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D Schlessinger

Publications and source records attributed to D Schlessinger.

At least 145 records · Page 8Linked to original sources
PubMed1988

Coregulation of processing and translation: mature 5' termini of Escherichia coli 23S ribosomal RNA form in polysomes.

In Escherichia coli, the final maturation of rRNA occurs in precursor particles, and recent experiments have suggested that ongoing protein synthesis may somehow be required for maturation to occur. The protein synthesis requirement for the formation of the 5' terminus of 23S rRNA has been clarified in vitro by varying the substrate of the reaction. In cell extracts, pre-23S rRNA in free ribosomes was not matured, but that in polysomes was efficiently processed. The reaction occurred in polysomes without the need for an energy source or other additives required for protein synthesis. Furthermore, when polysomes were dissociated into ribosomal subunits, they were no longer substrates for maturation; but the ribosomes became substrates again when they once more were incubated in the conditions for protein synthesis. All of these results are consistent with the notion that protein synthesis serves to form a polysomal complex that is the true substrate for maturation. Ribosomes in polysomes, possibly in the form of 70S initiation complexes, may more easily adopt a conformation that facilitates maturation cleavage. As a result, the rates of ribosome formation and protein synthesis could be coregulated.

Base Sequence↗
PubMed1988

Failure of aminoglycoside antibiotics to kill anaerobic, low-pH, and resistant cultures.

The critical inhibition of ribosome function by aminoglycosides has long been established. But the binding of drug to ribosomes is reversible: why then are aminoglycosides bactericidal? Several groups have shown that irreversible action (lethality) results from irreversible uptake into susceptible cells; conversely, resistance in cases such as anaerobiosis is associated with the failure of uptake. Oddly, the pattern of results excludes all traditional transport mechanisms; most unusual is the apparent dependence of uptake on the interaction of drug with ribosomes. A traditional view that ribosomes may function during uptake as a "sink" for aminoglycosides cannot explain all the data. Instead, the alternative is considered that cycling ribosomes at the cell membrane help to induce "one-way endocytic pores." Although no detailed mechanism is formulated, the results do suggest a way that the permeation of antibiotics might be systematically controllable to render them more cidal.

Anaerobiosis↗
PubMed1987

Processing of Escherichia coli 16S rRNA with bacteriophage lambda leader sequences.

To test whether any specific 5' precursor sequences are required for the processing of pre-16S rRNA, constructs were studied in which large parts of the 5' leader sequence were replaced by the coliphage lambda pL promoter and adjacent sequences. Unexpectedly, few full-length transcripts of the rRNA were detected after the pL promoter was induced, implying that either transcription was poor or most of the rRNA chains with lambda leader sequences were unstable. Nevertheless, sufficient transcription occurred to permit the detection of processing by S1 nuclease analysis. RNA transcripts in which 2/3 of the normal rRNA leader was deleted (from the promoter up to the normal RNase III cleavage site) were processed to form the normal 5' terminus. Thus, most of the double-stranded stem that forms from sequences bracketing wild-type 16S pre-rRNA is apparently not required for proper processing; the expression of such modified transcripts, however, must be increased before the efficiency of processing of the 16S rRNA formed can be assessed.

Bacteriophage lambda↗
PubMed1986

Proteins and RNA in mouse L cell core nucleoli and nucleolar matrix.

When intact nucleoli were prepared in the presence of enough leupeptin and phenylmethanesulfonyl fluoride to inhibit protease action, electrophoretic patterns of their constituent proteins were reproducible and very similar for L, HeLa, CHO, and rat hepatoma cells. "Core nucleoli", defined as that nucleolar fraction which remains after extensive DNase I action, had a protein composition similar to that of crude intact nucleoli, but were enriched for snRNA U3. Core nucleolar proteins included all of the histones, ribosomal proteins, and phosphorylated proteins with mobilities corresponding to 110 (protein C23) and 160 kilodaltons (kDa). The presence of protein C23 and of lamins A and C in nucleoli and core nucleoli was further verified by reaction with specific antibodies after one- or two-dimensional electrophoresis. A class of higher molecular weight proteins, ranging from 70 to greater than 200 kDa by mobility, was observed. It included at least 25 specific proteins, almost all of them highly acidic (pI less than 3.5). Treatment of core nucleoli with ethylenediaminetetraacetic acid/hypotonic buffer solubilized 30-35% of the small and large molecular weight proteins. In contrast, washing core nucleoli with 2 M NaCl selectively released U3 snRNA, 95% of the ribosomal RNA, and about half of the proteins, including C23 and most of the histones, ribosomal proteins, and other lower molecular weight proteins. The fraction remaining insoluble, "nucleolar matrix", was enriched for proteins of 34 and 57 kDa, lamins A and C, and most higher molecular weight proteins, as well as a portion of ribosomal spacer DNA.

Animals↗
PubMed1986

Irreversible block of the mycelial-to-yeast phase transition of Histoplasma capsulatum.

p-Chloromercuriphenylsulfonic acid (PCMS), a sulfhydryl inhibitor, prevented the mycelial-to-yeast transition of the dimorphic fungal pathogen, Histoplasma capsulatum. The effect of PCMS was specific for the mycelial-to-yeast transformation; it had no effect on growth of either the yeast or mycelial forms or on the yeast-to-mycelial transition. The failure of PCMS-treated mycelia to transform to yeast was permanent and irreversible. PCMS-treated mycelia could not infect mice but could stimulate resistance to infection by a pathogenic strain of Histoplasma capsulatum. These results suggest a new general strategy for vaccine development in diseases caused by dimorphic pathogens.

4-Chloromercuribenzenesulfonate↗
PubMed1985

Why is processing of 23 S ribosomal RNA in Escherichia coli not obligate for its function?

In an RNase III-deficient mutant of Escherichia coli, all 23 S ribosomal RNA in ribosomes is present in an unprocessed form with a double-stranded stem at the base of the molecule stable enough to be detected by electron microscopy under conditions where all other secondary structure is denatured. Molecules with variable stem lengths enter freely into polysomes, consistent with the existence of a similar but much shorter stem in mature 23 S rRNA in wild-type ribosomes.

Escherichia coli↗
PubMed1985

Ordered processing of Escherichia coli 23S rRNA in vitro.

In an RNase III-deficient strain of E. coli 23S pre-rRNA accumulates unprocessed in 50S ribosomes and in polysomes. These ribosomes provide a substrate for the analysis of rRNA maturation in vitro. S1 nuclease protection analysis of the products obtained in in vitro processing reactions demonstrates that 23S rRNA processing is ordered. The double stranded stem of 23S rRNA is cleaved by RNase III in vitro to two intermediate RNAs at the 5' end and one at the 3' end. Mature termini are then produced by other enzyme(s) in a soluble protein fraction from wild-type cells. The nature of the reaction at the 5' end is not clear, but the reaction at the 3' end is exonucleolytic, producing three heterogeneous mature termini. The two reactions are coordinated; 3' end maturation progresses concurrently with cleavages at the 5' end. Two results suggest a possible link between final maturation and translation: in vitro, mature termini are formed efficiently in the presence of additives required for protein synthesis; and all the processing intermediates detected from in vitro reactions are also found in polysomes from wild-type cells.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Electron microscopy of secondary structure in partially denatured precursor and mature Escherichia coli 16 S and 23 S rRNA.

The secondary structure of 16 S and 23 s rRNA sequences in 30 S preribosomal RNA of Escherichia coli was analyzed by electron microscopy after partial denaturation and compared to mature 16 S and 23 S rRNA examined under the same conditions. The sequences in the pre-rRNA notably lack the specific loops that dominate the 5'-terminal regions of mature 16 S and 23 S rRNA. In other respects, the sizes and locations of loops in the 23 S rRNA sequence are qualitatively very similar in mature and pre-rRNA. Eleven of 12 loops outside of the 5'-terminal domain correspond, with the most frequent features in the 3'-half of the molecule. In contrast, the sizes and locations of loops in the 16 S rRNA sequence differ between precursor and mature forms. In the pre-rRNA, instead of the 370-nucleotide 5'-terminal loop of mature rRNA, some 1000-nucleotide terminal loops are observed. The pre-rRNA also shows a frequent 610-nucleotide central loop and a large 1240-nucleotide loop not seen in the mature rRNA. Also, in the 3'-region of the sequence, the largest loops in pre-rRNA are 120 nucleotides shorter than in mature rRNA. We suggest that the structure of pre-rRNA may promote some alternate conformational features, and that these could be important during ribosome formation or function.

Escherichia coli↗
PubMed1985

Escherichia coli 23S ribosomal RNA truncated at its 5' terminus.

In a strain of E. coli deficient in RNase III (ABL1), 23S rRNA has been shown to be present in incompletely processed form with extra nucleotides at both the 5' and 3' ends (King et al., 1984, Proc. Natl. Acad. Sci. U.S. 81, 185-188). RNA molecules with four different termini at the 5' end are observed in vivo, and are all found in polysomes. The shortest of these ("C3") is four nucleotides shorter than the accepted mature terminus. In growing cells of both wild-type and mutant strains up to 10% of the 23S rRNA chains contain the 5' C3 terminus. In stationary phase cells, the proportion of C3 termini remains the same in the wild-type cells; but C3 becomes the dominant terminus in the mutant. Species C3 is also one of the 5' termini of 23S rRNA generated in vitro from larger precursors by the action of purified RNase III. We therefore suggest that some form of RNase III may still exist in the mutant; and since no cleavage is detectable at any other RNase III-specific site, the remaining enzyme would have a particular affinity for the C3 cleavage site, especially in stationary phase cells. We raise the question whether the C3 terminus has a special role in cellular metabolism.

Base Sequence↗
PubMed1985

Alternative conformations in Escherichia coli 16S ribosomal RNA.

Partially denatured 16S rRNA from 30S ribosomes shows features of secondary structure in electron microscopy that correspond to the well accepted secondary structure model derived from chemical modification and phylogenetic data. However, a very different conformation is seen in precursor 16S rRNA sequences contained within 30S pre-rRNA transcripts: the major 5'-terminal loop is absent, and several additional quite stable large loops, symmetrically placed in the molecule, are present. Features of the alternative structure are also seen in mature 16S rRNA from Escherichia coli and from two Bacillus species when heated in certain buffers. Microscopy thus reveals specific features of alternative conformations and their relative stabilities, suggesting a possible transition during ribosome formation.

Bacillus↗
PubMed1985

DNase I-resistant nontranscribed spacer segments of mouse ribosomal DNA contain poly(dG-dT).poly(dA-dC).

The nontranscribed spacer regions (NTS) that adjoin the coding portion of mouse ribosomal DNA are protected in nucleoli against exhaustive DNase I digestion. Since these sequences are degraded by the enzyme after they are extracted by phenol, the protection is suggested to result from the binding of specific proteins. The nucleolar structure would thus be organized to protect NTS sequences and expose the coding sequences for transcription. We show here that these protected sequences include tracts of poly(dG-dT).poly(dA-dC). We also report that these sequences are localized in regions flanking the rRNA transcription unit. These sequences can potentially form Z-DNA. The organized DNase I-resistant NTS structure in which they participate could be involved in structuring the nucleolus or in regulating transcription because poly(dG-dT).poly(dA-dC) sequences and portions of spacer rDNA can serve as transcriptional enhancer elements.

Animals↗
PubMed1985

Involvement of oxidative damage in erythrocyte lysis induced by amphotericin B.

Lysis of human erythrocytes induced by amphotericin B was retarded when the oxygen tension of the incubation mixture was reduced or when the antioxidant catalase was added; lysis was accelerated when cells were preincubated with the prooxidant ascorbate. In the atmosphere of reduced oxygen tension, the erythrocytes containing carboxyhemoglobin lysed at a slower rate than did the cells containing oxyhemoglobin. Consistent with a role for oxidative damage in lysis, the mixture of erythrocytes and amphotericin B showed an increase in malonyldialdehyde, the product of peroxidation, which paralleled the progression of hemolysis. In contrast, the permeabilizing effect of amphotericin B, measured as a decrease in intracellular K+, was not affected by changes in oxygen tension, catalase, or ascorbate treatment. These results imply that oxidant damage is involved in the lytic, but not in the permeabilizing, action of amphotericin B.

Amphotericin B↗
PubMed1985

Electron microscopy of the secondary structure in partially denatured rRNAs of Escherichia coli and Bacillus stearothermophilus.

Partially denatured 16S and 23S rRNAs from the thermophile Bacillus stearothermophilus show characteristic loop patterns when observed by electron microscopy. The patterns are very similar to those seen in rRNAs from Escherichia coli. At least 2 of 4 most stable interactions in 16S rRNA and 8 of 12 interactions in 23S rRNA are in common for the two species. These interactions correspond well to features of secondary structure in models inferred for rRNA from phylogenetic sequence comparisons and chemical modification studies. However, two additional large loops, enclosing large portions of the 23S rRNA, have been detected in B. stearothermophilus for the first time, and even though other loops are similar, their relative frequencies vary in the two species. Much of the variation is consistent with relative delta G degree values for putative base-paired stems at the base of different loops; but the 5'-terminal loops in 23S rRNA, for example, are unaccountably far less stable in B. stearothermophilus. Also, in general, structural features are not differentially stabilized in B. stearothermophilus; the relative stability of secondary structure in its ribosomes at elevated growth temperatures must involve interactions with ribosomal proteins or other cellular components.

Escherichia coli↗
PubMed1985

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse satellite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.

Animals↗
PubMed1984

Electron microscopy of secondary structure in partially denatured Escherichia coli 16S rRNA and 30S subunits.

Loops observed in partially denatured 16S rRNA lie within three domains, each about 500 nucleotides long. The loops observed in the 5' and central domains agree well with features of the model proposed by Woese et al. [Woese, C. R., Gutell, R., Gupta, R., & Noller, H. F. (1983) Microbiol. Rev. 47, 621-669]. The structure in the 3' domain is more complex and variable but is still consistent with the model. Published psoralen cross-linking studies have reported one of the observed loops but have also identified loops other than those observed here or predicted by any secondary structure model. These loops are stabilized by increasing concentrations of Mg2+ ions and by bound ribosomal proteins. For example, protein S4 in LiCl core particles stabilizes a loop of 370 nucleotides which forms part of its putative binding site on rRNA. The loop structures are characteristic enough to permit an overall comparison of the most stable of the predicted and observed loops in 16S and 23S rRNAs. Both rRNAs show a stable 5'-terminal loop and a set of subterminal nested loops near the 3' end.

Chlorides↗