Transformation of Claviceps purpurea using a bleomycin resistance gene

Summary To develop a DNA-mediated transformation system for Claviceps purpurea a vector was constructed using a bleomycin-resistance gene (bleo ^R) fused in frame to the Aspergillus nidulans trp C promoter as a dominant selection marker. The construct was shown to be functional in Aspergillus nidulans and Aspergillus niger and used to transform a wild strain of Claviceps purpurea. Transformats were obtained at low frequencies; they were shown to contain transforming DNA integrated into the chromosomal DNA, probably in multimeric copies and at multiple sites. Combined Southern, Northern and resistance level analysis indicate that the A. nidulans promoter is functional in C. purpurea.Dedicated to Professor Dr. Dr. h. c. K. Esser on the occasion of his 65^th birthday     

Isolation and partial characterization of heparan sulphate proteoglycan from the human glomerular basement membrane.

Heparan sulphate proteoglycan was solubilized from human glomerular basement membranes by guanidine extraction and purified by ion-exchange chromatography and gel filtration. The yield of proteoglycan was approx. 2 mg/g of basement membrane. The glycoconjugate had an apparent molecular mass of 200-400 kDa and consisted of about 75% protein and 25% heparan sulphate. The amino acid composition was characterized by a high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulphonic acid yielded core proteins of 160 and 110 kDa (and minor bands of 90 and 60 kDa). Alkaline NaBH4 treatment of the proteoglycan released heparan sulphate chains with an average molecular mass of 18 kDa. HNO2 oxidation of these chains yielded oligosaccharides of about 5 kDa, whereas heparitinase digestion resulted in a more complete degradation. The data suggest a clustering of N-sulphate groups in the peripheral regions of the glycosaminoglycan chains. A polyclonal antiserum raised against the intact proteoglycan showed reactivity against the core protein. It stained all basement membranes in an intense linear fashion in immunohistochemical studies on frozen kidney sections from man and various mammalian species.     

A stochastic model for the membrane potential of a stimulated neuron

We present a simple model describing the transition between the prefiring, firing and postfiring phases of a single neuron in a large neural net. Using typical values for the physiological parameters that enter the model, we find average interspike times that are close to those reported in experimental measurements.     

Involvement of ATP-dependent aminophospholipid translocation in maintaining phospholipid asymmetry in diamide-treated human erythrocytes

Crosslinking of membrane skeletal proteins such as spectrin by oxidation of their SH-groups can be provoked by treatment of intact erythrocytes with diamide. Shortly after exposure of human erythrocytes to diamide and despite the transverse destabilization of the lipid bilayer that was observed in these cells (Franck, P.F.H., Op den Kamp, J.A.F., Roelofsen, B. and Van Deenen, L.L.M. (1986) Biochim. Biophys. Acta 857, 127-130), no abnormalities could be detected regarding the asymmetric distribution of the phospholipids when probed by either the prothrombinase assay or brief exposure of the cells to a modified phospholipase A2 with enhanced membrane penetrating capacity. This asymmetry appeared to undergo dramatic changes however, when the ATP content of the cytosol had decreased to less than 10% of its original level during prolonged incubation of the treated cells. These observations indicate that the initial maintenance of phospholipid asymmetry in diamide-treated erythrocytes can be solely ascribed to the action of the ATP-dependent aminophospholipid translocase. This view is supported by experiments involving radiolabeled phospholipids of which trace amounts had been inserted into the outer membrane leaflet of diamide-treated red cells and which still showed a preferential translocation of both aminophospholipids in favour of the inner monolayer, be it that the efficiency of the translocase was found to be impaired when compared to control cells.     

Stimulation by glycerate 2,3-bisphosphate: a common property of cytosolic IMP-GMP 5'-nucleotidase in rat and human tissues

Glycerate 2,3-bisphosphate, a potent stimulator of the cytosolic 5'-nucleotidase which preferentially hydrolyzes IMP and GMP in human erythrocytes (Bontemps et al., 1988, Biochem. J. 250, 687-696), also stimulates the dephosphorylation of IMP in cytosol fractions of rat heart, liver, brain, kidney, spleen and erythrocytes, and of human polymorphonuclear leucocytes, mixed peripheral blood lymphocytes, platelets and fibroblasts. Depending on the cell type, stimulation by 5 mM glycerate 2,3-bisphosphate varied from 1.5- to 12-fold. Where investigated, glycerate 2,3-bisphosphate had an approx. 5-fold higher affinity for the enzyme than its other stimulator, ATP. These observations provide a useful tool to distinguish IMP-GMP 5'-nucleotidase from other 5'-nucleotidases, and suggest a common origin of the cytosolic IMP-GMP 5'-nucleotidase in various tissues.     

Normal phenotype and slight mental retardation in de novo distal 8p deletion (8pter----8p23.1:)

In this report we present a 9-year-old boy with mental retardation, behavioural problems and terminal deletion of the short arm of chromosome 8(8pter----8p23.1:). In contrast with previously reported patients with larger terminal and interstitial 8p deletions he did not present major phenotypic abnormalities.     

Microscopic observations on the interaction of the mycoparasite Verticillium biguttatum with Rhizoctonia solani and other soil-borne fungi

The mycoparasitic interactions of Verticillium biguttatum with Rhizoctonia solani and with a variety of other soil-borne fungi were investigated in dual cultures. V. biguttatum interacted with various soil fungi by appressed growth along the host hyphae and infrequent penetrations. Intracellular growth and subsequent sporulation, however, only occurred with R. solani, a few binucleate Rhizoctonia and Ceratobasidium spp., and Sclerotinia sclerotiorum. Effective mycoparasitism on sclerotia was restricted to those belonging to R. solani.Electron-microscopic observations revealed that V. biguttatum can penetrate the host cell with infection tubes. This process is probably mediated by enzymatic hydrolysis of the cell wall. Subsequently, trophic hyphae develop within the host cytoplasm, ultimately resulting in death of the host cell.     

Partial duplication of the short arm of chromosome 2 (dup(2)(p13----p21) associated with mental retardation and an Aarskog-like phenotype

In this report we describe a 3-year-old boy with partial trisomy of the short arm of chromosome 2 due to a de novo tandem duplication 2(dup(2)(p13----p21)). In addition to severe growth retardation and moderate psychomotor delay he presented a dysmorphic syndrome compatible with the clinical diagnostic of Aarskog syndrome.     

Choroideremia and deafness with stapes fixation: a contiguous gene deletion syndrome in Xq21.

The study of contiguous gene deletion syndromes by using reverse genetic techniques provides a powerful tool for precisely defining the map location of the genes involved. We have made use of individuals with overlapping deletions producing choroideremia as part of a complex phenotype, to define the boundaries on the X chromosome for this gene, as well as for X-linked mixed deafness with perilymphatic gusher (DFN3). Two patients with deletions and choroideremia are affected by an X-linked mixed conductive/sensorineural deafness; one patient, XL-62, was confirmed at surgery to have DFN3, while the other patient, XL-45, is suspected clinically to have the same disorder. A third choroideremia deletion patient, MBU, has normal hearing. Patient XL-62 has a cytogenetically detectable deletion that was measured to be 7.7% of the X chromosome by dual laser flow cytometry; the other patient, XL-45, has a cytogenetically undetectable deletion that measures only 3.3% of the X chromosome. We have produced a physical map of the X-chromosome region containing choroideremia and DFN3 by using routine Southern blotting, chromosome walking and jumping techniques, and long-range restriction mapping to generate and link anonymous DNA sequences in this region. DXS232 and DXS233 are located within 450 kb of each other on the same SfiI and MluI fragments and share partial SalI fragments of 750 and greater than 1,000 kb but are separated by at least one SalI site. In addition, DXS232, which lies outside the MBU deletion, detects the proximal breakpoint of this deletion. We have isolated two new anonymous DNA sequences by chromosome jumping from DXS233; one of these detects a new SfiI fragment distal to DXS233 in the direction of the choroideremia gene, while the other jump clone is proximal to DXS233 and detects a new polymorphism. These data refine the map around the loci for choroideremia and for mixed deafness with stapes fixation and will provide points from which to isolate candidate gene sequences for these disorders.     

An Xp22 microdeletion associated with ocular albinism and ichthyosis: approximation of breakpoints and estimation of deletion size by using cloned DNA probes and flow cytometry.

Ocular albinism of the Nettleship-Falls type (OA1) and X-linked ichthyosis (XI) due to steroid sulfatase (STS) deficiency are cosegregating in three cytogenetically normal half-brothers. The mother has patchy fundal hypopigmentation consistent with random X inactivation in an OA1 carrier. Additional phenotypic abnormalities that have been observed in other STS "deletion syndromes" are not present in this family. STS is entirely deleted on Southern blot in the affected males, but the loci MIC2X, DXS31, DXS143, DXS85, DXS43, DXS9, and DXS41 are not deleted. At least part of DXS278 is retained. Flow cytometric analysis of cultured lymphoblasts from one of the XI/OA1 males and his mother detected a deletion of about 3.5 million bp or about 2% of the X chromosome. Southern blot and RFLP analysis in the XI/OA1 family support the order tel-[STS-OA1-DXS278]-DXS9-DXS41-cen. An unrelated patient with the karyotype 46,X,t(X;Y) (p22;q11) retains the DXS143 locus on the derivative X chromosome but loses DXS278, suggesting that DXS278 is the more distal locus and is close to an XI/OA1 deletion boundary. If a contiguous gene deletion is responsible for the observed XI/OA1 phenotype, it localizes OA1 to the Xp22.3 region.