An Entry/Gateway? cloning system for general expression of genes with molecular tags in Drosophila melanogaster

Background  

Tagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. However, over-expression of fusion proteins sometimes leads to the unwanted lethality or developmental defects. Therefore, vectors that can express tagged proteins at physiological levels are desirable tools for studying dosage-sensitive proteins. We developed a set of Entry/Gateway^? vectors for expressing fluorescent fusion proteins in Drosophila melanogaster. The vectors were used to generate fluorescent CP190 which is a component of the gypsy chromatin insulator. We used the fluorescent CP190 to study the dynamic movement of related chromatin insulators in living cells.     

Entwicklungsphysiologische und biometrische Untersuchungen an DDT-sensiblen und resistenten St?mmen von Drosophila melanogaster

Zusammenfassung 1. Zwischen 2 DDT-sensiblen und 3 resistenten Stämmen von Drosophila melanogaster, die durch unabhängige Selektionszucht auseinander abgeleitet waren, ergaben sich entwicklungsphysiologische und morphologische Differenzen in gleicher Richtung, aber unterschiedlicher Expressivität.2. Die larvale Entwicklungsdauer war bei allen resistenten Stämmen verlängert. In Übereinstimmung damit schlüpften die resistenten Imagines später, ihr Lebendgewicht war erhöht.3. Die Schlüpfraten der Imagines, bezogen auf eine bestimmte Eizahl, sowie die Fertilität der Stämme zeigten keine Unterschiede.4. Mehrere Körperteile der resistenten Stämme ließen im Vergleich zu den sensiblen Rassen eine allometrische Wachstumszunahme erkennen. Der prozentuale Zuwachs der einzelnen Merkmale zeigte starke Unterschiede. Nur bei einem resistenten Stamm fand sich in einem Merkmal — den Tibien — eine Verkürzung gegenüber der sensiblen Vergleichsgruppe.5. Es wird diskutiert, welche möglichen Zusammenhänge zwischen der DDT-Resistenz und den abgeänderten Eigenschaften bestehen und welche Ursachen ihnen zugrunde liegen.Mit 1 Textabbildung     

Bestimmung des Heterochromatisierungsstadiums beim white-Positionseffekt mittels r?ntgeninduzierter mitotischer Rekombination in der Augenanlage von Drosophila melanogaster

Summary Position-effect variegation of eye pigmentation in the examined Dp(1;3)N ^264-58 females is due to an insertion of a X-chromosome section including the white-locus into the proximal heterochromatic region of the third chromosome. The light and dark pigmented areas have a cell lineage basis (Fig. 2). Flies bearing the w ⁺-duplication had two X-chromosomes marked with w ^a lz ⁵⁰ e and w ^a rb rux ² respectively (Fig. 3). X-ray induced mitotic recombination in presumptive eye cells of larvae resulted in w ^a lz ^50e /w ^a rb rux ² twin mosaic spots in the adult eyes. After young larvae were treated twin spots appeared, which had one partner light colored and one dark. Such combinations were rarely found after older larvae were treated. Treatment of young larvae in addition produced twin spots with one or both partners variegated (Figs. 5 and 6). Sometime after the stage at which younger larvae were treated and before the stage at which older larvae were treated the translocated w ⁺-gene in each cell was determined for function or no function. As a result the progeny of each of these cells synthesized pigment or not during the pupal stage. At a temperature of 25.5° C the developmental phase during which determination, i.e. heterochromatization of the white gene, takes place, begins not earlier than 39 hours after egg laying and ends about 8 hours later (Fig. 7). In females heterozygous for the short arm of the heterochromatic Y-chromosome linked distally to the X-chromosome (Y ^S X/X) one twin spot partners is homozygous for this arm (Y ^SX/Y^S X), the other lacks it (X/X; Fig.4a). The Y ^SX/Y^S X-partner were more frequently dark pigmented than the X/X-partners (Tables 3 and 4). This shows that heterochromatization of the translocated w ⁺-genes is markedly influenced by the genotype of the single cell. When two genotypes with varied amounts of heterochromatin were compared (Fig. 4) no difference in the phases of heterochromatization could be observed (Table 5). Therefore, when position-effect variegation is modified by varying the amount of heterochromatin in the genome the modification is probably not due to a shift in the phase of heterochromatization.

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Vorgelegt von E. Hadorn     

Dynamic properties of pH-dependent structural organization of the amyloidogenic β-protein (1-40)

The structural organization of the amyloidogenic β-proteins containing 40 amino acid residues (Aβ40) was studied by the high temperature molecular dynamics simulations in the acidic (pH~3) and basic (pH~8) pH regions. The obtained data suggest that the central Ala21-Gly29 segment of Aβ40, can adopt folded and partially unfolded structures. At the basic pH, this segment forms folded structures, stabilized by electrostatic interactions and hydrogen bonds. At the acidic pH, it forms partially unfolded structures. Two other segments flanking to the central segment exhibit the propensity to adopt unstable inter-converting α-helical, 310-helical and turn-like structures. One of these segments is comprised of the Ala30-Val36 residues at both of the considered pHs. The second segment is comprised of the Glu11-Phe20 at the basic pH and of the Glu11-Val24 residues at the acidic pHs. The revealed pH-dependent structuration of the Aβ40 allowed us to suggest a possible scenario for initial Aβ aggregation. According to this scenario, the occurrence of the partially unfolded states of the Ala21-Gly29 segment plays main role in the Aβ oligomerization process.     

Sexual conflict over the duration of copulation in Drosophila montana : why is longer better?

Background  

Conflicts of interest between the sexes are increasingly recognized as an engine driving the (co-)evolution of reproductive traits. The reproductive behaviour of Drosophila montana suggests the occurrence of sexual conflict over the duration of copulation. During the last stages of copulation, females vigorously attempt to dislodge the mounting male, while males struggle to maintain genital contact and often successfully extend copulations far beyond the females' preferred duration.     

The localization of “ordinary” sex-linked genes in section 20 of the polytene X chromosome of Drosophila melanogaster

A cytogenetic procedure is described whereby a combination of polytene chromosome analysis and complementation mapping has permitted the unequivocal localization of ordinary sex-linked genes (those not covered by the Y-chromosome) in Section 20, the most proximal region of Bridges' (1938) map of the polytene X-chromosome. Thus far, eleven functional units in Section 20 distal to the bobbed locus, but none proximal, have been resolved. We suggest that the polytenized portion of Section 20, which heretofore has traditionally been considered as heterochromatic, corresponds, in fact, with the euchromatic portion of the mitotic X-chromosome.     

Characteristics of βA chromosome haplotypes in Japanese

DNA polymorphism patterns linked to the ^A-globin gene were analyzed in healthy Japanese using four different restriction endonucleases. The chromosomes with the ^A-globin gene were mapped through an evaluation of the presence of seven different restriction sites (HincII 5 to ; HindIII in ^G and ^A; HincII in, and 3 to, ₁; AvaII in ; Bam-HI 3 to ). Among 36 chromosomes analyzed, 20 chromosomes had a haplotype of [+–––––+]. Among 55 individuals examined, 7 possessed a homozygous haplotye of [+–––––+]. All Japanese with the ^AT-globin gene had a subhaplotype of [–++–+] 5 to the -globin gene. Their major haplotypes were [–++–+–+] and [–++–++–]. It was expected that the presence of the ^AT-globin gene in Japanese may be deduced from subhaplotypes 5 to the -globin gene.     

Tandem organization of StarkB element (22.8 kb) in the maize B chromosome

A very large repetitive element (StarkB, 22.8 kb) is present in the maize B chromosome, presumably not organized as tandem arrays. Results of the current study are contrary to this notion. Out of eighteen StarkB-carrying sequences, nine were the expected internal fragment of StarkB, and nine others were fragments spanning two StarkB elements. One of the two StarkB components, GrandeB, was flanked in all clones with identical target sequences, as opposed to other Grandes that are associated with different target sequences. Also observed was a prominent Southern signal associated with a fragment representing the junction of two adjacent StarkB units. A clone possessing a structure inverse to that of the second component of StarkB is proposed to be the initial element into which a GrandeB inserted to derive StarkB. Most, if not all, isolated StarkB arrays were not the original form, being disrupted by the invasion of various mobile elements intertwined with various stages of amplification. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the GenBank database under the accession numbers EF468500 to EF468511, EU528676, and FJ386410 to FJ386429.     

Gene organization and plasticity of the β-lactam genes in different filamentous fungi

The genes pcbAB, pcbC and penDE encoding enzymes that catalyze the three steps of the penicillin biosynthesis have been cloned from Penicillium chrysogenum and Aspergillus nidulans. They are located in a cluster in Penicillium chrysogenum, Penicillium notatum, Aspergillus nidulans and Penicillium nalgiovense. The three genes are clustered in chromosome I (10.4 Mb) of P. chrysogenum, in chromosome II of P. notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. The cluster of the penicillin biosynthetic genes is amplified in strains with high level of antibiotic production. About five to six copies of the cluster are present in the AS-P-78 strain and 11 to 14 copies in the E1 strain (an industrial isolate), whereas only one copy is present in the wild type (NRRL 1951) strain and in the low producer Wis 54-1255 strain. The amplified region in strains AS-P-78 and E1 is arranged in tandem repeats of 106.5 or 57.6-kb units, respectively. In Acremonium chrysogenum the genes involved in cephalosporin biosynthesis are separated in at least two clusters. The pcbAB and pcbC genes are linked in the so-called early cluster of genes involved in the cephalosporin biosynthesis. The late cluster, which includes the cefEF and cefG genes, is involved in the last steps of cephalosporin biosynthesis. The early cluster was located in chromosome VII (4.6 Mb) in the C10 strain and the late cluster in chromosome I (2.2 Mb). Both clusters are present in a single copy in the A. chrysogenum genome, in the wild-type and in the high cephalosporin-producing C10 strains.     

Characterization of two moderately repetitive DNA components localized within the β-heterochromatin of Drosophila hydei

Two nuclear DNA fractions from Drosophila hydei were isolated by silver ion and actinomycin D binding and centrifugation in isopycnic salt gradients. Neither fraction is composed of nor does it contain any highly repetitive simple sequence DNA, as shown by melting and reassociation studies. — One fraction has a CsCl density of 1.702 g/cm³ and hybridizes in situ with the -heterochromatin of the chromocenter in polytene cells. This DNA fraction was found to be replicated during polytenization. In diploid cells this 1.702 g/cm³ component hybridizes to the heterochromatin of all four large autosome pairs, the middle part of the long arm of the Y-chromosome, but not to the X-heterochromatin. — A second fraction has a CsCl density of 1.697 g/cm³ and hybridizes in situ with polytene cells to the chromocenter and the nucleolus, but on metaphase chromosomes only to the nucleolus organizer regions.     

Frequent loss of heterozygosity in the β2-microglobulin region of chromosome 15 in primary human tumors

Downregulation or total loss of HLA class I expression on tumor cells is known as a mechanism of cancer immune escape. Alterations of the HLA phenotype are frequently due to mutations affecting genes encoding the HLA class I heavy chains located on chromosome 6p21 or the β2-microglobulin (β2m) gene encoding the light chain of the HLA complex located on chromosome 15q21. Frequently irreversible total loss of HLA class I molecules is due to the coincidence of two molecular events, the mutation of one β2m gene and the loss of the second copy. The latter is detectable as loss of heterozygosity (LOH) of microsatellite markers in the β2m region on chromosome 15q21 (LOH-15q21). Thus, LOH-15q21 might be an important event in the processes of HLA class I downregulation and total loss. Here we studied the frequency of LOH-15q21 in tumor tissues of different entities. By determining the status of heterozygosity of two microsatellite markers we detected LOH-15q21 in 44% of bladder carcinomas (n = 69), in 35% of colon carcinomas (n = 95), in 16% of melanomas (n = 70) but only in 7% of renal cancers (n = 45). Moreover, we observed a frequent coincidence of LOH-15q21 and LOH-6p21 in colorectal carcinoma, bladder carcinoma and melanoma, but not for renal carcinoma. We believe that the high incidence of LOH-15q21 in some malignancies and especially the coincidence of LOH-15q21 and LOH-6p21 might have a strong impact on tumor immunogenicity and on the efficiency of cancer immunotherapy.     

Mutagenic specificity of N′-methyl-N′-nitro-N-nitrosoguanidine in the gpt gene on a chromosome of Chinese hamster ovary cells and of Escherichia coli cells

Summary DNA base sequence changes induced by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis have been determined for the Escherichia coli gpt gene stably incorporated in a chromosome of Chinese hamster ovary cells and in the chromosome of both growing and starving E. coli cells, instead of on a plasmid as in most previous studies. In the three cases, nearly all mutations were G: C to A: T transitions, with a 2-to 4-fold higher mutation rate, compared to other sites, at guanines flanked on the 5 side by another guanine. Mutagenic hot spots in these experiments were less prominent than in published results for MNNG mutagenesis of gpt and of other genes. A suggested explanation involves repair of O⁶meG. At low levels of mutagenic products, most are repaired and even small differences in the repair rates leads to large differences in the relative amounts of residual O⁶meG at various sites; in contrast, at high levels of mutagenic products there is little effect of repair on the distribution.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - O⁶meG O⁶-methylguanine - N7meG N7-methylguanine - CHO Chinese hamster ovary     

The essentiality of B chain in stabilizing the structure of the A chain in β1-bungarotoxin fromBungarus multicinctus venom

The dynamic of Trp residue in ₁-bungarotoxin (gb ₁-Bgt), the A chain of ₁-Bgt and phospholipase A₂ (PLA₂) was assessed by fluorescence measurement. Acrylamide quenching studies showed that the exposure degree of the Trp in PLA₂ is higher than the Trp in ₁-Bgt. The Trp of ₁-Bgt had a higher accessibility for iodide, reflecting that the basic nature of the B chain might exert an attractive electrostatic force for iodide and increase the susceptibility of Trp in the A chain to iodide. Removal of the B chain of ₁-Bgt did not significantly affect the exposure degree of Trp in the A chain. Alternatively, the polarity of the environment around the Trp and the hydrophobic character of ANS and substrate binding sites in the separated A chain changed. Measurement of Trp fluorescence with increasing temperature showed that the stability of structure of ₁-Bgt was higher than those of the separated A chain and PLA₂. These results suggest that the B chain might interact with the A chain and stabilize the conformation of the A chain in ₁-Bgt.     

Induction of a Streptomyces cacaoi β-lactamase gene cloned in S. lividans

Summary The previously cloned class A -lactamase gene (bla) of Streptomyces cacaoi was shown to be inducible by -lactam compounds in the host organism S. lividans. A regulatory region of 2.75 kb was identified and the nucleotide sequence determined. It contained four open reading frames (ORFs) of which only two were complete and required for induction. ORF1-ORF2 exerted a positive regulatory effect on the expression of bla. Inactivation of ORF1 or of ORF2 resulted not only in the loss of induction, but also in a 30- to 60-fold decrease in the basal (non-induced) level of -lactamase production. ORF1 codes for a DNA-binding protein related to the AmpR repressor/activator, which controls the expression of ampC (class C -lactamase) genes in several Enterobacteria.