Conformation of methylated sequences in HeLa cell 18-S ribosomal RNA: nuclease S1 as a probe

18-S rRNA from HeLa cells was digested with nuclease S1. Under the conditions employed 15% of the total nucleotides and some 50% of the methylated nucleotides were released as low-molecular-weight products. The material which was precipitable by 70% ethanol after nuclease S1 digestion was subjected to further digestion by combined T1 plus pancreatic ribonucleases or by T1 ribonuclease alone, and fingerprints were prepared. It was found that the four sites which are modified late during ribosome maturation, and which contain base modifications, were all accessible to nuclease S1. By contrast fewer than one-half of the sites which are modified early during ribosome maturation, and which contain 2'-O-methyl groups, were accessible to nuclease S1; the remainder were protected, presumably by secondary or tertiary interactions within 18-S rRNA.     

Heterogeneous staining of rat hepatocyte nuclei by HBFP technique

Synopsis The non-enzymatic histochemical technique Haematoxylin-Basic Fuchsinpicric acid (HBFP) was studied in fresh-frozen and Carnoy-fixed, paraffin-embedded rat liver sections. The hepatocyte nuclei fell into two populations and showed either a crimson red or purple staining in frozen as well as paraffin sections. The heterogeneous staining of the rat heptocyte nuclei was also present when the tissue sections were stained by Methyl Green-Pyronin stain. The differing nuclear staining was present in the isolated nuclei also. The HBFP technique, therefore, appears potentially useful when applied to liver and other tissues as well.     

Preliminary observations on plant-rock type relationships in the Shiraz-Dashte Arjan area,South-West Iran

Summary In the Dashte-Arjan area near Shiraz, rock formations appear to control the distribution of various plant species. The Fars formation (Miocene) and the Asmari-Jahrom formation (Eocene-Oligocen) sediments are characterized by distinet plant species and life forms. Among the characteristic calciphytes of Asmari-Jahrom limestones are Astragalus acutus, Amygdalus lyciodes, Cerasus microcarpa, and Fraxinus rotundifolia. The Fars formation limestones are characterized by Astragulus gossypinus, Acantholimon flexuosum, Noaea mucronata and Phlomis bruguieri, Except for Glycrrhiza glabra, white and red marls seem to have similar species, such as Alhagi maurorum and Carthamus oxyacantha. Gypsum of Fars formation has Berberis integerrima and Rosa beggeriana. Various life forms have been distinguished and were found to be confined to different rock types as well.Nomenclature of species is given in table 1.We are greately indebted to Dr. P.H. Davis and Prof. G. Pontecorvo F.R.S. for critically reading this paper and making some invaluable suggestions. We are grateful to the University Research Grant Commission for providing us a grant for this work. We are also indebted to the Dean, College of Arts & Sciences, Pahlavi University, Shiraz, Iran for providing us with a vehicle during this work.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

Staining of Some Specific Regions of Human Chromosomes,particularly the Secondary Constriction of No. 9

SEVERAL procedures have been described recently which produce specific patterns of differential staining in human chromosomes^1–9. Techniques which involve DNA denaturation and reannealing reveal deeply stained areas on centromere and secondary constriction regions which have been equated with constitutive heterochromatin⁹.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

The mode of action of thidiazuron: auxins,indoleamines, and ion channels in the regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.

The biochemical mechanisms underlying thidiazuron (TDZ)-induced regeneration in plant cells have not been clearly elucidated. Exposure of leaf explants of Echinacea purpurea to a medium containing TDZ results in undifferentiated cell proliferation and differentiated growth as mixed shoot organogenesis and somatic embryogenesis. The current studies were undertaken to determine the potential roles of auxin, indoleamines, and ion signaling in the dedifferentiation and redifferentiation of plant cells. E. purpurea leaf explants were found to contain auxin and the related indoleamine neurotransmitters, melatonin, and serotonin. The levels of these endogenous indoleamines were increased by exposure to TDZ associated with the induction of regeneration. The auxin-transport inhibitor 2,3,5-triiodobenzoic acid and auxin action inhibitor, p-chlorophenoxyisobutyric acid decreased the TDZ-induced regeneration but increased concentrations of endogenous serotonin and melatonin. As well, inhibitors of calcium and sodium transport significantly reduced TDZ-induced morphogenesis while increasing endogenous indoleamine content. These data indicate that TDZ-induced regeneration is the manifestation of a metabolic cascade that includes an initial signaling event, accumulation, and transport of endogenous plant signals such as auxin and melatonin, a system of secondary messengers, and a concurrent stress response.