The Drosophila Dpit47 protein is a nuclear Hsp90 co-chaperone that interacts with DNA polymerase alpha.

Hsp90 is gaining increasing importance as a protein involved in controlling the normal functioning of the cell. To do this it apparently interacts with a battery of co-chaperone proteins that are involved in both substrate recognition and the progression of the Hsp90 catalytic pathway. In this report we have identified the Drosophila Dpit47 protein (DNA polymerase interacting tpr containing protein of 47 kDa) through its interaction with the DNA polymerase alpha. This protein is a predominantly nuclear protein, which forms a tight and stoichiometric interaction with Hsp90 and shows interaction with Hsp70. It also has substantial homology to other known Hsp90 co-chaperones, e.g. CNS1 and hop1, making it likely that this protein also functions as an Hsp90 co-chaperone. The interaction with the DNA polymerase alpha is not related to the special situation in early embryos where there are large amounts of maternal protein stockpiles of the polymerase, as it occurs to the same level in early and late embryos and also in proliferating cell culture. However, it does not occur in quiescent cells, making it likely that the protein is related to proliferation. This is also consistent with Dpit47 expression being higher in proliferating cells. The interaction between the Dpit47 and the polymerase takes place predominantly in the nucleoplasm, and seems to involve several subunits of the polymerase in comparable amounts, making it unlikely that it is solely required for the assembly of the polymerase complex. The polymerase can also be seen to interact with Hsp90, and the interaction between Dpit47 and the polymerase is increased by the specific Hsp90 inhibitor geldanamycin. This suggests that a complex of the Dpit47, Hsp90 and DNA polymerase exists in the cell. The interaction between DNA polymerase alpha and Dpit47 completely inhibits the activity of the polymerase. These results suggest that Hsp90 acts as a chaperone for DNA polymerase alpha and that this interaction is mediated through the novel co-chaperone Dpit47. This provides the first suggestion of a role for chaperones in DNA replication in higher eukaryotes.

Df31 is a novel nuclear protein involved in chromatin structure in Drosophila melanogaster.

We originally isolated the Df31 protein from Drosophila embryo extracts as a factor which could decondense Xenopus sperm, by removing the sperm specific proteins and interacting with histones to facilitate their loading onto DNA. We now believe that this protein has a more general function in cellular DNA metabolism. The Df31 gene encodes a very hydrophilic protein with a predicted molecular mass of 18.5 kDa. Immunostaining showed that Df31 was present in a wide range of cell types throughout differentiation and in both dividing and non-dividing cells. In all cases the protein is present in large amounts, comparable with the level of nucleosomes. Injection of antisense oligonucleotides to lower the level of Df31 in embryos caused severe disruption of the nuclear structure. Large irregular clumps of DNA were formed, and in most cases the amount of DNA associated with each clump was more than that found in a normal nucleus. Immunofluorescence, cell fractionation, and formaldehyde cross-linking show that Df31 is associated with chromatin and that a significant fraction of it binds very tightly. It also shows the same binding characteristics when loaded onto chromatin in vitro. Chromatin fractionation shows that Df31 is tightly associated with nucleosomes, preferentially with oligonucleosomes. Despite this no differences were observed in the properties of nucleosomes loaded in the in vitro system in the presence and absence of Df31. These results suggest that Df31 has a role in chromosomal structure, most likely acting as a structural protein at levels of folding higher than that of nucleosomes.

Localisation of the DmCdc45 DNA replication factor in the mitotic cycle and during chorion gene amplification.

The cdc45 protein was originally identified in Saccharomyces cerevisiae and shown to be essential for initiation of eukaryotic DNA replication. Subsequent isolation and characterisation of the corresponding genes from fission yeast, Xenopus and mammals also support a replication role for the protein in these species. They further suggest that during the course of its function cdc45 interacts with a number of other replication proteins, including minichromosome maintenance proteins, the origin recognition complex and DNA polymerase alpha. We have cloned the gene coding for cdc45 protein from Drosophila melanogaster. We have analysed the expression pattern of the cdc45 protein throughout the cell cycle and the life cycle using a combination of indirect immunofluorescence and subcellular fractionation. Our data show that cellular localisation and developmental regulation of the protein is consistent with a role in DNA replication. DmCdc45 is predominantly expressed in proliferating cells. In addition, its subcellular location is nuclear during interphase and the protein shows association with chromatin. The chromatin-associated form of the protein shows a post-translational modification, which may be involved in control of the action of the protein. DmCdc45 shows interactions with mcm proteins, however, the interactions detected show some specificity, perhaps suggesting a preferential association with particular mcm proteins. In addition we show that a stoichiometric mcm interaction may not be obligatory for the function of cdc45 in follicle cell replication, because, unlike the mcm proteins, DmCdc45 localises to the chorion amplification foci in the follicle cells of the ovary.

Importin-alpha3 is required at multiple stages of Drosophila development and has a role in the completion of oogenesis.

The Drosophila importin-alpha3 gene was isolated through its interaction with the large subunit of the DNA polymerase alpha in a two-hybrid screen. The predicted protein sequence of Importin-alpha3 is 65-66% identical to those of the human and mouse importin-alpha3 and alpha4 and 42.7% identical to that of Importin-alpha2 (Oho31/Pendulin), the previously reported Drosophila homologue. Both Importin-alpha3 and Importin-alpha2 interact with similar subsets of proteins in vitro, one of which is Ketel, the importin-beta homologue of Drosophila. importin-alpha3 is an essential gene, whose encoded protein is expressed throughout development. During early embryogenesis, Importin-alpha3 accumulates at the nuclear membrane of cleavage nuclei, whereas after blastoderm formation it is characteristically found within the interphase nuclei. Nuclear localisation is seen in several tissues throughout subsequent development. During oogenesis its concentration within the nurse cell nuclei increases during stages 7-10, concomitant with a decline in levels in the oocyte nucleus. Mutation of importin-alpha3 results in lethality throughout pupal development. Surviving females are sterile and show arrest of oogenesis at stages 7-10. Thus, Importin-alpha3-mediated nuclear transport is essential for completion of oogenesis and becomes limiting during pupal development. Since they have different expression patterns and subcellular localisation profiles, we suggest that the two importin-alpha homologues are not redundant in the context of normal Drosophila development.

Cloning and characterisation of the gene for the large subunit of the DNA primase from Drosophila melanogaster.

We have cloned the gene for the large subunit of the DNA primase from Drosophila melanogaster, and mapped it to position 77b on chromosome 3. The central region of the protein shows high similarity with homologues from other species, but the N- and C-termini diverge. The protein is enriched in replicating tissues, and consistent with this the region upstream of the gene contains close matches to the sites of two transcription factors - Dref and E2f - which have been implicated in controlling proliferation-associated genes.

Molecular and cellular characterization of CRP1, a Drosophila chromatin decondensation protein.

CRP1, a Drosophila nuclear protein that can catalyze decondensation of demembranated Xenopus sperm chromatin was cloned and its primary structure was deduced from cDNA sequence. Alignment of deduced amino acid sequence with published sequences of other proteins revealed strong homologies to Xenopus nucleoplasmin and NO38. CRP1 is encoded by one or several closely related genes found at a single locus, position 99A on the right arm of chromosome 3. CRP1 mRNA is expressed throughout Drosophila development; it is highest during oogenesis and early embryogenesis. mRNA levels correlate closely with levels of protein expression measured previously. Results of chemical crosslinking indicate that CRP1 is either tetrameric or pentameric; similar ambiguity was revealed by direct visualization using scanning transmission electron microscopy. Consistent with previously published results, parallel crosslinking studies of Xenopus nucleoplasmin suggested a pentameric structure. Scanning transmission electron microscopic examination after negative staining revealed that CRP1 and Xenopus nucleoplasmin are morphologically similar. CRP1 is able to substitute for nucleoplasmin in Xenopus egg extract-mediated sperm chromatin decondensation. In vitro, CRP1-induced decondensation is accompanied by direct binding of CRP1 to chromatin.

Regulation of scute function by extramacrochaete in vitro and in vivo.

The pattern of adult sensilla in Drosophila is established by the dosage-sensitive interaction of two antagonistic groups of genes. Sensilla development is promoted by members of the achaete-scute complex and the daughterless gene whereas it is suppressed by whereas extramacrochaete (emc) and hairy. All these genes encode helix-loop-helix proteins. The products of the achaete-scute complex and daughterless interact to form heterodimers able to activate transcription. In this report, we show that (1) extra-macrochaete forms heterodimers with the achaete, scute, lethal of scute and daughterless products; (2) extramacrochaete inhibits DNA-binding of Achaete, Scute and Lethal of Scute/Daughterless heterodimers and Daughterless homodimers and (3) extramacrochaete inhibits transcription activation by heterodimers in a yeast assay system. In addition, we have studied the expression patterns of scute in wild-type and extramacrochaete mutant imaginal discs. Expression of scute RNA during imaginal development occurs in groups of cells, but high levels of protein accumulate in the nuclei of only a subset of the RNA-expressing cells. The pattern is dynamic and results in a small number of protein-containing cells that correspond to sensillum precursors. extramacrochaete loss-of-function alleles develop extra sensilla and correspondingly display a larger number of cells with scute protein. These cells appear to arise from those that in the wild type already express scute RNA; hence, extramacrochaete is a repressor of scute function whose action may take place post-transcriptionally.

Spot test for detection of dapsone in urine: an assessment of its validity and interpretation in monitoring dapsone self-administration.

This study aimed to assess the validity and interpretation of the spot test for monitoring dapsone self-administration that employs filter paper impregnated with a modified Ehrlich's reagent. Urine specimens obtained from 20 volunteers, who took 100 mg dapsone for four days in succession, were investigated by this test. Findings indicate that spot tests will be negative after an average of three missed doses of dapsone, if compared with a standard of 5 micrograms dapsone per ml of urine. No negative spots are expected in fully compliant patients, and no positive spots are expected in patients who did not take dapsone for a week or longer. In the context of the treatment goal, it is argued that this degree of sensitivity makes the spot test a valid tool for the monitoring and management of patient compliance in leprosy control programs.

A simple urine spot test for monitoring dapsone self-administration in leprosy treatment.

A simple urine spot test for monitoring patient compliance to dapsone self-administration in leprosy therapy was recommended by WHO but later abandoned. The present article describes some important improvements to the test, which is characterized by its validity and straightforwardness.

ELISA tests for dapsone and pyrimethamine and their application in a malaria chemoprophylaxis programme.

Enzyme-linked immunosorbent asays (ELISAs) are described for determining levels of dapsone and pyrimethamine in urine. Both assays have a sensitivity of about 20 mug/l and are reproducible, but each produces some false positives. The problem of false positive reactions was partially obviated by requiring positive results in both assays. In a pilot study involving 50 children aged 3 months to 4 years who were given a single dose of Maloprim (pyrimethamine + dapsone), 75% were positive for dapsone 7 days after administration of the drug, while 25% were still positive 15 days after its administration. The corresponding proportions for pyrimethamine were 73% and 30%, respectively. Comparison of the results obtained in a larger chemoprophylaxis trial with those from the pilot study indicated that the assays described could be used to investigate whether antimalarials had been taken.

Relation between nutritional requirements and susceptibilities to antibiotics of strains of Neisseria gonorrhoeae from pharyngeal and anogenital sites.

Auxotyping and antibiotic susceptibility testing was carried out on 100 consecutive isolates of non-penicillinase producing strains of Neisseria gonorrhoeae (non-PPNG) taken on the same occasion from throat and anogenital sites, 100 non-PPNG strains isolated from the throat only, and 100 non-PPNG strains from anogenital sites only. Non-requiring, non-requiring and phenylalanine inhibited, proline requiring, amino acid group requiring, and arginine requiring auxotypes predominated in all groups of patients. Strains of the arginine requiring type found in anogenital sites tended to have additional requirements. The auxotypes and susceptibility to antibiotics of 93 of the 100 paired cultures from the throat and anogenital sites were identical. There appeared to be a slight preponderance of moderately susceptible strains in isolates from the throat. A strong correlation was found between nutritional requirements and sensitivity to antibiotics. Auxotypes of and minimum inhibitory concentrations (MICs) for N gonorrhoeae isolated from the throat were mostly the same as the auxotypes of and MICs for strains that were circulating during the study period in Amsterdam.

Intramuscular injection of dapsone in therapy for leprosy: a new approach.

Dapsone is the drug of first choice in the treatment of leprosy. Although the oral route of administration has been mostly used, recent studies of patient compliance revealed that only about 50% of the tables received by the patients are actually taken. It is generally assumed that irregular self-medication favors the development of dapsone resistance. The need for a more reliable route of administration led us to investigate the possibility of an i.m. dapsone depot injection. To achieve effective blood levels for 3-4 weeks, suspensions of large dapsone particles in an aqueous vehicle were made. In a trial with 20 leprosy patients in Nigeria, injection of 900 mg dapsone i.m. as a mixture of particle sizes less than 90 micrometer (20%) and 90-125 micrometer (80%) resulted in a serum level above 0.5 microgram/ml for 18 +/- 5 days with a mean peak concentration of 3.1 +/- 0.9 microgram/ml (n = 10). Injection of 1200 mg of the same particle-size mixture led to peak concentrations of 2.7 +/- 1.0 microgram/ml and maintenance of the level above 0.5 microgram/ml for 25 +/- 3 days (n = 5). After injection of 1200 mg (particle size less than 90 micrometer), serum levels were kept above 0.5 microgram/ml for 21 +/- 5 days with a maximum concentration of 3.9 +/- 1.2 microgram/ml (n = 5). Serum levels were measured using a rapid non-extractive HPLC method. The injections were very well tolerated. Due to these encouraging results, the dosage and formulation will be further optimized.