Cloning and sequencing the genes encoding uptake-hydrogenase subunits of Rhodocyclus gelatinosus

Summary Rhodocyclus gelatinosus grew photosynthetically in the light and consumed H₂ at a rate of about 665 nmol/min per mg protein. The uptake-hydrogenase (H₂ase) was found to be membrane bound and insensitive to inhibition by CO. The structural genes of R. gelatinosus uptake-H₂ase were isolated from a 40 kb cosmid gene library of R. gelatinosus DNA by hybridization with the structural genes of uptake-H₂ase of Bradyrhizobium japonicum and Rhodobacter capsulatus. The R. gelatinosus genes were localized on two overlapping DNA restriction fragments subcloned into pUC18. Two open reading frames (ORF1 and ORF2) were observed. ORF1 contained 1080 nucleotides and encoded a 39.4 kDa protein. ORF2 had 1854 nucleotides and encoded a 68.5 kDa protein. Amino acid sequence analysis suggested that ORF1 and ORF2 corresponded to the small (HupS) and large (HupL) subunits, respectively, of R. gelatinosus uptake-H₂ase. ORF1 was approximately 80% homologous with the small, and ORF2 was maximally 68% homologous with the large subunit of typical membrane-bound uptake-H₂ases.     

Shuttle-like movements of Golgi vesicles in the tip of growingChara rhizoids

Summary In tip-growingChara rhizoids, the in-vivo saltatory movements of Golgi vesicles were recorded. The movements in radial direction back and forth between the ER aggregate and the plasma membrane occurred three times more often than movements passing the ER aggregate tangentially. The mean velocity of the class of Golgi vesicles observed (0.4–1 m in diameter) was approx. 0.3 m/s. Higher speed of 1–1.5 m/s occurred only in radial directions. Possibly, the ER aggregate is involved in guidance of the Golgi vesicles.Abbreviations DIC differential interference contrast - ER endoplasmic reticulum - OsFeCN osmium tetroxide-potassium ferricyanide Dedicated to the memory of Professor O. Kiermayer     

Qualitative importance of the microbial loop and plankton community structure in a eutrophic lake during a bloom of cyanobacteria

Plankton community structure and major pools and fluxes of carbon were observed before and after culmination of a bloom of cyanobacteria in eutrophic Frederiksborg Slotssø, Denmark. Biomass changes of heterotrophic nanoflagellates, ciliates, microzooplankton (50 to 140 μm), and macrozooplankton (larger than 140 μm) were compared to phytoplankton and bacterial production as well as micro- and macrozooplankton ingestion rates of phytoplankton and bacteria. The carbon budget was used as a means to examine causal relationships in the plankton community. Phytoplankton biomass decreased and algae smaller than 20 μm replacedAphanizomenon after the culmination of cyanobacteria. Bacterial net production peaked shortly after the culmination of the bloom (510 μg C liter^?1 d^?1 and decreased thereafter to a level of approximately 124 μg C liter^?1 d^?1. Phytoplankton extracellular release of organic carbon accounted for only 4–9% of bacterial carbon demand. Cyclopoid copepods and small-sized cladocerans started to grow after the culmination, but food limitation probably controlled the biomass after the collapse of the bloom. Grazing of micro- and macrozooplankton were estimated from in situ experiments using labeled bacteria and algae. Macrozooplankton grazed 22% of bacterial net production during the bloom and 86% after the bloom, while microzooplankton (nauplii, rotifers and ciliates larger than 50 μm) ingested low amounts of bacteria and removed 10–16% of bacterial carbon. Both macro-and microzooplankton grazed algae smaller than 20 μm, although they did not control algal biomass. From calculated clearance rates it was found that heterotrophic nanoflagellates (40–440 ml^?1) grazed 3–4% of the bacterial production, while ciliates smaller than 50 μm removed 19–39% of bacterial production, supporting the idea that ciliates are an important link between bacteria and higher trophic levels. During and after the bloom ofAphanizomenon, major fluxes of carbon between bacteria, ciliates and crustaceans were observed, and heterotrophic nanoflagellates played a minor role in the pelagic food web.     

14. Are the impacts of events in the earth's history discernable in the current distributions of freshwater algae?

Freshwater microalgae, lacking a fossil record, have contributed little to the study of historical biogeography. Some of the innate difficulties are discussed, as well as some of the more hopeful possibilities, if distribution records, morphology and DNA sequence analysis are combined with knowledge of the earth's history. Examples of species within the same family showing quite different distributions are given, along with suggested explanations. These include possible examples of the role played by waterfowl in dissemination of freshwater algae.     

Xp-duplications with and without sex reversal

Duplications in Xp including the DSS (dosage sensitive sex reversal) region cause male to female sex reversal. We investigated two patients from families with Xp duplications. The first case was one of two sisters with karyotype 46,XY, der(22), t(X;22)(p11.3;p11)mat and unambiguous female genitalia. The living sister was developmentally retarded, and showed multiple dysmorphic features and an acrocallosal syndrome. The second case was a boy with a maternally inherited direct duplication of Xp21.3-pter with the breakpoint close to the DSS locus. He had multiple abnormalities and micropenis, but otherwise unambiguous male genitalia. We performed quantitative Southern blot analysis with probes from Xp22.13 to p21.2 to define the duplicated region. Clinical, cytogenetic, and molecular data from both patients were compared with those of previously reported related cases. A comparison of the extragenital symptoms revealed no differences between patients with or without sex reversal. In both cases, the symptoms were non-specific. Among 22 patients with a duplication in Xp, nine had unambiguous female genitalia and a well-documented duplication of the DSS region. Two patients with duplication of DSS showed ambiguous external genitalia. From these data, we conclude that induction of testicular tissue may start in these patients, but that the type of genitalia depends on the degree of subsequent degeneration by a gene in DSS.     

Ac/Ds transposon mutagenesis in Arabidopsis thaliana: mutant spectrum and frequency of Ds insertion mutants

Using a two-component Ac/Ds system consisting of a stabilized Ac element (Ac_c1) and a non-autonomous element (Ds_A), 650 families of plants carrying independent germinal Ds_A excisions/transpositions were isolated. Progenies of 559 of these Ac_c1/Ds_A families, together with 43 families of plants selected for excision/transposition of wild-type (wt)Ac, were subjected to a broad screening program for mutants exhibiting visible alterations. This resulted in the identification of 48 mutants showing a wide variety of mutant phenotypes, including embryo lethality (24 mutants), chlorophyll defects (5 mutants), defective seedlings (2 mutants), reduced fertility (5 mutants), reduced size (3 mutants), altered leaf morphology (2 mutants), dark green, unexpanded rosette leaves (3 mutants), and aberrant flower or shoot morphology (4 mutants). To test whether these mutants were due to transposon insertions, a series of Southern blot experiments was performed on 28 families, comparing in each case several mutant plants with others showing the wild-type phenotype. A preliminary analysis revealed in 4 of the 28 families analyzed a common, novel Ds_A fragment in all mutant plants, which was present only in heterozygous plants with wt phenotype, as expected for Ds_A insertion mutations. These four mutants included two showing embryo lethality, one with dark green, unexpanded rosette leaves and stunted inflorescences, and one with curly growth of stems, leaves and siliques. Further evidence for Ds_A insertion mutations was obtained for one embryo lethal mutant and for the stunted mutant, while in case of the second embryo lethal mutant, the Ds_A insertion could be separated from the mutant locus by genetic recombination.     

Anchor-linked intermediates in peptide amide synthesis are caused by dimeric anchors on the solid supports

Cleavage and kinetic studies have been carried out using commercially obtained H-Tyr(tBu)-5-(4′-aminomethyl-3′,5′-dimethoxyphenoxy)valeric acid-TentaGelS (H-Tyr(tBu)-4-ADPV-TentaGelS) and H-Tyr (tBu)-4-ADPV-Ala-aminomethyl-resin (H-Tyr(tBu)-4-ADPV-AM-resin) prepared from commercially available resin and loaded with commercially available Fmoc-4-ADPV-OH amide anchor. Cleavage with pure trifluoroacetic acid (TFA) gave the intermediate H-Tyr-4-ADPV-NH₂, which was then degraded to H-Tyr-NH₂, and cleavage with TFA/dichloromethane (1:9) yielded H-Tyr-4-ADPV-NH₂ which could be isolated in preparative amounts. Cleavage reactions with ¹⁵N-labelled H-Ala-4-ADPV-[¹⁵N]-Gly-AM-resin yielded the intermediate H-Ala-4-ADPV-NH₂, which contained no ¹⁵N as demonstrated by ¹H-NMR. The analysis of the commercial Fmoc-4-ADPV-OH amide anchor showed the presence of Fmoc-4-ADPV-4-ADPV-OH as an impurity in high amounts. This dimeric anchor molecule is the cause of formation of the anchor-linked peptide intermediate obtained during the cleavage from the resin. The particularly high acid-lability of the amide bond between the two ADPV moieties was utilized to synthesize sidechain and C-terminally 4-ADPV protected pentagastrin on a double-anchor resin, and to cleave it using 5% trifluoroacetic acid in dichloromethane. This method may offer a new way for the synthesis of protected peptide amides with improved solubility to be used in fragment condensation.     

Side reactions in the synthesis of phosphotyrosine-containing peptides

Summary A series of phosphopeptides Tyr(PO₃H₂)-Val-Pro-Xxx-Leu (Xxx=Met, Met(O), Nle, Dab or Cys), derived from the native platelet-derived growth factor- receptor (PDGF-) sequence, has been prepared to study their interaction with the src-homology 2 (SH2) domains of the p85 subunit of PI3 kinase. The phosphopeptides were synthesized using Fmoc methodology incorporating N-Boc dibenzyl-protected phosphotyrosine (Boc-Tyr[PO₃(Bzl)₂]) as the N-terminal amino acid, since the benzyl groups can be removed during resin cleavage with TFA. Only peptides containing methionine were found to exist partially as S-benzyl sulfonium salts after TFA cleavage from the resin. The desired peptide could be obtained from the S-benzyl sulfonium salt by hydrogenolysis.     

Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var. tenebrionis

NB176 is a Bacillus thuringiensis mutant derived by λ-irradiation of NB125 Bacillus thuringiensis var. tenebrionis (Krieg). It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein. Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment. Each cryIIIA gene-bearing HindIII fragment was cloned from NB176. The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers. Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn 1000. These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by γ-irradiation. Integration of additional copies of the cryIIIA gene into the native 90MDa plasmid of the wild-type B. thuringiensis var. tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity.     

Germline and somatic mosaicism in a female carrier of Duchenne muscular dystrophy

The family of a male with Duchenne muscular dystrophy (DMD) and a deletion within the dystrophin gene has been studied. Polymerase chain reaction analysis of ectopic mRNA from peripheral blood T+B lymphocytes and the use of (CA) _n repeat polymorphisms in and around the deleted region showed the proband's mother to be both a germline mosaic and a somatic mosaic for the deletion seen in her son. The mutation therefore occurred as a mitotic event early in embryogenesis.