Polytene chromosomes from ovarian pseudonurse cells of the Drosophila melanogaster otu mutant

Certain mutant alleles of the otu locus in Drosophila melanogaster produce abnormal nurse cells in the ovaries. These cells are called pseudonurse cells (PNC), since they generate polytene chromosomes instead of endopolyploid ones and do not normally have an oocyte to nurse. The banding pattern of polytene chromosome 3 from the salivary glands (SG) and from PNCs of homozygous otu ¹ females was compared and a detailed photomap of PNC chromosomes with different degrees of polyteny is presented. The banding pattern was found to be strikingly similiar in the two tissues. The puffing pattern of the PNC chromosomes was also studied and the function of the PNC chromosomes is discussed. No constrictions or breaks were found in the PNC chromosomes which seems to indicate that these sites, which are known to be underreplicated in the SG chromosomes, are equally replicated along with the rest of the chromosomes in the PNC nuclei.     

Cytophotometric studies on cells from the ovaries of otu mutants of Drosophila melanogaster

Summary Amounts of chromosomal DNA were estimated for Feulgen-stained, ovarian cells from flies carrying certain mutant alleles of the otu (ovarian tumor) gene. Epithelial sheath cells and lumen cells were found to contain the diploid (2C) amount of DNA and therefore served as internal, cytophotometric standards. Mitotically active follicle cells over young tumors-from homozygous otu ¹ females contained either the 2C or 4C amounts of DNA; whereas, the tumor cell population contained 2C, 4C and 8C nuclei and many intermediate values. Egg chambers also occur in homozygous otu ⁷ females. Follicle cells above these oocytes undergo a maximum of four cycles of endomitotic DNA replication. The accompanying nurse cells (PNC) contain polytene chromosomes. These undergo a maximum of 12 endonuclear replication cycles. The PNCs show the expected levels of DNA for the first 6 cycles and the fraction failing to replicate during subsequent cycles may be as small as 10%. Lower than expected levels of DNA were detected in PNCs from an otu ¹/otu ³ ovary, reflecting roughly 20% underreplication. The latter PNCs may have been interrupted before DNA synthesis was concluded. No simple model of genomic underreplication accounts for the several different patterns of DNA behavior observed for various otu mutants.     

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.     

Nurse cell polytene chromosomes of Drosophila melanogaster otu mutants: Morphological changes accompanying interallelic complementation and position effect variegation

Combinations of certain mutant alleles of the ovarian tumor gene permit the production of viable eggs. Two alleles that behave in this way are otu⁷ and otu¹. Females homozygous for either allele are sterile, and their ovarian nurse cells (NC) contain giant polytene chromosomes of various morphologies. Fertile flies (otu⁺ / otu⁺, otu⁻ / otu⁷, otu⁺ / otu¹¹) have endopolyploid nurse cells with typical dispersed chromosomes. Fertile hybrids (otu⁷ / otu¹¹) produce large numbers of polytene chromosomes comparable to, and often larger than, classic salivary gland (SG) chromosomes. Therefore, these otu hybrids provide a unique system for studying, at the chromosomal level, the activation and expression of genes functioning during oogenesis. The otu gene encodes a long and a short isoform. The normal long isoform appears to be responsible for the dispersion of chromosomes during the endomitotic DNA replications occurring in ovarian NCs. The genetic inactivation of euchromatic genes placed next to pericentric heterochromatin by a chromosomal rearrangement is accompanied by the compaction of corresponding chromosome regions. A comparative study of the manifestation of position-effect variegation for the polytene chromosomes of SG cells and NCs was made using the Dp(1;1)pn2b and Dp(1;f)1337 rearrangements. The percentage frequencies of block formation in the SG and NC nuclei for Dp (1;1) pn2b rearrangement were 92.6% vs. 15.8%, respectively; for Dp(1;f) 1337, these values were 56.8% vs. 9.7%. Therefore heterochromatin belonging to germ line chromosomes is in a configuration that is far less likely to inactivate inserted segments of euchromatin than is heterachromatin from somatic chromosomes. Dev. Genet. 20:163–174. 1997. © 1997 Wiley-Liss, Inc.     

Ovarian pathologies generated by various alleles of the otu locus in Drosophila melanogaster

A comparative cytological study was made of oogenesis in flies carrying various mutant alleles of the female sterile gene otu. It resides at 22.7 on the genetic map and within subdivision 7F of the cytological map of the X-chromosome. Each of the five ethyl methane sulfonate-induced mutations observed falls into one of three classes. In class 1, most mutant ovarioles lack germ cells; in class 2, most mutant ovarioles contain tumorous chambers; and in class 3 mutants, chambers occur that possess defective oocytes. The otu² allele belongs to class 1; otu¹ to class 2; and otu³, otu⁴, and otu⁵ to class 3. The mutations have no effects upon female viability or upon the viability and fertility of hemizygous males. Heterozygous females are fertile and have cytologically normal ovaries. In otu⁵ homozygotes, all ovarioles contain egg chambers, but oogenesis is prematurely terminated to produce a pseudo-stage 12 oocyte. Ovarioles from otu³ and from otu⁴ homozygotes contain both ovarian tumors and oocytes. Pseudonurse cells (PNC), which are cystocytes that have stopped dividing and have entered the nurse cell mode of development, are also abundant. PNCs contain polytene chromosomes. Since the homologs are paired, each nucleus has the haploid number of chromosomes. In chambers lacking an oocyte, the number of PNCs is less than the normal number of nurse cells. In chambers containing an oocyte, the number of accompanying nurse cells may be 15, or above or below normal. In vitellogenic chambers, the chromosomes in the nurse cells connected directly to the oocyte are more expanded than those in more distant nurse cells. The KA14 deficiency lacks the plus allele of otu. KA14 heterozygotes are fertile and have cytologically normal ovaries. When females carry KA14 and otu¹, otu³, otu⁴, or otu⁵, 80% of their ovarioles are agametic. When females carry otu² and one of the other mutant alleles, the ovarioles proceed further in development. So otu² produces a product that has a beneficial effect on the test allele. When two different otu alleles are combined in a single fly, the phenotype of the hybrid ovary usually most resembles that of the ovary homozygous for the “stronger” allele (the otu mutant that allows oogenesis to proceed farthest). The results indicate that the product of the otu⁺ locus functions at least three different times during oogenesis; first to permit oogonia to proliferate, second to control the division and differentiation of germarial cystocytes, and third to facilitate the normal growth of the ooplasm. The gene product appears to be required in higher concentrations at each developmental period. The lesions produced by the mutations are thought to interfere with the stability or functioning of the gene product, and the ovarian phenotype produced by a given genotype depends upon the concentration of functional gene product available to the germ cells.     

Alpha and beta heterochromatin in polytene chromosome 2 ofDrosophila melanogaster

The formation of alpha and beta heterochromatin in chromosomes of Drosophila melanogaster was studied in salivary glands (SGs) and pseudonurse cells (PNCs). In SGs of X0, XY, XYY, XX and XXY individuals the amounts of alpha heterochromatin were similar, suggesting that the Y chromosome does not substantially contribute to alpha heterochromatin formation. Pericentric heterochromatin developed a linear sequence of blocks in PNCs, showing morphology of both alpha and beta heterochromatin. In situ hybridization with Rsp sequences (H _o clone) revealed that the most proximal heterochromatic segment of the mitotic map (region h39) formed a polytenized block in PNCs. Dot analysis showed that the clone had a hybridization rate with PNC-DNA very close to that with DNA from mainly diploid head cells, whereas the homologous SG-DNA was dramatically underrepresented. A similar increase of DNA representation in PNC was found for AAGAC satellite DNA. The mitotic region h44 was found not to polytenize in the SG chromosome, whereas in PNC chromosome 2 this region was partly polytenized and presented as an array of several blocks of alpha and beta heterochromatin. The mapping of deficiencies with proximal breakpoints in the most distal heterochromatin segments h35 in arm 2L and h46 in 2R showed that the mitotic eu-heterochromatin transitions were located in SG chromosomes distally to the polytene 40E and 41C regions, respectively. Thus, the transition zones between mitotic hetero- and euchromatin are located in banded polytene euchromatin. A scheme for dynamic organization of pericentric heterochromatin in nuclei with polytene chromosomes is proposed. Received: 17 November 1995; in revised form: 10 April 1996 / Accepted: 18 September 1996     

Study of the organization of polytene chromosomes in the nuclei of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> pseudonurse cells with the use of the DNA library of the <Emphasis Type="Italic">Drosophila orena</Emphasis> chromocenter

The location of the Drosophila orena chromocenter in polytene chromosomes of pseudonurse cells of the D. melanogaster ovaries (the otu11 mutation) and salivary glands has been studied. Numerous sites of location of the D. orena chromocenter DNA have been found throughout the length of D. melanogaster chromosomes. The specific distribution of the binding sites for the DNA probe has made it possible to identify chromosomes and analyze their mutual positions in the three-dimensional space of the nuclei of pseudonurse cells. The mutual positions of chromosomes have been found to vary, the pericentromeric regions of different chromosomes differing from one another in associative ratios.     

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.     

Cytophotometric studies on cells from the ovaries of otu mutants of Drosophila melanogaster

Amounts of chromosomal DNA were estimated for Feulgen-stained, ovarian cells from flies carrying certain mutant alleles of the otu (ovarian tumor) gene. Epithelial sheath cells and lumen cells were found to contain the diploid (2C) amount of DNA and therefore served as internal, cytophotometric standards. Mitotically active follicle cells over young tumors from homozygous otu1 females contained either the 2C or 4C amounts of DNA; whereas, the tumor cell population contained 2C, 4C and 8C nuclei and many intermediate values. Egg chambers also occur in homozygous otu7 females. Follicle cells above these oocytes undergo a maximum of four cycles of endomitotic DNA replication. The accompanying nurse cells (PNC) contain polytene chromosomes. These undergo a maximum of 12 endonuclear replication cycles. The PNCs show the expected levels of DNA for the first 6 cycles and the fraction failing to replicate during subsequent cycles may be as small as 10%. Lower than expected levels of DNA were detected in PNCs from an otu1/otu3 ovary, reflecting roughly 20% underreplication. The latter PNCs may have been interrupted before DNA synthesis was concluded. No simple model of genomic underreplication accounts for the several different patterns of DNA behavior observed for various otu mutants.     

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.     

Fertile heteroallelic combinations of mutant alleles of the otu locus of Drosophila melanogaster

Summary This paper describes the ovarian pathologies observed when 108 different heteroallelic combinations were made involving 17 independent mutations at the ovarian tumor (otu) locus. Most of the mutant phenotypes can be explained as graded responses by individual germ cells to different levels of functionally active otu gene product (OGP) synthesized by the mutant cells themselves. The lowest and highest levels of OGP appear to be produced by otu ¹⁰ and otu ¹⁴, respectively. In most heteroallelic ovaries the alleles have additive effects, and hybrid germ cells reach a developmental stage more advanced than the weaker homozygote but less advanced than the stronger homozygote. However, examples of both positive and negative complementation also have been found, and these suggest that the products encoded by different mutant alleles can combine to form dimers or multimers which may be superior or inferior to the homodimers. In flies homozygous for otu ¹¹ most ovarioles contain tumors, but some germ cells are able to develop further than those in otu ¹⁴ homozygotes. This suggests that, while otu ¹¹ produces intermediate levels of OGP, it also produces a second product (which otu ¹⁴ cannot make) that is utilized at the period in oogenesis when development in cells homozygous for otu ¹⁴ is blocked. When otu ¹¹ is combined with any one of eight specific alleles, it allows oocyte/nurse cell syncytia to differentiate that can complete development and undergo embryogenesis, if fertilized. The endopolyploid nurse cells of these hybrids have giant polytene chromosomes, and the presence of GPCs in functionally active, germ-line derived cells provides an interesting new system for experimental study. Analysis of the characteristic ovarian pathologies produced by flies of different genotypes leads to the conclusion that the products of the otu ⁺ gene are utilized during at least six different periods in Drosophila oogenesis.     

The organization of macronuclear DNA sequences associated with C4A4 repeats in the polytene chromosomes of Stylonychia lemnae

Gene libraries of the micronucleus and the macronuclear anlagen of the polytene chromosome stage of Stylonychia lemnae were screened for internal C₄A₄ repeats. The number of these internal repeats was shown to be identical in both kinds of nuclei. Analysis of macronuclear sequences associated with C₄A₄ in the polytene chromosomes showed that several macronuclear DNA sequences are clustered. However, interspersed between short exons of one gene are located exons of several other genes, i.e. the exon of one gene is an intron for several other genes.by M. Trendelenburg     

Dynamic organization of the β-heterochromatin in the Drosophila melanogaster polytene X chromosome

Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25° C and β-heterochromatic in X0 males at 14° C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(1LR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin. Received: 1 July 1998 / Accepted: 7 September 1998     

Cytophotometric evidence for the transformation of oocytes into nurse cells inDrosophila melanogaster

Summary A small proportion of ovarian chambers from females homozygous for theotu ⁷ (forovariantumor) mutation contain an oocyte that in its nuclear morphology resembles a nurse cell. Such transformed oocytes also appear in colchicine-poisoned wild type ovaries. Cytophotometric estimates demonstrate that these oocytes have undergone 3–4 additional DNA replications, but that they lag behind the adjacent nurse cells by an average of 1.3 replication cycles. It follows that, under certain circumstances, the definitive oocyte can switch to the nurse cell developmental pathway and therefore that a mechanism normally exists for preventing the further replication of its DNA. In the case ofotu ⁷, oocytes sometimes restart their endocyclic DNA replications and produce paired, polytene, homologous chromosomes.     

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     

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.     

Cytogenetic analysis of the Ethiopian fruit fly <Emphasis Type="Italic">Dacus ciliatus</Emphasis> (Diptera: Tephritidae)

The Ethiopian fruit fly, Dacus ciliatus, is an important pest of cucurbits, which recently invaded the Middle East. The genetics and cytogenetics of D. ciliatus have been scarcely studied. Such information is, however, an essential basis for understanding the biology of insect pests, as well as for the design of modern control strategies. We report here the mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this species. The mitotic metaphase complement consists of six pairs of chromosomes, including one pair of heteromorphic sex (XX/XY) chromosomes. The heterogametic sex is ascribed to the male. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms), which correspond to the five autosomes of the mitotic nuclei, and a heterochromatic mass corresponding to the sex chromosomes. Banding patterns, as well as the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed. Chromosomal homologies between D. ciliatus and Bactrocera oleae are proposed by comparing chromosome banding patterns and by in situ hybridization of the hsp70 gene.     

Polyploidy and polyteny induced by a hymenopteran parasite in Dasyneura urticae (Diptera,Cecidomyiidae)

Summary Larvae of Dasyneura urticae parasitized by an unidentified species of the Platygasteridae were found to contain giant polyploid nuclei of a polytene type. Comparison of polytene chromosomes of the giant nuclei with those of salivary-gland nuclei of D. urticae has shown that the giant nuclei are derived from the host nuclei, polyploidy and polyteny of these nuclei being, therefore, induced by the parasite.