The Saccharomyces cerevisiae SGE1 gene product: a novel drug-resistance protein within the major facilitator superfamily

Several pleiotropic drug sensitivities have been described in yeast. Some involve the loss of putative drug efflux pumps analogous to mammalian P-glycoproteins, others are caused by defects in sterol synthesis resulting in higher plasma membrane permeability. We have constructed a Saccharomyces cerevisiae strain that exhibits a strong crystal violet-sensitive phenotype. By selecting cells of the supersensitive strain for normal sensitivity after transformation with a wild-type yeast genomic library, a complementing 10-kb DNA fragment was isolated, a 3.4-kb subfragment of which was sufficient for complementation. DNA sequence analysis revealed that the complementing fragment comprised the recently sequenced SGE1 gene, a partial multicopy suppressor of gal11 mutations. The supersensitive strain was found to be a sge1 null mutant. Overexpression of SGE1 on a high-copy-number plasmid increased the resistance of the supersensitive strain. Disruption of SGE1 in a wild-type strain increased the sensitivity of the strain. These features of the SGE1 phenotype, as well as sequence homologies of SGE1 at the amino acid level, confirm that the Sge1 protein is a member of the drug-resistance protein family within the major facilitator superfamily (MFS).     

Tyrosine attack by free radicals derived from catalytic decomposition of carbon tetrachloride

The interaction between free radicals derived from the catalytic decomposition of carbon tetrachloride and tyrosine (the N-acetyl tyrosine ethyl ester, ATEE) under anaerobic and aerobic conditions was studied. The structure of the reaction products formed was desciphered by the GLC/MS analysis of their trimethylsilyl derivatives. Under anaerobic conditions the formation of the following products was found: (1) an unsaturated derivative of the amino acid; (2) the trimethylsilyl derivative of N-acetyl chloro tyrosine ethyl ester; (3) a hydroxyl adduct of ATEE ; (4) an ATEE adduct having a chlorine and a CCl₃ group in the molecule (it is suggested that CCl₃ is attached to the benzyl carbon and the chlorine located in the benzene ring); (5) an ATEE adduct having only a CCl₃ group tentatively assigned to be located on the benzyl carbon; and (6) and (7) were found to be two isomers of an ATEE having one CCl₃ on the aromatic ring. Under aerobic conditions the following reaction products were identified: Two products which were similar to those numbered (1) and (2) and formed anaerobically; (8) and (11) two isomeric dichlorinated adducts of ATEE; (9) and (10) two isomeric dichlorinated monohydroxylated derivatives of ATEE. Concerning the potential relevance of these findings, we consider that if similar interactions to those here reported occurred during CCl₄ poisoning, the activity of enzymes having tyrosine in their active center might result in impairment. Further, enzymes operating on tyrosine moieties in proteins might be perturbed in their action if tyrosine groups were attacked by the free radicals arising from catalytic decomposition of CCl₄ evidenced here.     

Purification,and characterization of a β-mannanase of Trichoderma reesei C-30

Trichoderma reesei was studied for its ability to produce -mannanase activity on a variety of carbon sources. The highest -mannanase activity was produced on cellulose, whereas -mannan-containing carbon sources (such as kojac powder or locust bean gum) gave lower enzyme titres. The enzyme responsible for the major -mannanolytic activity from T. reesei was purified to physical homogeneity by preparative chromatofocusing and anion exchange fast protein liquid chromatography. This -mannanase is a glycoprotein, with a molecular mass of 46 (±2) kDa and an isoelectric point of 5.2. It has an optimal pH at 5.0 and broad pH stability (2.5–7.0). It is stable for 60 min at 55° C, and has an optimal temperature for activity at 75° C. During incubation with locust bean gum, the enzyme releases mainly tri- and disaccharides. Correspondence to: C. P. Kubicek     

The human complement component C8B gene: structure and phylogenetic relationship

The eighth component of human complement (C8) is a serum protein that consists of three chains (, and ), encoded by three separate genes, viz., C8A, C8B, and C8G. In serum, the -subunit is non-covalently bound to the disulfide-linked - subunit. Using a full-length C8 cDNA probe, we isolated several clones from human genomic DNA libraries. Four clones covering the complete cDNA sequence were characterized by TaqI restriction mapping and were shotgun subcloned into M13. C8-cDNA-positive clones were partially sequenced to characterize the 12 exons of the gene with sizes from 69 to 347 bp. All intron-exon junctions followed the GT-AG rule. By using polymerase chain reaction (PCR) primers located in the adjacent intron sequences, all 12 exons of the C8B gene could be amplified from genomic DNA. All fragments showed the expected sizes. The sizes of eight introns could be determined by using primer pairs that amplified two exons and the enclosed intron, and by restriction mapping. These analyses and the insert sizes of the genomic clones indicate that the C8B gene has a total size of approximately 40 kb. The polymorphic TaqI site of the C8B gene localized in intron 11 could be demonstrated by direct restriction fragment analysis of a PCR fragment containing exons 11 and 12, and the enclosed intron 11. Homology comparison of the C8B gene with C8A and C9 on the basis of the exon structure confirmed the ancestral relationship known from the protein level.     

Apios americana,a fly-pollinated papilionaceous flower?

In the flowers ofApios americana the vexillum remains ± in its bud position. In this way, a dark cavity is formed displaying a bright window at its base. Insect visitors are attracted to the hidden inflorescences by scent. In their attempt to reach the window they trigger an explosive release of stigma and pollen. Stigmatic fluid glues pollen to the visitor. All characters of the flower fit well into the myiophilous syndrome and thus flies are expected to be the proper pollinators.Apios americana seems to be the only myiophilous exception within the predominantly melittophilousFabaceae.     

Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus. Sequence analysis and identification of two hyp regulatory mutants

A 25kbp DNA fragment from the chromosome of Rhodobacter capsulatus B10 carrying hydrogenase (hup) determinants was completely sequenced. Coding regions corresponding to 20 open reading frames were identified. The R. capsulatus hydrogenase-specific gene (hup and hyp) products bear significant structural identity to hydrogenase gene products from Escherichia coli (13), from Rhizobium liguminosarum (16), from Azotobacter vinelandii (10) and from Alcaligenes eutrophus (11). The sequential arrangement of the R. capsulatus genes is: hupR2-hupU-hypF -hupS-hupL-hupM-hupD -hupF -hupG -hupH -huoJ -hupK -hypA-hypB-hupR1-hypC -hypD -hypE -ORF19 -ORF20 , all contiguous and transcribed from the same DNA strand. The last two potential genes do not encode products that are related to identified hydrogenase-specific gene products in other species. The sequence of the 12 R. capsulatus genes underlined above is presented. The mutation site in two of the Hup^? mutants used in this study, RS13 and RCC12, was identified in the hypF gene (deletion of one G) and in the hypD qene (deletion of 54 bp), respectively. The hypF gene product shares 45% identity with the product of hydA from E. coli and the product of hypF from R. leguminosarum. Those products present at their N-terminus a Cys arrangement typical of zinc-finger proteins. The G deletion in the C-terminal region of hypF in the RS13 mutant     

The synthesis and biochemical evaluation of new A1 selective adenosine receptor agonists containing 6-hydrazinopurine moieties

The synthesis and SAR of a series of novel derivatives of N-aminoadenosine is described, along with their in vitro effects in biochemical assays. The rat brain A₁ adenosine receptor binding of these compounds is very dependent upon the purine 2-substituent. The novel agonist, 2-chloro-N-[4-(phenylthio)-1-piperidinyl]adenosine, exhibits a L_i value for A₁ receptor binding of <1 nM.     

RECURRENT FORMATION AND POLYPHYLY OF NORDIC POLYPLOIDS IN DRABA (BRASSICACEAE)

Draba (Brassicaceae) is well known for its taxonomic complexity in arctic and alpine floras, and the polyploids in particular present vexing taxonomic problems. It has been suggested that polyploids in Draba may have formed recurrently from different populations of the parental species (polytopy), and it is also possible that a given taxonomic species may actually comprise several polyploid races, each originating from different progenitor species (polyphyly). To unravel the taxonomic complexity of polyploid Draba in the Nordic area, we investigated three of the most morphologically variable species and their possible progenitors using enzyme electrophoresis and restriction site analysis of chloroplast DNA (cpDNA) and nuclear ribosomal RNA genes (rDNA): D. norvegica (6x), D. lactea (6x), and D. corymbosa (16x). Electrophoretic analyses of progeny showed high levels of fixed heterozygosity in all three polyploids, demonstrating that all are genetic alloploids. Electrophoretic and rDNA data indicate that polytopic and/or polyphyletic origins have contributed to the complexity of these polyploids. However, a lack of cpDNA variation among the species limited the usefulness of this molecule for analysis of polyploid origins. The considerable electrophoretic variation observed in D. norvegica necessitates a minimum of three and probably 13 independent origins. Electrophoretic and rDNA data suggest that D. lactea and D. corymbosa are polyphyletic polyploids. Crossing data also support that D. corymbosa is polyphyletic. Given the widespread geographic distributions of these species and their possible progenitors, and that the populations analyzed represent only a small fraction of their geographic distributions, it is likely that these species have formed numerous times in different areas. As more molecular analyses of polyploids are completed, the data continue to suggest that multiple origins of polyploids are the rule rather than the exception.     

Assessing the impact of ozone on photosynthesis of European beech (Fergus sylvatica L.) in environmental chambers

An exposure — response study with proportionalto-ambient ozone levels was conducted in closed chambers on 3-year-old European beech (Fagus sylvatica L.) of montane origin. The fumigation started in April 1990 and lasted for a single growing season. Climate data and ozone concentrations monitored at an experimental station of the Institute for Applied Plant Biology, Schönenbuch, Switzerland were simulated in the exposure chambers 12 days later (1*O₃). To test exposure-response relations three additional treatments were applied, subambient (0.2*O₃) and two proportionally increased ozone treatments (1.5*O₃ and 2*O₃). The photosynthetic behaviour of the trees in August revealed the light reactions to be less affected than parameters which are related to the dark reactions of photosynthesis. Assimilation (A₃₅₀), apparent carboxylation efficiency (CE), and maximum photosynthetic capacity (A₂₅₀₀) were reduced with increasing ozone concentration. For the ozone response of CE and A₂₅₀₀ Critical Levels were calculated.