A microsatellite linkage map of Drosophila mojavensis

Background

Drosophila mojavensishas been a model system for genetic studies of ecological adaptation and speciation. However, despite its use for over half a century, no linkage map has been produced for this species or its close relatives.

Results

We have developed and mapped 90 microsatellites in D. mojavensis, and we present a detailed recombinational linkage map of 34 of these microsatellites. A slight excess of repetitive sequence was observed on the X-chromosome relative to the autosomes, and the linkage groups have a greater recombinational length than the homologous D. melanogaster chromosome arms. We also confirmed the conservation of Muller's elements in 23 sequences between D. melanogaster and D. mojavensis.

Conclusions

The microsatellite primer sequences and localizations are presented here and made available to the public. This map will facilitate future quantitative trait locus mapping studies of phenotypes involved in adaptation or reproductive isolation using this species.     

Cytochrome f translation in Chlamydomonas chloroplast is autoregulated by its carboxyl-terminal domain

The rate of synthesis of cytochrome f is decreased approximately 10-fold when it does not assemble with the other subunits of the cytochrome b(6)f complex in Chlamydomonas reinhardtii chloroplasts. This assembly-mediated regulation of cytochrome f synthesis corresponds to a regulation of petA mRNA initiation of translation. Here, we demonstrate that cytochrome f translation is autoregulated by its C-terminal domain. Five cytochrome f residues conserved throughout all chloroplast genomes-residue Gln-297 in the transmembrane helix and a cluster of four amino acids, Lys-Gln-Phe-Glu, at positions 305 to 308, in the stromal extension-participate in the formation of a translation repressor motif. By contrast, positively charged residues in the stromal extension have little influence on the autoregulation process. These results do not favor a direct interaction between the repressor motif and the petA 5' untranslated region but suggest the participation of a membrane-bound ternary effector.     

Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum

Sulfatase modifying factor 1 (SUMF1) is the gene mutated in multiple sulfatase deficiency (MSD) that encodes the formylglycine-generating enzyme, an essential activator of all the sulfatases. SUMF1 is a glycosylated enzyme that is resident in the endoplasmic reticulum (ER), although it is also secreted. Here, we demonstrate that upon secretion, SUMF1 can be taken up from the medium by several cell lines. Furthermore, the in vivo engineering of mice liver to produce SUMF1 shows its secretion into the blood serum and its uptake into different tissues. Additionally, we show that non-glycosylated forms of SUMF1 can still be secreted, while only the glycosylated SUMF1 enters cells, via a receptor-mediated mechanism. Surprisingly, following its uptake, SUMF1 shuttles from the plasma membrane to the ER, a route that has to date only been well characterized for some of the toxins. Remarkably, once taken up and relocalized into the ER, SUMF1 is still active, enhancing the sulfatase activities in both cultured cells and mice tissues.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Conformation of bovine myelin basic protein purified with bound lipids

The basic protein of myelin (called MBP) is an extrinsic protein of the myelin membrane. Its structure and function are still unknown. MBP has been extensively studied in its water-soluble form, but it is also known in a detergent-soluble form, which is purified with endogenous myelin lipids and should correspond to the native form of the protein in the membrane. In order to acquire insight into the structure of MBP, we have carried out circular dichroism (CD) experiments on the protein both in the lipid-free and in the lipid-bound form. Our data clearly show that lipid-free MBP is mainly disordered with only a small amount having α-helix and β-sheet motifs. On the other hand, the lipid-bound form of MBP appears to have a consistent amount of ordered secondary structure. Theoretical predictions, made using different computational methods, substantially confirm the tendency of the protein to assume an ordered secondary structure in accordance with our CD results. Received: 13 November 1998 / Accepted: 1 February 1999     

TLR2, TLR4 and the MyD88 Signaling Are Crucial For the In Vivo Generation and the Longevity of Long-Lived Antibody-Secreting Cells

This study was undertaken to gain better insights into the role of TLRs and MyD88 in the development and differentiation of memory B cells, especially of ASC, during the Th2 polarized memory response induced by Natterins. Our in vivo findings demonstrated that the anaphylactic IgG1 production is dependent on TLR2 and MyD88 signaling, and that TLR4 acts as adjuvant accelerating the synthesis of high affinity-IgE. Also, TLR4 (MyD88-independent) modulated the migration of innate-like B cells (B1a and B2) out of the peritoneal cavity, and the emigration from the spleen of B1b and B2 cells. TLR4 (MyD88-independent) modulated the emigration from the spleen of Bmem as well as ASC B220^pos. TLR2 triggered to the egress from the peritoneum of Bmem (MyD88-dependent) and ASC B220^pos (MyD88-independent). We showed that TLR4 regulates the degree of expansion of Bmem in the peritoneum (MyD88-dependent) and in BM (MyD88-independent) as well as of ASC B220^neg in the spleen (MyD88-independent). TLR2 regulated the intensity of the expansion of Bmem (MyD88-independent) and ASC B220^pos (MyD88-dependent) in BM. Finally, TLR4 signals sustained the longevity of ASC B220^pos (MyD88-independent) and ASC B220^neg into the peritoneum (MyD88-dependent) and TLR2 MyD88-dependent signaling supported the persistence of B2 cells in BM, Bmem in the spleen and ASC B220^neg in peritoneum and BM. Terminally differentiated ASC B220^neg required the cooperation of both signals through TLR2/TLR4 via MyD88 for longevity in peritoneum, whereas Bmem required only TLR2/MyD88 to stay in spleen, and ASC B220^pos rested in peritoneum dependent on TLR4 signaling. Our data sustain that earlier events on memory B cells differentiation induced in secondary immune response against Natterins, after secondary lymph organs influx and egress, may be the key to determining peripheral localization of innate-like B cells and memory B cells as ASC B220^pos and ASC B220^neg.     

The second National Toxicology Program comparative exercise on the prediction of rodent carcinogenicity: definitive results

Chemical carcinogenicity has been the target of a large array of attempts to create alternative predictive models, ranging from short-term biological assays (e.g. mutagenicity tests) to theoretical models. Among the theoretical models, the application of the science of structure-activity relationships (SAR) has earned special prominence. A crucial element is the independent evaluation of the predictive ability. In the past decade, there have been two fundamental comparative exercises on the prediction of chemical carcinogenicity, held under the aegis to the US National Toxicology Program (NTP). In both exercises, the predictions were published before the animal data were known, thus using a most stringent criterion of predictivity. We analyzed the results of the first comparative exercise in a previous paper [Mutat. Res. 387 (1997) 35]; here, we present the complete results of the second exercise, and we analyze and compare the prediction sets. The range of accuracy values was quite large: the systems that performed best in this prediction exercise were in the range 60-65% accuracy. They included various human experts approaches (e.g. Oncologic) and biologically based approaches (e.g. the experimental transformation assay in Syrian hamster embryo (SHE) cells). The main difficulty for the structure-activity relationship-based approaches was the discrimination between real carcinogens, and non-carcinogens containing structural alerts (SA) for genotoxic carcinogenicity. It is shown that the use of quantitative structure-activity relationship models, when possible, can contribute to overcome the above problem. Overall, given the uncertainty linked to the predictions, the predictions for the individual chemicals cannot be taken at face value; however, the general level of knowledge available today (especially for genotoxic carcinogens) allows qualified human experts to operate a very efficient priority setting of large sets of chemicals.