DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin.

Summary We have determined the sequence of the lamB gene from Klebsiella pneumoniae. It encodes the precursor to the LamB protein, a 429 amino acid polypeptide with maltoporin function. Comparison with the Escherichia coli LamB protein reveals a high degree of homology, with 325 residues strictly identical. The N-terminal third of the protein is the most conserved part of the molecule (1 change in the signal sequence, and 13 changes up to residue 146 of the mature protein). Differences between the two mature proteins are clustered mainly in six regions comprising residues 145–167, 173–187, 197–226, 237–300, 311–329, and 367–387 (K. pneumoniae LamB sequence). The most important changes were found in regions predicted by the two-dimensional model of LamB folding to form loops on the cell surface. In vivo maltose and maltodextrin transport properties of E. coli K 12 and K. pneumoniae strains were identical. However, none of the E. coli K12 LamB-specific phages was able to plaque onto K. pneumoniae. Native K. pneumoniae LamB protein forms highly stable trimers. The protein could be purified by affinity chromatography on starch-Sepharose as efficiently as the E. coli K12 LamB protein, indicating a conservation of the binding site for dextrins. However, none of the monoclonal antibodies directed against native E. coli K12 LamB protein recognized native purified K. pneumoniae LamB protein. These data indicate that most of the variability occurs within exposed regions of the protein and provide additional support for the proposed model of LamB folding. The fact that the N-terminal third of the protein is highly conserved is in agreement with the idea that it is part of, or constitutes, the pore domain located within the transmembranous channel and that it is not accessible from the cell surface.     

Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa

Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5–2 m) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.Abbreviations BrdU 5-bromodeoxyuridine - DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether) - N,N,N,N tetraacetic acid We thank Ann Cork for technical assistance, Professor B.E.S. Gunning (Australian National University) and Drs. A.R. Hardham (A.N.U.) and R.E. Williamson (A.N.U.) for intellectual and material support, Dr D. McCurdy (A.N.U.) for the purified anti-actin antibody, and Professor B. Stone (La Trobe University, Melbourne, Australia) for generously providing the anti-callose antibody. We also thank the Electron Microscopy Unit of A.N.U. for the use of facilities. L.C.F. gratefully acknowledges financial support from the National Sciences and Engineering Research Council of Canada.     

The activation process of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1α is conserved among angiosperms

In Arabidopsis thaliana, the activation process of the A1 EF-1 gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis-acting elements of the A1 promoter, or the deletion of an intron located within the 5 non-coding region, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5 intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculentum EF-1 promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-1 genes have been conserved among angiosperms and provide interesting data on the functional structure of the TEF 1 box.     

The carbohydrases of Bacillus stearothermophilus NCIB 11412 and NCIB 10814

Summary Two strains (NCIB 11412 and NCIB 10814) of the thermophilic organism Bacillus stearothermophilus were found to produce complex carbohydrase systems. The enzyme activities in each system include -amylase as the major component, maltase, pullulanase, a minor amylase and cyclodextrinase. The latter three activities are produced in low yield in both strains. A crude enzyme preparation from each strain possessed maltogenic properties on hydrolysis of soluble starch. Following rigorous purification procedures, the purified major -amylase from either strain did not produce maltose as a major end-product of starch hydrolysis. However, a partially purified mixture of pullulanase, minor amylase and cyclodextrinase activities from NCIB 11412 and NCIB 10814 produced 56.4% and 62.0% maltose, respectively, from soluble starch.     

Two frameshift mutations in the cystic fibrosis gene

Cystic fibrosis (CF) is a recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have identified in exon 7 two frameshift mutations, one caused by a two-nucleotide insertion and the other caused by a one-nucleotide deletion; these mutations--CF1154insTC and CF1213delT, respectively, are predicted to shift the reading frame of the protein and to introduce UAA(ochre) termination codons at residues 369 and 368.     

Algal pigment diversity in coastal sediments from Kerguelen (sub-Antarctic Islands) reflecting local dominance of green algae,euglenoids and diatoms

Summary High Performance Liquid Chromatography analysis of algal pigments from inter- and subtidal (deep and shallow) sediments from the Kerguelen Islands showed clear differences in the pigment composition at the different stations. High concentrations of chlorophyll c and fucoxanthin were present at all locations, indicating significant diatom densities, chlorophyll b was detected at all sites. At one station the other green algal pigments were also present; here green algae contributed more to chlorophyll a concentrations than diatoms, as estimated by using pigment ratios and microscopic observations. At another location chlorophyll b was associated with a high concentration of diadinoxanthin, indicating an abundance of euglenoids. This indicates that chemotaxonomy can be powerful tool in microphytobenthos studies since enumeration of living cells are difficult as many algae are attached to sediment particles (epipsammic algae). Ways of diagenesis, carotenoid degradation and the role of grazing are briefly mentioned. Phaeophorbide a-like pigments were the most significant chlorophyll a degradation products, with concentrations up to 110 g · g^–1 dry weight sediment, i.e. 10 times the chlorophyll a concentration. Some taxonomic estimations, based on pigments ratios, and their limits, are discussed.