DNA supercoiling factor localizes to puffs on polytene chromosomes in Drosophila melanogaster

DNA supercoiling factor (SCF) was first identified in silkworm as a protein that generates negative supercoils in DNA in conjunction with eukaryotic topoisomerase II. To analyze the in vivo role of the factor, we cloned a cDNA encoding Drosophila melanogaster SCF. Northern analysis revealed 1.6- and 1.8-kb mRNAs throughout development. The longer mRNA contains an open reading frame that shares homology with mouse reticulocalbin whereas the shorter one encodes a truncated version lacking the N-terminal signal peptide-like sequence. An antibody against SCF detected a 45-kDa protein in the cytoplasmic fraction and a 30-kDa protein in the nuclear fraction of embryonic extracts. Immunoprecipitation suggests that the 30-kDa protein interacts with topoisomerase II in the nucleus, and hence that it is a functional form of SCF. Immunostaining of blastoderm embryos showed that SCF is present in nuclei during interphase but is excluded from mitotic chromosomes. In larvae, the antibody stained the nuclei of several tissues including a posterior part of the salivary gland. This latter staining was associated with natural or ecdysteroid-induced puffs on polytene chromosomes. Upon heat treatment of larvae, the staining on the endogenous puffs disappeared, and strong staining appeared on heat shock puffs. These results implicate SCF in gene expression.     

Structure of the smallest salivary-gland secretory protein gene in Chironomus tentans

The salivary gland secretion in the dipteran Chironomus tentans is composed of approximately 15 different secretory proteins. The most well known of the corresponding genes are the four closely related Balbiani ring (BR) genes, in which the main part of each approximately 40-kb gene is composed of tandemly arranged repetitive units. Six of the seven additional secretory protein genes described share structural similarities with the BR genes and are members of the same BR multigene family. Here we report the identification of a new secretory protein gene, the sp12 gene, encoding the smallest component of the C. tentans salivary gland secretion. The gene has a corresponding mRNA length of approximately 0.7 kb and codes for a protein with a calculated molecular weight of 7,619 Da. The sp12 gene was characterized in seven Chironomus species. Based on a comparison of the orthologous gene sequences, we conclude that the sp12 gene has a repetitive structure consisting of diverged 21-bp-long repeats. The repeat structure and the codon composition are similar to the so-called SR regions of the BR genes and the sp12 gene may represent a diverged member of the BR multigene family.     

Hybridization of tRNAs of Drosophila melanogaster to polytene chromosomes

Highly purified tRNAs from Drosophila melanogaster were iodinated with 125I and hybridized to squashes of polytene chromosomes of Drosophila silivary glands followed by autoradiography to localize binding sites. Most tRNAs hybridize strongly to more than one site and weakly to one or more additional sites. The major sites for various tRNAs are the following: tRNA2Arg, 42A, 84F1,2; tRNA2Asp, 29DE; tRNA3Gly, 22BC, 35BC, 57BC, tRNA2Lys, 42A, 42E; tRNA5Lys, 84AB, 87B; tRNA2Met, 48B5-7, 72F1-2, 83F-84A; tRNA3Met, 46A1-2, 61D1-2, 70F1-2; tRNA4Ser, 12DE, 23E; tRNA7Ser, 12DE, 23E; tRNA3aVal, 64D; tRNA3bVal, 84d3-4, 92b1-9; tRNA4Val, 56D3-7, 70BC.     

Su(UR)ES: a gene suppressing DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster polytene chromosomes

A genetic locus suppressing DNA underreplication in intercalary heterochromatin (IH) and pericentric heterochromatin (PH) of the polytene chromosomes of Drosophila melanogaster salivary glands, has been described. Found in the In(1)scV2 strain, the mutation, designated as Su(UR)ES, was located on chromosome 3L at position 34. 8 and cytologically mapped to region 68A3-B4. A cytological phenotype was observed in the salivary gland chromosomes of larvae homozygous and hemizygous for Su(UR)ES: (i) in the IH regions, that normally are incompletely polytenized and so they often break to form "weak points," underreplication is suppressed, breaks and ectopic contacts disappear; (ii) the degree of polytenization in PH grows higher. That is why the regions in chromosome arm basements, normally beta-heterochromatic, acquire a distinct banding pattern, i. e., become euchromatic by morphological criteria; (iii) an additional bulk of polytenized material arises between the arms of chromosome 3 to form a fragment with a typical banding pattern. Chromosome 2 PH reveals additional alpha-heterochromatin. Su(UR)ES does not affect the viability, fertility, or morphological characters of the imago, and has semidominant expression in the heterozygote and distinct maternal effect. The results obtained provide evidence that the processes leading to DNA underreplication in IH and PH are affected by the same genetic mechanism.     

Structural and functional properties of linker histones and high mobility group proteins in polytene chromosomes

Variants of histone H1 and high mobility group (HMG) proteins and their genes in Dipteran insects are being studied in our laboratory and have revealed different properties of DNA binding and intrachromosomal distribution. One of the H1 variants of Chironomus is found only in a minority of polytene chromosome bands and differs from the other H1 proteins of the same organism by genomic organization and by an inserted structural motif, the KAPKAP repeat, that is present also in single H1 variants of other, evolutionarily remote organisms. NH2-terminal peptides containing the KAPKAP repeat were found in vitro to interact with DNA, whereas no DNA interaction was observed with the homologous peptide of another H1 variant that does not contain the inserted KAPKAP repeat. We assume that H1 variants containing the KAP motif may interact with a stretch of linker DNA and package chromatin more tightly than other H1 variants. A large series of antibodies directed against different sites in all regions of the H1 molecule is being applied in studying the sites of interaction of the H1 molecule with other molecules in interphase chromatin in terms of antibody epitope accessibility. A search for insect proteins that share properties of the mammalian HMG proteins resulted in isolation and sequencing of two different HMG1 proteins and an HMGI protein. The HMG1 protein of the midge, Chironomus tentans, show a differential distribution in chromosomes. The more abundant cHMG1a protein appears uniformly distributed, whereas the less abundant cHMG1b protein could be localized only in chromosomal puffs. This strongly indicates that these highly similar proteins have different functions in chromatin. The Chironomus HMGI protein and the intron/exon organization of its gene were found to be very similar to human HMGI/Y proteins that are highly abundant in rapidly proliferating cells. Common properties of HMG1 and HMGI proteins include high affinity interaction with AT-rich DNA, irregular DNA structures, and the capacity to bend DNA. These properties suggest that the HMG proteins may have an architectural role in assembling different types of chromatin.     

Stretched polytene chromosomes--model for studying the functional organization of eukaryotic chromosomes

To localize functional loci on cytological maps of polytene chromosomes we propose to use 10-100 times stretched chromosomes. Three different ways of stretchening are briefly considered: the squash tissue preparation, when chromosomes are stretched by hydrodynamical forces; the treatment of isolated polytene chromosomes in 10-minus 4M EDTA OR 0.8M NaCL with subsequent change of these solution for saline when abrupt structural changes occur in chromosomes and they become morphologically homogeneous threads (Gruzdev and Belaya, 1973); and, finally, the use of microneedles of the micromanipulator. After an intense (ca. 100 times) stretchening, the autoradiography is sufficient to localize the loci within one micron length of double helical DNA molecule.     

Ecdysteroid receptor and ultraspiracle from Chironomus tentans (Insecta) are phosphoproteins and are regulated differently by molting hormone

A consists of berberine chloride and an extract from geranium herb. To clarify mechanisms of the antidiarrheal effect of Phelloberin-A, we investigated the astringent action by determining its binding activity to rabbit hemoglobin and effects on active transport, which was indicated by short-circuit current (Isc), in rat jejunum by the Ussing chamber technique. The effects of berberine chloride and geranium herb on both the binding activity to hemoglobin and the electrophysiological parameters such as Isc were compared with those of the antidiarrhoeicas, tannic acid, albumin tannate and bismuth subnitrate. Geranium herb, tannic acid and bismuth subnitrate increased significantly the binding activity to hemoglobin at concentrations of > 1 mg/ml, > 0.3 mg/ml and 10 mg/ml, respectively, but berberine or albumin tannate did not. Geranium herb and tannic acid dose-dependently and moderately increased Isc in rat jejunal mucosa and the increase became significant at a concentration of 10 mg/ml. Neither berberine chloride, albumin tannate nor bismuth subnitrate affected Isc. In contrast, cholera toxin, which increases the secretion from intestinal mucosa to the lumen and induces diarrhea, decreased Isc at a concentration of 0.1 mg/ml. The decrease of Isc induced by cholera toxin was antagonized by pretreatment with geranium herb (10 mg/ml), indicating that geranium herb inhibited the toxin-induced increase in secretion. These results suggest that geranium herb possesses an astringent action and moderately increases Isc across the intestinal mucosa. Therefore, the effects may support an antidiarrheal effect of both geranium herb and Phelloberin-A.     

Electron spectroscopic imaging analyses of the distribution of phosphorus in Balbiani ring granules and in the surrounding nucleoplasm

When they were introduced to the world market in the 1980s, levonorgestrel subdermal implants offered the promise of an exciting alternative to traditional hormonal contraception. They provide highly effective, long-acting protection from pregnancy, without the need for user compliance. Broad acceptability of the drug has been reported throughout the world. Recently, however, the implants have met with opposition. The drug is associated with a variety of adverse effects, and removal of implants can be problematic. Serious events have been reported in women using levonorgestrel subdermal implants, although causal relationships have not been demonstrated. Additionally, concerns have been raised over the potential for coercive use of the drug. Numerous law suits have been filed alleging serious problems with implants. As a result, the drug has received considerable negative media attention. Before the controversy over levonorgestrel subdermal implants erupted, contraceptive development had declined, resulting from limitations to profits and funding, legal threats, and changes in the insurance industry. The levonorgestrel subdermal implant experience may serve to accelerate this trend. While the introduction of levonorgestrel subdermal implants offered an alternative to the current array of medical contraception, its experience may serve to dampen future contraceptive development efforts. Costly litigation and much controversy involving the implants have acted to create disincentives to further research and development of new methods of medical contraception.     

Structure variations of short polytene chromosomes in 2 pyrenean populations of Bilobella aurantiaca Caroli (Collemboles)

Structural variations of the short polytene chromosomes VI and VII occur in two Pyrenean populations of Bilobella aurantiaca. They are the result of longitudinal cleavage and also of variations in the number of the thin bands. They are not dependent on ecological factors, but often correlate with the chromosome polytenic stage.     

Alphoid repetitive DNA in human chromosomes

The thesis describes the first extensive DNA sequence analysis that demonstrated that the tandemly repeated alphoid DNA in the centromere of the human chromosomes consists of distinct subfamilies and in a number equal to or exceeding the number of chromosomes. The expected presence of only one or a few distinct subfamily on individual chromosomes was supported by the characterization of an extremely well-defined subfamily specific for chromosome 7 and represented in the original collection of subfamilies. The pattern of chromosome-specificity breaks down among the acrocentric chromosomes where chromosomes 13 and 21 were found to share one and chromosomes 14 and 22 to share another specific subfamily. By in situ hybridization these subfamilies were shown not to be shared by other chromosomes. The remarkable pairwise pattern of sequence homogenization was present also in the chimpanzee genome raising the question of its biological role. However, the subfamilies on these human and chimpanzee chromosomes are not orthologous but were shown to originate from two evolutionarily different repeat families. It follows that dramatic sequence evolution has occurred in one or both species during or after separation. The sequence evolution might even occur at a higher rate in humans. This possibility was studied in orthologous alphoid sequences on the X chromosome of humans and the great apes. The analysis supports the general view that our closest relative is the chimpanzee and indicates that the rate of recombination is increased in the human repeat DNA. A "molecular clock" running faster in this DNA may have evolutionary implications. Finally, the usefulness of alphoid subfamilies as chromosome-specific markers is illustrated in a cytogenetic dissection of the centromeric region of Robertsonian translocations. The breakpoints were located to satellite III DNA leaving these chromosomes dicentric. The order of the different tandem DNAs on the p-arm of the acrocentric chromosomes could also be established.     

Polymorphism of pericentromere heterochromatin of polytene chromosomes of ovarian trophocytes in natural populations of the malaria mosquito Anopheles messeae Fall

The HBsAg status and demographic data of 2480 pregnant women who attended antenatal clinics at Maternal and Child Health Centres in Hong Kong were collected by means of a self-administered questionnaire over a 1-week period in July 1996, to explore the underlying reason of a higher than expected HBsAg prevalence. Local women constituted 49.2% of the sample, mainland Chinese 39.7% and others 11.1%. The overall HBsAg prevalence was 10.0%. When related to place of birth, those born in Hong Kong had a prevalence of 8.4% whereas the prevalence of those born in mainland China was 13.1% (P < 0.001). The overall HBsAg carriage rate is high because of a higher rate in immigrants in the community. It is apparent that the HBsAg prevalence of local people in Hong Kong has been decreasing in the past decade. Overall, the current HBsAg carriage rate in the local adult population is estimated to have declined to about 8%.     

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

The effect of platelet factor 4 (PF4) on myoblast cultures with or without basic fibroblast growth factor (bFGF) or other growth factors was investigated in the present in vitro experiments, with reference to bFGF binding to myoblast membrane fraction. When PF4 was added to the culture medium 1 day after myoblast cultivation, the nuclei of both myoblasts and myotubes were markedly reduced in number in a dose-dependent manner, whereas the inhibitory effect of PF4 on myoblast development was not observed when PF4 was added to the culture medium 3, 7, or 14 days after myoblast cultivation. In contrast, bFGF significantly increased the numbers of myoblast and myotube nuclei. When bFGF and PF4 were simultaneously added to the culture medium, PF4 abolished the facilitatory effects of bFGF on myogenesis. The real-time biospecific interaction analysis (BLA) core system showed that the myoblast membrane fraction at 1 day after cultivation contains bFGF-binding elements which are blocked by PF4 in a dose-dependent manner. Moreover, [126I]-bFGF binding experiments indicated the existence of both high and low affinity binding sites on myoblast membranes, although the high affinity binding sites decreased in number and the dissociation constant increased in value as the culture period was prolonged. Among the six other growth factors examined, acidic fibroblast growth factor and platelet-derived growth factor-BB stimulated myogenesis, and their effects were blocked by PF4 treatment. These findings suggest that: 1) PF4 inhibits myoblast proliferation and myotube formation only for a limited initial period of cultivation, possibly because of the time-dependent down-regulation of high affinity bFGF receptors: and 2) PF4 may be used as a tool to investigate the function of endogenous heparin-binding growth factors upregulated transiently at a certain developmental stage or in case of tissue damage and repair, even though it is not monospecific to bFGF.     

The terminal DNA structure of mammalian chromosomes

In virtually all eukaryotic organisms, telomeric DNA is composed of a variable number of short direct repeats. While the primary sequence of telomeric repeats has been determined for a great variety of species, the actual physical DNA structure at the ends of a bona fide metazoan chromosome with a centromere is unknown. It is shown here that an overhang of the strand forming the 3' ends of the chromosomes, the G-rich strand, is found at mammalian chromosome ends. Moreover, on at least some telomeres, the overhangs are > or = 45 bases long. Such surprisingly long overhangs were present on chromosomes derived from fully transformed tissue culture cells and normal G0-arrested peripheral leukocytes. Thus, irrespective of whether the cells were actively dividing or arrested, a very similar terminal DNA arrangement was found. These data suggest that the ends of mammalian and possibly all vertebrate chromosomes consist of an overhang of the G-rich strand and that these overhangs may be considerably larger than previously anticipated.     

Retrotransposon-related DNA sequences in the centromeres of grass chromosomes

Several distinct DNA fragments were subcloned from a sorghum (Sorghum bicolor) bacterial artificial chromosome clone 13I16 that was derived from a centromere. Three fragments showed significant sequence identity to either Ty3/gypsy- or Ty1/copia-like retrotransposons. Fluorescence in situ hybridization (FISH) analysis revealed that the Ty1/copia-related DNA sequences are not specific to the centromeric regions. However, the Ty3/gypsy-related sequences were present exclusively in the centromeres of all sorghum chromosomes. FISH and gel-blot hybridization showed that these sequences are also conserved in the centromeric regions of all species within Gramineae. Thus, we report a new retrotransposon that is conserved in specific chromosomal regions of distantly related eukaryotic species. We propose that the Ty3/gypsy-like retrotransposons in the grass centromeres may be ancient insertions and are likely to have been amplified during centromere evolution. The possible role of centromeric retrotransposons in plant centromere function is discussed.