytospectrophotometry as described by Mendelsohn (1966) and PoIlister et al. (1969). The spherical inclusions were located and thei r image aligned within

Extrachromosomal Inclusion in Nuclei of Tricholioproctia 51

Fig. 1. Photomicrograph of a living dorsal foot-pad nucleus showing the presence and morphology of spherical nuclear inclusions (interference contrast)

the boundaries of the measuring diaphragm. :For more exact movement of the microscope stage the operating controls were replaced by knurled knobs some 5 cm in diameter. Absorption measurements were made from selected areas of the larger inclusions and the two wavelengths chosen such t ha t the ext inct ion at one wavelength was half the ext inct ion at the other. Due to the relatively high optical density of the inclusions, excitat ion values were not chosen from the region of highest absorption (560 nm); instead 520 nm was selected as the higher wave- l eng th The second wavelength varied from 487-489 nm. The DNA of individual inclusions was averaged from three separate readings.

Results

1. General Morphology

Fig. 1 shows the typical appearance of a living dorsal foot-pad nucleus; it clearly demonstrates the presence of small spherical bodies lying free within the nncleoplasm. The bodies were observed to move within the living nucleus. I t will also be observed that other spherical inclusions are attached to each other and to chromosomal bands.

The bodies were also present in Feulgen squash and Azure B preparations, but many were damaged or lost during the various histological stages of processing the tissues. Stains with Azure B showed that the bodies possessed a ribonucleoprotein sheath. The latter was routinely lost during Feulgen staining thereby exposing the central core of DNA.

When examined by interference contrast optics, the DNA of the unattached bodies appeared spherical (Fig. 2a) and possessed similar optical characteristics to images of polystyrene latex spheres. With transmitted brightfield illumination the inclusions appeared as distinct circles as shown in Fig. 2b. Darkfield illu- mination proved to give the finest resolution (Fig. 2 c); the image produced was that of a torus. Polystyrene latex spheres produced similar images. In the bead- like chains of granules attached to specific chromosomal bands some also showed the optical characteristics of spheres, the largest being furthest from the site of at tachment (Fig. 3).

4*

52 B. Roberts

Fig. 2a--c. Morphology of the DNA of the inclusions located in the dorsal foot-pad nuclei of T. impatiens (a) interference contrast, (b) brightfield, (c) darkfield

Fig. 3a and b. Photomicrograph of attached bodies (a) brightfield (b) darkfield. Feulgen stain. The arrows indicate attached spherical bodies

2. Volume o /DNA o/the Spherical Inclusions

Exploiting the increased image enhancement at tained by darkfield illumination in a non-matching refractive index oil, I was able to calculate the diameter of the DNA by comparison with the known diameter of the polystyrene latex spheres. Under darkfield illumination the spheres produced rings of light, with the dimen- sions of the boundary wave varying with focus (Martin, 1949). Fig. 4 illustrates the method of calculation where the corrected diameter of a sphere equals half the sum of the external and internal diameter of the boundary wave. Using this method and calibrating from the micrometer measurements also photographed with darkfield illumination, the polystyrene latex spheres (n = 10) were found to have a diamter of 0.557 ~ 0.038 ~z.

The mean diameters of the DNA core of the spherical inclusions ranged from 0.445 ~ to 1.389 ~ (Table 1). The volume of the DNA (n =42) fell into six clearly

Extraehromosomal Inclusion in Nuclei of Tricholioproctia 53

Fig. 4. Diagramatic representation of the method of calculation of the diameter of polystyrene latex spheres and the DNA of spherical inclusions in the dorsal polytene nuclei when viewed with darkfield illumi- nation. The corrected diameter (D) equals half the sum of the external diameter (A) and the internal diameter (B) of the boundary wave

D

A

defined classes in a 2 : 4 : 8 ... ratio. The volume of the DNA spheres is shown in Fig. 5 arranged in r ank order and plot ted on a logari thmic scale. The his togram shows the d i s t r ibu t ion of the DNA and the six designated classes.

3. D N A Content o/Spherical Inclusions

The DNA conten t of the larger Feu]gen s ta ined spherical inclusions (classes I I I to VI) was measured (n = 32) by the two wavelength method of cy~ospectrophoto- metry. Classes I and I I were no t uti l ized since results would be subject to error (Casperson, 1950; Moses, 1952).

The DNA of classes I I I to VI fell into four precisely defined sub-classes in a 2 : 4 : 8 : 16 geometric progression (Table 1). No t rue interclass values were observed. The results are summarized in Fig. 6 where DNA values are expressed in a rb i t ra ry un i t s and are arranged in r ank order on a logari thmic scale. The his togram shows the occurrence of DNA classes.

Table 1. The DNA content and the diameter and volume of the DNA of spherical inclusions in the dorsal foot-pad nuclei of T. imTatiens

Designated Number Mean Mean Mean DNA content class~ measured diameter (tz) volume (~) (expressed in

arbitrary units)

I 2 0.445 0.046 __b II 8 0.556 0.091 _ b III 8 0.702 0.182 0.465 IV 10 0.881 0.359 0.969 V 6 1.093 0.688 1.866 VI 8 1.389 1.407 3.806

a See Figs 5 and 6. b Estimates of DNA subject to error.

5 4 B . Roberts

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Fig. 5. The volume of DNA of spherical inclusions in dorsal footpad nuclei of T. impatiens. Measurements are arranged in rank order on a logarithmic scale and the histogram shows the distribution of values into geometric multiples. The open circles represent polystyrene latex

spheres

8.0

~ 4 D - -

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Fig. 6. Feulgen-DNA measurements of classes III to VI of spherical inclusions in the dorsal nuclei of T. impatiens. Values are expressed in arbitrary units arranged in rank order and are plotted on a logarithmic scale. The histogram shows the distribution of values into geometric

multiples

6. Relationship Between DNA Volume and D N A Content

The relationship between DNA volume and DNA content of spherules of classes III to VI (n = 3 2 ) is shown in Fig. 7. The data cluster into four distinct groups.

A regression line was fitted by the method of least squares (Sokal and Rohlf, 1969). This yielded the following relationship: log DNA~- -0 .424+ l .017 log Volume. The slope of the regression line proves to be highly significant (P < 0.0001).

Extrachromosomal Inclusion in Nuclei of Tricholioproctia 55

G.4

5.2

2 ~ (D c-

x: ~ 1.6 O -

"" "~ 0.8 7 ~ C:I

0.4

0.2

/ /

(x) ~,g

/ (/K)**4~

/ / - -

1 I I J I I 0.1 0.2 0.4 0.8 1.6 5.2

Volume (,u. 5)

Fig. 7. The relationship between DNA volume and DNA content of individual spherules from classes I I I to VI. Scales are logarithmic

D i s c u s s i o n

Oarkfield illumination with the compound microscope is fundamentally a method of enhancing image contrast (Loveland, 1972). Indeed, the first arrangement for darkfield microscopy was termed the ultramicroscope (Gage, 1932). The darkfield optical system func- tions in illuminating the obj e ct field with rays of light that will never enter the obj e ctive aperature to form part of the image beam except when they are reflected or diffracted into it by the ele- ments of the specimen. The overall result is that the object is highly resolved and appears self luminous on a black background (Needham, 1958). Preparations for darkfield illumination must be mounted or suspended in a medium of different refractive index (Moses, 1952).

I n t he p resen t inves t iga t ion i t was observed t h a t po ly s ty r ene la tex spheres and the D N A of the inclusions d i f f rac ted l ight to form the image of a torus . B y app l ica t ion of t he formula D = (A ~-B) /2 (Fig. 4), ex t r eme ly accura te measure- men t s were obta ined . The average d i ame te r of po lys ty rene la tex spheres of specif ied size 0.557 • ~ was found to be 0.557 ~:0.038 ~ when ca lcu la ted wi th a Lei tz mic romete r scale p h o t o g r a p h e d and processed unde r iden t ica l con- di t ions. I t would appea r t h a t th is m e t h o d of m e a s u r e m e n t of spher ica l objec ts has po ten t i a l app l i ca t ion in t he fields of medica l and biological research.

I n recent t imes , error of absorp t ion measu remen t is usua l ly no t a t t r i b u t a b l e to t he a p p a r a t u s (Sandr i t t e r , 1969). Since 1960, r e m a r k a b l e improve me n t s in opt ica l a p p a r a t u s and components have been made. The per iod has seen the con- s t ruc t ion of h igh qua l i ty apoch roma t i e objec t ives of su i tab le numer ica l ape ra tu re , the genera l ava i l ab i l i t y of powerfu l and s tab i l ized d ischarge lamps, excel lent monoch roma to r s and the deve lopmen t of pho tomul t ip l i e r tubes of increased sens i t iv i ty .

56 B. Roberts

The ability to discern fine detail with a microscope is termed resolving power and is best measured by its reciprocal, the resolution of the system (Loveland, 1970). In the present study the resolution of the optical system using light of 500 nm was found to be 0.189 ~. Thus the diameter of the DNA of the measured inclusions of classes I I I to VI was some 4 to 7 times greater than the resolution. Caspersson (1950) has shown that the minimal object for measurement of ab- sorption closely approaches the lower limit conditioned by the properties of the microscope objective, but should be greater than the resolving power of the system. The DNA of the inclusions of classes I and I I was not measured as their dimensions approached or were smaller than the wavelength of light.

The introduction of a biological object in a photometric system leads to disruption of the light beam. If the object is spherical light may be disturbed by refraction, absorption and reflection (Sandritter, 1966). Caspersson (1950) has shown that with transmitted light a sphere, suspended in a medium of differing refractive index, causes light to form a light scattering pattern which varies with the diameter of the particle. If the diameter of the particle approximates and exceeds 0.2 ~ the light scatter is overcome by the correct matching of refractive indexes of the object and the mounting medium. If correctly matched almost no darkfield image of the specimen should be evident (Moses, 1952; Pollister et al., 1969). In the present s tudy refractive indices of 1.528-1.566 proved suitable.

This study has shown that in the dorsal foot-pad nuclei of T. impatiens extra- chromosomal bodies are present in isolated living nuclei and in squashed prep- arations. Staining with Azure B and Schiff's Feulgen reagent revealed that the inclusions were composed of a central core of DNA surrounded by a ribonucleo- protein coat. Small nuclear inclusions such as the micronucleoli of the oocytes of the newt Triturus (Miller, 1966), the micronucleoli of the salivary glands of Hybosciara /ragilis (da Cunha et al., 1969) and Sciara coprophila (Gabrusewycz- Garcia and Kleinfeld, 1966), the "H-bodies" of the salivary glands of Chironomus melanotus (Keyl and Hi~gele, 1966), and the DNA granules of Sarcophaga bullata (Whitten, 1965; l~oberts, 1968) also consist of a central core of DNA surrounded by a ribonucleoprotein sheath or nueleolonema. In S. bullata these bodies have been shown to incorporate tri t iated thymidine and tri t iated uridine (Roberts, 1968)--an indication of replicative and synthesizing function.

In the foot-pad nuclei of T. impatiens the spherical geometry of both the un- fixed inclusion and of the DNA core is of interest (Figs. 1 and 2). In the salivary gland nuclei of H. /ragilis the majority of free micronucleoli are spheres having a distinct spherical Feulgen positive core at all stages of development (da Cunha et al., 1969). Electron mierographic studies by Gabrusewycz-Garcia (1972) have shown that the salivary glands of Sciara coprophila also contain spherical micro- nucleoli as well as many irregularly shaped masses. The chromosomes of the sali- vary glands of Chironomus melanotus produce "H-bodies" from heteroehromatic kinetechore regions which appear very similar to the inclusions described for T. impatiens since they appear circular in transmitted light (Keyl and H~gele, 1966). Also the dorsal giant cells of Sarcophaga bullata produce spherical inclusions which are similar, if not the same, as those described for T. impatiens (Roberts, unpub- lished observation). I t would appear that in all the examples cited the polytene

Extrachromosomal Inclusion in Nuclei of Tricholioproctia 57

nuclei a t some s tage of deve lopmen t possess inclusions ly ing free in the nncleoplasm which have a spher ica l form. This s t u d y has shown t h a t in T. impatiens both the inclusions and the centra l core of D N A are spheres.

The f inding t h a t bo th the vo lume of D N A and the D N A content , where measured , fell into d i s t inc t classes of a doubl ing of t he pre-exis t ing a m o u n t is of biological impor tance . I n th is s t u d y i t was shown t h a t four d i s t inc t D N A volume/ D N A content classes were p resen t and doubt less o thers would be observed if t he resolu t ion of t he techniques were improved .

No d a t a are avai lab le on the D N A content of the var ious nuclear inclusions of the po ly t enc nuclei so far discussed. However , Miller (1966) has made electron microscopic measurements of pe r iphera l micronucleolar D N A isola ted f rom Triturus oocytes. I n p r epa ra t i ons to ana lyze the contents of t he mieronucleolus Miller found the D N A in t he form of closed loops in which the length of a popu- la t ion of such D N A loops va r ied according to a regula r geometr ic progression. Miller descr ibed loops wi th t he D N A rep l i ca ted in uni ts of 11.25 ~x in length. I t seems possible t h a t t he D N A of the spher ica l inclusions of the dorsa l nuclei of T. impatiens m a y have a s imi lar s t ruc ture to t h a t descr ibed above.

Though the presen t inves t iga t ion does no t e luc ida te t he source of the spher ica l bodies, i t appears l ike ly t h a t t h e y are syn thes ized b y specific ehrotnosomal bands (Fig. 3) and then f reed in to t he nucleoplasm. Such a mode of synthes is would help expla in the observed classes of D N A v o l u m e / D N A content .

Acknowledgements. I thank Mr. V. Salanitri for his assistance in rearing the fly cultures and Professor C.M. Williams for his helpful comments.

References

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Cosse, J. J. De, Aiello, N. : Feulgen hydrolysis: Effect of acid and temperature. J. Histochem. Cytochem. 14, 601-604 (1966)

Cunha, A.B. Da, Pavan, C., Morgante, J.S., Carrido, M.C.: Studies on cytology and dif- ferentiation in Sciaridae. II. DNA redundancy in salivary gland cells of Hybosciara fragilis (Diptera, Sciaridae). Genetics 61, Suppl., 335-349 (1969)

Flax, M.H., Himes, M.H.: 3/iicrospectrophotometric analysis of metachromatic staining of nucleic acids. Physiol. Zool. 25, 297-311 (1952)

Gabrusewycz-Garcia, N. : Further studies of the nucleolar material in salivary gland nuclei of Seiara coprophila. Chromosoma (Berl.) 38, 237454 (1972)

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)JIiller, O.L. : Extrachromosomal nucleolar DNA in amphibian oocytes. J. Cell Biol. 23, 60A (1964)

58 ]3. Roberts

Miller, O.L.: Structure and composition of peripheral nucleoli of salamander oocytes. Nat. Cancer. Inst. Monogr. 28, 53-66 (1966)

Miller, O.L., Beatty, ]3. R. : Extrachromosomal nucleolar genes in amphibian oocyLes. Genetics 61, Suppl., 133 143 (1969)

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Pollister, A.W., Swift, H., Rasch, E.M.: Microphotometry with visible light. In: Physical techniques in biological research (G. Oster and A. W. Pollister, eds.), p. 201-250. New York: Academic Press, Inc. 1969

Roberts, B.: DNA granule synthesis in the giant foot-pad nuclei of Sarcophaga bullata. J. Cell Biol. 89, l12A (1968)

Sanch'itter, W.: Methods and results in quantitative eytochemistry. In: Introduction to quantitative cytochemistry (G. L. Wied, ed.), p. 159-201. New York: Academic Press, Inc. 1966

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Received June 3, 1975 / Accepted June 11, 1975 by J. G. Gall Ready for press June 21, 1975