ThegrpD55 locus ofEscherichia coli appears to be an allele ofdnaB

Attempts to characterize thegrpD55 mutation ofEscherichia coli have led us to conclude that the gene had been assigned an incorrect map position. The mutation was found to cotransduce withmalF3089:: Tn10 (at 91.5 min) and adnaB-expressing plasmid was able to complement fully thegrpD55 defect in replication. These studies strongly suggest thatgrpD55 is an allele ofdnaB and is localized near 92 min on theE. coli linkage map.     

The Effect of Salicylic Acid on Peroxidase Activity in Wheat Calli Cocultured with the Bunt Pathogen Tilletia caries

The effect of salicylic acid (SA) on peroxidase activity in wheat (Triticum aestivum L.) calli cocultured with the bunt pathogen Tilletia caries was studied. Fungal infection was shown to activate cytoplasmic peroxidase. SA suppressed total peroxidase activity but did not inhibit the peroxidase with pI 9.8. A novel chitin-specific peroxidase with pI 3.5 appeared after the SA treatment. The infection of SA-treated cells with Tilletia caries activated the isoenzymes with pI 3.5, 4.8, and 7.5 and stimulated their secretion into the culture medium. The ability of SA to control wheat peroxidase activity during pathogenesis is discussed. The important role of this control in plant defense responses to the bunt pathogen is emphasized.     

Subdomain organization ofBacillus thuringiensis entomocidal proteins'' N-terminal domains

N-Terminal domain (65 kD) of -endotoxin produced byBacillus thuringiensis ssp.alesti, as shown by limited proteolysis, consists of two subdomains of molecular mass 30 and 33 kD that correspond, respectively, to conservative and variable regions of the -endotoxin primary structure. Furthermore, proteolysis of these subdomains leads to their conversion into at least two fragments of molecular mass 10 kD stable to proteinase action. Such a pattern of molecular organization appears to be common for several structurally related -endotoxins that belong to thekurstaki group. Entomicidal protein produced by ssp.israelensis (70 kD), which differs strongly fromalesti and otherkurstaki group -endotoxins, retains a similar type of molecular organization and consists of two subdomains with molecular mass of 35 kD. Apparently, the characteristic pattern of the -endotoxins' molecular structure reflects separation of functions (e.g., host recognition and toxicityper se) between domains and subdomains of these proteins.     

Pheromone puffs suppress mating by Plodia interpunctella and Sitotroga cerealella in an infested corn store

The efficacy of pheromone mating disruption was investigated in a 7×6×3 m corn storage room harboring a high population density of Indian meal moth, Plodia interpunctella (Hübner) and Angoumois grain moth, Sitotroga cerealella (Olivier). Pheromones were released from a controlled release dispenser, the metered semiochemical timed release system (MSTRS^TM) at emission rates of 0.6 g min^–1 (Z9,E12:14:Ac for Indian meal moth) and 0.2 g min^–1 (Z7,E11-16:Ac for Angouimois grain moth). Mating disruption efficacy was evaluated using three parameters: male capture in pheromone traps, visual examination of mating behavior, and the incidence and frequency of mating as measured by spermatophores. In three trials, comparisons were made between data collected before pheromone treatment and during treatment. Disruption of pheromone source location by males averaged 70% and 40% for P. interpunctella and S. cerealella, respectively, in the three trials. In addition, reduced levels of copulation by both species were recorded during pheromone treatment. More importantly, significant reductions were recorded in the incidence and frequency of mating by females of both species collected during the treatment period. While 85% of P. interpunctella females collected before pheromone treatment in three trials had mated at least once, only 50% of the females collected during treatment had mated. The mean number of matings, as measured by spermatophores, ranged between 0.8–1.1 and 0.5–0.7 before and during pheromone treatment, respectively. Similarly, a 20–30% reduction in the proportion of mated S. cerealella females was recorded during pheromone treatment. In the three trials, mean number of spermatophores per S. cerealella female averaged 1.0 and 0.7 during the pretreatment and treatment periods, respectively. Additional tests conducted in small boxes also recorded significant mating disruption of both species.     

Further studies on carbon catabolite regulation of β-lactam antibiotic synthesis inCephalosporium acremonium

A high glucose concentration (6%) interfered with production of -lactam antibiotics byCephalosporium acremonium. Production rate of the pathway intermediate, penicillin N, by resting cells harvested from a high glucose fermentation, peaked and declined early in the fermentation. When cells were grown in the standard medium (2.7% glucose + 3.6% sucrose), penicillin N productivity was prolonged, showing two peaks, the first during trophophase and the second afterwards. The decline in productivity was not prevented by addition of the amino acid precursors of -lactam antibiotics. The addition of glucose to resting cells drastically decreased formation of the end product, cephalosporin C, but had only a moderate effect on penicillin N production. Glucose markedly repressed the ring-expansion enzyme (deacetoxy-cephalosporin C synthetase) but had a lesser effect on the tripeptide cyclization enzyme (isopenicillin N synthetase). We conclude that the major effect of a low (2%) or a high (6%) concentration of a rapidly used carbon source (e.g., glucose, glycerol, maltose) onC. acremonium fermentations is repression of the metabolically unstable ring-expansion enzyme and hence of formation of cephalosporins. On the other hand, the lesser degree of repression of the cyclization enzyme and itsin vivo stability allow penicillin N to accumulate normally or even at increased rates except at high carbon source concentrations.     

A nonconjugative mobilizable broad host range plasmid of Acinetobacter sp. that determines HgCl2 resistance

Summary HgCl₂-resistant strains of Acinetobacter sp. obtained from the soil at the Khaidarkan mercury mine (Kirghiz SSR) were found to contain, apart from large plasmids (60 kb), a small plasmid (7.5 kb) designated pKL1. It was established by conjugative crosses and transformation that pKL1 is a broad host range mobilizable plasmid and that it carries the Hg^r determinant. The restriction map of pKL1 was constructed; the site of the Hg^r determinant and the regions essential to replication were localized. A comparison of these results with earlier data suggests that microorganisms belonging to one microbiocenosis may carry Hg^r determinants on plasmids with highly different structures and properties.Deceased on July 16, 1985     

Semper's (zoanthid) larvae: pelagic life,parentage and other problems

Semper's larvae were obtained from <300 out of 1800 plankton tows taken in the world's oceans (1964–1993). Zoanthellae (larvae of Sphenopidae) occurred at 217 stations and zoanthinae (larvae of Zoanthidae) at 86, the two larval types showing distributions clearly delimited by a minimum sea temperature (22 °C for zoanthellae, 18 °C for zoanthinae; a statistically significant difference, P<0.001). Length of formalin-fixed zoanthellae was 2–8.6 mm and of zoanthinae 1.5–5.9 mm. Endodermal zooxanthellae were present in 9/24 zoanthinae but in no zoanthellae (of 19). Three larvae contained an endo-commensal/parasitic amphipod. Septa were externally visible in larger zoanthinae and were counted in transverse sections of other larvae, a majority of which (both kinds) had 12 septa, the normal maximum. The pattern was brachycnemic in 40/43 larvae and anomalous (but non-macrocnemic) in three. If macrocnemic genera reproduce by Semper's larvae, they should have been represented in such a large sample. The distribution of adult Epizoanthus was examined: many species are deep sea (recorded down to 5000 m) but shallow-water species are relatively plentiful in, for example, the Adriatic and North Seas. No Semper's larva has ever been recorded from either. Some Parazoanthus species also occur in shallow water, especially associated with western Atlantic reef sponges. If they produce Semper's larvae, these have never been found. It is probable that macrocnemic zoanthids settle from planulae that do not develop into recognizable zoanthellae or zoanthinae.     

Preferential transposition of aDrosophila P element to the corresponding region of the homologous chromosome

Genetic and physical analyses have demonstrated an intimate interaction or pairing of homologous chromosomes in the nuclei of manyDrosophila cell types. Experiments were performed to determine whether P elements transposing from a given chromosome to its homolog would preferentially insert in the region corresponding to the donor site, perhaps due to such a proximity. AP[lacZ;ry ⁺] element at thecactus locus (35F) on the second chromosome was mobilized and 96 insertions on the homolog were recovered. The distribution of these new insertions was determined by recombination mapping and molecular analysis, and compared with a control set of 93 second-chromosome insertions originating from theX chromosome. A nearly threefold preference was observed for re-insertion in a region of two to three number divisions aroundcactus on the homolog. However, none of these local insertions was actually within 50 kb of the site atcactus corresponding to the starting site. This is in marked contrast to the previously described phenomenon of intrachromosomal local transposition, where the majority of local transpositions are within 10 kb. The data suggest that the mechanisms for intrachromosomal and interchromosomal local transposition are distinct, and are consistent with a model for interchromosomal local transposition involving proximity of homologous chromosomal regions in the nuclei of the germline cells.     

Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage

Summary Most of the inducible mutagenesis observed in Escherichia coli after treatment with many DNA damaging agents is dependent upon the products of the umuD,C operon. RecA-mediated proteolytic processing of UmuD yields a carboxyl-terminal fragment (UmuD) that is active for mutagenesis. Processing of UmuD is therefore a critical step in the fixation of mutations. In this paper we have analyzed the requirements for UmuD processing in vivo. Standard immuno-detection assays, coupled with a sensitive chemiluminescence detection assay, have been utilized to probe levels of chromosomally encoded Umu proteins from whole-cell E. coli extracts. We found that the derepression of additional SOS gene products, other than RecA, was not required for UmuD processing. Moreover, efficient cleavage of UmuD was observed only in the presence of elevated levels of activated RecA, suggesting that efficient processing would occur only under conditions of severe DNA damage. Detection of chromosomally encoded Umu proteins has allowed us, for the first time, to measure directly the cellular steady-state levels of these proteins under various SOS inducing conditions. UmuD was present at 180 copies per uninduced cell and was measured at 2400 copies per cell in strains that lacked a functional repressor. Induced levels of UmuC were approximately 12-fold lower than UmuD with 200 molecules per cell. These levels of cellular UmuC protein suggest that it functions through specific protein-DNA or protein-protein interactions, possibly as a lesion recognition protein or by interacting with DNA polymerase III.     

Heterogeneity of calcium compartmentation: Electron probe analysis of renal tubules

Summary The objective of this study has been to determine the intracellular localization of calcium in cryofixed, cryosectioned suspensions of kidney proximal tubules using quantitative electron probe X-ray microanalysis. Two populations of cells have been identified: 1) Viable cells, representing the majority of cells probed, are defined by their relatively normal K/Na concentration ratio of 41. Their measured Ca content is 4.1±1.4 (sem) mmol/kg dry wt in the cytoplasm and 3.1 ± 1.1 mmol/kg dry wt in the mitochondria, or an average cell calcium content of 3.8 mmol/kg dry wt. 2) Nonviable cells, defined by the presence of dense inclusions in their mitochondria and a K/Na concentration ratio of 1. The Ca content is 15±2 mmol/kg dry wt in the cytoplasm and 685±139 mmol/kg dry wt in the mitochondria of such cells. Assuming 25 to 30% of the cell volume is mitochondrial, the overall calcium content of such nonviable cells is 210 mmol/kg dry wt. The presence of these inclusions in 4 to 5% of the cells would account for the average total Ca content measured in perchloric acid extracts of isolated proximal tubule suspensions ( 18 nmol/mg protein or 12.6 mmol/kg dry wt). Whole kidney tissues display a large variability in toal Ca content (4.5 to 18 nmol/mg protein, or 3.4 to 13.5 mmol/kg dry wt), which could be accounted for by inclusion in 0 to 4% of the cells. The electron probe X-ray microanalysis (EPXMA) data conclusively demonstrate that thein situ mitochondrial Ca content of viable cells from the kidney, proximal tubule is low and support the idea that mitochondrial Ca may regulate dehydrogenase activity but probably does not normally control cytosolic free Ca.     

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.     

Na,K-ATPase in several tissues of the rat: Tissue-specific expression of subunit mRNAs and enzyme activity

Summary The relative contents of Na,K-ATPase subunit mRNAs in rat renal cortex; ventricular myocardium, skeletal muscle (hind limb), liver and brain (cerebrum) were measured. Expressed per unit DNA, mRNA₁ content was 2-fold greater in the kidney and brain as compared to either heart, skeletal muscle or liver. The hierarchy of mRNA₂ expression was brain > skeletal muscle > heart, whereas mRNA₃ was restricted to brain. Betal subunit mRNA content in both kidney and brain exceeded the abundance of liver mRNA ₁ by 7-fold. In all tissues examined, the combined abundances of the alpha subunit mRNAs exceeded the content of mRNA ₁ The hierarchy of Na,K-ATPase activity expressed per unit. DNA was brain > kidney > skeletal muscle = heart > liver. The sum of mRNA as well as mRNA ₁ content, expressed per g of tissue, was highest in brain and kidney. A statistically significant correlation between mRNA ₁ content and Na,K-ATPase activity was evident.     

Endothelin-1 and insulin induce cellular inactivation of protein kinase FA/glycogen synthase kinase-3α in a common signaling pathway

In this study, we investigate the effects of endothelin-1 (ET-1) and insulin on the cellular activity of protein kinase F_A/glycogen synthase kinase-3 (kinase F_A/GSK-3) in rat adipocytes. The cellular activity of kinase F_A/GSK-3 is inhibited to 50% of control within 30 min when cells are treated with 1 nM ET-1 at 37°C; in addition, significant inhibition to 60% of control is observed at as low as 1 pM ET-1. Conversely, ET-1 at concentrations up to 1 nM has no direct effect on purified kinase F_A/GSK-3 in vitro. Immunoblotting analysis further reveals that the protein level of this kinase is not significantly changed when treated with 1 nM ET-1 for 30 min. Similar to ET-1, insulin as low as 10 nM can also induce inactivation of kinase F_A/GSK-3 to 50% of control in adipocytes when processed under identical conditions. Most importantly, when treated with both insulin and ET-1, the activity of kinase F_A/GSK-3 can be decreased only to 50% of control. Taken together, the results provide initial evidence that ET-1 and insulin may regulate this important multisubstrate/multifunctional protein kinase in a common signaling pathway in cells.     

The structure of cyanobacterial phycobilisomes: a model

Phycobilisomes, supramolecular complexes of water-soluble accessory pigments, serve as the major light-harvesting antennae in cyanobacteria and red algae. Regular arrays of these organelles are found on the surface of the thylakoid membranes of these organisms. In the present study, the hemi-discoidal phycobilisomes of several species of cyanobacteria were examined in thin sections of cells and by negative staining after isolation and fixation. Their fundamental structures were found to be the same. Isolated phycobilisomes possessed a triangular core assembled from three stacks of disc-shaped subunits. Each stack contained two discs which were 12 nm in diameter and 6–7 nm thick. Each of these discs was probably subdivided into halves 3–3.5 nm thick. Radiating from each of two sides of the triangular core were three rods 12 nm in diameter. Each rod consisted of stacks of 2 to 6 disc-shaped subunits 6 nm thick. These discs were subdivided into halves 3 nm thick.The average number of discs of 6 nm thickness forming the peripheral rods varied among the strains studied. For certain chromatically adapting strains, the average rod length was dependent upon the wavelength of light to which cells were exposed during growth. Analyses of phycobilisomes by spectroscopic techniques, polyacrylamide gel electrophoresis, and electron microscopy were compared. These analyses suggested that the triangular core was composed of allophycocyanin and that the peripheral rods contained phycocyanin and phycoerythrin (when present). A detailed model of the hemi-discoidal phycobilisome is proposed. This model can account for many aspects of phycobiliprotein assembly and energy transfer.Abbreviations PBS phycobilisome(s) - PBP phycobiliprotein(s) - AP allophycocyanin - PC phycocyanin - PE phycoerythrin - PEC phycoerythrocyanin - AP-B allophycocyanin B - C- cyanobacterial - R- rhodophytan - B- Bangiophycean - SDS sodium dodecyl sulfate - LPP Lyngbya-Plectonema-Phormidium group - Na-KPO₄ buffers NaH₂PO₄ titrated with a solution of KH₂PO₄ of equivalent molarity to a given pH     

Pepsin fragmentation of botulinum type E neurotoxin: Isolation and characterization of 112, 48, 46, and 16 kD fragments

Controlled digestion of 150 kD single chain botulinum type E neurotoxin with pepsin atpH 6.0 produced 112, 48, 46, and 16 kD fragments. These were chromatographically purified; their locations in the 1300 amino acid residue long neurotoxin were determined by identifying the amino terminal 10 residues of 112 and 48 kD fragments, 50 residues of 46 kD fragment, and 59 residues of 16 kD fragment. The 48 and 112 kD fragments contain the N-terminal segment of the neurotoxin (i.e., residue no. 1 to 425 and 1 to 990, respectively), the 46 kD fragment corresponds to 407 residues of the C-terminal region, and the 16 kD fragment contains the 140 residues from a segment nearer to the C-terminus. The 48 kD fragment is similar to the 50 kD N-terminal light chain of the 150 kD dichain neurotoxin, which is generated by tryptic cleavage of the 150 kD single chain neurotoxin, and is separated from the 100 kD C-terminal heavy chain by dithiothreitol (DTT) reduction of an intrachain disulfide bond in the presence of 2 M urea (Sathyamoorthy and DasGupta,J. Biol. Chem. 260, 10461, 1985). The pepsin-generated 48 kD fragment, unlike the light chain, was isolated without exposure to DTT and urea. The single chain 112 kD fragment following trypsin digestion yielded 48 and 60 kD fragments that were separable after DTT reduction of the intrachain disulfide which links them. The N-terminal residues of the smaller fragment were identical to that of the single chain 150 kD neurotoxin; the single chain 112 kD fragment is therefore the neurotoxin minus the 50 kD C-terminal half of the heavy chain. The biological activities of the 48 and 112 kD fragments can be demonstrated in permeabilized PC12 cells (Lomnethet al., J. Neurochem. 57, 1413, 1991); they inhibit norepinephrine release.     

Summer egg production rates of paracalanid copepods in subtropical waters adjacent to Australia's North West Cape

The biological oceanography of waters adjacent to Australia's North West Cape (21° 49 S, 114° 14 E) was studied during the austral summers of 1997/98 and 1998/99. We measured egg production rate (EPR) by the small paracalanid copepods that dominated the calanoid community. Bottle incubation experiments were conducted at a shallow (20 m) station in the mouth of Exmouth Gulf, and at a shelf-break station (80 m). In 1997/98, we measured EPR by Paracalanus aculeatus, P. indicus, Acrocalanus gracilis and Bestiolina similis, but in 1998/99, we concentrated on P. indicus. Maximal observed EPRs by Paracalanus and Acrocalanus species were 30 eggs female^–1 d^–1, but B. similis attained only 17 eggs female^–1 d^–1. Sporadic measurements of EPR by P. aculeatus minor (maximum 4 eggs female^–1 d^–1) and Parvocalanus crassirostris ( 9 eggs female^–1 d^–1) were also made. However, maximal EPRs were seldom achieved and were often less than 10 eggs female^–1 d^–1. There was no difference between EPR of either P. indicus or B. similis in 1997/98 and 1998/99, despite differences in temperature. Trophic resources severely limit copepod egg production in this area. We suggest that variability and skewness of egg production data derived from individual incubations may be used to judge the degree of food limitation of the population and the variability in feeding success between individuals. The dominance of small copepods and the invariance in their EPR suggest that pulses in physical forcing and subsequent primary production will be severely damped by trophodynamic processes before reaching larval fish.     

Cloning and analysis of DNA sequences from Streptomyces hygroscopicus encoding geldanamycin biosynthesis

A gene library constructed from large (20 kb) fragments of total DNA from the geldananmycin-producing strain Streptomyces hygroscopicus 3602 cloned in the plasmid vector pIJ61 were used to transform S. lividans TK24. Three transformants of about 800 tested were found to have acquired the ability to produce an antibiotic lethal to a geldanamycin-sensitive strain of Bacillus subtilis. The plasmids isolated from these transformants, pIA101, pIA102 and pIA103, each contained an insert of 15 kb. A 4.5 kb DNA fragment from the insert in pIA102 hybridised to DNA from S. hygroscopicus 3602 and to DNA encoding part of the erythromycin polyketide synthase but not to S. lividans TK24 DNA. The integration-defective phage vector C31 KC515 containing this 4.5 kb fragment was able to lysogenise S. hygroscopicus 3602 to produce lysogens defective in geldanamycin production. Loss of the prophage restored the ability to produce geldanamycin. Extracts of fermentation broth cultures of S. lividans containing pIA101, pIA102 and pIA102 and pIA103 analysed by thin-layer chromatography (TLC) contained compounds identical or very similar to purified geldanamycin, which were not present in S. lividans. These compounds showed a mass spectrum indistinguishable from geldanamycin. The evidence suggests that the clones contain DNA sequences encoding functions required for geldanamycin biosynthesis including components of the polyketide synthase.     

Resistance to Spodoptera exigua in Apium prostratum

Two Apium accessions were compared with the commercial cultivar Tall Utah 52–70R (A. graveolens [L.]) for resistance to Spodoptera exigua (Hübner)(Lepidoptera: Noctuidae). Oviposition rate was not significantly different between the three genotypes. In all accessions, eggs were usually placed on the upper half of the plants. Implications of this oviposition pattern on S. exigua management in celery are discussed. The wild species A. prostratum ssp prostratum var filiform (A230) showed a significantly higher resistance to S. exigua than 52–70R. The levels of carcinogenic and mutagenic linear furanocoumarins in the commercial cultivar 52–70R (1.41 g/g in the petioles; 5.85 g/g in the leaves) and in the plant accession A. nodiflorum (5.40 g/g in the petioles; 2.99 g/g in the leaves) were far below the concentration reported to produce acute contact dermatitis (18.0 g/g). The levels of furanocoumarins in A. prostratum petioles (186.14 g/g) and leaves (326.45 g/g) were 10 and 18 times higher, respectively, than the concentration known to cause contact dermatitis. However, resistance in A. prostratum was primarily due to non-preference and the linear furanocoumarins did not induce non-preference. Therefore, the resistance shown by this plant accession does not appear to be furanocoumarin-based and may be suitable for transfer to commercial celery for use in S. exigua management.     

Gene-sized DNA molecules of the macronuclei in three species of hypotrichs: Size distributions and absence of nicks

Macronuclear DNAs from three related hypotrichous ciliated protozoans were compared by agarose gel electrophoresis. Each was shown to be composed of DNA duplexes that yielded a unique pattern of bands overlying a continuous distribution of DNA sizes ranging from 400 bp to 20,000 bp. By EM, the number average molecular sizes for doublestranded DNA were 2,200 bp for Oxytricha sp., 2,514 bp for Stylonychia pustulata and 1,836 bp for Euplotes aediculatus. Contrary to previous reports we present evidence that the macronuclear DNAs in each of these three organisms lack single-stranded interruptions.