Metabolic interlock between the acetolactate synthase isoenzymes and lysine biosynthesis in Escherichia coli K-12

Summary Some of the strains containing mutations in the genes for the acetolactate synthase isoenzymes are temperature sensitive (ts). Suppression of the acetolactate synthase defect due to one of these mutations suppresses also the ts phenotype; moreover, a genetic cross shows that the two phenotypes cannot be dissociated.The ts phenotype is accompanied by a decreased efficiency of transduction with Pl phage. Observations at the light microscope show formation of abnormal cells. Under specific conditions diaminopimelate stimulates growth and restores normal transduction efficiency. The rate of diaminopimelate formed and excreted by non-growing cells decreases when an acetolactate synthase mutation is present.We give evidence that the ts phenotype is due to an increased formation of lysine from diaminopimelate; this causes a starvation for the latter and therefore cell wall abnormalities. In fact, even at the permissive temperature, the lysine pool is 8x increased in a strain with an acetolactate synthase defect, while a slight decrease in the diaminopimelate pool is observed. Moreover, introduction into a ts strain of a mutation in lysA (the gene coding for diaminopimelate decarboxylase) cures the ts phenotype. Finally among the temperature resistant revertants we found some lysine auxotrophs.     

Immunohistochemische Lokalisierung der Isoenzyme der Creatinkinase im menschlichen Gewebe

Zusammenfassung In Gefrierschnitten lassen sich die Isoenzyme der Creatinkinase MM und BB durch spezifische Antikörper selektiv präzipitieren. Nach Sättigung der freien Antikörpervalenzen durch Zugabe von exogenem Antigen wird das im Schnitt fixierte Isoenzym durch eine histochemische Technik lokalisiert. Die Methode ermöglicht eine isoenzymspezifische Histochemie der Creatinkinase.Die Anwendung dieser Technik am menschlichen Gewebe führte zu folgenden Ergebnissen: CK-BB ließ sich sowohl in der Muskulatur als auch in der Mucosa des Colons nachweisen, während im Skelettmuskel zwischen cytoplasmatischer CK-MM und membrangebundenem Enzym im sarkoplasmatischen Reticulum und im Sarkolemm differenziert werdn konnte. In der Tonsille wurden CK-BB in epithelialem Gewebe, CK-MM in Muskelfasern lokalisiert. Das Isoenzymmuster von benachbarten einzelnen Schnitten wurde parallel immunologisch analysiert. Auf den Vorteil der Kombination von Immuntitration und Histchemie an gleichen Schnitten wird verwiesen.

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Immunohistochemical localization of creatinkinase isoenzymes in human tissue

Summary The use of an immunohistochemical method permits the localization of creatine kinase isoenzymes MM and BB in tissue sections. Frozen sections are first incubated with the specific antiserum and secondly with the soluble antigen under investigation. The antibody fixed creatine kinase can then be visualized by the tetrazolium-salt linked histochemical reaction.In this way CK-BB was found in the smooth muscle and the mucosa of the human colon. In sections of skeletal muscle CK-MM was predominantly localized in the intermyofibrillar space. Membrane bound activity could be demonstrated in the sarcoplasmic reticulum and the surface membrane after elution of the cytoplasmic enzyme.In the human tonsilla CK-BB was localized in lymphatic and epithelial tissues, CK-MM in the muscle fibers. The isoenzyme patterns in single sections of tonsilla were in parallel determined by the immunotitration assay. The results indicate the usefulness of the combined application of histochemistry and immunotitration in serial tissue sections.

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Unterstützt durch das Schwerpunktprogramm Enzymdiagnostik Pf 32/36 der Deutschen Forschungsgemeinschaft, 5300 Bonn-Bad Godesberg     

Chemical inhibition of cell wall formation and cytokinesis,but not of nuclear division,in protoplasts of Nicotiana tabacum L. cultivated in vitro

The effect of cytochalasin B, colchicine, coumarin and 2,6-dichlorobenzonitrile on cell wall formation and cellular division was studied by light and electron microscopy with tobacco mesophyll protoplasts cultivated in vitro. 2,6-dichlorobenzonitrile was found to be the most effective and reversible inhibitor of cell wall formation. The other inhibitors caused irreversible damage and/or inhibited mitosis. In protoplasts cultivated in the presence of 2,6-dichlorobenzonitrile the total inhibition of cell wall formation had no effect on nuclear division, but cytokinesis was totally inhibited so that multinucleate protoplasts were obtained.Abbreviations DB 2,6-dichlorobenzonitrile=dichlobenil - CB cytochalasin B     

Cytologic evidence for three human X-chromosomal segments escaping inactivation

Summary Early replication of prometaphasic human sex chromosomes was studied with the bromodeoxyuridine (BrdU)-replication technique. The studies reveal that two distal segments of Xp, including bands Xp 22.13 and Xp 22.3, replicate early in S-phase and therefore may not be subject to random inactivation. Furthermore, the replication of these distal segments of Xp occurs synchronously with those of the short arm of the Y chromosome including bands Yp 11.2 and Yp 11.32. These segments of Xp and Yp correspond well to the pairing segment of the X and Y chromosomes where a synaptonemal complex forms at early pachytene of human spermatogenesis. The homologous early replication of Yp and the distal portion of Xp may be interpreted as a remnant left untouched by the differentiation of heteromorphic sex chromosomes from originally homomorphic autosomes. A third early replicating segment is situated on the long arm of the X chromosome and corresponds to band Xq 13.1. This segment may be correlated with the X-inactivation center postulated by Therman et al. (1979).     

Isolation and visualisation of alkali stable protein/DNA complexes

Distinct polypeptides, 54,000–68,000 daltons in size, are alkali-stably bound to eukaryotic DNA. DNA fragments several hundred base pairs in length associated with these polypeptides are preferentially retained on glass fibre filters from solutions containing 1 M sodium chloride. About 50 percent of the protein/DNA complexes present in total DNA are retained on filters together with about 2 percent of the DNA. This preferential binding is demonstrated (a) by the ratio of ³H and ³⁵S radioactivity retained on filters after filtration of DNA from [³H]thymidine and L-[³⁵S]methionine labelled cells, (b) radioiodination of the material retained on filters and passing filters respectively and (c) by electron microscopical visualisation of the polypeptide component in the complexes after chemical modification with dinitrofluorobenzene (DNFB) followed by incubation with dinitrophenyl (DNP) specific antibodies.     

Utilization of limiting concentrations of ortho-phosphate and production of extracellular organic phosphates in cultures of marine diatoms

The utilization of ortho-phosphate by two coastal marine diatomspecies, Nitzschia closterium and Cyclotella cryptica, was studiedin batch cultures. The hypothesis was tested that thresholdconcentrations in the phosphate uptake determine the lower limitof environmental phosphate, permitting the existence of species.The turn-over time of residual medium phosphate in culturesis {small tilde}10 min, indicating a rapid equilibration ofconcentration dependent on uptake with leakage of ortho-phosphate.Increasing phosphate starvation in cultures diminished the residualortho-phosphate in the range of {small tilde}60–<2nmol l^–1, as measured radiochemically after elution onSephadex® G-10 gel. These concentrations encompass the rangeof limiting phosphate concentration in continuous cultures ofthe few microalgae, for which these concentrations are actuallymeasured. The diatoms excreted {small tilde}20–100 nmolI^–1 of organic phosphate. One dominating compound, probablyan unusual nucleotide, was incompletely or not resorbed underphosphate starvation. In contrast, Nitzschia closterium excretedunder ample phosphate supply a series of three related compounds,probably phospholipids, that were resorbed under depletion.The association of the organic phosphates with macromolecularexudates is interpreted, along with the other observations,as an indication for a hardly explored periplasmatic phosphatemetabolism in these algae. ³Dedicated to Prof. Dr. H.-A. von Stosch in honour of his 75thbirthday. ⁴This study was conducted at the University of Marburg undersupport of the Humboldt Foundation Publication no. 64 of theproject "Biological Research of the Eems-Dollard Estuary".     

The organization of the gene for the human cerebroside sulfate activator protein

The organization of 14 exons covering 97% of the cDNA sequence of human cerebroside sulfate activator protein precursor has been determined from two overlapping EMBL-4 human genomic clones extending over 17kb. All exons and exon/intron splice junctions and five introns were sequenced. Exon 8 consists of only 9 bp and is involved in alternative splicing which generates three different mRNAs of cerebroside sulfate activator precursor.