Triplex DNA in plasmids and chromosomes

Circular plasmids containing pyrimidine purine tracts can form both inter-and intramolecular triplexes. Addition of poly(dTC) to plasmid pTC45, which contains a (TC)45.(GA)45 insert, results in intermolecular triplex formation. Agarose-gel electrophoresis gives rise to many well-resolved bands, which correspond to 1, 2, 3, 4... plasmid molecules attached to the added pyrimidine strand. In the electron microscope these complexes appear as a rosette of petals. The mobility of these triplex-containing complexes can be retarded by the addition of a triplex-specific monoclonal antibody, Jel318. Intramolecular triplex formation can be demonstrated at pH 5 in pTC45 and also in pT463-I, a plasmid containing a segment of a crab satellite DNA with both (G)n.(C)n and (TCC)n.(GGA)n inserts. However, although the intermolecular triplex remains stable for some time at pH 8, intramolecular triplex formation only occurs at low pH. Triplexes can also be detected by an immunoblotting procedure with Jel318. This unfamiliar structure is readily demonstrated in eukaryotic extracts, but not in cell extracts from Escherichia coli. Triplexes may thus be an inherent feature of eukaryotic chromosome structure.     

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.     

Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.     

Energy metabolism of Chironomus anthracinus (Diptera: Chironomidae) from the profundal zone of Lake Esrom,Denmark, as a function of body size,temperature and oxygen concentration

The profundal zone of Lake Esrom, Denmark has a dense population of Chironomus anthracinus, which survives 2–4 months of oxygen depletion each summer during stratification. The metabolism of 3rd and 4th instar larvae was examined in regard to variation in biomass and temperature. Respiration at air saturation was described by a curvilinear multiple regression relating oxygen consumption to individual AFDW and temperature. At 10 °C and varying oxygen regimes the O₂ consumption and CO₂ production of 4th instar larvae were almost unaltered from saturation to about 3 mg O₂ l^–1, but decreased steeply below this level. The respiratory quotient increased from 0.82 at saturation to about 3.4 at oxygen concentrations near 0.5 mg O₂ l^–1. This implied a shift from aerobic to partially anaerobic metabolism. At 0.5 mg O₂ l^–1 the total energy production equalled 20% of the rate at saturation of which more than one third was accounted for by anaerobic degradation of glycogen. This corresponded to a daily loss of 12 µg mg AFDW^–1 or approximately 5% of the body reserves. At unchanged metabolic rate the glycogen store would last three weeks, but long term oxygen deficiency causes a further suppression of the energy metabolism in C. anthracinus.     

A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli.

A 1.5-kb genomic fragment isolated from Streptomyces avermitilis that directs the synthesis of a brown pigment in Escherichia coli was characterized. Since pigment production in recombinant E. coli was enhanced by the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated with melanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitilis was unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototype of melanin genes in Streptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single open reading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic and eukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressed in E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), which is the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brown pigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients. Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations of this acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, the production of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitilis gene encoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated in the medium by this clone.     

Pool sequencing of natural HLA-DR,DQ, and DP ligands reveals detailed peptide motifs,constraints of processing,and general rules

We have approached the problem of MHC class II ligand motifs by pool sequencing natural peptides eluted from HLA-DR, DQ, and DP molecules. The results indicate surprisingly clear patterns, although not quite as clear as with natural class I ligands. The most striking feature is a highly dominant Proline at position 2. We interpret this to be a consequence of aminopeptidase N-like activity in processing. Another general aspect is the existence of three to four hydrophobic or aromatic anchors, whereby the first and the last are separated by five to eight residues. The peptide motifs for HLA-DR1, DR5, DQ7, and DPw4 are allele-specific and differ by spacing and occupancy of anchors. The anchors tend to be flanked by clusters of charged residues, and small residues, especially Ala, are frequent in the motif centers. These detailed motifs allow one to interpret most previous (DR-) motifs as fitting one or more of the anchors or conserved clusters. The relative motif symmetry suggests the possibility of bidirectional binding of peptides in the class II groove.     

A hydroxyproline-containing class IV chitinase of sugar beet is glycosylated with xylose

Two acidic chitinase isoforms, SP1 and SP2, have been purified to homogeneity from leaves of sugar beet (Beta vulgaris) infected with Cercospora beticola. SP1 and SP2 are extracellular proteins with an apparent molecular mass of 35 kDa and an approximate pI of 4.2. Since the only major difference was slightly diverging M _r's, only the SP2 chitinase was further characterized. Partial amino acid sequence data for SP2 was used to generate a polymerase chain reaction (PCR) clone employed for the isolation of a cDNA clone encoding SP2. SP2 exhibits significant structural identity with the class IV chitinases from sugar beet, rapeseed, bean and maize, but differs from the other members of this class in having a longer hinge region, comprising 22 amino acid residues, with a repeated TTP motif. Western blotting analyses, using antibody raised against SP2, demonstrated an induction of SP protein during infection with C. beticola. The induction was very local, with high protein accumulation found close to the infection site only. Amino acid compositional analysis of SP2 revealed that five out of fourteen prolines are hydroxylated. No glucosamine or galactosamine residues are present. Evidence was obtained that SP2 is glycosylated with a limited number (7) of xylose residues: (1) SP2 was stained with the periodic acid-Schiff (PAS) reagent, (2) electrospray mass spectrometry on SP2 gave a series of M _r's with a consistent increase between two molecular masses of 132 Da, (3) SP2 was recognized by an antibody specific for -1,4-D-xylopyranose. The vacuolar class I chitinases A and B in tobacco have recently been shown to comprise a new class of hydroxyproline-containing proteins (Sticher et al., Science 257 (1992) 655–657). The SP2 chitinase differs from these in being glycosylated and, thus, represents a novel type of hydroxyproline-containing glycoproteins in plants.     

Mechanisms for oscillation and frequency control in reciprocally inhibitory model neural networks

We describe four different mechanisms that lead to oscillations in a network of two reciprocally inhibitory cells. In two cases (intrinsic release and intrinsic escape) the frequency of the network oscillation is insensitive to the threshold voltage of the synaptic potentials. In the other two cases (synaptic release and synaptic escape) the network frequency is strongly determined by the threshold voltage of the synaptic connections. The distinction between the different mechanisms blurs as the function describing synaptic activation becomes less steep and as the model neurons are removed from the relaxation regime. These mechanisms provide insight into the parameters that control network frequency in motor systems that depend on reciprocal inhibition.