Characterization of the effectors required for stable inheritance of Streptococcus pyogenes pSM19035-derived plasmids in Bacillus subtilis

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (or) [ and ]. Inactivation or deletion of or results in SegA^– plasmids. Better than random segregation requires an active segB region. The segB region contains two ors (or and or). Inactivation of either of the orfs does not lead to an increase in cell death, but or^– plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.     

Testis-specific β2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous β1 tubulin

In Drosophila as in many organisms tubulins are encoded by a gene family. We have determined the complete nucleotide sequences coding for the 1 and 2 tubulins of Drosophila melanogaster and the 2 tubulin of D. hydei, and found these insect tubulins to be highly conserved and like tubulins of other organisms. This is discussed with reference to the possible functional domains of these proteins. — The 1 tubulin gene of Drosophila is constitutively expressed, whereas the 2 tubulin is expressed specifically in the testes. In D. melanogaster the amino acid sequences of these proteins are 95% homologous, differing at only 25 positions. In the testes the 2 tubulin participates in different microtubules as shown by genetic analysis (Kemphues et al. 1982). Interestingly, all of the amino acids characteristic of the testis-specific 2 tubulin are also present in the corresponding gene of D. hydei. Of special interest is the high degree of conservation of the carboxy-terminal domain in these functionally equivalent tubulins.     

Synthesis of Aminoethyl Glycosides of the Ganglioside GM1 and Asialo-GM1 Oligosaccharide Chains

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside, in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in a tetrasaccharide, 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, in 69% yield. The complete removal of O-protecting groups in the tetrasaccharide, the replacement of N-trichloroacetyl by N-acetyl group, and the reduction of the aglycone azide group to amine led to the target aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of asialo-GM₁ ganglioside in 72% overall yield. Selective 3-O-glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,6-di-O-benzyl--D-galactopyranosyl)--D-glucopyranoside with thioglycoside methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero--D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid afforded 2-azidoethyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)oate]-(2 3)-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, the selectively protected derivative of the oligosaccharide chain of GM₃ ganglioside, in 79% yield. Its 4-O-glycosylation with a disaccharide glycosyl donor, (4-trichloroacetophenyl-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl) 1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid gave 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-{[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)onate]-(2 3)}-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside in 85% yield. The resulting pentasaccharide was O-deprotected, its N-trichloroacetyl group was replaced by N-acetyl group, and the aglycone azide group was reduced to afford in 85% overall yield aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)-[-D-Neu5Ac-(2 3)]--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of GM₁ ganglioside.     

Isolation of a rat immune response gene identical to an alleged mouse A class II β-chain pseudogene

A human HLA-DQ -chain cDNA was used as a probe to identify and isolate a rat major histocompatibility antigen -chain gene from a genomic library constructed in the vector Charon 28 using Wistar rat DNA (RT1 ^u). The isolated exon of the rat gene (RT1.B ₂) encoding a -chain second domain was found to share 93% nucleotide homology with a mouse A ₂ exon. Although the genomic organization of this gene is consistent with the hypothesis that it represents a pseudogene, the remarkable preservation of a specific sequence favors the view that this class II antigen -chain gene has retained its coding function.     

Structure and evolution of the HLA Class II SXβ gene

The class II major histocompatibility complex antigens are cell-surface heterodimers consisting of an a and a chain. Cosmid cloning has shown that the three families of clas II antigens, DR, DQ, and DP, are encoded within the HLA-D region of chromosome 6 as a series of discrete gene clusters. The DP cluster contains two pairs of a and genes, one of which encodes the biochemically-defined DP antigen. In order to assess whether the other two genes, SXa and SX, are also expressed, potential coding regions have been subcloned and sequenced. The SX3 gene is shown to contain region closely homologous to all six exons of DP. A 1 bp deletion in the 2 exon, also observed for the SX4 allele, causes a translation frameshift, suggesting that SX is a pseudogene. However, all the other exons, as well as their splice sites and the putative promoter region, appear to be intact.     

O-Glycosylhydrolases of Marine Filamentous Fungi: β-1,3-Glucanases of Trichoderma aureviride

The ability to produce extracellular O-glycosylhydrolases was studied in 14 strains of marine filamentous fungi sampled from the bottom sediments of the South China Sea. The following activities were detected in the culture liquids of the fungi: N-acetyl--D-glucosaminidase, -D-glucosidase, -D-galactosidase, -1,3-glucanase, amylase, and pustulanase. -1,3-Glucanases were isolated by ultrafiltration, hydrophobic interaction chromatography, and ion exchange chromatography, and their properties were studied. Data on products of enzymatic digestion of laminaran, absence of transglycosylation activity, and the pattern of action of natural inhibitors confirmed that -1,3-glucanase belonged to the exo type. Inhibitor analysis demonstrated the role of a thiol group and tryptophan and tyrosine residues in the catalytic activity.     

Polymorphism of the HLA DR1 haplotype in the Israeli population investigated at the serological,cellular, and genomic levels

In the present report, we used serological, cellular, and restriction fragment length polymorphism (RFLP) to investigate the DR1 haplotype in the Israeli population. We describe an Israeli homozygous typing cell (HTC), HLA-DwLVA, which defines a new lymphocyte-activating determinant associated with Bw65, DR1 and distinct from Dwl. The parents of this donor, non-Ashkenazi Algerian Jews, are first cousins and share HLA-Cw8, Bw65, BfS, DR1, DQw1, DPw4. No specificity could be assigned to HLA-DwLVA using the 91 Ninth Workshop HTCs. Two families and forty unrelated DR1 individuals were studied with DwLVA and a panel of DR1/Dw1 HTCs. HLA-DwLVA showed segregation as a single determinant within families. This new specificity was present in 24 out of 40 (60%) unrelated DR1 individuals, indicating that in the Israeli population DwLVA is the main lymphocyte-defined determinant associated with the serologically defined DRI specificity, in contrast to non-Jewish Caucasoids where DR1 is significantly associated with Dw1. The vast majority of DwLVA-positive carriers were also Bw65 carriers, indicating that Bw65, DR1, DwLVA may represent a typical allele combination in the Israeli population. The RFLP analysis established the correlation of certain RFLPs with Dw1 and DwLVA. In addition, we describe a cluster of RFLPs that may correspond to a new Dw subtype associated with DR1, for which no serological and cellular reagents have been described so far.     

Einige Bemerkungen über die Ersatzgeschlechtstiere vonReticulitermes

Zusammenfassung An Laboratoriumszuchten vonReticulitermes lucifugus Rossi undReticulitermes flavipes Kollar (aus den Hamburger Befallsgebieten) werden Beobachtungen zur Biologie der Ersatzgeschlechtstiere mitgeteilt. Für die Durchführung dieser Beobachtungen wurden besondere Schaunester eingerichtet. Folgende Punkte der beobachteten Verhaltensweisen werden hier hervorgehoben: Die Nymphen der Ersatzgeschlechtstiere sammeln sich stets in Herden von 80–100 Einzeltieren und werden von einer Gruppe Arbeiter (als Hütehunde) bewacht. Die Arbeiter halten die Nymphen auf einem engen Raum zusammen und versuchen andere Nestgenossen von den Nymphen fernzuhalten. Alle ein bis zwei Tage wechseln die Herden ihren Standort. Das Weiterziehen geschieht gleichfalls in Form von Herden.Die Ernährung der Nymphen geschieht auf stomodealem Wege durch die Arbeiter. *** DIRECT SUPPORT *** A0180089 00007     

Interaction of cytochrome c L with methanol dehydrogenase from Methylophaga marina 42:

The reaction of methanol dehydrogenase with cytochrome c _L from Methylophaga marina and the reactions of the non-physiological substrates, Wurster's blue and ascorbic acid, with both proteins were studied as a function of temperature (4–32 °C), pressure (1–2000 bar) and ionic strength using the optical high pressure stopped-flow method. The thermodynamic parameters H, S and V were determined for all reactions where electron transfers are involved. These data allowed the determination of the Maxwell relationships which proved the internal thermodynamic consistency of the system under study. A conformational change on the cytochrome c _L level was deduced from both breaks in the Arrhenius plots and the variation of the V with temperature.Abbreviations MOPS 4-morpholinepropanesulfonic acid - CHES 2-(cyclohexylamino)ethanesulfonic acid - MDH methanol dehydrogenase - EDTA ethylenedinitrilotetraacetic acid disodium salt - BTB bromothymol blue (3,3-dibromothymolsulfoneph-thalein) - PQQ 2,7,9-tricarboxy-lH-pyrrolo-[2,3f]quinoline-4,5-dione - cytochrome c _HH mammalian horse heart cytochrome c     

Nuclear behavior in multinucleate Schizophyllum commune germlings

Summary The ultrastructure of a fir morphological mutant containing multinucleate cells is described in Schizophyllum commune. The germlings of basidiospores which arose from mating fir with wild-type mycelium were studied in culture by phase contrast microscopy to elucidate behavior of multinucleate cells. Nuclear division appeared synchronous from two nuclei yielding four progeny through six nuclei producing twelve products, beyond which loss of synchrony was indicated. Compensatory nuclear migration into an anucleate cell was presumed during synchronous division of nuclear aggregates in the adjacent cell of an individual germling. The migrant nucleus eventually returned to the cell of origin. However, the return route was not via the central pore of the septum but rather occurred at the juncture of the cross-wall with the germling-periphery. Ultrastructure of a partial septum in fir which could accommodate nuclear passage of this sort is described.     

Yeast ribosomal proteins

Summary Homology maps between bacteriophages 81, 80 and were constructed on the basis of electron microscope observation of DNA heteroduplexes. In 81/80 heteroduplex, the left half and the right terminal region of 13% the total molecular length were highly homologous, while the remaining region covering the early gene cluster was entirely nonhomologous. In 81/ heteroduplex, high-degree homologies were detected at the left 14% terminal region covering the head gene cluster, the central 3.8% region covering the att-int-xis region and the 1.3% Q homology region. Low-degree homologies of shorter length were scattered at the tail gene cluster, b2 region, cIII region, PQ region and SR region. Comparing our results with the homology maps of other lambdoid phages reported by Simon et al. (1971) and Fiandt et al. (1971), a phylogenic relation of 81 to other lambdoid phages and the role of recombination in the course of divergence of lambdoid phages are discussed.     

Charles Darwin's theory of evolution: A review of our present understanding

The paper characterizes Darwin's theory, providing a synthesis of recent historical investigations in this area. Darwin's reading of Malthus led him to appreciate the importance of population pressures, and subsequently of natural selection, with the help of the wedge metaphor. But, in itself, natural selection did not furnish an adequate account of the origin of species, for which a principle of divergence was needed. Initially, Darwin attributed this to geographical isolation, but later, following his work on barnacles which underscored the significance of variation, and arising from his work on botanical arithmetic, he supposed that diversity allowed more places to be occupied in a given region. So isolation was not regarded as essential. Large regions with intense competition, and with ample variation spread by blending, would facilitate speciation. The notion of place was different from niche, and it is questioned whether Darwin's views on ecology were as modern as is commonly supposed. Two notions of struggle are found in Darwin's theory; and three notions of variation. Criticisms of his theory led him to emphasize the importance of variation over a range of forms. Hence the theory was populational rather than typological. The theory required a Lamarckian notion of inheritable changes initiated by the environment as a source of variation. Also, Darwin deployed a use/habit theory; and the notion of sexual selection. Selection normally acted at the level of the individual, though kin selection was possible. Group selection was hinted at for man. Darwin's thinking (and also the exposition of his theory) was generally guided by the domestic-organism analogy, which satisfied his methodological requirement of a vera causa principle.     

Cloning and expression in Saccharomyces cerevisiae of a β-amylase gene from the oomycete Saprolegnia ferax

Genomic DNA and cDNA encoding the -amylase from the oomycete, Saprolegnia ferax, were cloned into Saccharomyces cerevisiae and analyzed. The Spl. ferax -amylase gene consisted of a 1350 bp open reading frame, encoding a protein of 450 amino acids with a calculated mass of 49353 Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 42% similarity to the -amylase of Arabidopsis thaliana. The -amylase gene was expressed in Sacc. cerevisiae and its product was secreted into the culture medium.     

Comparative study of the antitumor effect of two types of murine recombinant interferons, (β) and (γ), against B16-F10 melanoma

Summary A comparative study of the antitumor effect of murine recombinant interferon() Mu-rIFN() and murine recombinant interferon() Mu-rIFN() on B16-F10 melanoma was conducted. Administration of Mu-rIFN() i.p. into C57BL/6 mice on days 1 to 7 produced a higher suppressive effect than Mu-rIFN() both on the growth of s.c. implanted tumor and on the formation of artificial pulmonary metastasis. Pharmacokinetic study of Mu-rIFN() demonstrated that high plasma levels were retained for a long time. In clonogenic assay, Mu-rIFN() at 1000 units/ml showed about 80% inhibition of colonies of B16-F10 melanoma. However, Mu-rIFN() hardly inhibited the colonies, even at 1000 units/ml. Augmentation of natural killer (NK) cytotoxicity was much greater with Mu-rIFN() than Mu-rIFN(), whereas Mu-rIFN() enhanced the cytotoxicity of peritoneal macrophages more strongly than Mu-rIFN(). Injection of Mu-rIFN() i.p. 1 day before tumor challenge also inhibited the formation of pulmonary metastasis of B16-F10 melanoma. However, pretreatment of mice with carrageenan significantly suppressed the inhibitory effect of Mu-rIFN(). From these results, it is suggested that the inhibitory effect of Mu-rIFN() on the tumor growth and metastases of B16-F10 melanoma is mediated partly by direct antitumor effect and partly by the activation of macrophages, and that the augmentation of NK activity contributes mainly to the antitumor effect of Mu-rIFN().     

Establishment and characterization of Rubus tissue culture systems for in vitro bioassays against phytotoxins from Rubus fungal pathogens

The Rubus species R. parviflorus, R. spectabilis and R. strigosus interfere with conifer seedling establishment on forest regeneration sites in Canada and the United States. As a first step towards microbial metabolite-based control, callus and cell suspension cultures of the Rubus species were developed as a bioassay system to detect phytotoxic compounds that may have relevance in a vegetation control context. Rapidly growing friable callus and suspension cultures were obtained from leaf disks of the three weedy Rubus species using similar culture media conditions (modified Murashige and Skoog) but required different plant growth regulators (R. parviflorus, 4.5 M 2,4-D; R. spectabilis, 26.9 M NAA/0.5 M zeatin; and R. strigosus, 12.4 M picloram). Cell growth and health attributes including callus circumference, degree of browning and suspension culture cell viability as measured by the TTC vital stain assay were developed and were rapid and convenient to use. We have established Rubus tissue culture systems that will make it possible for large scale screening of phytotoxic metabolites.     

Genetic characterization of a new splice variant of the β2 subunit of the voltage-dependent calcium channel

This study reports a novel splice variant form of the voltage-dependent calcium channel ₂ subunit (_2g). This variant is composed of the conserved amino-terminal sequences of the _2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the ₁ subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the ₂ subunit was similar in exon-intron organization to the murine ₃ and human ₄ subunits. Electrophysiological evaluation revealed that _2a and _2g affected channel properties in different ways. The _2a subunit increased the peak amplitude, but failed to increase channel inactivation, while _2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as ₃ and ₄, significantly increased the peak current and accelerated current inactivation.     

Quantitative resistance to barley leaf stripe (Pyrenophora graminea) is dominated by one major locus

A major gene underlying quantitative resistance of barley against Pyrenophora graminea, a seedborne pathogen causing leaf stripe, was mapped with molecular markers in a barley doubled haploid (DH) population derived from the cross Proctor x Nudinka. This quantitative trait locus (QTL) accounts for r ²= 58.5% and was mapped on barley chromosome 1, tightly linked to the naked gene. A second resistance QTL accounting for 29.3% of the variation in the trait was identified on the P arm of barley chromosome 2. Another two minor QTLs were detected by further analysis. None of the QTLs was found in the barley chromosome 2 Vada region studied by Giese et al. (1993).     

Zur Brutbiologie des Seggenrohrs?ngers(Acrocephalus paludicola)

Zusammenfassung In der vorliegenden Arbeit werden Beobachtungen ausgewertet, die in den letzten Jahren in einer Seggenrohrsängerpopulation der Mark Brandenburg gesammelt wurden. Brutbiotop ist eine Großseggenwiese. und haben getrennte Reviere, wobei sich -Reviere, -Reviere und auch - und -Reviere zum Teil sehr erheblich überlagern. Keinem konnte ein bestimmtes zugeordnet werden, sie leben also in Keinehe. versuchen, aus der Nestnähe zu vertreiben. Aggressive Handlungen zwischen benachbarten konnten nicht festgestellt werden. Ihre Singflüge unterscheiden sich von denen der Schilfrohrsänger. 28 Eier maßen im Durchschnitt 17,76×13,14 mm. Nur die brüten und Füttern. Die durchschnittliche Fütterungsfrequenz liegt bei 19,7 Fütterungen pro Stunde, die tägliche Aktivitätszeit der ist im Juli etwa 1 Stunde kürzer als im Juni. 13–14 Tage nach dem Schlüpfen verlassen die Jungen das Nest. In der Nestlingsnahrung ist bei Julibruten der hohe Heuschreckenanteil auffällig. Nachdem die Jungen selbständig sind, streunen sie im Brutbiotop umher. Zweitbruten sind sehr wahrscheinlich, konnten aber noch nicht nachgewiesen werden.

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Summary Observations made during the last years on a population of the Aquatic Warbler (Acrocephalus paludicola) in Brandenburg, Germany, gave the following results:In a sedge meadow, withCarex elata the dominant species, males and females ofAcrocephalus paludicola held separate territories, those of males as well of females sometimes considerably overlapping territories held by other individuals of the same or the opposite sex. In no case a certain male could be considered as having formed a firm pair bond with a certain female: on the contrary, the relations between the sexes were very loose, and probably the birds are even promiscuous. Females try to chase away males from the neighbourhood of their nests. Aggressiveness between neighbouring males has not been observed. The display flights of the males are different from those of the Sedge Warbler(A. schoenobaenus). 28 eggs measure on the average 17,76×13,14 mm. Females alone incubate and feed the young, at an average rate of 19,7 times per hour. The young leave the nest 13–14 days after hatching. Young reared in July are fed a great amount of grasshoppers. Second broods probably occur, but could not be proved.

     

Biotransformation of 5βH-pregnan-3βol-20-one and cardenolides in cell suspension cultures of Nerium oleander L.

Summary In order to demonstrate enzyme activities playing a role in the biosynthesis of cardenolides and 2,6-dideoxysugars, 5H-pregnan-3ol-20-one and cardenolides (digitoxigenin, oleandrigenin/L-oleandrose, oleandrin, neriifolin, digitoxigeninmonodigitoxoside and strospeside) were fed to cell suspension cultures of Nerium oleander L.. It could be shown that cell suspension cultures of Nerium oleander L. are able to oxidize, isomerize and glucosylate 5H-steroidaglycones at C-3. The respective glucosides of the 5H-steroid-aglycones are the main biotransformation products. These cell cultures are an appropriate tool for the production of labelled 5H-steroidglucosides.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - EtOAc ethylacetate - MeOH methanol - MS Murashige & Skoog     

The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.