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Biomedical subjects

D Schlessinger

Publications and source records attributed to D Schlessinger.

At least 163 records · Page 9Linked to original sources
PubMed1984

RNase III cleavage is obligate for maturation but not for function of Escherichia coli pre-23S rRNA.

RNase III makes the initial cleavages that excise Escherichia coli precursor 16S and 23S rRNA from a single large primary transcript. In mutants deficient in RNase III, no species cleaved by RNase III are detected and the processing of 23S rRNA precursors to form mature 23S rRNA fails entirely. Instead, 50S ribosomes are formed with rRNAs up to several hundred nucleotides longer than mature 23S rRNA. Unexpectedly, these aberrant subunits function well enough to participate in protein synthesis and permit cell growth. Consistent with the inference that RNase III cleavages are absolutely required for 23S rRNA maturation, when 50S ribosomes from a strain deficient in RNase III were incubated with a ribosome-free extract from a RNase III+ strain, rRNA species processed by RNase III and species with normal mature 23S rRNA termini were produced.

Base Sequence↗
PubMed1984

Interaction of plasma proteins and lipoproteins with amphotericin B.

Amphotericin B (AmB) binds to the cholesterol in lipoproteins, as determined by comigration in density gradient ultracentrifugation and changes in the circular dichroic spectrum. The saturation curve and Scatchard plots obtained with circular dichroism suggest that four to 10 cholesterol molecules in low-density lipoproteins bind to one molecule of AmB. AmB interacts more rapidly with low- and very-low-density lipoproteins than with high-density lipoproteins, but the circular dichroic spectrum of the complexed species is the same in all three cases. AmB also binds to other proteins in blood, but much higher concentrations of these proteins than of lipoproteins are needed for comparable binding. Interaction with lipoproteins stabilizes the antifungal activity of AmB. Interaction with lipoproteins and with much higher concentrations of other proteins in blood can also inhibit the effects of AmB on red blood cells, which contain cholesterol in their plasma membranes, but not the effects on Candida albicans, whose membranes contain ergosterol. An appropriate inference is that, when used clinically, AmB circulates in blood bound to lipoproteins and other proteins. The toxic and therapeutic effects of AmB in clinical situations are thus contingent on competitive interactions between sterol-containing cellular membranes of the host and the parasite and components of blood, such as lipoproteins and proteins.

Amphotericin B↗
PubMed1984

Stimulatory, permeabilizing, and toxic effects of amphotericin B on L cells.

High concentrations of amphotericin B (AmB) killed mouse L cells, but low concentrations increased plating efficiency and stimulated the incorporation of labeled precursors into DNA and RNA. Thus, there were two disparate effects of AmB on L cells, stimulatory and toxic, and they occurred in distinct dose-related stages. AmB also affected the permeability of L cells. In dose-response studies, increases in cell membrane permeability, measured as the loss of K+ ions, occurred along with the stimulation of [3H]uridine incorporation into RNA. In contrast, stimulation of [3H]thymidine incorporation into DNA was only observed in cells recuperating from AmB-induced permeability changes. When the K+ concentration in the medium was lowered to 0.5 from 4.5 mM, or when 1 mM ouabain was added to the cultures, cell killing was potentiated, but the stimulatory and permeabilizing effects of subtoxic concentrations of AmB were unaffected. Furthermore, etruscomycin, a polyene antibiotic without any permeabilizing effects, nevertheless induced an enhancement of plating efficiency and of incorporation of [3H]uridine into RNA and [3H]thymidine into DNA. Our results suggest that the dose-related stimulatory, permeabilizing, and toxic effects of AmB most probably have distinct mechanisms of action and may be independent of one another.

Amphotericin B↗
PubMed1983

S1 nuclease mapping analysis of ribosomal RNA processing in wild type and processing deficient Escherichia coli.

S1 nuclease mapping was used to assess rRNA processing in Escherichia coli. Single-stranded DNA probes complementary to the sequences bordering each terminus of 16 S and 23 S rRNA were end-labeled, hybridized to total E. coli RNA, and treated with S1 nuclease. The resultant DNA fragments were then displayed on denaturing polyacrylamide gels. Measurements of steady state levels of precursor rRNA species and measurements of the rates of decay of precursors after transcription arrest by rifampicin gave consistent results. 1) The rRNA precursor species identified in wild type cells corresponded to those previously identified by other means. 2) In RNase III-deficient strains, mature 16 S rRNA termini form at the same rate as in wild type cells; but the normal mature termini of 23 S rRNA are never generated. 3) RNase III cleavage at the 5' end of 23 S rRNA can occur before the 3' end of the same molecule is synthesized. 4) The cleavages that generate the mature termini of 16 S rRNA are interdependent; in the BUMMER strain, slow processing at the 5' end is accompanied by slow processing at the 3' end. Thus, the kinetically observed order of processing reactions is obligate for some cleavages but not for others, and the assumption that complete rRNA processing is required for function fails for 23 S rRNA.

Base Sequence↗
PubMed1983

Structure of partially denatured Escherichia coli 23 S ribosomal RNA determined by electron microscopy.

The secondary structure of 23 S ribosomal RNA was analyzed by electron microscopy after partial denaturation. A reproducible pattern of loops was seen when molecules were spread for electron microscopy in 50% formamide solutions containing various concentrations of Mg2+ and Na+. Some loops were stabilized more than others by Na+ or by Mg2+; but in general, small amounts of Mg2+ (0.5 to 1.0 mM) markedly stabilized all the major loops, as did much greater amounts of Na+ (100 mM). However, at all levels of Mg2+ examined, increasing levels of Na+ destabilized loop structures. These data are consistent with the known salt dependence of double-stranded DNA and transfer RNA structure. The four most frequently observed loops correspond, within the limits of measurement error, to the major loops in the secondary structure models of Noller et al. (1981) and Glotz et al. (1981). These four loops are, in length and position of their midpoints along the 23 S rRNA molecule: 490 +/- 50 at 250 +/- 40; 350 +/- 50 at 1860 +/- 80; 400 +/- 70 at 2330 +/- 150; and 570 +/- 100 at 2350 +/- 100. Three of the four have base-paired stems with delta G0 values among the lowest of all the loops in the two indirect models. At least two are also among the most stable loops found in computer searches of the 23 S rRNA sequence for dyad symmetry. These results demonstrate that partial denaturation mapping can both identify prominent features of secondary structure in rRNA and estimate their relative stability.

Escherichia coli↗
PubMed1983

Regulation of glucose 6-phosphate dehydrogenase expression in CHO-human fibroblast somatic cell hybrids.

Human--hamster somatic cell hybrids have been obtained by fusion of a CHO line (NA31) doubly deficient in hypoxanthine guanine phosphoribosyltransferase and glucose 6-phosphate dehydrogenase (G6PD) with normal G6PD(+) human fibroblasts. Analysis of NA31 extracts has revealed that, although G6PD activity is nearly absent, significant activity can be detected with 2-deoxyglucose 6-phosphate as substrate, so that the mutant and normal forms of the enzyme can both be easily detected. The cell hybrids obtained express human G6PD. The human G6PD subunits are distributed in homodimeric molecules as well as in human--hamster heterodimeric molecules. However, whereas the amount of hamster G6PD subunits present in the hybrid is similar to that in the hamster parental cells, the amount of human G6PD subunits is decreased by 3- to 10-fold when compared to the human parental cell. These results indicate that either the expression of the G6PD gene or the stability of the gene product is altered in the hybrid. By mutagenesis and selection in diamide (a substance that oxidizes intracellular glutathione), we have isolated a clone with a 3- to 5-fold increase in human G6PD activity. This derivative may have an increased rate of expression of the human G6PD structural gene.

Animals↗
PubMed1983

Electrophoretic elution of nucleic acids from acrylamide and agarose gels.

A simple method for electrophoretic elution of nucleic acids from gel slices is described. The procedure utilizes a standard tube gel system and can be completed in as little as one hour. Nucleic acids are recovered in a small volume with almost 100% efficiency. The procedure is applicable equally to acrylamide and agarose gels, and small as well as large RNA and DNA molecules. The eluted nucleic acids are essentially undegraded and are suitable for a variety of structural and biological analyses.

DNA↗
PubMed1983

Mouse rDNA: sequences and evolutionary analysis of spacer and mature RNA regions.

Two regions of mouse rDNA were sequenced. One contained the last 323 nucleotides of the external transcribed spacer and the first 595 nucleotides of 18S rRNA; the other spanned the entire internal transcribed spacer and included the 3' end of 18S rRNA, 5.8S rRNA, and the 5' end of 28S rRNA. The mature rRNA sequences are very highly conserved from yeast to mouse (unit evolutionary period, the time required for a 1% divergence of sequence, was 30 X 10(6) to 100 X 10(6) years). In 18S rRNA, at least some of the evolutionary expansion and increase in G + C content is due to a progressive accretion of discrete G + C-rich insertions. Spacer sequence comparisons between mouse and rat rRNA reveal much more extensive and frequent insertions and substitutions of G + C-rich segments. As a result, spacers conserve overall G + C richness but not sequence (UEP, 0.3 X 10(6) years) or specific base-paired stems. Although no stems analogous to those bracketing 16S and 23S rRNA in Escherichia coli pre-rRNA are evident, certain features of the spacer regions flanking eucaryotic mature rRNAs are conserved and could be involved in rRNA processing or ribosome formation. These conserved regions include some short homologous sequence patterns and closely spaced direct repeats.

Animals↗
PubMed1983

Location of the initial cleavage sites in mouse pre-rRNA.

The locations of three cleavages that can occur in mouse 45S pre-rRNA were determined by Northern blot hybridization and S1 nuclease mapping techniques. These experiments indicate that an initial cleavage of 45S pre-rRNA can directly generate the mature 5' terminus of 18S rRNA. Initial cleavage of 45S pre-rRNA can also generate the mature 5' terminus of 5.8S rRNA, but in this case cleavage can occur at two different locations, one at the known 5' terminus of 5.8S rRNA and another 6 or 7 nucleotides upstream. This pattern of cleavage results in the formation of cytoplasmic 5.8S rRNA with heterogeneous 5' termini. Further, our results indicate that one pathway for the formation of the mature 5' terminus of 28S rRNA involves initial cleavages within spacer sequences followed by cleavages which generate the mature 5' terminus of 28S rRNA. Comparison of these different patterns of cleavage for mouse pre-rRNA with that for Escherichia coli pre-rRNA implies that there are fundamental differences in the two processing mechanisms. Further, several possible cleavage signals have been identified by comparing the cleavage sites with the primary and secondary structure of mouse rRNA (see W. E. Goldman, G. Goldberg, L. H. Bowman, D. Steinmetz, and D. Schlessinger, Mol. Cell. Biol. 3:1488-1500, 1983).

Animals↗
PubMed1982

lac Transcription in Escherichia coli cells treated with chloramphenicol.

When protein synthesis was blocked by chloramphenicol in vivo, transcription initiation of lac mRNA was severely inhibited. In a promoter mutant (L8-UV5) or in wild-type cells supplemented with adenosine 3',5'-phosphate (greater than or equal to 5 mM), nearly normal initiation could be achieved, and when the mRNA chains formed were extracted, they coded for the 5'-terminal alpha-peptide of the lacZ gene in vitro. However, even under such conditions, only a fraction of RNA polymerases proceeded to the end of the Z gene in the presence of chloramphenicol; as a consequence, a wide range of sizes of mRNA was produced, and very few transcripts were formed all the way to the natural termination site of the operon. In other words, premature transcription termination occurred in chloramphenicol-treated cells, as current models predict, but terminations occurred to variable extents at several intragenic sites and apparently at least one intergenic site. Termination at intragenic sites occurred far less in cells bearing a mutation in the transcription termination factor rho.

Chloramphenicol↗
PubMed1981

Multiple ribosomal RNA cleavage pathways in mammalian cells.

The sequence content of mouse L cell pre-rRNA was examined by RNA gel transfer and blot hybridization. Nuclear RNAs were separated by agarose gel electrophoresis, transferred to diazo-paper, and hybridized to twelve different restriction fragments that are complementary to various sections of 45S pre-rRNA. An abundant new 34S pre-rRNA and less abundant new 37S, 26S and 17S pre-rRNAs were detected. The presence of these new pre-rRNAs suggests the existence of at least two new pre-rRNA cleavage pathways. 34S and 26S pre-rRNAs were also detected in HeLa cells suggesting that these new cleavage pathways are characteristic of mammalian cells. Further, an abundant new 12S precursor to 5.8S rRNA was also detected and is common to all the proposed cleavage pathways. The previously identified 45S, 41S, 32S and 20S pre-rRNAs were readily detected and their general structure confirmed. The 20S pre-rRNA is characteristic of the known pathway used by HeLa and other cells, and its presence suggests that growing mouse L cells use this pre-rRNA cleavage pathway. The 36S pre-rRNA characteristic of the previously described mouse L cell cleavage pathway was not detected. In all these cleavage pathways pre-rRNA cleavage sites are apparently identical and occur at or near the termini of the mature 18S, 5.8S and 28S rRNA sequences. The pathways differ only in the temporal order of cleavage at these sites. The position of the 5.8S rRNA sequence was located within the internal transcribed spacer. The known and conserved sequence of 5.8S rRNA from several organisms predicts a characteristic pattern of restriction enzyme sites for 5.8S rDNA. Internal transcribed spacer rDNA was mapped with restriction enzymes, and the characteristic pattern was found near the midpoint of the internal transcribed spacer. This places the 5.8S rRNA sequence at or near the 5' terminus of 32S pre-rRNA.

Animals↗
PubMed1981

Functional inactivation of lac alpha-peptide mRNA by a factor that purifies that Escherichia coli RNase III.

Using RNA-directed synthesis of the alpha-peptide of beta-galactosidase as an assay, a factor was purified that inactivated further function of the mRNA. In the presence of Ca2+ ions to inhibit most nuclease activity, inactivation of mRNA occurred during incubation with ribosomes or with a 1 M KCl wash of ribosomes. The inactivation activity required Mg2+ ions, and purified as a single factor which did not bind to DEAE-cellulose, but bound reversibly to phosphocellulose. The factor eluted from Sephadex G-150 with an apparent molecular weight of about 43,000. Purified 700-fold, it showed no detectable exonuclease activity, and little or no cleavage of a variety of single-stranded substrates, including full length lac operon mRNA; but repurified inactivated mRNA was still inactive for protein synthesis. The factor did not inhibit poly(U)-directed polyphenylalanine synthesis. When proteins isolated from the ribosomal wash were individually tested, highly purified RNase III, which purifies in the same way and has the same size, also inactivated lac mRNA. The ribosomal wash from an RNase III- strain showed little if any activity compared to that from an isogenic RNase III+ strain. The possibility of a site-specific inactivating cleavage of mRNA by RNase III at or near the 5' end is considered.

Calcium↗
PubMed1981

Escherichia coli ribosome unfolding in low Mg2+ solutions observed by laser Raman spectroscopy and electron microscopy.

Ribosomes unfolded by the removal of Mg2+ at 25 degrees C were studied by Raman spectroscopy and electron microscopy. Raman spectra showed a reduction in the 813 cm-1 phosphodiester signal of 30S and 50S ribosomes compared to intact ribosomes, suggesting that a fraction of the ribose moieties had shifted from the 3' endo (ordered) to the 3' exo (disordered) conformation. The maximum diameters of unfolded 30S and 50S ribosomes, judged by electron microscopy, were 1.8 and 2.5-fold greater, respectively, than those of intact ribosomes. Most unfolded 30S ribosomes had three distinct structural domains and appeared "Y-shaped"; whereas most unfolded 50S ribosomes had four distinct domains and appeared "X-shaped". When ribosomes were partially unfolded (by brief exposure to 0.04 mM Mg2+ or EDTA), several possible intermediates in the unfolding process were observed. Both the shapes of particles and their Raman spectra reached the same final state in 0.04 mM Mg2+, where more than 50% of the rRNA phosphates are discharged by Mg2+, as in 10 mM EDTA, where less than 1% are discharged.

Escherichia coli↗
PubMed1981

Lysosomal enzyme activities and RNA turnover rates in growing and nongrowing WI-38 and HeLa cells.

Activities of three lysosomal enzymes--acid RNase. N-acetyl-beta-D-glucosaminidase and acid phosphatase--were determined during the growth cycles of WI-38 and HeLa cells, as well as in radiation-arrested WI-38 cells. In confluent and growth-arrested cultures of WI-38 cells, the lysosomal RNase increased six- to sevenfold; glucosaminidase, four- to fivefold; and phosphatase, two- to threefold. In HeLa cells, the lysosomal enzymes also increased in confluent cultures, but less than twofold; and the RNase level increased only transiently. In both WI-38 and HeLa cells, the rate of RNA breakdown also increased as cultures approached confluency. The rate of turnover of RNA, like the level of acid RNase, was higher in WI-38 cells than in HeLa cells (4 d half-life compared to 8 d). The increase in acid RNase could be prevented by incubation of cells in NH4Cl, but the rate of turnover in the presence of NH4Cl increased just as much when cells became confluent or stopped growth. The content of acid RNase could be changed more than 10-fold without altering the rate of RNA turnover. It is suggested that the increase in enzyme level is more important for possible autophagy or increased digestion of engulfed RNA, rather than for normal RNA turnover, when growth stops.

Acetylglucosaminidase↗
PubMed1981

Dual pathways for ribonucleic acid turnover in WI-38 but not in I-cell human diploid fibroblasts.

The turnover rates of 3H-labeled 18S ribosomal ribonucleic acid (RNA), 28S ribosomal RNA, transfer RNA, and total cytoplasmic RNA were very similar in growing WI-38 diploid fibroblasts. The rate of turnover was at least twofold greater when cell growth stopped due to cell confluence, 3H irradiation, or treatment with 20 mM NaN3 or 2 mM NaF. In contrast, the rate of total 3H-protein turnover was the same in growing and nongrowing cells. Both RNA and protein turnovers were accelerated at least twofold in WI-38 cells deprived of serum, and this increase in turnover was inhibited by NH4Cl. These results are consistent with two pathways for RNA turnover, one of them being nonlysosomal and the other being lysosome mediated (NH4Cl sensitive), as has been suggested for protein turnover. Also consistent with the notion of two pathways for RNA turnover were findings with I-cells, which are deficient for many lysosomal enzymes, and in which all RNA turnover was nonlysosomal (NH4Cl resistant).

Adenosine Triphosphate↗