Five gentamicin-resistant clinical isolates of Staphylococcus aureus were found to contain self-transmissible plassaids of 32 to 37 megadaltons in size. Restriction endonuclease digests of the plasmids were markedly similar to those of reference plasmids of unrelated geographical origin, thus suggesting the significant contribution of common conjugal plasmids to the emergence of gentamicin resistance in S. aureus populations.Recent reports have documented the conjugal transfer of gentamicin resistance (Gm') plasmids in Staphylococcus aureus (4,7, 11). The proportion of staphylococcal Gm' plasmids which are self-transmissible (Tra+), however, and the extent to which conjugation may account for the emergence of Gm' S. aureus, is as yet unknown. To determine whether self-transmissible plasmids might be contributing toward Gmr S. aureus encountered sporadically at a local hospital, 10 clinical isolates of Gm' S. aureus, obtained over a 6-month period, were screened for the presence of Tra+ Gm' plasmids. The results from five strains (Table 1), representative of the entire group, are reported here.Isolates were screened for plasmid content by the rapid boiling method of Holmes and Quigley (5), modified for S. aureus. Cells grown overnight on brain heart infusion (BHI) agar were suspended in 10 ml of saline (0.5% NaCl) to an optical density of 1.0 at 540 nm. The cells were harvested by centrifuigation, suspended in 0.4 ml of STET buffer (8.0%O sucrose, 5.0% Triton X-100, 50 mM EDTA, 50 mM Tris; pH 8.0), and transferred to 1.5-mi Eppendorf tubes. After the addition of 12 ,ul of lysostaphin (Sigma Chemical Co.; 10 mg/ml in 0.05 M Tris [pH 7.5}-0.145 M NaCl), the tubes were placed in boiling water for 50 s and immediately centrifuged (15,000 x g) for 8 tnin, and the supernatent fluid was removed to a fresh Eppendorf tube. The nicleic acid was precipitated by the addition of 2 volumes of 95% ethanol at ambient temperature, followed by centrifugation for 5 min. The precipitated DNA was suspended in 20 ,ul of TES buffer (30 mM Tris [pH 8.0], 5 mM EDTA, 50 mM NaCl) and analyzed on 0.7% agarose minigels (7 by 10 cm) in Tris-borate buffer (8) at 9 V/cpm for 1.5 to 2 h. For res*tiction endonuclease digestion, DNA was prepared as described above from cells grown overnight on BHI agarose (0.7%). After ethanol precipitation, the DNA was resuspended in 250 ILI of TE buffer (10 mM Tris [pH 7.5], 1 mM EDTA) and treated with a 20 ,ug/ml concentration of preboiled RNase 1A (1 mg/ml in 50 mM sodium acetate, pH 5.0) for 30 min at 37°C. A 50-,u volume of 5 M potassium acetate was added, and the mixture was clarified (2) by centrifugation (15,000 x g) for 2 min. The DNA was reprecipitated from the supernatent fluid with 2 volumes of ethanol, cleaved with EcoRI restriction endonuclease as outlined by the supplier, and analyzed on 0.7% agarose gels (15 by 20 cm) at 2.5 V/cm for 18 h.Bacterial matings were performed by a modification of the procedure described by Forbes and Schaberg (4). Overnight BHI agar cultures were adjusted in saline to...
Three small RNA species were detected in human cells, and their cDNAs were synthesized and cloned. These RNAs are nucleolar, are 207, 154, and 135 nucleotides long, and are named El, E2, and E3, respectively, and their unique nucleotide sequences suggest that they may belong to an additional family of small nucleolar RNAs. The 5' ends of these three RNAs do not appear to have a trimethylguanosine cap or another type of cap. Apparent homologs of these three RNAs were detected in mouse, rabbit, and frog cells, suggesting their universal importance. They are housekeeping RNA species, since they are present in all rabbit tissues analyzed.The formation of cytoplasmic ribosomes in eukaryotic cells involves a complex series of synthesis, processing, assembly, and transport steps, many of which are poorly understood (reviewed recently in refs. 1 and 2). Some small nucleolar RNA (snoRNA) species, such as U3 RNA and yeast snR10 and U14 RNA, are required for rRNA precursor (pre-rRNA) processing (3-7). It has been difficult to study some of the low-abundance small nuclear RNAs in the absence of specific DNA or antibody probes. We have made cDNA probes for three snoRNAs and report some of the properties of these snoRNAs.t MATERIALS AND METHODS General Methods. The following procedures were done as described: preparation of nuclear and cytoplasmic fractions from HeLa cells (8); isolation ofnucleoli (9); isolation ofRNA (10); hybrid selection of RNA (8); 10%o polyacrylamide gel electrophoresis (8); electrophoresis of RNA in 1% agarose and 2.2 M formaldehyde (11); RNA sequencing by the dideoxynucleotide method, using HeLa cell nucleolar RNA, avian myeloblastosis virus reverse transcriptase, and sequence-specific, antisense radioactive primers (12); and immunoprecipitation assays (13). The recommendations of the corresponding manufacturers were followed for (i) hybridization with ZetaProbe membranes (Bio-Rad) and (ii) Radioactive Hybridization Probes. The El, E2, and E3 sequences in the corresponding cDNA clones, minus the tails that had been added for cDNA synthesis and cloning, were amplified by the polymerase chain reaction (14). The amplified products were then used as templates for extension of El-, E2-, and E3-specific, antisense 3' end primers with the Klenow fragment of DNA polymerase I in the presence of [a-32PldATP to generate the respective labeled, sequencespecific DNA probes. The radioactive DNA probe for Ul RNA was made by extension of a Ul-specific primer using as template the human Ul gene plasmid, linearized near the 5' end ofthe Ul gene. The radiolabeled DNA probe for U3 RNA was made by random primer labeling (15) using the rat U3 RNA sequence plasmid as template. RESULTSThere were some minor small RNA bands in HeLa cell nuclear RNA (bands labeled 1, 2, and 3 in lane 1 of Fig. 1) that could not be hybrid selected with any of the available DNA clones for small RNAs. RNA from these three sections of the gel was used to make three cDNA libraries. Clones from these libraries were used to probe Northern bl...
We have found earlier three small nucleolar RNA (snoRNA) species, named El, E2, and E3, that have unique nudeotide sequences and may participate in ribosome formation. The present report shows that there is a monophosphate at the 5' end of each of these three snoRNAs, suggesting that their 5' termini are formed by RNA processing. El, E2, and E3 human genomic sequences were isolated. Apparently, the E2 and E3 loci are genes for the main E2 and E3 RNA species, based on their full homology, while the El locus is a gene for an El RNA sequence variant in HeLa cells. These loci do not have any of the intragenic or flanking sequences known to be functional in other genes. The El gene is located within the first intron of the gene for RCC1, a protein that regulates onset of mitosis. There is subsntial sequence homology between the human E3 gene and flanking regions, and intron 8 and neighboring exons of the gene for mouse translation initiion factor 4AII. Injection of the human El, E2, and E3 genes into Xenopus oocytes generated sequence-specific transcripts of the approximate sizes of the respective snoRNAs. We discus why the available results are compatible with specific transcription and prosing occurring in frog oocytes.Some of the small nuclear RNA genes, such as Ul, U2, U3, U4 and U5, constitute an additional subfamily of genes transcribed by RNA polymerase II, while others, such as U6 and 7SK, represent another class of genes transcribed by RNA polymerase III (1-3). Seven small nucleolar RNAs (snoRNAs) have been described in metazoa (U3, U8, U13, U14, X, Y, and 7-2/MRP) and 12 snoRNAs have been characterized in yeast (U3, U14, snR3, snR4, snR5, snR8, snR9, snR10, snRll, snR30, snR189, and snR190) (4). We recently found three additional snoRNAs in human cells, which were named El, E2, and E3 (5). Their unique sequences imply that they are members of another class of snoRNAs, their 5' termini are not capped, they are present at low levels in vertebrate cells, and they appear to be "housekeeping" RNAs, expressed in all tissues examined (5). They may function in some aspect of ribosome formation, since in addition to being detected only in the nucleolar fraction, they are psoralen-photocrosslinked in vivo to unique segments of pre-rRNA (6). The the snoRNA plasmids when indicated) were injected into the nucleus of oocytes (20 nl per oocyte), which were then incubated at 19°C for 24 hr. When indicated, 2 hr after the plasmid injection [a-32P]GTP was injected into the cytoplasm of half of the oocytes, and then all oocytes were incubated at 19°C for 24 hr. When indicated, the solution containing one or two plasmids also included a-amanitin at 2 or 400 pg/ml ("low" or "high" level, respectively). The deproteinized samples were digested with DNase I [Worthington (code DPFF) or BRL] at 100 pg/ml, with or without RNase A (Sigma) at 40 pg/ml for either 15 min at 22°C or 30 min at 37°C. Nonradioactive RNA was fractionated by 5% (10% for radioactive RNA) polyacrylamide gel electrophoresis and electroblotted to Zeta-Pr...
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