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The expression of the c-src gene in embryonie and adult tissue of the teleost fish Xiphophorus helleri was analyzed by in-situ hybridization. The highly conserved fish c-src gene was found to be expressed at high levels in midterm embryos, where c-src mRNA was localized in developing neurons of the sensory layer of the differentiating retina and in the developing brain. In adult tissues the expression of c-src was found to persist in certain cell types of the brain and the neural retina, especially in the bipolar cells of the inner nuclear layer, which are postmitotic, fully differentiated mature neurons. Thus c-src in Xiphophorus appears to be a developmentally regulated proto-oncogene which is important for neuronal differentiation during organogenesis, but whose persistence of expression in certain terminally differentiated neurons strongly suggests a particular maintenance function for c-src in these cells as well.
It has been suggested that the proto-oncogene c-src plays a functional role in developing neurons, and in the mature nerve cells of higher vertebrales. The coelenterate Hydra represents tbe most primitive known organism possessing nerve cells. With Southern blot hybridizations we have demonstrated src-related sequences in Hydra. Antisera specific for the c-src gene product (pp60 c-src) of birds and mammals precipitate a protein from Hydra cell extracts with a tyrosine-specific protein kinase activity. Studies of tissues and cells fractionated from a temperature sensitive mutant of Hydra which is depleted of interstitial (including nerve) cells at tbe non-permissive temperature, have indicated the src-like kinase of Hydra to be preferentially expressed in nerve cells. The high conservation of structural features and of the expression pattern indicates a basic function for pp60c-src in neurons.
The observation of a slower migrating form of pp6oc-src in neural tissue of chicken and mouse has recently been shown to be due to an alternative transcript form of tbe c-src gene (Martinez et al.: Science 237:411-415, 1987; Levy et al.: Mol Cell Bio17:4142- 4145, 1987). An insertion of 18 basepairs between exons 3 and 4, presumed to be due to alternative splicing of a mini-exon, gives rise to six amino acid residues not found in the non-neuronal (termed flbroblastic) form of pp60\(^{c-src}\). Wehave addressed the question of the evolutionary origin of the c-src neuronal insert · and its functional signiflcance regarding neural-speciflc expression of the c-src gene. To this end we have investigated whether the c-src gene of a lower verlebrate (the teleost fish Xiphophorus) gives rise to a neural-specific transcript in an analogous manner. We could show that the fish c-src gene does encode for a "fibroblastic" and a "neuronal" form of transcript and that the neuronal transcript does indeed arise by way of alternative splicing of a mini-exon. The miniexon is also 18 basepairs long and we could demoostrate directly that this exon lies within the intron separating exons 3 and 4. For comparative purposes we have examined whether the fish c-yes gene, the member of the src gene family most closely related to c-src, also encodes a neural tissue-specific transcript. No evidence for a second transcript form in brain was obtained. This result suggests that the mini-exon arose within the c-src gene lineage sometime between the srclyes gene duplication event and the divergence of the evolutionary lineage giving rise to the teleost fish. Published genomic sequence of src-related genes in Drosophila and our own results with Hydra demoostrate no intron in these species at the analogous location, consistent with first appearance of this mini-exon sometime between 550 and 400 million years ago.
A cloned cytolytic determinant from the genome of Bacillus cereus GP-4 has been characterized at the molecular Ievel. Nucleotide sequence determination revealed the presence of two open reading frames. 8oth open reading frames were found by deletion and complementation analysis to be necessary for expression of the hemolytic phenotype by Bacillus subtilis and Escherichia coli hosts. The 5' open reading frame was found to be nearly identical to a recently reported phospholipase C gene derived from a mutant B. cereus strain which overexpresses the respective protein, and it conferred a lecithinase-positive phenotype to the B. subtilis host. The 3' open reading frame encoded a sphingomyelinase. The two tandemly encoded activities, phospholipase C and sphingomyelinase, constitute a biologically functional cytolytic determinant of B. cereus termed cereolysin AB.
In culture supematants of both Listeria ivanovii and Listeria monocytogenes Sv4b, for the first time a hemolysin of molecular weight 58 kDa was identified, which had all the characteristics of an SH-activated cytolysin, and which was therefore identified as Iisteriolysin 0 (LLO). In the case of L. ivanovii a second major supematant protein of molecular weight 24 kDa co-purified with LLO. However, the function of this protein has to be determined. In culture supematants of L. ivanovii a sphingomyelinase and a Iecithinase activity could be detected, both enzymatic activities together contributing to the pronounced hemolysis caused by L. ivanovii. The N-tenninal amino acid sequences of LLO and the 24 kDa from L. ivanovii are shown.
The WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) region has been assigned to chromosome 11p13 on the basis of overlapping constitutional deletions found in affected individuals. We have utilized 31 DNA probes which map to the WAGR deletion region, together with six reference loci and 13 WAGR-related deletions, to subdivide this area into 16 intervals. Specific intervals have been correlated with phenotypic features, leading to the identification of individual subregions for the aniridia and Wilms tumor loci. Delineation, by specific probes, of multiple intervals above and below the critical region and of five intervals within the overlap area provides a framework map for molecular characterization of WAGR gene loci and of deletion boundary regions.