A fast and sensitive multiple sequence alignment algorithm

A two-step multiple alignment strategy is presented that allowsrapid alignment of a set of homologous sequences and comparisonof pre-aligned groups of sequences. Examples are given demonstratingthe improvement in the quality of alignments when comparingentire groups instead of single sequences. The modular designof computer programs based on this algorithm allows for storageof aligned sequences and successive alignment of any numberof sequences. Received on August 23, 1988; accepted on December 6, 1988     

Preparation and characterization of cell-free protein synthesis systems from oocytes and eggs of Xenopus laevis

During the maturation of the oocytes of the frog Xenopus laevis, the rate of protein synthesis shows a twofold increase. Studies of the mechanisms involved in this stimulation have been seriously limited by the lack of an active cell-free translation system. We have now prepared such systems from oocytes, progesterone-matured oocytes and eggs of Xenopus laevis by induction of lysis by centrifugation of whole cells. The extracts are highly active in incorporation of labelled amino acids and, in the progesterone-matured and egg extracts, a substantial proportion of this is due to reinitiation on endogenous mRNA, as shown by the use of inhibitors. The increased rate of protein synthesis previously observed in intact oocytes following progesterone-induced maturation is reflected in the relative activities of the extracts. The difference in activity is not due to the presence of a dominant inhibitor of translation in the extracts from unstimulated oocytes. Labelling studies with initiator tRNA ([35S]Met-tRNAf) indicate a higher concentration of 43S preinitiation complexes in the extracts from unstimulated oocytes, suggesting an impairment of initiation of translation at or after the mRNA-binding step. Extracts from both oocytes and progesterone-matured oocytes translated endogenous mRNAs to give products ranging over a wide spectrum of molecular weight. However, significant translation of exogenous (globin) mRNA required the presence of reticulocyte postribosomal supernatant, suggesting that one or more factors required for mRNA recruitment is limiting in these extracts.     

Perspectives of taste reception

Summary Ion: solute cotransporters frequency are incapable of achieving equilibrium between the solute accumulation and the transmembrane difference of the electrochemical potential of the ion. The presence of uncoupled flows of ion and solutes (leaks) is often advanced as an explanation. Here an alternative is discussed. The net accumulation of solute may be so slow that equilibrium can never be attained at finite times (e.g., several hours). Cotransporters may exhibit strong product inhibition, and the net influx of solute approaches zero far from equilibrium. The inherent slowness of net transport under these conditions is termed catalytic inefficiency. The likelihood that galactoside: H⁺ cotransport inEscherichia coli, hexose: H⁺ cotransport inChlorella vulgaris, andd-glucose: Na⁺ cotransport in brush-border membranes exhibit catalytic inefficiency is examined. The existence of strong product inhibition complicates the determination of the stoichiometry of cotransport and the characterization of chemically modified or mutant cotransporters.     

Molecular genetic study of human arginase deficiency

We have explored the molecular pathology in 28 individuals homozygous or heterozygous for liver arginase deficiency (hyperargininemia) by a combination of Southern analysis, western blotting, DNA sequencing, and PCR. This cohort represents the majority of arginase-deficient individuals worldwide. Only 2 of 15 homozygous patients on whom red blood cells were available had antigenically cross-reacting material as ascertained by western blot analysis using anti-liver arginase antibody. Southern blots of patient genomic DNAs, cut with a variety of restriction enzymes and probed with a near-full-length (1,450-bp) human liver arginase cDNA clone, detected no gross gene deletions. Loss of a TaqI cleavage site was identified in three individuals: in a homozygous state in a Saudi Arabian patient at one site, at a different site in homozygosity in a German patient, and in heterozygosity in a patient from Australia. The changes in the latter two were localized to exon 8, through amplification of this region by PCR and electrophoretic analysis of the amplified fragment after treatment with TaqI; the precise base changes (Arg291X and Thr290Ser) were confirmed by sequencing. It is interesting that the latter nucleotide variant (Thr290Ser) was found to lie adjacent to the TaqI site rather than within it, though whether such a conservative amino acid substitution represents a true pathologic mutation remains to be determined. We conclude that arginase deficiency, though rare, is a heterogeneous disorder at the genotypic level, generally encompassing a variety of point mutations rather than substantial structural gene deletions.     

Effects of abscisic acid on somatic embryo maturation of hybrid larch

Somatic embryos of hybrid larch (Larixleptoeuropaea) whichhad been matured for 4 weeks on maturation medium, developednormally on medium supplemented with 60 µM ABA, but abnormallyon medium with no ABA. A comparative structural and histochemicalinvestigation was carried out on these two types of mature embryos.At the light microscope level, differences between both treatmentswere visible only after 2–3 weeks of maturation. At aroundthis time, abnormal development becomes evident macroscopically:ABA-minus embryos remain rather stubby as opposed to the morecylindrically shaped ABA-plus embryos. Whereas somatic embryosmatured with ABA consist of densely cytoplasmic cells showinga high rate of cell division, ABA-minus embryos are largelymade up of expanded and highly vacuolate cells, indicating thatgrowth in the latter is mainly due to cell expansion and notdivision. After 4 weeks of maturation, ABA-minus embryos beginto elongate in the hypocotyl region, and precocious germinationwas observed frequently. Again, these morphogenetic events werelargely due to abnormal timing of cell expansion. Histochemically,storage proteins were found only in somatic embryos maturedfor 4 weeks with ABA. This observation is in line with resultsobtained by total protein analysis, yielding significantly lowertotal protein contents in ABA-minus embryos both on a freshweight and a per embryo basis after 4–5 weeks of maturation.Deposition of starch grains mainly in the cortex tissue of thehypocotyl region was observed within 2 weeks of maturation invarying amounts regardless of ABA supply. Polyphenols, in particularcatechins and proanthocyanidins, were present in all embryosfrom the very onset of development. They were localized preferentiallyin the proximal suspensor cells and the basal region of theembryo. However, accumulation of polyphenols was generally muchmore pronounced in embryos matured without ABA, indicating alack of biochemical regulatory competence in those embryos. Key words: Abscisic acid, embryonal development, somatic embryo, storage protein, polyphenols     

Linkage disequilibrium analysis in Machado-Joseph disease patients of different ethnic origins

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration originally described in families of Portuguese-Azorean ancestry. The hypothesis that its present world distribution could result from the spread of an original founder mutation has been raised. To test this possibility we have conducted a linkage disequilibrium study of markers segregating with the MJD1 locus in a total of 64 unrelated families of different geographical origins. Significant association was detected between the MJD1 locus and marker alleles at loci D14S280, D14S1050 and D14S81. All affected individuals, except one Chinese family, had allele 3 (237 bp) at D14S280. This finding is consistent with a founder effect in our MJD population. However, distinct haplotypes were observed in patients originating from the two Azorean islands showing the highest disease prevalence; therefore, the possible existence of more than one founder mutation can not be excluded with the markers currently available. Received: 27 February 1996 / Revised: 4 June 1996     

Dynamics of the sit-to-stand movement

The strategies of the sit-to-stand movement are investigated by describing the movement in terms of the topology of an associated phase diagram. Kinematic constraints are applied to describe movement sequences, thus reducing the dimension of the phase space. This dimensional reduction allows us to apply theorems of topological dynamics for two-dimensional systems to arrive at a classification of six possible movement strategies, distinguished by the topology of their corresponding phase portrait. Since movement is treated in terms of topological structure rather than specific trajectories, individual variations are automatically included, and the approach is by nature model independent. Pathological movement is investigated, and this method clarifies how subtle abnormalities in movement lead to difficulties in achieving a stable stance upon rising from a seated position. This article was processed by the author using the LAT_EX style file pljour2 from Springer-Verlag.     

Differential Expression of a Mycobacterium tuberculosis Protein Recognized by a Mouse Monoclonal Antibody

Murine monoclonal antibodies were produced against Mycobacterium tuberculosis (Mtb) using standard hybridoma procedures. By a whole cell enzyme-linked immunosorbent assay (ELISA), one monoclonal antibody (mAb), HB28, demonstrated high level specific reactivity to Mtb. Western blot analysis demonstrated reactivity to a single 65 kDa Mtb protein in the cell wall extract and culture filtrate. HB28 mAb appears to be recognizing a 65 kDa Mtb protein that is over-expressed by Mtb but not other species under certain culture conditions. Differential expression and detection of this protein by HB28 mAb may have potential for diagnostic applications.     

Immunodetection of cytoskeletal structures and the Eg5 motor protein on deep-etch replicas of Xenopus egg cortices isolated during the cortical rotation

Summary— We have developed a new method for immunogold detection on deep-etch replicas of isolated Xenopus egg cortices in order to examine the interactions of different cortical elements in three dimensions at high resolution. We have applied this technique to vegetal cortices isolated during the second half of the first cell cycle. The vegetal cortical region at this time is the site of cellular machinery responsible for the ‘cortical rotation’. The entire cortex translocates with respect to the inner cytoplasm, relocating dorsalising determinants to the future dorsal side of the egg. The aligned microtubules in the shear zone between cytoplasm and cortex, implicated in the cortical rotation, were found to be organised as interweaving loose bundles. Interleaved amongst these aligned microtubules were extensive sheets of ER lying in layers parallel to the egg surface. Cytokeratin filaments were found to associate closely with the microtubules over short stretches. Putative actin filaments were present in the shear zone and in the cortex. Eg5, an abundant kinesin-related microtubule motor protein, and candidate for a role in generating cortical rotation movement, showed an almost exclusive localisation to microtubules. Immunofluorescence studies of cortices treated with detergent to disrupt ER or cold to depolymerise microtubules confirmed that Eg5 associates primarily with microtubules. We propose revised models for the mechanism of cortical rotation based on these observations and conclude that Eg5 is unlikely to move ER relative to microtubules during the cortical rotation.     

Metabolism of halogenated compounds in the white rot fungus Bjerkandera adusta studied by membrane inlet mass spectrometry and tandem mass spectrometry

Membrane inlet mass spectrometry has been used for the characterization of halogenated organic compounds produced by the fungus Bjerkandera adusta. Using this technique we obtained electron impact-, chemical ionization-, electron capture negative chemical ionization-mass spectra and tandem mass spectra directly from the growth medium. Through this direct analysis of the samples we identified novel bioconversion products and confirmed recently published data on the production of both chlorinated and brominated methoxybenzaldehyde metabolites. Growth profiles of the culture grown on a defined medium showed that the production of secondary metabolites starts after approximately 6 days and reaches maximal concentrations of 25-250 muM after 15-20 days. Although delayed, the production of secondary metabolites paralleled a depletion of glucose from the medium and stopped shortly after all glucose had been consumed. Experiments in which fluoro- and bromo-labeled 4-methoxybenzaldehydes were added to the medium at day 8 showed biotransformation of these compounds into chloro-3-fluoro-4-methoxy-benzaldehyde and chloro-3-bromo-4-methoxybenzaldehyde, respectively. No dichlorinated products were observed, suggesting that halogenation takes place only at the meta position on the 4-methoxybenzaldehydes. These experiments are the first to bring direct evidence of a halogenation mechanism, where the enzymatic attack takes place directly on the 4-methoxybenzaldehyde intermediates. (c) John Wiley & Sons, Inc.