The gene for the ataxia-telangiectasia variant,Nijmegen breakage syndrome,maps to a 1-cM interval on chromosome 8q21.

Nijmegen breakage syndrome (NBS; Seemanová II syndrome) and Berlin breakage syndrome (BBS), also known as ataxia-telangiectasia variants, are two clinically indistinguishable autosomal recessive familial cancer syndromes that share with ataxia-telangiectasia similar cellular, immunological, and chromosomal but not clinical findings. Classification in NBS and BBS was based on complementation of their hypersensitivity to ionizing radiation in cell-fusion experiments. Recent investigations have questioned the former classification into two different disease entities, suggesting that NBS/BBS is caused by mutations in a single radiosensitivity gene. We now have performed a whole-genome screen in 14 NBS/BBS families and have localized the gene for NBS/BBS to a 1-cM interval on chromosome 8q21, between markers D8S271 and D8S270, with a peak LOD score of 6.86 at D8S1811. This marker also shows strong allelic association to both Slavic NBS and German BBS patients, suggesting the existence of one major mutation of Slavic origin. Since the same allele is seen in both former complementation groups, genetic homogeneity of NBS/BBS can be considered as proved.     

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     

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Kinetic evidence for insertion of actin monomers between the barbed ends of actin filaments and barbed end-bound insertin, a protein purified from smooth muscle

An actin polymerization-retarding protein was isolated from chicken gizzard smooth muscle. This protein copurified with vinculin on DEAE-cellulose and gel filtration columns. The polymerization-retarding protein could be separated from vinculin by hydroxylapatite chromatography. The isolated polymerization-retarding protein lost its activity within a few days, but was stable for weeks when it was not separated from vinculin. We termed the polymerization-retarding protein "insertin". Because of the instability of the isolated insertin, we investigated the effect of insertin-vinculin on actin polymerization. Insertin-vinculin retarded nucleated actin polymerization maximally fivefold. Polymerization at the pointed ends of gelsolin-capped actin filaments was not affected by insertin-vinculin, suggesting that insertin-vinculin binds to the barbed ends, but not to the pointed ends, of actin filaments. Retarded polymerization was observed even if the actin monomer concentration was between the critical concentrations of the ends of treadmilling actin filaments. As at this low monomer concentration the pointed ends depolymerize, monomers appeared to be inserted at the barbed ends between the terminal subunit and barbed end-bound insertin molecules. Insertin-vinculin was found not to increase the actin monomer concentration to the value of the pointed ends. These observations support the conclusion that insertin is not a barbed end-capping protein but an actin monomer-inserting protein. According to a quantitative analysis of the kinetic data, all observations could be explained by a model in which two insertin molecules were assumed to bind co-operatively to the barbed ends of actin filaments. Actin monomers were found to be inserted between the barbed ends and barbed end-bound insertin molecules at a rate of about 1 x 10(6) M-1 s-1. Insertin may be an essential part of the machinery of molecules that permit treadmilling of actin filaments in living cells by insertion of actin molecules between membranes and actin filaments.     

Effect of the state of oxidation of cysteine 190 of tropomyosin on the assembly of the actin-tropomyosin complex

Tropomyosin, cross-linked at cysteine 190, was found to bind more weakly to actin filaments than uncross-linked tropomyosin. Cross-linking of tropomyosin can cause actin filaments nearly completely covered with tropomyosin to be uncovered almost completely. The critical monomer concentration of actin is not significantly changed by binding of cross-linked or uncross-linked tropomyosin to actin filaments. The binding curves were analyzed quantitatively, thereby taking into account the polar end-to-end contact of tropomyosin molecules bound by actin and the overlap of the seven subunit binding sites along the actin filament. Under the conditions of the experiment (80 mM KCl, 1 mM MgCl2, pH 7.5, 38-42 degrees C), the equilibrium constant for isolated binding of tropomyosin to actin filaments is in the range 1 x 10(3)-3 x 10(3) M-1. The equilibrium constants for binding of tropomyosin to binding sites along the actin filament with one or two neighbouring tropomyosin molecules are in the range of 10(6) or 10(8) to 10(9) M-1, respectively. The equilibrium constants for binding of tropomyosin to binding sites along the actin filament with one or two neighbouring tropomyosin molecules are in the range of 10(6) or 10(8) to 10(9) M-1, respectively. The equilibrium constants for cross-linked and uncross-linked tropomyosin differ by a factor of only about two. Owing to the highly cooperative binding, these differences are sufficient so that actin filaments nearly completely covered with uncross-linked tropomyosin are uncovered almost completely by cross-linking tropomyosin at cysteine 190.