Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger.

A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes of Aspergillus niger. The corresponding proteins, rhamnogalacturonan hydrolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme. In A. niger cultures which were shifted from growth on sucrose to growth on apple pectin as a carbon source, the expression of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently induced after 3 h of growth on apple pectin. The rhamnogalacturonan hydrolase B gene was not induced by apple pectin, but the rhgB gene was derepressed after 18 h of growth on either apple pectin or sucrose. Gene fusions of the A. niger rhgA and rhgB coding regions with the strong and inducible Aspergillus awamori exlA promoter were used to obtain high-producing A. awamori transformants which were then used for the purification of the two A. niger rhamnogalacturonan hydrolases. High-performance anion-exchange chromatography of oligomeric degradation products showed that optimal degradation of an isolated highly branched pectin fraction by A. niger rhamnogalacturonan hydrolases A and B occurred at pH 3.6 and 4.1, respectively. The specific activities of rhamnogalacturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively, which is significantly lower than the specific activity of A. aculeatus rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5). Compared to the A enzymes, the A. niger B enzyme appears to have a different substrate specificity, since additional oligomers are formed.     

Two inward K+ channels in the xylem parenchyma cells of barley roots are regulated by G-protein modulators through a membrane-delimited pathway

In order to study the mechanism and regulation of K⁺ resorption from the xylem by the cells that border the xylem vessels (the xylem parenchyma cells), K⁺ inward-rectifying channels (KIRCs) in the plasma membrane of xylem parenchyma cells from Hordeum vulgare L. cv. Apex were studied using the patch-clamp technique. In the inside-out configuration, three different types of K⁺ channel and a further K⁺ conductance could be identified. Two of these channels, named KIRC1 and KIRC2, were activated by guanosine 5′-[β,γ-imido]triphosphate (Gpp(NH)p; 150 μM), a non-hydrolyzable derivative of GTP, indicating that channel activity was up-regulated by G-proteins; modulation of channel activity occurred via a membrane-delimited pathway, since the effect could be demonstrated in cell-free patches. At 100 mM external K⁺, KIRC1 had a conductance of 8 pS. There was no effect of ATP on channel activity. Likewise, addition of 150 μM guanosine 5′-[β-thio]diphosphate (GDPβS) or adenosine 5′-[γ-thio]triphosphate (ATPγS) failed to activate KIRC1, indicating nucleotide specificity of the effect. A second K⁺ channel, activated by Gpp(NH)p (KIRC2) with gating properties clearly different from the first one was less frequently observed. Four different substates could be identified; the main level had a conductance of about 2 pS. Gating below the Nernst potential of K⁺ (E_K) was voltage-independent. The channel closed at potentials more positive than E_K. A third, hyperpolarization-activated K⁺ channel, KIRC3, with a low open probability was encountered in inside-out patches. It had a conductance of 45 pS in 100 mM K⁺. Channel activity was not affected by the addition of G-protein modulators. Moreover, slowly activating inward currents carried by K⁺ were recorded in several patches that are ascribed to a `subpicosiemens conductance'. Neither GDPβS nor Gpp(NH)p appeared to have an effect on the currents. Whole-cell measurements with these G-protein modulators included in the pipette solution were in general agreement with the results obtained on cell-free patches. A statistical evaluation revealed that time-dependent inward currents were larger when the G-protein activator Gpp(NH)p was included in the pipette medium compared to measurements with the inhibitor GDPβS. With the GTP analogue, an additional instantaneous component was elicited that was ascribed to KIRC2 activity. Data are discussed with respect to the putative role of G-proteins in conveying hormonal signals. Regulation by G-protein may either serve to fine-tune K⁺ uptake by xylem parenchyma cells or to initiate depolarization, followed by salt-efflux through depolarization-activated cation and anion channels. Received 11 October 1996 / Accepted: 21 April 1997     

A unique PE_PGRS protein inhibiting host cell cytosolic defenses and sustaining full virulence of Mycobacterium marinum in multiple hosts

Despite intense research, PE_PGRS proteins still represent an intriguing aspect of mycobacterial pathogenesis. These cell surface proteins influence virulence in several pathogenic species, but their diverse and exact functions remain unclear. Herein, we focussed on a PE_PGRS member from Mycobacterium marinum, MMAR_0242, characterized by an extended and unique C‐terminal domain. We demonstrate that an M. marinum mutant carrying a transposon insertion in MMAR_0242 is highly impaired in its ability to replicate in macrophages and amoebae, because of its inability to inhibit lysosomal fusion. As a consequence, this mutant failed to survive intracellularly as evidenced by a reduced number of cytosolic actin tail‐forming bacteria and by quantitative electron microscopy, which mainly localized MMAR_0242::Tn within membrane‐defined vacuoles. Functional complementation studies indicated that the C‐terminus, but not the N‐terminal PE_PGRS domain, is required for intracellular growth/survival. In line with these findings, disruption of MMAR_0242 resulted in a highly attenuated virulence phenotype in zebrafish embryos, characterized by restricted bacterial loads and a failure to produce granulomas. Furthermore, expression of MMAR_0242 in Mycobacterium smegmatis, a non‐pathogenic species naturally deficient in PE_PGRS production, resulted in increased survival in amoebae with enhanced cytotoxic cell death and increased survival in infected mice with splenomegaly. Overall, these results indicate that MMAR_0242 is required for full virulence of M. marinum and sufficient to confer pathogenic properties to M. smegmatis.     

The role of recombination in the evolvement of the fragile X mutation

Summary The frequency of recombination in the regions adjacent to the fragile X locus was studied in two groups of carriers: daughters of transmitting males and transmitters of maternally inherited fragile X chromosomes. Approximately one-half of the offspring of the former and one quarter of the off-spring of the latter are recombinant. Recombinants and parentals are equally distributed among affected and normal off-spring in the two groups. These results indicate that crossing-over at or around the fragile X locus occurs in every meiosis in doughters of transmitting males, although the recombinant chromatids do not necessarily carry the fragile X mutation. Hence, crossing-over is unequivocally associated with, but is not the direct cause of, the transition from the primary genetic lesion to the final mutation.     

Flow sorting in the study of cell-cell interaction

Undifferentiated mouse teratocarcinoma cells were cocultivated with differentiated mouse endoderm cells in order to study the possible induction of teratocarcinoma cell differentiation. A difference in DNA content between the two cell types was experimentally introduced to enable the reisolation of the teratocarcinoma cells after cocultivation. Pseudotetraploid (2s) endoderm cell lines were produced from pseudodiploid (1s) cells by treatment of these cells with cytochalasin B and flow sorting of tetraploid cells, using Hoechst 33342 as a viable DNA stain, with subsequent cloning of sorted single cells. In model experiments, where mixtures of 1s teratocarcinoma and 2s endoderm cells were stained with Hoechst 33342, the teratocarcinoma cells could be reisolated with a purity of about 97%. After a cocultivation period of 24 days viable teratocarcinoma cells could be isolated from the cocultivation mixture with a purity of 95%. Two dimensional analysis of the protein pattern of these cells indicated that cocultivation did not induce a differentiated (endoderm) pattern. Therefore according to this analysis the teratocarcinoma cells were not induced to differentiate during a 24 day cocultivation period. The method described offers excellent possibilities for studying cell-cell interaction in vitro.     

Cytological evidence of defective template in the fragile X chromosome

In cells of fragile X patients, the changed X segment may appear as a poorly staining region or a gap, or as a deletion, involving one or both chromatids. To find out whether the fragile site represents ah incompletely replicated DNA sequence, as has been suggested recently, we analyzed the four chromatids of methotrexate-induced endoreduplicated fragile X chromosomes. Our main observations were: (1) a deleted chromatid was never internal to a poorly staining one; (2) an endoreduplicated X chromosome with a fragile site never included a normal chromatid. These results can be explained by assuming that DNA at the fragile site, when replicated in the presence of methotrexate, may undergo defective replication and give rise to improperly packaged chromatin, appearing as a chromatid with a poorly staining region or a gap in the following metaphase. The same DNA may fail to function as a template in the following S-phase and give rise to a chromatid with a single-stranded segment, appearing as a deleted chromatid in the following metaphase.Dedicated to the memory of Professor Menashe Marcus, teacher, colleague, arid friend     

Multiple effects of differentiation-inducing factor on prespore differentiation and cyclic-AMP signal transduction in Dictyostelium

Abstract. The effects of the differentiation inducing factor (DIF) on several cAMP-induced responses in Dictyostelium were investigated. It was found that DIF reduces the apparent affinity of cell-surface cAMP receptors. DIF does not affect the cAMP-induced cGMP response, but it is a potent inhibitor of the cAMP-relay response. DIF also inhibits the induction of prespore differentiation by cAMP in aggregation-competent cells. We also compared the effects of DIF on cAMP-induced responses with those of the relay inhibitor, caffeine, and the morphogen, adenosine.