Chromosomal Organization of Rrna Operons in Bacillus Subtilis

Integrative mapping with vectors containing ribosomal DNA sequences were used to complete the mapping of the 10 rRNA gene sets in the endospore forming bacterium Bacillus subtilis. Southern hybridizations allowed the assignment of nine operons to distinct BclI restriction fragments and their genetic locus identified by transductional crosses. Nine of the ten rRNA gene sets are located between 0 and 70 degrees on the genomic map. In the region surrounding cysA14, two sets of closely spaced tandem clusters are present. The first (rrnJ and rrnW) is located between purA16 and cysA14 closely linked to the latter; the second (rrnI, rrnH and rrnG) previously mapped within this area is located between attSPO2 and glpT6. The operons at or near the origin of replication (rrnO,rrnA and rrnJ,rrnW) represent "hot spots" of plasmid insertion.     

The Differential Contribution by Individual Enzymes of Glycolysis and Protein Catabolism to the Relationship between Heterozygosity and Growth Rate in the Coot Clam, Mulinia Lateralis

The locus-specific effects of heterozygosity upon individual growth rate were determined for 15 polymorphic enzymes among 1906 individuals from a single cohort sample of the marine bivalve Mulinia lateralis. Two measures of individual growth rate (total wet weight and shell length) were made at collection and after a period of growth in the laboratory. The correlation between heterozygosity and growth rate was independently determined for each locus using multiple linear regression, thereby providing a rank of individual locus effects; these differed significantly. The four estimated rankings of relative locus effects (initial length, initial weight, length added in the laboratory, and added weight) were not statistically different. That is, a locus with a large effect of heterozygosity on growth rate in nature had a similarly large effect on laboratory growth rate. The effect of a locus was not related to heterozygosity per se; some highly heterozygous loci had no detectable correlation with growth rate. The data contained two pairs of relatively tightly linked loci; in both cases one locus of a pair had significant effects on growth rate, while the other had no effect. Loci with large and significant correlations with growth rate synthesize enzymes which function in protein catabolism or glycolysis; heterozygosity in enzymes of the pentose shunt, redox balance, or other miscellaneous metabolic roles was not correlated with growth rate. Since the metabolic basis for the correlation is known to derive from individual differences in net energy status, particularly energetic costs of whole-body protein turnover, these data indicate that phenotypic effects (e.g., variation in growth rate) are determined by heterozygosity at the studied genes, not other linked loci.     

Role of self carriers in the immune response and tolerance. XII. Effect of epitope density and antigen-presenting cell phenotype on the presentation of hapten-modified self for the induction of immunity or tolerance in vitro

The data presented in the accompanying paper (J. P. Cogswell, R. P. Phipps, and D. W. Scott, Cell. Immunol. 114, 55-70, 1988) indicate that certain macrophage-like and lymphoid dendritic-like (P388AD.2) tumor lines which express major histocompatibility encoded class II (Ia) antigens and produce interleukin 1 (IL-1) are uniquely able to present hapten-modified self (HMS) in an immunogenic fashion in vivo. In the current study, the relationship between phenotype and function has been confirmed utilizing a completely in vitro system. This investigation revealed that B-cell priming required T cells restricted to P388AD.2's I-A antigens. In addition, exogenous IL-1 reconstituted the response of an IL-1-deficient tumor (P388AD.2-ILd), although it had no effect on the other nonimmunogenic Ia+ tumor lines. Unlike the in vivo system, effective B-cell tolerance was induced when P388AD.2 was modified with high concentrations (10 mM) of hapten or when highly haptenated tumor was added to 0.1 mM TNBS-modified P388AD.2. These results suggest that positive regulation of in vitro immune responses to HMS is dependent upon the phenotype of the accessory cell carrier (with lymphoid dendritic-like cells being unusually potent), while negative regulation is associated with high epitope density. This system now allows the dissection of the properties of different accessory cells and the signals required for B-cell priming or tolerance induction.     

Role of self carriers in the immune response and tolerance. XI. Correlation of Ia expression and interleukin-1 production with delivery of immunogenic signals in vivo by hapten-modified accessory cell-like tumor lines

It was recently demonstrated that a lymphoid dendritic-like tumor, P388AD.2, presented hapten-modified self (HMS) in an immunogenic fashion even after injection via the normally "tolerogenic" intravenous (iv) route. To determine whether this property was unique to the P388AD.2 line, other hapten-modified tumors were administered iv and the result of their presentation was measured by changes in the number of splenic plaque-forming cells (PFC) following in vitro challenge with thymic-independent antigens. Of the six tumors tested, two (P388 and J774.5R) primed for augmented PFC responses, while four others (P388NA.10, P388D1, WEHI-231, and 70Z/3) did not. When these tumors were compared for Ia expression and production of interleukin-1 (IL-1), it was discovered that (1) all of the immunogenic tumors were Ia+ and IL-1 producing (IL-1+), although not all Ia+,IL-1+ tumors could elicit augmented PFC responses; (2) none of the tumors that were deficient in either Ia expression or IL-1 production could prime B-cell responses in vivo; and (3) the ability to augment PFC responses was proportional to the density of Ia on the immunogenic tumors. These results demonstrated that P388AD.2 was not the only tumor line capable of presenting HMS iv as an immunogen, and that the accessory cell phenotype is critical for the induction of an immunogenic response in vivo.     

Do motilin and pancreatic polypeptide regulate duodenal bile acid delivery?

The plasma levels of the enteric hormones, motilin and pancreatic polypeptide, cycle in association with fasting intestinal motility and are altered by feeding. Intravenous administration of motilin causes gallbladder contraction and increased sphincter of Oddi phasic motor activity, whereas pancreatic polypeptide causes gallbladder relaxation. To determine if endogenous plasma levels of motilin and pancreatic polypeptide control sphincter of Oddi and gallbladder motility, and regulate duodenal bile acid delivery, we measured during fasting and after feeding the correlation between (a) changes in plasma motilin or pancreatic polypeptide, and (b) the duodenal delivery of a steady-state hepatic output of radiolabelled bile acid. Four dogs were prepared with duodenal cannulas. Duodenal motility was recorded manometrically. Plasma levels of pancreatic polypeptide and motilin were determined during a full cycle of the migrating myoelectric complex for 20 min before and 40 min after ingestion of a standard meal. To assess the effect of the sphincter of Oddi and the gallbladder together, or the gallbladder alone on duodenal bile acid delivery, the dogs received a continuous i.v. infusion of [14C]taurocholic acid (TCA); duodenal delivery of TCA was quantitated with the sphincter of Oddi intact using duodenal marker perfusion, or with the sphincter of Oddi cannulated and zero outflow resistance. In the interdigestive period with the sphincter of Oddi intact, only 0.1 (r2) of the variance of duodenal bile acid delivery can be predicted from the variance of motilin, and the correlation of plasma pancreatic polypeptide with duodenal TCA delivery is opposite that expected if pancreatic polypeptide caused gallbladder relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment

Filaggrin is a specific epidermal protein which is the precursor of the free amino acids, urocanic acid and pyrrolidone carboxylic acid which are largely responsible for the ability of the stratum corneum of the skin to remain hydrated at low environmental humidity. The distribution of filaggrin shown by immunofluorescence in the stratum corneum of the rat changed dramatically during the first hours of postnatal life. During late foetal development, filaggrin accumulated through the entire thickness of the stratum corneum, indicating that there was a block on the subsequent processing of the protein which normally would convert it to free amino acids. Immediately after birth this block was lifted and normal proteolysis of the filaggrin took place in the outer part of the stratum corneum, leaving the normal adult pattern of a thin zone of cells containing filaggrin at the bottom of the stratum corneum. This activation of filaggrin proteolysis was dependent on the drop in external water activity caused by the transition from an aqueous environment in utero to a dryer environment after birth and it could be blocked by maintaining a 100% humidity atmosphere around the newborn rat after birth. In isolated stratum corneum in vitro, filaggrin proteolysis took place only between 80 and 95% relative humidity, both higher and lower relative humidity blocked the proteolysis. Application of occlusive patches to adult rats prevented the normal proteolysis of filaggrin, indicating that this mechanism controls not only the massive filaggrin proteolysis occurring after birth but also the proteolysis occurring during normal stratum corneum maturation. The stratum corneum therefore has the ability to respond to changes in external humidity by altering the level of the stratum corneum where it converts its reserves of filaggrin into water binding amino acids, such that under humid conditions water binding components will be produced in only the most superficial stratum corneum, or even not produced at all.     

Distribution of Hydrogen-Metabolizing Bacteria in Alfalfa Field Soil

H₂ evolved by alfalfa root nodules during the process of N₂ fixation may be an important factor influencing the distribution of soil bacteria. To test this hypothesis under field conditions, over 700 bacterial isolates were obtained from fallow soil or from the 3-mm layer of soil surrounding alfalfa (Medicago sativa L.) root nodules, alfalfa roots, or bindweed (Convolvulus arvensis L.) roots. Bacteria were isolated under either aerobic or microaerophilic conditions and were tested for their capacity to metabolize H₂. Isolates showing net H₂ uptake and ³H₂ incorporation activity under laboratory conditions were assigned a Hup⁺ phenotype, whereas organisms with significant H₂ output capacity were designated as a Hout⁺ phenotype. Under aerobic isolation conditions two Hup⁺ isolates were obtained, whereas under microaerophilic conditions five Hup⁺ and two Hout⁺ isolates were found. The nine isolates differed on the basis of 24 standard bacteriological characteristics or fatty acid composition. Five of the nine organisms were isolated from soil around root nodules, whereas the other four were found distributed among the other three soil environments. On the basis of the microaerophilic isolations, 4.8% of the total procaryotic isolates from soil around root nodules were capable of oxidizing H₂, and 1.2% could produce H₂. Two of the Hup⁺ isolates were identified as Rhizobium meliloti by root nodulation tests, but the fact that none of the isolates reduced C₂H₂ under the assay conditions suggested that the H₂ metabolism traits were associated with various hydrogenase systems rather than with nitrogenase activity. Results from this study support the concept that H₂ evolution by alfalfa root nodules has a significant effect on the surrounding microenvironment and influences the number and diversity of bacteria occupying that region.     

Comparison of growth,exogenous auxin sensitivity,and endogenous indole-3-acetic acid content in roots ofHordeum vulgare L. and an agravitropic mutant

The chemically induced barley (Hordeum vulgare L.) mutation, agr, was found to be a simple recessive trait resulting in agravitropic roots and normal gravitropic shoots. The total seedling root growth was similar for mutant and wild-type roots, although the mutant had fewer roots per seed and greater elongation per root. Although the concentration of exogenous indole-3-acetic acid (IAA) required to reduce root growth by 50% (GR50) was 12 times greater for the agravitropic mutant, agravitropic and gravitropic roots were equally sensitive to exogenous applications of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA). Root IAA contents, determined by high-pressure liquid chromatography (HPLC), were not different for gravitropes and agravitropes. The greater root elongation rates, lack of sensitivity to exogenous IAA, and normal endogenous IAA levels indicate that auxin-controlled growth regulation may be altered in the mutant.     

Biosynthesis of porphyrins and corrins. 1. 1H and 13C NMR spectra of (hydroxymethyl)bilane and uroporphyrinogens I and III

High-field NMR spectroscopic methods have been applied to study the reactions catalyzed by porphobilinogen (PBG) deaminase and uroporphyrinogen III (uro'gen III) cosynthase, which are the enzymes responsible for the formation of the porphyrin macrocycle. The action of these enzymes in the conversion of PBG, [2,11-13C]PBG, and [3,5-13C]PBG to uro'gens I and III has been followed by 1H and 13C NMR, and assignments are presented. The principal intermediate that accumulated was the correspondingly labeled (hydroxymethyl)bilane (HMB), the assignments for which are also presented.     

The equine protease inhibitory system (Pi): Abnormal expressions of PiF,PiL, and PiS

Three cases of abnormal expression of the equine protease inhibitory alleles, Pi F, L, and S₁, were observed following the examination of 30,000 plasma samples by one-dimensional acid (pH 4.6) polyacrylamide gel electrophoresis. Characterization of the abnormal proteins in terms of isoelectric point, molecular mass, inhibitory spectra, and sialic acid content was performed using one- and two-dimensional electrophoretic techniques. The Pi F and S₁ abnormalities were postulated to be the result of amino acid substitutions causing alterations in the processing of the carbohydrate side chains. No explanation could be offered for the Pi L abnormality other than a charge shift mutation. Abnormal types, F*, L*, and S*₁ behaved as alleles but the distribution of L* in offspring from one stallion (present in only 6 of 83 offspring) differed significantly from expectation.This work was supported by a grant from the Australian Stud Book, Alison Road, Randwick, N.S.W. 2031.