Translational regulation of the L11 ribosomal protein operon of Escherichia coli: mutations that define the target site for repression by L1.

The L11 ribosomal protein operon of Escherichia coli contains the genes for L11 and L1 and is feedback regulated by the translational repressor L1. The mRNA target site for this repression is located close to the Shine-Dalgarno sequence for the first cistron, rp1K (L11). By use of a random mutagenesis procedure we have isolated and characterized a series of point mutations in the L11 leader mRNA which eliminate or greatly diminish the regulation by L1. The mutations define a region essential for translational regulation upstream of the L11 Shine-Dalgarno sequence and identify a region of structural homology with the L1 binding site on 23S rRNA. These results are also consistent with the previously proposed model for the secondary structure of the L11 leader mRNA.     

Alteration of the specificity of PvuII restriction endonuclease.

The restriction endonuclease PvuII which cleaves the sequence CAGCTG, at the position indicated by the arrow, was found to decrease its substrate specificity in the presence of organic solvents. Thirty-three sites, that we have named PvuII sites, were identified on the nucleotide sequence of pBR322 DNA. The new recognition sequences cleaved in pBR322 DNA, at the positions indicated by the arrows, were shown to be AAGCTG, GAGCTG, CNGCTG, CANCTG, CAGNTG, CAGCNG, CAGCTC and CAGCTT. (TAGCTG and the complementary sequence CAGCTA are not present in pBR322 DNA). From these recognition sequences, we deduced that PvuII activity recognizes and cleaves degenerate sequences which differ from the standard PvuII sequence CAGCTG at only one of the recognition site. Any substitution can occur at any one of the six positions in the hexanucleotide sequence. The optimum incubation medium for PvuII activity was found to be: 10-50 mM Tris-HCl, pH 8.5, 12-15 mM MgCl2, 50 mM NaCl, 10% ethanol + 10% dimethylsulfoxide (DMSO).     

GABA-mediated synaptic inhibition of projection neurons in the antennal lobes of the sphinx moth,Manduca sexta

Responses of neurons in the antennal lobe (AL) of the moth Manduca sexta to stimulation of the ipsilateral antenna by odors consist of excitatory and inhibitory synaptic potentials. Stimulation of primary afferent fibers by electrical shock of the antennal nerve causes a characteristic IPSP-EPSP synaptic response in AL projection neurons. The IPSP in projection neurons reverses below the resting potential, is sensitive to changes in external and internal chloride concentration, and thus is apparently mediated by an increase in chloride conductance. The IPSP is reversibly blocked by 100 microM picrotoxin or bicuculline. Many AL neurons respond to application of GABA with a strong hyperpolarization and an inhibition of spontaneous spiking activity. GABA responses are associated with an increase in neuronal input conductance and a reversal potential below the resting potential. Application of GABA blocks inhibitory synaptic inputs and reduces or blocks excitatory inputs. EPSPs can be protected from depression by application of GABA. Muscimol, a GABA analog that mimics GABA responses at GABAA receptors but not at GABAB receptors in the vertebrate CNS, inhibits many AL neurons in the moth.     

A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector

A set of genomic plasmid banks was constructed using the centromere-containing yeast shuttle vector YCp50. The centromere-containing vector is useful for the isolation of genes that are toxic to yeast when present in high copy number. Fourteen independent banks were prepared each with an average representation of two to three times the yeast genome. Any individual plasmid from a given bank is guaranteed to be of independent origin from plasmids obtained from each of the other banks. The banks were constructed from three different size classes of DNA fragments that resulted from varying conditions of partial digestion with Sau3A. This avoided the bias caused by differential sensitivity of sites to cleavage with Sau3A. Insert DNA is sufficiently large that most genes will be present in the set of plasmid banks at a frequency of about 0.1%.     

Organization of Methanobacterium thermoautotrophicum bacteriophage psi M1 DNA

Summary M1 is a virulent bacteriophage of Methanobacterium thermoautotrophicum strain Marburg. Restriction enzyme analysis of the linear, 30.4 kb phage DNA led to a circular map of the 27.1 kb M1 genome. M1 is thus circularly permuted and exhibits terminal redundancy of approximately 3 kb. Packaging of M1 DNA from a concatemeric precursor initiates at the pac site which was identified at coordinate 4.6 kb on the circular genome map. It proceeds clockwise for at least five packaging rounds. Headful packaging was also shown for M2, a phage variant with a 0.7 kb deletion at coordinate 23.25 on the map.     

In vitro and in vivo studies on the mitochondrial import of CBS1, a translational activator of cytochrome b in yeast

Summary Translation of mitochondrial cytochrome b mRNA in yeast is activated by the product of the nuclear gene CBS1. CBS1 encodes a 27 kDa precursor protein, which is cleaved to a 24 kDa mature protein during the import into isolated mitochondria. The sequences required for mitochondrial import reside in the amino-terminal end of the CBS1 precursor. Deletion of the 76 amino-terminal amino acids renders the protein incompetent for mitochondrial import in vitro and non-functional in vivo. When present on a high copy number plasmid and under the control of a strong yeast promoter, biological function can be restored by this truncated derivative. This observation indicates that the CBS1 protein devoid of mitochondrial targeting sequences can enter mitochondria in vivo, possibly due to a bypass of the mitochondrial import system.     

Foraminifera may structure meiobenthic communities

The cosmopolitan benthic foraminiferan, Ammonia beccarii, is a fervent microfloral predator which often forms densely-populated 2–4 cm² aggregates in the field. Sediments within aggregate patches become extensively pelletized, mucus bound and depleted in microfloral food. On a West German Wattenmeer mudflat, copepodite and naupliar densities of a predominant harpacticoid copepod, Amphiascoides limicola, were significantly depressed in sediments containing>100 A. beccarii·3 cm^-2 suggesting a possible foraminiferal: copepod amensalism. Therefore, I cultured A. beccarii and A. limicola separately in sediment microcosms and then tested if A. limicola's seemingly negative reaction to sediments containing A. beccarii occurs under controlled conditions, how various life stages of A. limicola are affected, and what the repulsive mechanisms of A. beccarii may be. In natural field sediments seeded with a latin-square dispersion of sterile sediment patches containing 0 or 100 A. beccarii, mean A. limicola naupliar and copepodite densities were 2 to 6 times lower in Ammonia-rich patches than Ammonia-poor patches (i.e. patches containing <100 A. beccarii·3 cm^-2). Choice experiments directly testing potential A. beccarii inhibitory mechanisms were conducted with A. limicola copepodites: Cubic microcosms containing a latin-square patch dispersion of (1) sterile sediments (SS) seeded with 100 A. beccarii (low microflora), (2) SS bound with sterile mucus (0.0001%) (low microflora), (3) SS seeded with pelletized sediments (high microflora), and (4) SS seeded with mucus and pellets (high microflora), showed that copepodites colonized 12 & 3, but 1 & 4 were not significantly different. Mucus addition by itself, in the absence of pelletization and microflora, strongly facilitated colonization—as did addition of microfloral-rich pelletized sediments. Pelletization and mucousbinding combined, but with low microflora, were least attractive to A. limicola. Pelletization and mucous-binding combined, but with high microflora, were more attractive to A. limicola than its complement, but not significantly so. Thus A. beccarii's inhibition of A. limicola is probably not caused by sediment pelletization and simple mucous exudates but by local microfloral depletion within aggregate foraminiferal patches.Contribution No 774 of the Belle W. Baruch Institute for Marine Biology and Coastal Research     

Seed Development in Phaseolus vulgaris L. cv Seminole: I. Developmental Independence of Seed Maturation

Phaseolus vulgaris L. cv Seminole pods removed from the plant continued their development when incubated in suitable conditions. Seeds continued to grow and develop and pods and seeds passed through an apparently normal developmental sequence to dryness. Seed growth was at the expense of pod dry weight (DW) reserves. Losses of pod DW paralleled DW gains by seeds in detached pods and in pod cylinders containing a seed. The transfer activity was apparent only within the period 10 to 30 days after anthesis (DAA) with maximal activity between 15 to 20 DAA. This period corresponds to maximum pod growth and the attainment of maximal DW. Seeds are in only the early phase of seed growth at this time. No DW transfer was observed at developmental stages beyond 30 to 35 DAA when normal senescence DW losses in pods became evident and seeds were in the later phase of seed fill. Pods or pod cylinders remained green and succulent over the transfer period, later passing through yellowing and drying phases characteristic of normal development. DW transfer was dependent on funicle integrity and was readily detectable in pod cylinders after 7 days incubation. The DW transfer activity may contribute to continuing nutrition of seeds under conditions where the normal assimilate supply to seeds becomes limiting. Defoliation and water stress treatments applied to Phaseolus plants reduced seed yields but allowed persistence of seed maturation processes such that all seeds developing to dryness were capable of germination.     

Localization and Pattern of Graviresponse across the Pulvinus of Barley Hordeum vulgare

Pulvini of excised stem segments from barley (Hordeum vulgare cv `Larker') were pretreated with 1 millimolar coumarin before gravistimulation to reduce longitudinal cell expansion and exaggerate radial cell enlargement. The cellular localization and pattern of graviresponse across individual pulvini were then evaluated by cutting the organ in cross-section, photographing the cross-section, and then measuring pulvinus thickness and the radial width of cortical and epidermal cells in enlargements of the photomicrographs. With respect to orientation during gravistimulation, we designated the uppermost point of the cross-section 0° and the lowermost point 180°. A gravity-induced increase in pulvinus thickness was observable within 40° of the vertical in coumarin-treated pulvini. In upper halves of coumarin-treated gravistimulated pulvini, cells in the inner cortex and inner epidermis had increased radial widths, relative to untreated gravistimulated pulvini. In lower halves of coumarin-treated pulvini, cells in the central and outer cortex and in the outer epidermis showed the greatest increase in radial width. Cells comprising the vascular bundles also increased in radial width, with this pattern following that of the central cortex. These results indicate (a) that all cell types are capable of showing a graviresponse, (b) that the graviresponse occurs in both the top and the bottom of the responding organ, and (c) that the magnitude of the response increases approximately linearly from the uppermost point to the lowermost. These results are also consistent with models of gravitropism that link the pattern and magnitude of the graviresponse to graviperception via statolith sedimentation.