Different behaviour of Xbb+ and Xbb chromosomes in D. melanogaster with only one nucleolus organizer.

Summary We have examined the rDNA content of male and female adult flies having only one nucleolus organizer (NO), using X chromosomes carrying wild or partially deleted bobbed loci (Xbb ⁺/O, Xbb ⁺/X_NO-and Xbb/O, Xbb/X_NO ^-).The results show that in Xbb ⁺/O and Xbb ⁺/X_NO ^-flies, where only somatic gene compensation is supposed to occur, the rDNA increase, although less pronounced than previously reported, is directly proportional to the number of rRNA genes initially present in the nucleolus organizer. In Xbb/O and in Xbb/X_NO ^-flies the rDNA increase is relatively much higher than that observed in flies carrying bb ⁺ instead of bb. It is suggested that this may be due to rDNA premagnification and somatic gene compensation occurring simultaneously in the former flies.On leave of absence from International Institute of Genetics and Biophysics, Naples, Italy     

Regulation of rRNA gene number in Drosophila melanogaster: new aspects resulting from the use of free duplications

Summary We have isolated a bobbed (bb) mutant on the free duplication Dp(1; f)122bb ⁺ and we have measured the rDNA content of the bb ⁺ and the bb loci in genetic combinations in which none of the phenomena involved in the change of the rDNA redundancy occurs. We have also measured the rDNA content of the two bb loci carried by the free duplications in two different genetic combinations: (1) and females in which there are two attached X chromosomes completely deleted for the nucleolus organizer (NO) regions and there-fore the only rDNA is contributed by the free duplication; (2) X/Dp122bb ⁺ and X/Dp122bb males, in which there are two bb loci, one on the X chromosome and the other on the X free duplication.The bb ⁺ and the bb duplications produced an overall increase of the rDNA content in the two genetic conditions tested.These results are not in favour of both a cis and trans effect of the regulator locus (cr ⁺ locus) hypothesised as being involved in the disproportionate replication of rRNA genes.     

Check of Gene Number during the Process of rDNA Magnification

THE multiple sequences of rDNA (DNA complementary to ribosomal RNA) of the Drosophila genome are localized at the bobbed locus, located in the X chromosome, position 66 and in the short arm of the Y chromosome^1,2. Wild bobbed (bb⁺) is that locus which, without a partner, gives rise to a normal phenotype. That locus which in similar conditions is incapable of giving rise to a normal phenotype is called a bobbed mutation (bb) and contains fewer genes for rRNA. The number of genes for rRNA in different individuals can vary considerably. One mechanism for rDNA variation is unequal crossing over³. Another mechanism, described by Tartof⁴, becomes apparent when individual flies, carrying only one bobbed locus, are constructed and only if such a locus is on the X chromosome; that is, if one constructs Xbb⁺/O males (and also Xbb/O males) or Xbb⁺/X_NO- females. Such individuals show a higher rDNA content than expected from the analysis of the same locus in Xbb⁺/Xbb⁺ females or in Xbb⁺/Ybb⁺ males. The increase of rDNA in this case is not inheritable⁴.     

Increased number of nucleoli in the salivary gland cells of Drosophila melanogaster under conditions of rDNA dose compensation

A considerable increase in the number of nucleoli non-associted with the nucleolar organizer (NO) was shown in the salivary gland cells of Drosophila melanogaster mutants, heterozygous for a deficiency of NO. The frequency of formation of additional nucleoli increased with the raising of the chromosome polyteny level. By means of in situ hybridization we showed that in the mutant and the wildtype polytene cells the ribosomal DNA (rDNA) of these unlawful nucleoli included ribosomal gene repeats (18S+28S) with two types of insertions: ivs-I and ivs-II Such additional nucleoli can be attached to varying sites of the polytene chromosomes containing type I insertion sequences.     

rDNA magnification in D. melanogaster: state of rDNA copies following the first step.

Summary D. melanogaster males of bb/O genetic constitution undergoing rDNA magnification were mated singly to XXbb ⁺/O females, yielding bb/O male progeny, and to X_NO-w sn bb ⁺ fameles, yielding bb/X_NO- females. The male and female offspring were scored for the bb ⁺ phenotype.Results show that there is a higher percentage of bb ⁺ flies in the bb/O male progeny than in bb/X_NO- females progeny, in single crosses as well as in the combined data. rRNA/DNA hybridization experiments agree with this observation, by showing that the rDNA content in the progeny of premagnified flies was higher in the sons than in the daughters.These data indicate that the increase of ribosomal RNA genes is not due to a stable event such as an unequal mitotic sister exchange, whereas they do not contrast with the extracopy model.     

Chromosomal organization of Drosophila tumours

In otu mutants of Drosophila melanogaster ovarian tumours develop because of the high mitotic activity of the mutant cystocytes; the latter are normally endopolyploid. In certain alleles of otu, however, a varying proportion of the mutant ovarian cystocytes undergo polyteny. Mutant cystocytes with polytene chromosomes are termed pseudonurse cells (PNC). Polytene chromosome morphology and banding patterns in PNC of otu ¹/otu³ flies were cytologically analysed. Extensive variability was noted in the quality of the banding pattern of the PNC chromosomes which ranged from highly condensed (condensed PNC chromosomes) to those with a banding pattern (banded PNC chromosomes) similar to that in larval salivary gland cells (SGC). Both the condensed and banded PNC chromosomes frequently enter into a diffuse state characterised by weakened synapsis of the polytene chromatids and alterations in their banding pattern (diffuse PNC chromosomes). Analysis of DNA synthesis patterns in the various morphological forms of PNC polytene chromosomes by ³H-thymidine autoradiography revealed a basic similarity to the pattern seen in polytene nuclei of larval SGC. Independently replicating sites, however, could be unambiguously identified only in banded PNC chromosomes. Comparison of late replicating sites in such PNC chromosomes with those of larval SGC showed a remarkable similarity in the two cell types. These results suggest a close correlation between the polytene chromosome banding pattern and its replicative organization.     

A Study of Rdna Magnification Phenomenon in a Repair-Recombination Deficient Mutant of DROSOPHILA MELANOGASTER

The rDNA magnification process consists of a rapid and inheritable rDNA increase occurring in bobbed males: in a few generations the bb loci acquire the wild-type rDNA value and reach a bb⁺ phenotype.—We have analyzed the rDNA magnification process in the repair-recombination-deficient mutant mei9^a, both at the phenotypical and rDNA content levels. In mei9^a bb double mutants the recovery of bb⁺ phenotype is strongly disturbed and the rDNA redundancy value fails to reach the wild-type level. The strong effect of this meiotic mutation on rDNA magnification suggests a close relationship between this phenomenon and the repair-recombination processes.     

Conservation of nucleolar structure in polytene tissues of Ceratitis capitata (Diptera: Tephritidae)

Nucleolar structure was studied in mitotic and three polytene tissues of the Mediterranean fruit fly, Ceratitis capitata using in situ hybridization with a tritium-labelled rDNA probe and silver staining. In mitotic metaphase chromosomes nucleolar organiser regions were localised in the short arms of both sex chromosomes. In polytene nuclei of trichogen cells, salivary glands and fat body rDNA was detected within nucleoli. Nucleoli in these tissues have a similar structure with rDNA labelling concentrated in a central core. Silver staining resulted in very heavy staining of polytene nucleoli and interphase nucleoli in diploid cells. Silver staining of nucleolar organisers in metaphase chromosomes is weak or absent although the X chromosome has numerous dark silver bands in other locations. The results suggest that nucleolar structure is conserved in polytene tissues contrasting with the variability of autosomal banding patterns and sex chromosome structure. They also indicate that silver staining is not necessarily specific for nucleolar regions.     

Mutual regulation of magnified bobbed loci in Drosophila melanogaster

Summary rDNA magnification in D. melanogaster increases the redundancy of that locus to a value higher than the wild type. A magnified locus (bb ^m) can lose the express copies according to rDNA content of partner sexual chromosome. This paper is a study of behaviour of two bb ^m loci put together to show their interaction.     

Two AT-rich satellite DNAs in the chironomid Glyptotendipes barbipes (Staeger)

Two AT-rich satellite DNAs are present in the genome of Glyptotendipes barbipes. The two satellites have densities of 1.680 g/cm³ (=21% GC) and of 1.673 g/cm³ (=13% GC) in neutral CsCl-density gradients. The main band DNA has a density of 1.691 g/cm³ (=32% GC). This value is in agreement with the 33% GC-content of G. barbipes DNA calculated from thermal denaturation (T_M=83° C). — In brain DNA as well as in salivary gland DNA the two satellite sequences together comprise 12–15% of the total G. barbipes DNA. Comparisons of the density profiles of DNA extracted from polytene and non-polytene larval tissue gave no hints for underreplication of the satellite DNAs during polytenization. — The two satellite DNAs have been isolated from total DNA by Hoechst 33258-CsCl density centrifugation and then localized in the polytene salivary gland chromosomes by in situ hybridization. Both satellite sequences hybridize to all heterochromatic centromere bands of all four chromosomes of G. barbipes. Satellite I (1.673 g/cm³) hybridizes mainly with the middle of the heterochromatin, satellite II (1.680 g/cm³) hybridizes with two bands at the margin of the heterochromatin. In situ hybridization with polytene chromosomes of Chironomus thummi revealed the presence of G. barbipes satellite sequences also in the Ch. thummi genome at various locations, mainly the centromere regions.     

Ribosomal RNA Cistrons of X Chromosomes Clonally Derived from D. MELANOGASTER Laboratory Populations: Redundancy, Organization and Stability

To determine whether heterogeneity exists in the organization or redundancy of the rRNA cistrons of inbred populations of Drosophila melanogaster, we have derived a number of sublines from the strains Oregon R and Canton S. These two strains were chosen because our previous studies have demonstrated a difference in the competence of these strains to exhibit a "compensatory response" of the rDNA. In each subline, the X chromosomes are descended from a single maternal X, that is, each line is homozygous for a particular nucleolus organizer (NO). These derivative lines have been characterized in terms of rDNA content and organization, using quantitative liquid hybridizations and Southern blot analyses, respectively. Our studies reveal that both of the highly inbred parent populations contained a heterogeneous array of X chromosomal rDNA contents. Once isogenized, the rDNA redundancy of a given X chromosomal NO can be shown to remain stable for at least 20 generations in culture. We detect no restriction pattern heterogeneity among X chromosomes isolated from a given strain, despite relatively large differences in their rDNA contents. This leads us to suggest that there is no significant clustering of intervening sequence-bearing (ivs ⁺) genes within the rDNA loci of chromosomes from the populations examined. Furthermore, we conclude that apparent alterations in rDNA redundancy known as the compensatory response are not related to the heterogeneity of rDNA content within a population.     

Extent of polyteny in the pericentric heterochromatin of polytene chromosomes of pseudonurse cells of otu (ovarian tumor) mutants of Drosophila melanogaster

In the polytene nuclei of germ-line cells (ovarian pseudonurse cells) of Drosophila melanogaster females mutant for otu ¹¹ (ovarian tumor), the pericentric heterochromatin is much more abundant than in somatic salivary gland cells. This is due to the degree of heterochromatin compaction (and consequently the level of underreplication) being lower in the nurse cells than in the salivary gland cells. The lower level of compaction probably results in a very low degree of position effect gene inactivation in the ovarian nurse cells.     

Comparison of the polytene chromosomes of the salivary gland,the fat body and the midgut nuclei of Drosophila auraria

Photo-maps of the fat body and midgut polytene chromosomes of Drosophila auraria were constructed. These photo-maps are compared with a new, more detailed photo-map of the salivary gland chromosomes of the same species. Seven, not previously described inverted tandem-duplications were detected, raising the number of such structures found in this species to 31. The constancy of the banding pattern based on the analysis of the above chromosomes is discussed.     

Ectopic expression of the<Emphasis Type="Italic"> Suppressor of Underreplication</Emphasis> gene inhibits endocycles but not the mitotic cell cycle in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis>

The Suppressor of Underreplication ( SuUR) gene contributes to the regulation of DNA replication in regions of intercalary heterochromatin in salivary gland polytene chromosomes. In the SuUR mutant these regions complete replication earlier than in wild type and, as a consequence, undergo full polytenization. Here we describe the effects of ectopic expression of SuUR using the GAL4-UAS system. We demonstrate that ectopically expressed SuUR exerts qualitatively distinct influences on polyploid and diploid tissues. Ectopic expression of SuUR inhibits DNA replication in polytene salivary gland nuclei, and reduces the degree of amplification of chorion protein genes that occurs in the follicle cell lineage. Effects caused by ectopic SuUR in diploid tissues vary considerably; there is no obvious effect on eye formation, but apoptosis is observed in the wing disc, and wing shape is distorted. The effect of ectopic SuUR expression is enhanced by mutations in the genes E2F and mus209 ( PCNA). Differential responses of polyploid and diploid cells to ectopic SuUR may reflect differences in the mechanisms underlying mitotic cell cycles and endocycles.Communicated by G. P. Georgiev     

Complete replication of DNA in polytene nuclei of salivary glands ofDrosophila melanogaster

Summary Combined cytophotometric and autoradiographic experiments are performed on individual polytene salivary gland nuclei of X/X-female and X/Y-male larvae ofDrosophila melanogaster, DNA measurements of unlabeled nuclei reveal complete douplings of all 4C DNA quantity during polytenization. These new data do not agree with the hypothesis of heterochromatic underreplication.     

Functional dissection of the Suppressor of UnderReplication protein of Drosophila melanogaster: identification of domains influencing chromosome binding and DNA replication

The Suppressor of UnderReplication (SuUR) gene controls the DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster salivary gland polytene chromosomes. In the present work, we investigate the functional importance of different regions of the SUUR protein by expressing truncations of the protein in an UAS–GAL4 system. We find that SUUR has at least two separate chromosome-binding regions that are able to recognize intercalary and pericentric heterochromatin specifically. The C-terminal part controls DNA underreplication in intercalary heterochromatin and partially in pericentric heterochromatin regions. The C-terminal half of SUUR suppresses endoreplication when ectopically expressed in the salivary gland. Ectopic expression of the N-terminal fragments of SUUR depletes endogenous SUUR from polytene chromosomes, causes the SuUR ⁻ phenotype and induces specific swellings in heterochromatin.     

Satellite DNA of Anopheles stephensi liston (Diptera: Culicidae)

Four satellite DNAs in the Anopheles stephensi genome have been defined on the basis of their banding properties in Hoechst 33258-CsCl density gradients. Two of these satellites, satellites I and II, are visible on neutral CsCl density gradients as a light density peak forming approximately 15% of total cellular DNA. Hoechst-CsCl density gradient profiles of DNA extracted from polytene tissues indicates that these satellites are underreplicated in larval salivary gland cells and adult female Malpighian tubules and possibly also in ovarian nurse cells. The chromosomal location of satellite I on mitotic and polytene chromosomes has been determined by in situ hybridisation. Sequences complementary to satellite I are present in approximately equal amounts on a heterochromatic arm of the X and Y chromosomes and are also present, in smaller amounts, at the centromere of chromosome 3. A quantitative analysis of the in situ hybridisation experiments indicates that sequences complementary to satellite I at these two sites differ in their replicative behaviour during polytenisation: heterosomal satellite I sequences are under-replicated relative to chromosome 3 sequences in polytene larval salivary gland and ovarian nurse cell nuclei.     

EM autoradiographic studies on polytene nuclei of Drosophila melanogaster

Replication in the chromocentre heterochromatin of salivary gland polytene nuclei of Drosophila melanogaster has been examined by ³H-thymidine EM autoradiography. In vitro pulse labelling of salivary glands from late third instar larvae showed that the chromocentre heterochromatin replicates in synchrony with the euchromatin in the nucleus. Within the chromocentre region, the central compact mass, identified earlier as the alpha heterochromatin, did not incorporate ³H-thymidine at any stage of the S, while the surrounding beta heterochromatin was always labelled in nuclei with labelled euchromatin. In a second set of experiments, growing larvae from just after hatching till late third instar stages, were fed on food containing ³H-thymidine, and at the end of larval life, the incorporation in salivary gland nuclei was examined by EM autoradiography. A grain density analysis of the EM autoradiographs revealed that the alpha heterochromatin does not replicate at all from after hatching till late third instar while the beta heterochromatin replicates as much as the euchromatin. Non-replication of the alpha heterochromatin provides the explanation for the lowered amount of heterochromatin in the polytene nuclei compared to their diploid counterparts. Implications of these observations on the organization of chromocentre heterochromatin in polytene nuclei and its homology to the heterochromatic regions in mitotic chromosomes are discussed.