Secondary structure as primary determinant of the efficiency of ribosomal binding sites in Escherichia coli.

Using a previously described vector (pKL203) we fused several heterologous ribosomal binding sites (RBSs) to the lacZ gene of E. coli and then studied the variation in expression of the fusions. The RBSs originated from bacteriophage Q beta and MS2 genes and the E. coli genes for elongation factor EF-Tu A and B and ribosomal protein L11 (rplK). The synthesis of the lacZ fusion proteins was measured by an immuno precipitation method and found to vary at least 100-fold. Lac-specific mRNA synthesis follows the variation in protein production. It appears that there is a correlation between the efficiency of an RBS to function in the expression of the fused gene and the lack of secondary structure, involving the Shine and Dalgarno nucleotides (SDnts) and/or the initiation codon. This efficiency is context dependent. The sequence of the SD nts and the length and sequence of the spacer region up to the initiation codon alone are not able to explain our results. Deletion mutations, created in the phage Q beta replicase RBS, reveal a complex pattern of control of expression, probably involving the use of a "false" initiation site.     

High-resolution proton magnetic resonance studies of the 3'-terminal colicin fragment of 16 S ribosomal RNA from Escherichia coli. Assignment of iminoproton resonances by nuclear Overhauser effect experiments and the influence of adenine dimethylation on the hairpin conformation

The "colicin" fragments comprising the 49 3'-terminal nucleotides of 16 S ribosomal RNA have been isolated from wild-type Escherichia coli and from a kasugamycin-resistant mutant that lacks methylation of two geminal adenine residues. Proton nuclear magnetic resonance (n.m.r.) spectra (500 MHz) were recorded at various temperatures. The low-field resonances arising from the hydrogen-bonded iminoprotons of paired bases were assigned using the nuclear Overhauser effect (n.o.e.). Crucial to the interpretation of the spectra are the resonances that originate from the two hydrogen-bonded iminoprotons of a U X G basepair. Combined with temperature-jump relaxation kinetics experiments the n.o.e.s lead to the conclusion that a conserved A X U/U X G junction in the hairpin is a thermolabile dislocation in the helix. The n.m.r. spectra of the wild-type and mutant fragment are only different with respect to the iminoproton resonances of the two base-pairs adjoining the hairpin loop. The spectra recorded at various temperatures tend to indicate that dimethylation of the adenosines labilizes these base-pairs, but no definitive conclusions are drawn. The results confirm our previous views that dimethylation of the adenosine residues affects the conformation of the hairpin loop.     

Immunological Studies on Dermatophytes IV. Chemical Structures and Serological Reactivities of Polysaccharides from Microsporum praecox, Trichophyton ferrugineum, Trichophyton sabouraudii, and Trichophyton tonsurans

The chemical structures and serological specificities of polysaccharides isolated from four species of dermatophytes, Microsporum praecox, Trichophyton ferrugineum, T. sabouraudii, and T. tonsurans, were investigated. Each of these species yielded a mixture of crude polysaccharides which could be separated into three water-soluble, neutral polysaccharides free of nitrogen. These were grouped as galactomannan I, galactomannan II, and glucan. The galactomannans I were quite similar in chemical structure. When measured by complement fixation, their serological cross-reactivities were similar with rabbit antisera to each of these species except T. sabouraudii. The differences in their relative reactivities with this antiserum could be correlated with differences in structure and specificity of this antiserum for galactofuranose end groups. The galactomannans II differed both in chemical structure and in their serological reactivities with antisera to each of these species. The galactomannan II from T. ferrugineum differed most in chemical structure and was the least reactive serologically. The glucans also differed in both structure and serological reactivities.     

Kinetics of Induction of the Lactose Operon on an Episome in Salmonella typhimurium

A kinetic study of induction of the enzymes of the lactose operon was carried out under conditions known to affect the kinetics of derepression of the enzymes of the histidine operon. The results show that the lactose system is similar to the histidine system in its responsiveness to conditions thought to affect the formylating capacity of the cell. This was demonstrated in the following ways: (i) trimethoprim, which is known to reduce the formylating capacity of the cell, gives rise to a relatively long interval between the times of induction of beta-galactosidase and transacetylase; (ii) under conditions in which the histidine operon is derepressed, chloramphenicol causes a prolongation of the interval between the times of induction of the two enzymes, and this prolongation is reversed by adenine, methionine, and serine, compounds known to enrich the one-carbon pool of the cell; and (iii) 4-amino-5-imidazolcarboxamide ribonucleoside, a compound which may act as a drain for formyl groups, reverses the effect of the latter compounds. The finding that the interval between the times of induction of the two enzymes is shortened under conditions expected to maintain a relatively high intracellular fo rmylating capacity suggests that under certain conditions translation of the polycistronic messenger ribonucleic acid of the lactose operon may be initiated at more than one site or may proceed more rapidly from the operator end.     

CHLOROPLAST DEVELOPMENT IN OCHROMONAS DANICA

When dark-grown cells of Ochromonas danica are placed in the light, the amount of chlorophyll a per cell increases 82-fold; the content of carotenoid pigment, 24-fold. Concomitantly with this increase in chlorophyll and carotenoid pigment, the small proplastid of dark-grown cells develops into a large lamellate chloroplast. During the first 12 hours in the light, vesicles appear within the loose clusters of dense chloroplast granules, enlarge, align themselves into rows (plates in three dimensions), and fuse into discs. Double discs may form from the more or less simultaneous fusion of two adjacent plates of vesicles or by the addition of vesicles to an already formed single disc. Three-disc bands arise by the addition of a disc to an already formed two-disc band through the approach and fusion of more vesicles. After 24 hours in the light, most of the chloroplast bands contain three discs, but the chloroplasts are still small. After 48 hours in the light, almost all the cells contain full-sized chloroplasts with a full complement of three-disc bands. However, at this time the amount of chlorophyll a and carotenoid pigment is only one-half of maximum. During the next 3 days in the light, as the number of chlorophyll and carotenoid molecules per chloroplast approximately doubles, there is a compression of the discs in each band (from 180 to 130 A) and a precise alignment of their membranes. Changes also occur in the nucleus when dark-grown cells are placed in the light. There is an increase in the number of small nucleolar bodies, many of which lie directly against the nuclear envelope, and in a few cells a dense mass of granules is seen between the two membranes of the nuclear envelope.     

An electron microscopic study of the cerebral blood vessels of the opossum

Summary Blood vessels of the opossum brain are paired, artery and vein, and end in a closed loop. Both anatomically and physiologically they are true end-arteries. The two limbs are enclosed within a single basement membrane and are separated from each other by an intercapillary cell thought to be analogous to the usual capillary pericytes. The similarity of this vascular arrangement to that in the rabbit placenta and in the medulla of the kidney is discussed in relation to the counter-current multiplier system.This work was supported in part by the Beaumont-May Institute of Neurology and by a grant, B-425, from the United States Public Health Service.Research Fellow of the National Multiple Sclerosis Society.     

FINE STRUCTURE OF THE NEUROHYPOPHYSIS OF THE OPOSSUM (DIDELPHIS VIRGINIANA)

The neurohypophysis of the opossum (Didelphis virginiana) was studied by electron microscopy in order to amplify Bodian''s classic light microscopic observations in which he demonstrated a definite lobular pattern. The lobule of the opossum neurohypophysis is divided into three regions: a hilar, a palisade, and a septal zone. The hilar portion contains bundles of nerve fibers, the extensions of the hypothalamo-hypophyseal tract containing neurofilaments but few neurosecretory granules. In the opossum, pituicytes have a densely fibrillar cytoplasm. Herring bodies are prominent in the hilar region. They are large bodies packed with neurosecretory granules that have been described as end bulb formations of axons. From the hilar region, axons fan out into a palisade zone where the nerve terminals packed with neurosecretory granules, mitochondria, and microvesicles abut upon basement membranes. The neurosecretory granules are similar to those present in the neurohypophysis of other mammals, except for an occasional huge granule of distinctive type. Material morphologically and histochemically resembling glycogen occurs as scattered particles and as aggregates within nerve fibers. The septal zone, containing collagen, fibroblasts, and numerous small capillaries, is separated from the adjacent glandular tissue by a basement membrane.     

Survey for mitochondrial-desmosome complexes in differentiating epithelia

Summary Mitochondria are frequently found to be closely associated with the plaques of desmosomes in a variety of columnar or cuboidal epithelia of fetal or early postnatal mammals (mouse, rat, human being). The organs in which mitochondrial-desmosome complexes were found include stomach, small intestine, pancreas, kidney, epididymis, seminal vesicle, coagulating gland, thyroid gland. The association has not been observed in simple squamous epithelium (vascular endothelium). Mitochondria lie quite close to desmosomes in the stratum spinosum of stratified squamous mucous epithelium of fetal animals and also to axo-dendritic synapses in still poorly differentiated central nervous system. Mitochondria have also been detected close to attachment sites in ectoderm of the early frog gastrulae. Here there is as yet no visible plaque material.We suggest that the mitochondria may provide energy or some chemical for the formation of the plaque. This hypothesis does not explain why the complexes are not found in poorly differentiated epithelia from older animals.Dedicated to Professor Berta V. Scharrer on her 60th birthday, with affection and admiration. — This study was supported by U.S.P.H.S. research grants NB-05219 and GM-10757 from the National Institutes of Health.     

High-frequency1H NMR studies of the effects of growth factors and phorbol esters on normal syrian hamster diploid fibroblast cells

The nuclear magnetic resonance (NMR) parameters, spin-lattice (T₁), and spin-spin (T₂) relaxation time, are usually longer for neoplastic cells than for normal cells of the same cell type. This has generally been true at low NMR frequencies (≤100 MHz) when comparisons have been made between normal and neoplastic cells that have both spent a short time in culture. We have previously demonstrated that although the T₁ values of paired normal and neoplastic Syrian hamster (SH) fibroblastic cells in culture are not significantly different when measured at 300 MHz, the 300 MHz T₂ values for the neoplastic cells are smaller than those of the normal cells. (Xin et al. (1986),Cell Biophysics 8, 213.) Since treatment of normal diploid cells with polypeptide growth factors or tumor promoters frequently results in reversible expression of neoplasia-associated phenotypes, T₁ and T₂ were obtained at 300 MHz for treated and untreated SH cells to see if these compounds could also produce smaller 300 MHz T₂ values. Secondary culture SH fetal fibroblast cells were treated with epidermal growth factor (EGF), fibroblast growth factor (FGF), phorbol-12,13-didecanoate (PDD) and 4-α-phorbol-12,13-didecanoate (4αPDD). Treatment with either growth factor resulted in smaller T₂ values, but a statistically significant decrease was not observed for PDD or 4αPDD. The observed reductions in T₂ values were correlated with the morphological and growth-stimulatory effects of these compounds on the cells.