Drosophila Genome Size

The C value for Drosophila melanogaster is not only useful in its relationship to the net amount of genetic information embedded in its chromosomes (1,4,12), but it is also needed to study changes in DNA levels associated with alterations in the total amount of genetic coding potential following changes in cell size and cell functions during larval and adult life in this species and its congeners (18,28-31). Sequencing of the Drosophila genome (4) has now set the future course for the field of molecular developmental biology.

There is a fundamental question in Drosophila cytogenetics that can be stated as follows: "What is the functional significance of the selective genome reduplication that is the signature of development in so many dipteran species?" During the life-span of many dipterans, organs other than just the larval salivary glands (Malpighian tubules, gastric folds, fat bodies, hindgut, rectum, and nurse cells of germ-line origin in the ovarioles) regularly show large, densely stained nuclei in certain tissues at certain times during growth and differentiation (20,25,26,32-34). Analysis of these phenomena requires, in part, the capacity to measure DNA amounts in individual nuclei from extremely small tissue samples, a task admirably suited to cytophotometry, using the sperm C value of 0.18 pg DNA (2-4). For example, we can now say, with reasonable assurance, that larval hemocytes of Drosophila are predominately at the 4C level (0.72 pg DNA), but are almost exclusively at the 2C level (0.36 pg DNA) in adult males (see Fig. 1). Also, we can say that oenocyte nuclei (see refs. 28-30) in females remain at the 4C level (0.72 pg DNA) throughout imaginal life (see Fig. 1), unlike cells of the female adult fat body (AFB), which show a progressive shift from 2C cells to 4C cells to 8C cells and even a few 16C cells that contain about 2.8 pg DNA per nucleus during maturation and senescence (see Fig. 2). AFB cells in males also show 4C and 8C nuclei during imaginal life. In contrast to the rather staid behavior of DNA in the AFB, analyses of DNA levels in cells of the larval fat body (LFB) show dramatic changes with time (31,34). The values shown in Fig. 3 were obtained by measuring the DNA-Feulgen content of individual fat body nuclei from the time of hatching on through the growth and maturation of the fat body in third instar larvae, some 92-96 h after hatching. Mature larvae show more than a hundred-fold increase in the DNA content of their largest nuclei, which may have DNA levels as high as 40 pg per nucleus at the peak of activity in this multifunctional tissue (see Fig. 3) (34; Rasch and Butterworth, unpublished work). As expected (29-31), there is a marked decrease in nuclear DNA content accompanying organ histolysis that occurs during pupation. Only a few remnant LFB nuclei can sometimes be recognized in the vicinity of the developing AFB in newly eclosed adults (see Fig. 3). The data in Fig. 3 were com-

Fig. 1. Histograms of Feulgen-DNA amounts per nucleus for larval and adult hemocytes and oenocytes of Drosophila melanogaster, expressed in arbitrary units of relative integrated absorbance, to estimate the extent of genome replication in different tissues at ongoing stages of development. Note differences in frequencies of 2C and 4C hemocyte nuclei at the larval and adult stages. Oenocytes from adult females show only 4C nuclei, presumably holding at an arrested G2 stage of the cell cycle (Rasch, unpublished data).

Fig. 1. Histograms of Feulgen-DNA amounts per nucleus for larval and adult hemocytes and oenocytes of Drosophila melanogaster, expressed in arbitrary units of relative integrated absorbance, to estimate the extent of genome replication in different tissues at ongoing stages of development. Note differences in frequencies of 2C and 4C hemocyte nuclei at the larval and adult stages. Oenocytes from adult females show only 4C nuclei, presumably holding at an arrested G2 stage of the cell cycle (Rasch, unpublished data).

puted from the average DNA content determined for populations of 50-75 nuclei in fat body cells measured in adipose tissue at each different stage of development. The data on these changes in DNA levels shown represent more than 3000 scans of individual fat body nuclei and are based on measurements of chicken blood cell nuclei used as a reference standard of 2.5 pg DNA (2,12,16,31,34).

Both cytological and biochemical evidence (19,22,23,27,35) appear to demonstrate that highly compacted chromatin (constitutive heterochromatin) is sig-

Fig. 2. Histograms of nuclear DNA measurements of fat body nuclei from 14-d, 28-d, and 67-d adult females and males of Drosophila melanogaster. The C value for sperm (0.18 pg DNA) was used to identify a shift in nuclear DNA contents from 2C to 4C to 8C during senescence in both female and male adults.

Fig. 2. Histograms of nuclear DNA measurements of fat body nuclei from 14-d, 28-d, and 67-d adult females and males of Drosophila melanogaster. The C value for sperm (0.18 pg DNA) was used to identify a shift in nuclear DNA contents from 2C to 4C to 8C during senescence in both female and male adults.

nificantly underreplicated in types of dipteran cells that involve differential replication patterns for (1) euchromatin, (2) compacted chromatin (i.e., heterochromatin), and (3) nucleolar-associated chromatin domains (14). The functional significance of these anomalies in DNA replication is not understood

Fig. 3. Changes in average nuclear DNA content of adipose cells during larval development in Drosophila melanogaster. Some of the large, somewhat densely stained fat body nuclei approximate 128C and approach up to the 256C DNA levels, suggesting that at least six to seven or more cycles of endoreduplication occur during the course of fat body maturation and senescence (31,34).

Fig. 3. Changes in average nuclear DNA content of adipose cells during larval development in Drosophila melanogaster. Some of the large, somewhat densely stained fat body nuclei approximate 128C and approach up to the 256C DNA levels, suggesting that at least six to seven or more cycles of endoreduplication occur during the course of fat body maturation and senescence (31,34).

at present, but they do point out that additional critical studies are needed to account, at least in part, for apparent discrepancies between expected DNA levels from replication of the entire genome and the amount of DNA that is detected by DNA-Feulgen cytophotometry in polysomatic dipteran tissues (1,10,12,23-25,33,36). Perhaps the elegant studies of Gerbi and her co-workers (37) to identify precise sites of origins of DNA replication for the giant poly-tene chromosomes of Sciara coprophila have now opened the way to apply their strategies to the persistent problem of heterochromatin underreplication in Drosophila.

Because manifestations of selective gene replication as well as differential gene activation and selective gene expression are of considerable interest to geneticists and developmental biologists, there is a pressing need to determine C values for a given species and to follow changes in the amounts of DNA per nucleus in particular types of tissue during differentiation. With the "return of the H-word (heterochromatin)," to quote Lohe and Hilliker (14), there is a growing interest in the potential role of highly repetitive and/or satellite DNAs and their associated proteins in molecular and cellular biology (15). In addition, comparisons of C values are of interest in phylogenetic studies of specia-tion and evolution in many groups of animals (1,10).

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