A STUDY OF THE NUCLEOLAR MATERIAL IN SCIARA COPROPHILA

In the polytene chromosomes of Sciara coprophila, in addition to a nucleolus, large numbers of nucleolarlike structures or micronucleoli are formed. A detailed mapping localized the nucleolar organizer at one end of the X chromosome and revealed that approximately 18% of the bands of each chromosome are potentially capable of producing micronucleoli. Most of these sites are in regions known from a previous study to show asynchronous DNA replication: DNA puffs and certain heterochromatic regions. Micronucleoli are rarely found in association with bulbs. The RNA metabolism of the polytene chromosomes during late fourth instar was studied using radioautographic techniques. Isolated glands were incubated in tritiated uridine for 10 to 30 min, and radioautographs were made of squash preparations. Despite the wide range of variation found among different larval cultures, the following pattern was observed. Just prior to and at the beginning of DNA puff formation, a period of intense extrachromosomal nucleolar and micronucleolar RNA synthesis occurs. After maximal development of the DNA puffs, the synthesis of extrachromosomal RNA is at a low point, while incorporation into bulbs and DNA puffs remains high. With the onset of the prepupal stage, all nuclear RNA synthesis ceases.     

Cytogenetic analysis of Malpighian tubule and salivary gland polytene chromosomes of Bactrocera oleae (Dacus oleae) (Diptera: Tephritidae).

Photomaps of the Malpighian tubule and the salivary gland polytene chromosomes of Bactrocera oleae (Dacus oleae) are presented and compared with those of the fat body. Five polytene chromosomes (10 polytene arms) corresponding to the five autosomes of the mitotic nuclei, as well as a heterochromatic mass corresponding to the sex chromosomes, are observed in the nuclei of the three somatic tissues. The most prominent features of each polytene chromosome, the reverse tandem duplications, as well as the rather unusual ectopic pairing of the telomeric regions of different chromosome arms, are described. The constancy of the banding pattern based on the analysis of the three larval tissues is discussed.     

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.     

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.     

Effects of gamma-radiation on the organisation of polytene chromosomes ofGlyptotendipes salinus Michailova (Chironomidae,Diptera)

A series of laboratory experiments onGlyptotendipes salinus were carried out in order to assess cytogenetic effects of different doses of gamma-radiation on polytene chromosomes, isolated from salivary glands. Chrinonomid larvae (III–IV larval stage) were irradiated with doses varying from 0.05 to 1.00 Gy (5–100 rad) and were bred under laboratory conditions until the fourth larval stage. Cytogenetic slides were analyzed for an estimation of occurrence of changes in the organization of the polytene chromosomes caused by gamma-radiation. A specific heterochromatin effect was found in certain chromosomes of the investigated species after 1.00 Gy irradiation. Decondensation of the centromeric heterochromatin and increased functional activity of Balbiany ring 2 were observed in the fourth (G) chromosome. Regression of the nucleolus of the first (AB) chromosome was detected.     

Polytene chromosomes of Oxytricha: Biochemical and morphological changes during macronuclear development in a ciliated protozoan

After conjugation in the ciliated protozoan, Oxytricha, polytene chromosomes are formed during the development of a macronucleus from a micronucleus. Here we report a microscopic study of these chromosomes and an analysis of their DNA. The polytene chromosomes of Oxytricha bear a strong morphological resemblance to the polytene chromosomes of the Dipteran salivary gland. The nucleus of a developing macronuclear anlage contains 120±2 polytene chromosomes and each chromosome has an average of 81 bands; a total of about 10,000 bands per nucleus. At a later stage in development, the number of bands per chromosome is reduced by a factor of four, presumably due to fusion of adjacent bands. The polytene chromosomes then break up into their constituent bands, each of which is encased in a vesicle. There are about 2,700 vesicles per nucleus. — During the growth of polytene chromosomes, there is a change in the relative proportion of sequences in the DNA. The DNA from polytene nuclei has a buoyant density of 1.695 g/cc, significantly lighter than the density of the original micronuclear DNA (1.698 g/cc to 1.702 g/cc). We interpret this buoyant density change to be the result of differential replication of DNA sequences during polytene chromosome growth. A second change in DNA composition occurs after the polytene stage of development, shown by a shift in buoyant density to 1.701 g/cc in the DNA of the mature macronucleus. During this second process, the molecular weight of the DNA is reduced from greater than 50×10⁶ daltons to about 2×10⁶ daltons.This paper is No. VI in the series, DNA of Ciliated Protozoa.     

Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera,Culicinae)

Dipteran polytene chromosomes provide an excellent model for understanding in species complexes, as well as for structural and functional cytogenetics. The status of species in the Culex pipiens complex is controversial and the use of polytene chromosomes for cytogenetic analysis in the subfamily Culicinae has been difficult because of methodological problems. In this study, Malpighian tubule polytene chromosomes were obtained from young (0 to 12 h, 20 C) and old (20 to 42 h, 28 C) laboratory-bred C. pipiens quinquefasciatus pupae. The chromosome maps for this species were constructed and compared with published data for C. pipiens pipiens and C. p. quinquefasciatus. Although the banding patterns were conserved between subspecies, analysis of the structural variations in the bands and interbands revealed differences apparently related to the physiological stage and ecogeographical strain. The organization of the centromeric regions in larval and pupal chromosomes showed greater similarity to each other than did those of pupal and adult chromosomes. The use of pupal polytene chromosomes for in situ hybridization with vector competence probes is discussed.     

Electron microscope mapping of the pericentric and intercalary heterochromatic regions of the polytene chromosomes of the mutant Suppressor of underreplication in Drosophila melanogaster

Breaks and ectopic contacts in the heterochromatic regions of Drosophila melanogaster polytene chromosomes are the manifestations of the cytological effects of DNA underreplication. Their appearance makes these regions difficult to map. The Su(UR)ES gene, which controls the phenomenon, has been described recently. Mutation of this locus gives rise to new blocks of material in the pericentric heterochromatic regions and causes the disappearance of breaks and ectopic contacts in the intercalary heterochromatic regions, thereby making the banding pattern distinct and providing better opportunities for mapping of the heterochromatic regions in polytene chromosomes. Here, we present the results of an electron microscope study of the heterochromatic regions. In the wild-type salivary glands, the pericentric regions correspond to the beta-heterochromatin and do not show the banding pattern. The most conspicuous cytological effect of the Su(UR)ES mutation is the formation of a large banded chromosome fragment comprising at least 25 bands at the site where the 3L and 3R proximal arms connect. In the other pericentric regions, 20CF, 40BF and 41BC, 15, 12 and 9 new bands were revealed, respectively. A large block of densely packed material appears in the most proximal part of the fourth chromosome. An electron microscope analysis of 26 polytene chromosome regions showing the characteristic features of intercalary heterochromatin was also performed. Suppression of DNA underreplication in the mutant transforms the bands with weak spots into large single bands.     

Photoinduced bonding of endogenous ecdysterone to salivary gland chromosomes of Chironomus tentans

Endogenous ecdysterone has been bonded to chromosomal loci by irradiation of Ch. tentans salivary glands. The hormone has been localized on the polytene chromosomes by indirect immunofluorescence microscopy. Hormone binding to chromosomes is stage-specific. Seven chromosomal loci could be identified which specifically bound hormone in larval salivary glands, and 21 chromosomal loci which specifically bound hormone in prepupal salivary glands. All puffs that have been described by Clever (1961) as being inducible by ecdysterone have been found to contain irreversibly bound ecdysterone in prepupal salivary gland chromosomes. A small number of puff sites in larval salivary gland chromosomes exhibited varying amounts of bound ecdysterone, (as judged by fluorescence intensity) most notably 117B and Balbiani rings 1 and 3 on chromosome IV. In addition to stage specific binding sites, there were many others showing equal binding of the hormone in both, larval and prepupal, stages of development. — Fluorescence intensities (reflecting the amount of bonded hormone) at puff sites along the tip section of the prepupal salivary gland chromosome arm IR have been computed indicating that differences between fluorescence intensities of different puffs can be expressed as multiples of a basic fluorescence intensity. Thus, the amount of fluorescence intensity (bonded hormone) in the various puffs may be quantized. — The data indicate that in Ch. tentans salivary glands ecdysterone acts, at the chromosomal level. The development of larvae into prepupae generates more puff sites and more hormone binding. This is discussed in the light of current models of hormone-receptor function.     

Tissue-specific schedule of selective replication in Drosophila nasutoides

Summary Dramatic changes in the DNA composition of post-mitotic versus mitotic and germ line nuclei occur during development in different organisms. Drosophila nasutoides possesses n=4 chromosomes which were quantified with a microphotometer in females. The diploid (2 C) DNA content was 0.79 pg or 7.7×10⁸ nucleotide pairs, calculated from brain metaphases and calibrated with hen erythrocyte nuclei. The individual elements comprised X=9%, 2=16%, 3=13%, and 4=62% of the total complement. In polytene nuclei of larval salivary glands which had undergone 11 endoreplication cycles, chromosome 4 contained only 1.55% of total Feulgen DNA. Thus, in contrast with other Drosophila genomes, where under-replicating material is dispersed to all elements, a huge quantity of non-endoreplicating DNA is restricted to a single chromosome. This permits accurate determination of the timing of under-replication in the single cell. The data presented here suggest that the schedule is tissue-specific. Larval hind gut and salivary duct nuclei begin under-replication during the first endocycle, whereas adult and larval salivary glands mainly begin during the second cycle. In Malpighian tubules the onset of selective DNA syntheses occurs during either the first or the second endocycle.     

Homologous banding patterns in the polytene chromosomes from the larval salivary glands and ovarian nurse cells of Anopheles stephensi liston (Culicidae)

A comparison of the banding patterns of two homologous polytene chromosome arms from the larval salivary gland and ovarian nurse cell complement of Anopheles stephensi is presented. The homologous chromosomes from the somatic larval salivary glands and germ-line derived ovarian nurse cells have essentially the same band-interband organisation. An analysis of the ³H-uridine labelling patterns of a small chromosome segment from the two tissues indicates that germ-line polytene chromosomes are not radically different from somatic polytene chromosomes in their patterns of gene expression.     

Differential staining of polytene chromosome bands in Chironomus by Giemsa banding methods

Two Giemsa banding methods (C banding and RB banding) are described which selectively stain the centromere bands of polytene salivary gland chromosomes in a number of Chironomus species. — By the C banding method the polytene chromosome appearance is changed grossly. Chromosome bands, as far as they are identifiable, are stained pale with the exception of the centromere bands and in some cases telomeres, which then are intensely stained reddish blue. — By the RB method the centromere bands are stained bright blue, whereas the remainder of the polytene bands stain red to red-violet. — Contrary to all other species examined, in Chironomus th. thummi numerous interstitial polytene chromosome bands, in addition to the centromere regions, are positively C banded and blue stained by RB banding. In the hybrid of Ch. th. thummi x Ch. th. piger only those interstitial thummi bands which are known to have a greater DNA content than their homologous piger bands are C banding positive and blue stained by the RB method whereas the homologous piger bands are C banding negative and red stained by RB banding. Ch. thummi and piger bands with an equal amount of DNA both show no C banding and stain red by RB banding. — It seems that the Giemsa banding methods used are capable of demonstrating, in addition to centromeric heterochromatin, heterochromatin in those interstitial polytene chromosome bands whose DNA content has been increased during chromosome evolution.     

Heterochromatic chromosomes and satellite DNAs of Drosophila nasutoides

Drosophila nasutoides is distinguished from other Drosophila species in that the metaphase karyotype shows a pair of very large V-shaped chromosomes. With Giemsa, a distinctive C-banding pattern is revealed along the arms of this large chromosome, indicating a largely heterochromatic nature. Furthermore, the banding patterns of the arms are symmetrical, indicating that it is an iso-chromosome. A comparison between the metaphase karyotype and polytene chromosomes suggests that the large V chromosome appears as the dot chromosome in polytene squash. One autosome has twice the arm length of typical Drosophila polytene chromosomes and arose either by centric fusion and a pericentric inversion, or by translocation connecting distal ends with a subsequent loss of one centromere. This chromosome appears to have a short arm which ectopically pairs with the proximal region of the long arm, representing a duplication of about ten bands. When the nuclear DNA is examined by neutral CsCl gradient, four satellites are observed. As much as sixty percent of the total DNA appears as satellites in the lysate of larval brains. No satellite was detectable in the lysate of salivary glands. These observations led us to suggest that the heterochromatic nature of the large V chromosome is due to the presence of all four satellites in this chromosome and that this large chromosome appears as the dot because of the under-reduplication of the satellites during polytenization.     

Veränderungen im Puffmuster und das Wachstum der Riesenchromosomen in Speicheldrüsen von Drosophila melanogaster aus spätlarvalen und embryonalen Spendern nach Kultur in vivo

Salivary glands from late third instar larvae of Drosophila melanogaster were transplanted into the abdomens of adult female and male flies and were kept in this medium from 6 to 120 h. Changes in the puffing pattern of chromosome arm III L were studied after the culture in vivo. Two noticeable puffs are induced. They are located in 68 B and 78 E. Neither of these loci show activity during normal development. — Front halves of embryos (6 to 9 h of age) were also transferred into adults. After 5 to 13 days in vivo they are able to develop and differentiate larval structures. Salivary glands, imaginal discs, fat body, Malpighian tubules and muscle fibers could be identified. Even 4 h old embryos can form polytene salivary gland chromosomes after a 13 day culture. These chromosomes can reach sizes comparable with the maximal size in normal development. In some nuclei an extensive growth leads to “supergiant” chromosomes. The puffs in 68B and 78E are formed in the polytenic chromosomes from embryonic implants as in cultured larval salivary gland chromosomes.     

Polytene chromosome maps of the melon fly Bactrocera cucurbitae (Diptera: Tephritidae).

Standard photographic maps of the polytene chromosomes are presented for the melon fly Bactrocera cucurbitae, a serious pest of fleshy fruits and vegetables. Five larval salivary gland polytene chromosomes (10 polytene arms) were isolated, and their characteristic features and landmarks have been recognized. Banding patterns of each of the polytene arms are presented, where variation in band intensity and puffs appear to reflect fundamental differences in chromosomes. The whole polytene genome has been typically mapped by dividing it into 100 sections and the subsections were lettered. The mitotic chromosomes of larval brain ganglia are also examined, five pairs of autosomes and an XX/XY sex chromosome pair. In addition, a heterochromatic mass corresponding to the sex chromosomes are observed in the polytene nuclei of salivary gland tissue. This investigation showed that B. cucurbitae has excellent cytological material for polytene chromosome analysis and proved to be very useful for obtaining more detailed genetic information on the pest's natural populations.     

兴义维蚋多线染色体研究（英文）

首次在国内对兴义维蚋Simulium (Wilhelmia) xingyiense的多线染色体进行研究, 并提供其多线染色体标准图。选取兴义维蚋的成熟幼虫, 用改良苯酚品红染色法进行唾腺多线染色体制备, 并进行测量、 描述及分析。结果表明： 兴义维蚋多线染色体数目为3对（2n=6）。Ⅰ号染色体具中央着丝粒, Ⅱ和Ⅲ号染色体均为亚中央着丝粒染色体。核仁组织者区位于Ⅰ号染色体短臂近着丝粒端。巴尔比尼氏环和双泡位于Ⅱ号染色体短臂近中央位置。3对染色体的着丝粒区可形成明显的染色中心。兴义维蚋多线染色体具有多态性的倒位, 倒位频率为0.64。兴义维蚋多线染色体的着丝粒、 核仁组织区、 巴氏环、 双泡等主要特征性结构的位置及形态恒定一致,可作为该种的重要鉴别特征。其多态性的倒位可为该蚋种在细胞水平上进行蚋类分类鉴别和系统发育等研究提供基础资料。     

An ovarian chromosome map of Anopheles stephensi

A polytene chromosome map of Anopheles stephensi has been prepared from the ovarian nurse cells of adult females. Many homologous regions can be recognized in comparisons between the ovarian and salivary gland chromosome maps but band for band homologies are not readily evident. The preparation of polytene chromosomes from ovarian nurse cells is easier than from the larval salivary glands and the results more consistent.