Targeted mutagenesis of the Sap47 gene of Drosophila: Flies lacking the synapse associated protein of 47 kDa are viable and fertile

BACKGROUND: Conserved proteins preferentially expressed in synaptic terminals of the nervous system are likely to play a significant role in brain function. We have previously identified and molecularly characterized the Sap47 gene which codes for a novel synapse associated protein of 47 kDa in Drosophila. Sequence comparison identifies homologous proteins in numerous species including C. elegans, fish, mouse and human. First hints as to the function of this novel protein family can be obtained by generating mutants for the Sap47 gene in Drosophila. RESULTS: Attempts to eliminate the Sap47 gene through targeted mutagenesis by homologous recombination were unsuccessful. However, several mutants were generated by transposon remobilization after an appropriate insertion line had become available from the Drosophila P-element screen of the Bellen/Hoskins/Rubin/Spradling labs. Characterization of various deletions in the Sap47 gene due to imprecise excision of the P-element identified three null mutants and three hypomorphic mutants. Null mutants are viable and fertile and show no gross structural or obvious behavioural deficits. For cell-specific over-expression and "rescue" of the knock-out flies a transgenic line was generated which expresses the most abundant transcript under the control of the yeast enhancer UAS. In addition, knock-down of the Sap47 gene was achieved by generating 31 transgenic lines expressing Sap47 RNAi constructs, again under UAS control. When driven by a ubiquitously expressed yeast transcription factor (GAL4), Sap47 gene suppression in several of these lines is highly efficient resulting in residual SAP47 protein concentrations in heads as low as 6% of wild type levels. CONCLUSION: The conserved synaptic protein SAP47 of Drosophila is not essential for basic synaptic function. The Sap47 gene region may be refractory to targeted mutagenesis by homologous recombination. RNAi using a construct linking genomic DNA to anti-sense cDNA in our hands is not more effective than using a cDNA-anti-sense cDNA construct. The tools developed in this study will now allow a detailed analysis of the molecular, cellular and systemic function of the SAP47 protein in Drosophila

Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells.

To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOH-terminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesin-like protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with translational regulation in certain mRNAs. After transient expression in CV-1 cells, the kinesin heavy chain showed both a diffuse distribution and a filamentous staining pattern that coaligned with microtubules but not vimentin intermediate filaments. Altering the number and distribution of microtubules with taxol or nocodazole produced corresponding changes in the localization of the expressed kinesin heavy chain. The expressed NH2-terminal motor and the COOH-terminal tail domains, but not the alpha-helical coiled coil rod domain, also colocalized with microtubules. The finding that both the kinesin motor and tail domains can interact with cytoplasmic microtubules raises the possibility that kinesin could crossbridge and induce sliding between microtubules under certain circumstances

Architecture of coatomer: Molecular characterization of delta-COP and protein interactions within the complex

Copyright © 2011 by The Rockefeller University Press.Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.This work was supported by The Deutsche Forschungsgemeinschaft (SFB 352), the Human Frontier Science Program, and the Swiss National Science Foundation No. 31-43366.95

In Situ Hybridisation for Human and Comparative Gene Mapping

The overall aim of this project was to develop the technique of in situ hybridisation (ISH) for the localisation of cloned DNA sequences on the Y and other chromosomes in man and other primates using both 3H- and biotin labelling. Using tritium labelling two problems were encountered and circumvented, high non-specific labelling and loss of chromosome morphology following hybridisation. Following this, seven anonymous DNA sequences cloned in plasmid vectors were localised to specific chromosome sites in the human: GMGY10 (DYS59) and GMGY7 (DYS58) to the short of the Y chromosome (Yp), P2F2 (DY25) to chromosome X band q21 and Yp, pY3.4 (DYZ1) to chromosome Y distal band Yq12, GMGY3 (DYS13) to Yp and chromosome 9 region p23-pter, GMGY4b (DYS51) to the pericentromeric region of chromosomes Y, 15, 21, 22, JG73 to pericentromeric region of chromosomes 19, 21, 22 , Y and (14, 15, and 20) and finally JG51 (D21S89) to the pericentromeric region of chromosomes 13, 14, 15, 20, 21, and 22. Under conditions of high stringency the last two probes were localised to chromosome 21, and chromosomes 13, 14 and 22 respectively, contrary to filter hybridisation data where these two sequences appear to be identical. Conventional in situ hybridisation techniques using radioactive probes suffer the serious disadvantages of prolonged autoradiographic exposure times and limited spatial resolution. To overcome these problems several non-isotopic methods have been introduced using a variety of different immunogenic, fluorescent and enzymatic labels. The possibility of using biotin-labelling for gene mapping was explored during this project using a recently described technique that employs a streptavidin-alkaline phosphatase detection system. The method was shown to be simple, reliable, rapid and sufficiently sensitive to detect single copy DNA sequences, as was indicated by the localisation of a 3.2kb DNA sequence, p72 (D21S92), to chromosome 21 proximal band q21. Results were obtained in 24 hours as compared with 1 week autoradiographic exposure for repetitive probe and 3 weeks for single copy probe. Another probe, GMGXY8 (DXYS34) was mapped to chromosome Yp. All probes mentioned above had been regionally mapped by Southern analysis using somatic cell hybrids. ISH not only confirmed but also extended the previous findings by indicating autosomal homologies for probes GMGY3, GMGY4b and regions of extended homology for JG73 and JG51. The homology between short arm of chromosomes Y and 9 revealed for probe GMGY3 is of special interest in view of the fact that GMGY3 resides in the sex-determining region of the Y chromosome. This finding coupled with observations that 9p monosomy is sometimes associated with anomalous sex differentiation could suggest a functional homology between Yp and distal 9p. ISH was also used to determine the chromosomal location of Y-specific sequences in eleven patients with paradoxical sex chromosome complements previously shown to possess such sequences. Eight XX males were studied with 3H-labelled GMGY10 and/or GMGY7 and in all cases it could be clearly demonstrated that these Y-specific sequences were located on distal short arm of the X chromosome (band Xp22.3) as predicted by the X-Y interchange hypothesis (Ferguson-Smith, 1966). The ninth XX male was investigated with probe P2F2 and although signal was recorded on the short arm of the X chromosome it could not be shown conclusively that this was due to an X-Y. interchange. Using GMGY10 or GMGY7 probes in two apparent XO males with an additional minute chromosomal fragment present in their peripheral blood lymphocytes it was demonstrated that the fragment had originated from a Y chromosome thus explaining the male phenotype of these individuals. Apart from its value in gene mapping studies, ISH has an enormous potential as a tool in clinical diagnosis. This was demonstrated in the present study by the confirmation of a suspected Yp:15q translocation in amniotic fluid cells and in paternal lymphocytes with a chromosome 15 polymorphism by using 3H-labelled probe pY3.4. The last part of the project involved comparative mapping in the gorilla (Gorilla gorilla), chimpanzee (Pan troglodytes) and orangutan (Pongo pygmaeus) of a human transfer RNA gene for glutamic acid (tRNA Glu ) and probes GMGY10 and GMGY7. The tRNA Glu gene was localised to distal long arm of chromosome 1 (band 1p36 according to ISCN nomenclature) in all three species, providing further evidence for homology between distal long arm of the ape chromosome 1 and distal short arm of human chromosome 1 where this gene has been previously localised

A new procedure to analyze RNA non-branching structures

RNA structure prediction and structural motifs analysis are challenging tasks in the investigation of RNA function. We propose a novel procedure to detect structural motifs shared between two RNAs (a reference and a target). In particular, we developed two core modules: (i) nbRSSP_extractor, to assign a unique structure to the reference RNA encoded by a set of non-branching structures; (ii) SSD_finder, to detect structural motifs that the target RNA shares with the reference, by means of a new score function that rewards the relative distance of the target non-branching structures compared to the reference ones. We integrated these algorithms with already existing software to reach a coherent pipeline able to perform the following two main tasks: prediction of RNA structures (integration of RNALfold and nbRSSP_extractor) and search for chains of matches (integration of Structator and SSD_finder)

Upstream stimulatory factors are involved in the P1 promoter directed transcription of the AbetaH-J-J locus

<p>Abstract</p> <p>Background</p> <p>Alternative splicing of the locus AβH-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) β-hydroxylase (AAH), truncated homologs of AAH with a role in calcium homeostasis humbug and junctate and a structural protein of the sarcoplasmic reticulum membranes junctin. AAH and humbug are over expressed in a broad range of malignant neoplasms. We have previously reported that this locus contains two promoters, P1 and P2. While AAH and humbug are expressed in most tissues under the regulation of the P1 promoter, AAH, junctin and junctate are predominantly expressed in excitable tissues under the control of the P2 promoter. We previously demonstrated that Sp transcription factors positively regulate the P1 promoter.</p> <p>Results</p> <p>In the present study, we extended the functional characterization of the P1 promoter of the AβH-J-J locus. We demonstrated by quantitative Real-time RT-PCR that mRNAs from the P1 promoter are actively transcribed in all the human cell lines analysed. To investigate the transcription mechanism we transiently transfected HeLa cells with sequentially deleted reporter constructs containing different regions of the -661/+81 P1 nucleotide sequence. Our results showed that (i) this promoter fragment is a powerful activator of the reporter gene in HeLa cell line, (ii) the region spanning 512 bp upstream of the transcription start site exhibits maximal level of transcriptional activity, (iii) progressive deletions from -512 gradually reduce reporter expression.</p> <p>The region responsible for maximal transcription contains an E-box site; we characterized the molecular interactions between USF1/2 with this E-box element by electrophoretic mobility shift assay and supershift analysis. In addition, our USF1 and USF2 chromatin immunoprecipitation results demonstrate that these transcription factors bind the P1 promoter <it>in vivo</it>.</p> <p>A functional role of USF1/USF2 in upregulating P1-directed transcription was demonstrated by analysis of the effects of (i) <it>in vitro </it>mutagenesis of the P1/E-box binding site, (ii) RNA interference targeting USF1 transcripts.</p> <p>Conclusion</p> <p>Our results suggest that USF factors positively regulate the core of P1 promoter, and, together with our previously data, we can conclude that both Sp and USF DNA interaction and transcription activity are involved in the P1 promoter dependent expression of AAH and humbug.</p

Scotin, a novel p53-inducible proapoptotic protein located in the ER and the nuclear membrane

p53 is a transcription factor that induces growth arrest or apoptosis in response to cellular stress. To identify new p53-inducible proapoptotic genes, we compared, by differential display, the expression of genes in spleen or thymus of normal and p53 nullizygote mice after γ-irradiation of whole animals. We report the identification and characterization of human and mouse Scotin homologues, a novel gene directly transactivated by p53. The Scotin protein is localized to the ER and the nuclear membrane. Scotin can induce apoptosis in a caspase-dependent manner. Inhibition of endogenous Scotin expression increases resistance to p53-dependent apoptosis induced by DNA damage, suggesting that Scotin plays a role in p53-dependent apoptosis. The discovery of Scotin brings to light a role of the ER in p53-dependent apoptosis