The Mrs1 Splicing Factor Binds the bI3 Group I Intron at Each of Two Tetraloop-Receptor Motifs

Most large ribozymes require protein cofactors in order to function efficiently. The yeast mitochondrial bI3 group I intron requires two proteins for efficient splicing, Mrs1 and the bI3 maturase. Mrs1 has evolved from DNA junction resolvases to function as an RNA cofactor for at least two group I introns; however, the RNA binding site and the mechanism by which Mrs1 facilitates splicing were unknown. Here we use high-throughput RNA structure analysis to show that Mrs1 binds a ubiquitous RNA tertiary structure motif, the GNRA tetraloop-receptor interaction, at two sites in the bI3 RNA. Mrs1 also interacts at similar tetraloop-receptor elements, as well as other structures, in the self-folding Azoarcus group I intron and in the RNase P enzyme. Thus, Mrs1 recognizes general features found in the tetraloop-receptor motif. Identification of the two Mrs1 binding sites now makes it possible to create a model of the complete six-component bI3 ribonucleoprotein. All protein cofactors bind at the periphery of the RNA such that every long-range RNA tertiary interaction is stabilized by protein binding, involving either Mrs1 or the bI3 maturase. This work emphasizes the strong evolutionary pressure to bolster RNA tertiary structure with RNA-binding interactions as seen in the ribosome, spliceosome, and other large RNA machines.     

Kinetic and thermodynamic framework for assembly of the six-component bI3 group I intron ribonucleoprotein catalyst

The yeast mitochondrial bI3 group I intron RNA splices in vitro as a six-component ribonucleoprotein complex with the bI3 maturase and Mrs1 proteins. We report a comprehensive framework for assembly of the catalytically active bI3 ribonucleoprotein. (1) In the absence of Mg(2+), two Mrs1 dimers bind independently to the bI3 RNA. The ratio of dissociation to association rate constants, k(off)/k(on), is approximately equal to the observed equilibrium K(1/2) of 0.12 nM. (2) At magnesium ion concentrations optimal for splicing (20 mM), two Mrs1 dimers bind with strong cooperativity to the bI3 RNA. k(off)/k(on) is 15-fold lower than the observed K(1/2) of 11 nM, which reflects formation of an obligate intermediate involving one Mrs1 dimer and the RNA in cooperative assembly of the Mrs1-RNA complex. (3) The bI3 maturase monomer binds to the bI3 RNA at almost the diffusion-controlled limit and dissociates with a half-life of 1 h. k(off)/k(on) is approximately equal to the equilibrium K(D) of 2.8 pM. The bI3 maturase thus represents a rare example of a group I intron protein cofactor whose binding is adequately characterized by a one-step mechanism under conditions that promote splicing. (4) Maturase and Mrs1 proteins each bind the bI3 RNA tightly, but with only modest coupling (approximately 1 kcal/mol), suggesting that the proteins interact at independent RNA binding sites. Maturase binding functions to slow dissociation of Mrs1; whereas prior Mrs1 binding increases the bI3 maturase k(on) right to the diffusion limit. (5) At effective concentrations plausibly present in yeast mitochondria, a predominant assembly pathway emerges involving rapid, tight binding by the bI3 maturase, followed by slower, cooperative assembly of two Mrs1 dimers. In the absence of other factors, disassembly of all protein subunits will occur in a single apparent step, governed by dissociation of the bI3 maturase.     

Immuno-electron-microscopic analysis of the basal route of secretion in the subcommissural organ of the rabbit

Summary Low-temperature-embedded tissue of the subcommissural organ (SCO) of the rabbit was analyzed for the basal route of secretory product by means of indirect immuno-metal cytochemistry (protein A-gold technique) at the electron-microscopic level. By use of (1) an antiserum against bovine Reissner's fibre (see Sterba et al. 1981) and, thereafter, (2) particulate gold-marker solution, immunoreactive sites could be clearly visualized within the extracellular matrix of both (a) the basal part of the ependymal cell layer, and (b) the hypendyma proper. Abundant secretory material was identified within (i) dilated intercellular spaces (a + b) as well as (ii) branching basal lamina labyrinths and distinct perivascular spaces (b). All these compartments are thought to belong to a system of extracellular channels, which may function in secretion directed toward hypendymal blood vessels.Supported by Grants from the Ministry for Sciences and Technology of the German Democratic RepublicThe expert technical assistance of Mrs. S. Mehnert, Mrs. E. Siebert, Mrs. Ch. Schneider, Mrs. I. Seifert and Mr. H. Wolf is gratefully acknowledgedDedicated to Prof. Dr.Dr.h.c. Andreas Oksche on the occasion of his 60th birthday     

The development of the human tonsilla palatina

Summary Tonsils of human fetuses at the 8th to the 28th gestational week (g.w.) were investigated by electron microscopy, enzyme histochemistry, and immunohistochemistry on cryostat sections. The development of the tonsilla palatina starts during the 14th g.w. when the mesenchyme underlying the mucous membrane of the tonsillar cavity becomes invaded by mononuclear wandering cells. In fetuses of about the 16th g.w. epithelial crypts grow down into the connective tissue and are infiltrated by T-lymphocytes. At the same time, precursors of interdigitating cells (IDC) can be identified among the epithelial cells. Frequently, lymphocytes and IDC-like cells are in close contact. From these findings it is concluded that the infiltrated crypt epithelium in the human tonsilla palatina represents a T-cell region. Primary follicles develop in earlier fetal stages than in all other secondary lymphoid organs. They contain precursors of dendritic reticulum cells and lymphoid cells that belong to the B-cell line. These primary follicles may be considered as the first assemblage of B-cell regions in human fetal lymphoid tissue. The present findings indicate that the formation of different stationary elements during the development of B-cell regions and T-cell regions is an important factor for the homing and antigen-dependent maturation of different subpopulations of immunocompetent lymphoid cells.This investigation was supported by grants from the Deutsche Forschungsgemeinschaft, particularly the Sonderforschungsbereich 111The authors appreciate the contribution of human fetal material from Dr. J. von Hollweg and Dr. J. Körner from the Hospital Heidberg c.o. Hamburg and the excellent technical assistance of Mrs. O.-M. Bracker, Mrs. H. Hansen, Mrs. I. Knauer, Mrs. R. Köpke, Mrs. I. König, Mrs. F. Müller, Mrs. H. Siebke and Mrs. H. Waluk     

Mrs2p is an essential component of the major electrophoretic Mg2+ influx system in mitochondria

Steady-state concentrations of mitochondrial Mg(2+) previously have been shown to vary with the expression of Mrs2p, a component of the inner mitochondrial membrane with two transmembrane domains. While its structural and functional similarity to the bacterial Mg(2+) transport protein CorA suggested a role for Mrs2p in Mg(2+) influx into the organelle, other functions in cation homeostasis could not be excluded. Making use of the fluorescent dye mag-fura 2 to measure free Mg(2+) concentrations continuously, we describe here a high capacity, rapid Mg(2+) influx system in isolated yeast mitochondria, driven by the mitochondrial membrane potential Deltapsi and inhibited by cobalt(III)hexaammine. Overexpression of Mrs2p increases influx rates 5-fold, while the deletion of the MRS2 gene abolishes this high capacity Mg(2+) influx. Mg(2+) efflux from isolated mitochondria, observed with low Deltapsi only, also requires the presence of Mrs2p. Cross-linking experiments revealed the presence of Mrs2p-containing complexes in the mitochondrial membrane, probably constituting Mrs2p homo- oligomers. Taken together, these findings characterize Mrs2p as the first molecularly identified metal ion channel protein in the inner mitochondrial membrane.     

Ontogeny and organization of the stationary non-lymphoid cells in the human thymus

Summary Ontogenetic differentiation of the human thymus was investigated in 50 embryos by means of light and electron microscopic methods in an attempt to clarify the morphogenesis of the complicated microecology of thymic tissue. At the 8th gestational week (g.w.), the primordium of the thymus contains almost exclusively undifferentiated epithelial cells. At the 10th g.w., the epithelial cells in the central part are spindle-shaped. During the subsequent weeks the cortical region of the thymus becomes separated into lobes by mesenchymal septa containing hemopoietic precursor cells and large electronlucent cells with irregularly shaped nuclei. The latter cells are also found in the deeper presumptive medullary regions of the thymus; they differentiate into interdigitating reticulum cells (IDC). The permeation of the medulla of the thymus by non-epithelial IDC occurs concurrently with the formation of cortical and medullary epithelial cells. Between the 12th and 14th g.w. the cortical and medullary differentiation is completed. At this time-stage cortical small lymphocytes differ in morphological shape from medullary lymphocytes, the latter acquiring the appearance of immunocompetent T cells and establishing intimate contact with the IDC.These findings indicate that the thymic cortex and medulla contain different epithelial cells. In addition, the thymic medulla displays cells characterized by the morphology of typical interdigitating reticulum cells of peripheral lymphoid tissue. The structural pattern of the thymus is correlated to morphologically differing lymphoid cell populations in the cortical and medullary regions.This investigation was supported by grants from the Deutsche Forschungsgemeinschaft and by the Sonderforschungsbereich 111The authors dedicate this paper to Professor Helmut Leonhardt on the occasion of his 60th birthday. The authors also appreciate the excellent technical assistance of Mrs. I. Knauer, Mrs. H. Waluk and Mrs. H. Siebke     

Preparation and discharge of secretion in the subcommissural organ of the rat

Summary The secretion of the subcommissural organ (SCO) of the rat was studied by means of immunocytochemistry at the electron-microscopic level with the use of (1) the polar embedding medium Lowicryl K4M at -30° C, (2) the protein A-gold technique, and (3) a rabbit antiserum against bovine Reissner's fiber (see Sterba et al. 1981).Two different substructures of the ependymal and the hypendymal SCO-cells display a positive immunocytochemical reaction: (1) sacs containing flocculent secretion, which originate from the granular endoplasmic reticulum, and (2) vacuoles filled with fine granular secretion, which are pinched off from the Golgi apparatus. The secretory material of the sacs and the vacuoles is discharged both (i) apically into the cerebrospinal fluid and (ii) basally into intercellular spaces of the SCO-hypendyma. The apically released secretion is condensed to a lamina-like formation, which more caudally assumes the form of Reissner's fiber. The route of the basally released secretion remains, however, vague. The periodically striated bodies, which were thought to be morphological mediators of the discharge of the secretion into the capillaries, are never labeled by gold particles.Supported by grants from the Ministry for Science and Technology of the German Democratic RepublicThe expert technical assistance of Mrs. B. Wolff, Mrs. S. Mehnert, Mrs. E. Siebert, Mrs. Ch. Schneider, and Mrs. I. Seifert is gratefully acknowledged     

Mutational analysis of functional domains in Mrs2p, the mitochondrial Mg2+ channel protein of Saccharomyces cerevisiae

The nuclear gene MRS2 in Saccharomyces cerevisiae encodes an integral protein (Mrs2p) of the inner mitochondrial membrane. It forms an ion channel mediating influx of Mg2+ into mitochondria. Orthologues of Mrs2p have been shown to exist in other lower eukaryotes, in vertebrates and in plants. Characteristic features of the Mrs2 protein family and the distantly related CorA proteins of bacteria are the presence of two adjacent transmembrane domains near the C terminus of Mrs2p one of which ends with a F/Y-G-M-N motif. Two coiled-coil domains and several conserved primary sequence blocks in the central part of Mrs2p are identified here as additional characteristics of the Mrs2p family. Gain-of-function mutations obtained upon random mutagenesis map to these conserved sequence blocks. They lead to moderate increases in mitochondrial Mg2+ concentrations and concomitant positive effects on splicing of mutant group II intron RNA. Site-directed mutations in several conserved sequences reduce Mrs2p-mediated Mg2+ uptake. Mutants with strong effects on mitochondrial Mg2+ concentrations also have decreased group II intron splicing. Deletion of a nonconserved basic region, previously invoked for interaction with mitochondrial introns, lowers intramitochondrial Mg2+ levels as well as group II intron splicing. Data presented support the notion that effects of mutations in Mrs2p on group II intron splicing are a consequence of changes in steady-state mitochondrial Mg2+ concentrations.     

Purification of peptide synthetases involved in pristinamycin I biosynthesis.

Several assays of pristinamycin I synthetases based on adenylate or thioester formation were developed. Purification to near homogeneity of these enzymatic activities from cell extracts of Streptomyces pristinaespiralis showed that three enzymes could activate all pristinamycin I precursors. SnbA, a 3-hydroxypicolinic acid: AMP ligase activating the first pristinamycin I residue, was purified 200-fold, using an ATP-pyrophosphate exchange assay. This enzyme was shown to be a monomer with an Mr of 67,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then a multifunctional enzyme, consisting of two identical subunits (SnbC) with Mrs of 240,000 and able to bind covalently L-threonine as a thioester, was purified 100-fold. This protein also activated L-aminobutyric acid, which is further epimerized to generate the third residue of the pristinamycin I macrocycle. A third protein, consisting of two identical subunits (SnbD) with Mrs estimated to be between 250,000 and 350,000, was purified 200-fold. This large enzyme catalyzed thioesterification and subsequent N-methylation of 4-dimethylamino-L-phenylalanine, the fifth pristinamycin I residue. SnbD could also activate L-proline, the fourth pristinamycin I residue, and some preparations retained a low but significant activity for the last two pristinamycin I precursors. Finally, a single polypeptide chain (SnbE) with an Mr of 170,000, catalyzing L-phenylglycine-dependent ATP-pyrophosphate exchange, was purified 3,000-fold and characterized. Stepwise Edman degradation of the entire polypeptides or some of their internal fragments provided amino acid sequences for the four isolated proteins. The purified SnbE protein was further shown to be a proteolytic fragment of SnbD.     

A cytochemical method for fixation and staining of lipids in frozen-dried mouse pancreas for study with light and electron microscopes

Summary A method is described for the cytochemical identification of lipids in acinar cells of the pancreas based chiefly on reactions of their carboxyl ester linkages and their double bonds. The method involves the reaction in vacuo of certain amine and hydrazine vapors with lipids in the solid state. The method is useful for studies with the light and electron microscopes.This research was supported in part by grants from the Public Health Service (GM 08328) and the Commonwealth Fund.I have been guided throughout by the perceptive advice of Professor Herbert S. Anker, Department of Biochemistry, University of Chicago. I am indebted also to the tireless work and support of Mrs. Faustina Manelis and Mrs. Elizabeth Vilkas.Dedicated to Professor Friedrich Wassermann with admiration and affection on the occasion of his eightieth birthday.     

Schoenus vegetation and environmental conditions in South and Southeast Sweden

Summary The relations between the Schoenus phytocoena and their site conditions are elucidated by stand and species ordinations (ORDINA, RA) and by comparisons of ordinations of environmental variables alone and ordination of the combined environmental and species variables (RA). Correlations between the environmental variables show that they may be gathered into two contrasting groups, the carbon and the carbonate groupings, respectively. This first direction of variation is related to hydrological conditions causing the differences in thickness of organic soils, content of organic carbon and dry weight of intact soil per unit volume. The second direction of variation is associated with a nutrient factor complex, in this study represented by available phosphate.The carbon grouping and a constantly high level of mainly topogeneous soil water is typical of sites of the Oxycoccus-Schoenus association. The Sesleria group of the Primula-Schoenus association is related to sites with a periodically low table of topogeneous or soligeneous soil water and mineral or mucky soils with high values of carbonate content, pH and CEC and a fairly low nutrient state, while the sites of the Bartsia-Ophrys and Valeriana groups have a fairly high level of mainly soligeneous soil water and mucky soils with variable carbonate content, intermediate pH and CEC and somewhat higher nutrient state. Periodically flooded sites with topogeneous soil water and mineral soils are typical of the Cladium-Schoenus nigricans, Schoenus intermedius-Schoenus ferrugineus and Glaux phytocoena.Nomenclature is the same as in Tyler (1980).This study is part of my doctor's thesis. I am most grateful to Professor Nils Malmer, Head of the Department, for variable advice and discussions. My thanks are also rendered to Dr. E. van der Maarel, University of Nijmegen, The Netherlands, Dr. R.S. Clymo, Westfield College, University of London, Great Britain, Fil kand. Stefan Persson, Dr. Germund Tyler, Mrs Maj-Britt Larsson, Mrs Anita Balogh, Mrs Mimmi Varga, Mrs Kerstin Richter, and Mr Robert Dewsnap.     

Effect of cyproterone acetate,d-norgestrel and progesterone on the canine mammary gland

Summary The effects of short-term (8 weeks) treatment with different doses of cyproterone acetate (CPA), d-norgestrel (d-N) and progesterone (PRO) on the mammary gland were studied in cycle-synchronized beagle bitches (first anoestrus). Mammary glands from non-treated primiparous beagle bitches in the 6th and 9th weeks of pregnancy were also included. The results were evaluated using the whole-mount technique, histologic, histochemical and biochemical methods. CPA, d-N and PRO were shown to cause dose-dependent mammary growth accompanied by an increase in the mammary weight, DNA content and activity of several histochemically demonstrable dehydrogenases. These changes resembled in some aspects mammary development observed in the last third of pregnancy. A single human oral contraceptive dose (HCD) of CPA as well as a dose as low as 1.0mg/kg/day subcutaneously of PRO was capable of stimulating complete mammary development. A comparable effect was first observed as a result of treatment with as much as 100 times the HCD of d-N. However, d-N and CPA were shown to be more effective than PRO in stimulating ductal proliferative activity. These structural and biochemical responses indicate that quantitative and/or qualitative differences may exist between PRO, the synthetic progesterone derivative CPA and the synthetic nortestosterone progestagen d-N with regard to their growth-promoting effect on the canine mammary gland. This may be explained by possible differences in their potency and range of biological activity, pharmacodynamics and effects on pituitary hormone secretion.The authors are grateful to Dr. Christel Schöbel and Mrs. P. Kurth for carrying out the experimental work on animals, to Dr. Y. Nishino and Mr. M. Leidecker for biochemical determination of DNA, to Dr. J. Kaufmann for statistical analysis, to Miss E. Fallenbacher, Mrs. B. Schilk, Mrs. G. Soulioti and Miss. U. Tüshaus for their excellent technical assistance, and to Dr. P. Günzel for his advice and encouragement     

Exocrine pancreas under experimental conditions

Summary After the application of parachlorophenylalanine (pCPA), an amino acid analogue, paracrystalline inclusions are observed in the exocrine pancreas of the rat. The formation of the paracrystalline structures varies according to the dose and the time of examination. Although the first alterations can be seen in the Golgi apparatus and the condensing vacuoles, the main localization of these structures is within the cisternae of the RER. At the same time as degenerative changes occur in the cells, involving autophagic and heterophagic processes, regneration also takes place. With the freeze-fracturing method, the paracrystalline inclusions are interpreted as lamellae or plates of probably altered secretory proteins in extremely extended RER-cisternae. The fracture surfaces of the paracrystals show a periodicity of about 80 Å running diagonally to the main axis of the paracrystalline structures, which are mainly oriented from the basal parts of the exocrine pancreatic cells to the cell apices.The mechanism of paracrystalline formation is discussed on the basis of the morphologic results. It could be shown that after pCPA administration the amylase content is decreased concomittantly with degranulation. pCPA seems not to be incorporated into secretory proteins; high intracellular concentrations, however, are required to induce the formation of the paracrystalline structures. This morphological study is the basis for other studies dealing with secretion and intracellular transport in the pancreatic acinar cell under experimental conditions.We are very grateful to Mrs. B. Brühl, Mrs. I. Stenull and cand. med. P. Zahn for technical assistence. We also gratefully acknowledge Prof. Dr. R. Taugner for the help with freeze-fracturing     

Ultrastructure of subcellular fractions of bull spermatozoa after hyamine treatment

Summary Ejaculated bull spermatozoa (SZ) were washed and incubated with a cationic surface active agent, Hyamine 2389, and centrifuged using 2-step discontinuous sucrose density gradient. The washed SZ, Hyamine-treated SZ and subcellular spermatozoal fractions obtained after centrifugation were prepared for electron microscopy. The washing did not cause any major structural changes in SZ. The Hyamine treatment of SZ disrupted the outer acrosome membranes. The anterior part of acrosome (the acrosomal cap) was detached retaining its integrity, or forming vesicles by fusing with the cell membrane as in true acrosome reaction. Because of this structural similarity in vesicle formation, Hyamine is assumed to be a suitable experimental initiator for acrosome reaction. The loosened acrosomal membranes were harvested almost totally by the centrifugation. The acrosomes were seen as loosened U-shaped unbroken acrosomal caps or as vesicles with fuzzy acrosomal material. The lightest particles were vesicles consisting of smooth membranes, formed evidently of sperm cell membrane. A negligible amount of fibrous sheaths were also among acrosomal membranes but no other sperm parts were encountered.The authors are thankful to Mrs. Marita Aaltonen, Mrs. Sirpa From, Miss Ulla Mäntylä, Mr. Mauno Lehtimäki and Mr. Urpo Reunanen for their skilful technical assistance.     

Ultrastructure of the age-involuted adult human thymus

Summary Age involuted thymus tissue from a middle aged (33 years) and an old (63 years) man have been examined by electron microscopy and compared with thymus tissue from children. Biopsies had been taken during surgical correction of congenital heart defects.The fine structural architecture of cortex, medulla and connective tissue in the remaining lymphatic islands in the adult thymus investigated was not different to the thymus of children. We were surprised to find vigorous lymphocytopoiesis in the cortical regions and to recognize extended areas of medulla with a cellular composition which obviously provides the same microenvironment for T-cell maturation as the medulla of the non involuted thymus. Our findings are discussed in relation to the increasing arguments that the human thymus serves an immunological function throughout life.This investigation was supported by grants from the Deutsche ForschungsgemeinschaftI express my thanks to Professor Dr. med. Alexander Bernhard (Kiel) for kindly providing the human thymus tissue. The author also appreciates the excellent technical assistance of Mrs. Knauer, Mrs. Parczany, Mrs. Siebke and Mrs. Waluk     

Developmental changes in the structural organization of the lectin discoidin I detected by limited proteolysis

Digestion of discoidin I with several proteolytic enzymes reveals the existence of structural domains in this lectin. Significative differences have been detected in the pattern of fragments generated by V8 protease on discoidin I of various developmental situations. The changes observed can be related to the presence of various types of tetrameric structures in discoidin I. Together with the presence of different types of isoforms in vegetative vs. differentiated cells, the results presented here suggest the involvement of different structural organizations in discoidin I which can be related to the biological functions of this lectin.     

Immunocytochemical localization of urotensin I neurons in the caudal neurosecretory system of the white sucker (Catostomus commersoni)

Summary The localization of urotensin I has been investigated in the caudal neurosecretory system of the white sucker (Catostomus commersoni). The peptide is present in all the cells of the system both large and small, in the large axons passing to the urophysis, and in fine beaded fibres not only within the urophysis but also in a fine plexus lateral to the large cells in the spinal cord proper. The possibility that the caudal neurosecretory system is not a functionally uniform system but rather a collection of dissimilar cells of different synaptic inputs with a common entity, urotensin I, is discussed. Moreover, the feasibility of a urotensin I feedback loop is described.Financial support for this investigation was provided in part by MRC (Canada). K.L. is MRC career investigator; K.L.W, was in receipt of an Alberta Heritage Foundation for Medical Research Fellowship. It is a pleasure to record the valuable technical assistance of Mrs. W. Ho and the dedicated assistance in the collection of the experimental animals by Mrs. Helen Wilson of Nanton, Alberta.     

Population structure, levels of selection, and the evolution of intracellular symbionts

Many obligately intracellular symbionts exhibit a characteristic set of genetic changes that include an increase in substitution rates, loss of many genes, and apparent destabilization of many proteins and structural RNAs. Authors have suggested that these changes are due to increased mutation rates, or, more commonly, decreased effective population size due to population bottlenecks at the symbiont or, perhaps, host level. I propose that the increase in substitution rates and accumulation of deleterious mutations is a consequence of the population structure imposed on the endosymbionts by strict host association, loss of horizontal transmission and potentially conflicting levels of selection. I analyze a population genetic model of endosymbiont evolution, and demonstrate that substitution rates will increase, and the effect of those substitutions on endosymbiont fitness will become more deleterious as horizontal transmission among hosts decreases. Additionally, I find that there is a critical level of horizontal transmission below which natural selection cannot effectively purge deleterious mutations, leading to an expected loss of fitness over time. This critical level varies across loci with the degree of correlation between host and endosymbiont fitness, and may help explain differential retention and loss of certain genes.