Showing papers by "Public Health Research Institute published in 1984"
TL;DR: The translational attenuation model, supported by sequencing data and by mutational analysis, proposes that binding of erythromycin causes stalling of a ribosome during translation of a "leader peptide", resulting in isomerization of the ermC transcript from an inactive to an active conformer.
Abstract: The regulation of ermC is described in detail as an example of regulation on the level of translation. ermC specifies a ribosomal RNA methylase which confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics. Synthesis of the ermC gene product is induced by erythromycin, a macrolide antibiotic. Stimulation of methylase synthesis is mediated by binding of erythromycin to an unmethylated ribosome. The translational attenuation model, supported by sequencing data and by mutational analysis, proposes that binding of erythromycin causes stalling of a ribosome during translation of a "leader peptide", resulting in isomerization of the ermC transcript from an inactive to an active conformer. The ermC system is analogous to the transcriptional attenuation systems described for certain biosynthetic operons. ermC is unique in that interaction with a small molecule inducer mediates regulation on the translational level. However, it is but one example of nontranscriptional -level control of protein synthesis. Other systems are discussed in which control is also exerted through alterations of RNA conformation and an attempt is made to understand ermC in this more general context. Finally, other positive examples of translational attenuation are presented.
173 citations
TL;DR: The nucleotide sequences surrounding the junctions of Tn554 are reported in three independent ‘primary’ insertions and two ‘secondary” insertions of the transposon, suggesting that the mechanism ofTn554 insertion may be significantly different from that of other transposons.
Abstract: Transposable elements from prokaryotic and eukaryotic organisms are discrete DNA segments bounded by inverted or directly repeated sequences that insert into non-homologous DNA in a reaction that is independent of the general recombination functions of the host. The mechanisms proposed generally involve a staggered double-stranded scission of the target DNA, ligation to the nicked ends of the transposable element, and replication of the element, resulting in the generation of a directly repeated oligonucleotide target sequence flanking the new copy of the element1–3. Most transposons have a relatively low degree of target site specificity coupled with a low insertion frequency. Tn554, a Staphylococcus aureus transposon which specifies resistances to erythromycin and spectinomycin, displays an unusually high degree of insertion specificity. Tn554 transposes with high efficiency to a unique (‘primary’) site in the S. aureus chromosome4,5 and only rarely (<10−6 per transductant) to other, secondary sites6. We report here the nucleotide sequences surrounding the junctions of Tn554 in three independent ‘primary’ insertions and two ‘secondary’ insertions of the transposon. Two unusual features are revealed: first, the termini of Tn554 contain neither inverted nor directly repeated sequences. Second, transposition of Tn554 does not generate the short direct repeats of the target DNA that are characteristic of other transposable elements. These results suggest that the mechanism of Tn554 insertion may be significantly different from that of other transposons.
76 citations
TL;DR: A 13-amino acid long sequence is proposed to be part of the catalytic domain of this class of enzymes, based on the observed homologies in the primary sequences of the other amidotransferases examined.
76 citations
TL;DR: Comparison of the ermD sequence and that of its product to two other sequenced MLS determinants reveals substantial phylogenetic relatedness, although the three genes are not homologous by the criterion of Southern blot hybridization.
Abstract: The DNA sequence of ermD, a macrolide-lincosamide-streptogramin B (MLS) resistance determinant cloned from the chromosome of Bacillus licheniformis, has been determined, ermD encodes an erythromycin inducible protein of molecular weight 32,796. S1 nuclease mapping of the ermD promoter has revealed the presence of an approximately 354 base leader sequence on the ermD transcript. This leader contains a short open reading frame sufficient to encode a 14 amino acid peptide, which is preceded by a potential ribosomal binding site. The leader sequence has the potential to fold into several base paired structures, in some of which the ribosomal binding site for the ermD product would be sequestered. Deletion analysis demonstrated that the leader contains regulatory sequences. Removal of the ermD promoter and fusion to an upstream promoter did not interfere with induction, strongly suggestion that ermD regulation is posttranscriptional. Based on these features it appears likely that ermD is regulated by a translational attenuation mechanism, analogous to that suggested for ermC, a resistance element from Staphylococcus aureus (Gryczan et al. 1980; Horinouchi and Weisblum 1980). Comparison of the ermD sequence and that of its product to two other sequenced MLS determinants reveals substantial phylogenetic relatedness, although the three genes are not homologous by the criterion of Southern blot hybridization.
74 citations
TL;DR: It is shown that the RS represent short regions of homology embedded in largely non-homologous contexts and that the crossovers take place within these homologous regions.
Abstract: Cointegrates involving pairs of compatible staphylococcal plasmids can be isolated either by co-selection during transduction (Novick et al. 1981) or by selection for survival at the restrictive temperature of a thermosensitive, replication defective plasmid in the presence of a stable one. Cointegrates are formed by recombination at two specific sites, RSA and RSB. RSB is present on each of six plasmids analyzed, namely pT181, pE194, pC194, pS194, pUB110, and pSN2, and RSA is present on two of these, pT181 and pE194. In this communication, it is shown that the RS represent short regions of homology (RSA is some 70 bp in length and RSB is about 30) embedded in largely non-homologous contexts and that the crossovers take place within these homologous regions. The pT181 and pE194 RSA sequences contain several mismatches which permit the localization of the crossover events to several different sites within the overall RS segment. The recombination system involved is therefore general (homology-specific) rather than site-specific (sequence-specific). Mismatches included within the crossover region are always corrected to the pT181 configuration. The cointegrates are therefore formed by a relatively efficient general rec system that recognizes short regions of homology and gives rise to Holliday junctions that probably involve very short heteroduplex overlaps. The sequence results are consistent with asymmetric single-strand invasion of a contralateral gap with nucleotide conversion by copying. It is noted that RSB has substantial homology with the par sequence of plasmid pSC101, suggesting that it may be involved in plasmid partitioning.
60 citations
Patent•
18 May 1984TL;DR: Expression and secretion vector which comprises the signal and promoter sequence of Bacillus cereus (herein "B. cereus") gene which codes for penicillinase and to the construction of vectors and the use of the vectors in the expression and secretion of one or more exogenous polypeptides in microorganisms for example, B. subtilis as mentioned in this paper.
Abstract: Expression and secretion vector which comprises the signal and promoter sequence of Bacillus cereus (herein "B. cereus") gene which codes for penicillinase and to the construction of vectors and the use of the vectors in the expression and secretion of one or more exogenous polypeptides in microorganisms for example, B. subtilis.
57 citations
TL;DR: Experiments are consistent with the translational attenuation model of ermC regulation, and together with other evidence, suggest that ermD is regulated by a similar mechanism.
Abstract: Plasmids were constructed containing the regulatory regions and N-terminal portions of ermC and of ermD , fused in phase with the coding sequence of the Escherichia coli lacZ gene. ermC and ermD are erythromycin (Em) inducible macrolide-lincosamide-streptogramin B resistance elements derived from Staphylococcus aureus and Bacillus licheniformis, respectively. The fusion plasmids were introduced into B. subtilis and used to study ermC and ermD regulation. In both cases, beta-galactosidase synthesis could be induced by low levels of Em. Induction was prevented by introduction of ole-2, a chromosomal mutation which decreases ribosomal affinity for Em. Induction also did not occur in the presence of intact copies of ermC , suggesting that prior or concomitant methylation of 23S rRNA, a treatment known to decrease ribosomal affinity for Em, was capable of interfering with ermC and ermD induction. These experiments are consistent with the translational attenuation model of ermC regulation, and together with other evidence, suggest that ermD is regulated by a similar mechanism.
42 citations
TL;DR: It was concluded that the physiologic role of the stalk is enhancement of phosphate acquisition, and the stimulatory role of calcium in this process was attributed to its support of stalk development and to its stabilization of internal membrane/cell envelope association within the cell-stalk juncture.
Abstract: Calcium was found to stimulate stalk development in Caulobacter crescentus and to relieve the inhibition of development long known to be caused by phosphate. This suggested that phosphate inhibition could be attributed to its interaction with Ca2+, thereby depriving the cells of a factor that promoted development. Calcium was also found to promote phosphate acquisition by the cells, observed as acceleration of growth at extremes of phosphate concentration, as promotion of carbon-source utilization rather than storage, and as support for phosphate-dependent resistance to arsenate inhibition of growth. Cytological studies of dividing cells revealed that stalked siblings had greater access to exogenous phosphate for use in growth or for storage as polyphosphate, and that access of non-stalked sibling to phosphate was dependent on the length of the stalk of the dividing cell. It was concluded that the physiologic role of the stalk is enhancement of phosphate acquisition. The stimulatory role of calcium in this process was attributed to its support of stalk development and to its stabilization of internal membrane/cell envelope association within the cell-stalk juncture.
32 citations
TL;DR: The Pf3 virus genome encoding its major coat protein and its single-stranded DNA-binding protein is organized somewhat like the corresponding region of the fd (M13, f1) genome, but the major Coat protein is unique among the major coat proteins of fd and the other filamentous phages studied.
Abstract: The region of the Pf3 virus genome encoding its major coat protein and its single-stranded DNA-binding protein is organized somewhat like the corresponding region of the fd (M13, f1) genome. Nevertheless, the major coat protein is unique among the major coat proteins of fd and the other filamentous phages studied in that it lacks a signal sequence and appears to be a direct translation product and in that it has fewer basic amino acid residues than its equivalent of DNA phosphates in the virion. These features are relevant to considerations of both protein insertion into membranes and DNA structure in filamentous viruses. The single-stranded DNA-binding protein also has a sequence that is different from the sequences of single-stranded DNA-binding proteins from other filamentous viruses.
28 citations
TL;DR: In vivo and in vitro transcription mapping studies indicate the size of complementary RNA to be around 1 kb with the 5' initiation site at base 79 and the 3' termination site in the area of base 1138, which indicates the presence of a 5' untranslated RNA and a fairly long 3' extension.
Abstract: We have determined the sequence of a 1228 base-pair cloned DNA fragment from Bacillus licheniformis capable of specifically complementing mutations in the spoOH gene, which is required for the early stage of sporulation in B. subtilis. The sequence has only one long open reading frame consisting of 168 codons. In vivo and in vitro transcription mapping studies indicate the size of complementary RNA to be around 1 kb with the 5' initiation site at base 79 and the 3' termination site in the area of base 1138. This indicates the presence of a 5' untranslated RNA and a fairly long 3' extension. The promoter sequence of this gene is 5'TATAAT3' at -10, and 5'TTGACG3' at -35, a typical E. coli-like promoter sequence, and is transcribed in vitro specifically only by RNA polymerase containing delta 55 and not delta 37-containing holoenzyme.
24 citations
TL;DR: Analysis of radiolabeled parasite-polypeptides on SDS-polyacrylamide gels indicated that pulse-labeled KP has the electrophoretic mobility of a 75 kDa polypeptide and is subsequently chased to an apparently 80-85 kDa form.
TL;DR: It is suggested that GST and GSH play important roles in DMBA inactivation in rats previously administered Sudan III and glutathione transferase (GST) activity toward 1-chloro-2,4-dinitrobenzene in the liver cytosol induced by these azo dyes.
Abstract: Noninbred Long-Evans rats fed the reduced form of glutathione (GSH) 2 hours before injection with 7,12-dimethylbenz[a]anthracene [(DMBA) CAS: 57-97-6)] showed a significant suppression of DMBA-induced chromosome aberrations (CA) in bone marrow cells. However, rats given injections of ethyl maleate [(EM) CAS: 141-05-9; (z)-2-butenedioic acid diethyl ester] 0.5 or 2 hours before being killed showed a remarkable fall in the hepatic GSH level. Furthermore, the administration of EM and bromosulfophthalein [CAS: 71-67-0; 3,3'-(4,5,6,7-tetrabromo-3-oxo-1(3H)-isobenzofuranylidene) bis(6-hydroxy)benzenesulfonic acid disodium salt] shortly before the DMBA injection inhibited the suppressive effects of Sudan III on DMBA-induced CA. A clear positive correlation was found between the capacity of Sudan III and related azo dyes to protect against DMBA-induced CA in bone marrow cells and glutathione transferase (GST) activity toward 1-chloro-2,4-dinitrobenzene in the liver cytosol induced by these azo dyes. DMBA activation with hepatic S-9 obtained from rats treated by polychlorinated biphenyls (PCB), phenobarbital, and Sudan III in the Ames system was high in this order as was also the case for the cytochrome P450 content. The addition of GSH to the Ames system containing S-9 from PCB-treated rats resulted in a substantial loss in the mutagenicity of DMBA. The present results suggest that GST and GSH play important roles in DMBA inactivation in rats previously administered Sudan III.
TL;DR: Intrapool variations in an incompatible heteroplasmid strain are inevitable and it is shown that these variations can be exploited by differential selection to amplify one plasmid at the expense of the other.
TL;DR: It is proposed that MLS resistance may have arisen in the Streptomyces and spread to B. licheniformis another gram positive bacterium found in soil and has spread and evolved further to yeild a variety of related Staphylococcal and Streptococcal MLS determinants.
Abstract: Naturally occurring erythromycin (Em) resistance was found in 11 of the 18 Bacillus licheniformis isolates tested but was absent from a wide variety of other Bacillus strains. The Em resistance elements confer inducible macrolide-lincosamide-streptogramin B (MLS) resistance and are related to ermD an MLS resistance element previously cloned from the chromosome of B. licheniformis 749. The MLS sensitive B. licheniformis strains and the other sensitive Bacillus strains tested, lack sequences with detectable homology to ermD. The sensitive B. licheniformis strains do exhibit homology to sequences which flank ermD in B. licheniformis 749. The relative sizes of the homologous DNA fragments suggest that the sensitive strains are lacking a 3.6 kb segment which contains ermD. It is shown that ermD is homologous to chromosomal DNA from Streptomyces erythreus ATCC 11635, an Em producing organism. These observations suggest to us that MLS resistance may have arisen in the Streptomyces and spread to B. licheniformis another gram positive bacterium found in soil. It is further proposed that ermD is or was located on a transposon-like element and has spread and evolved further to yeild a variety of related Staphylococcal and Streptococcal MLS determinants.
01 Jan 1984
TL;DR: A grossly oversimplified survey of the immune system found that immunocytes recognize and destroy virus-infected cells that display unorthodox surface viral antigens, whereas antibodies react with extracellularly disseminated viruses and neutralize their infective capability.
Abstract: Viral infection elicits an immune response consisting of multiple populations of immunologically differentiated cells (immunocytes) and multiple populations of soluble immunoglobulins (antibodies). Subject to genetic restrictions, immunocytes recognize and destroy virus-infected cells that display unorthodox surface viral antigens, whereas antibodies react with extracellularly disseminated viruses and neutralize their infective capability. Although each of these immunological elements has its own modus operandi, they are also interdependent: e.g., cytotoxic “killer” cells depend on antibody as a mediator; antibody is synthesized by B-type immunocytes; B cells are “helped” by T-type immunocytes; under certain conditions B cells are suppressed by T cells. Subsequent discussions will unravel the above grossly oversimplified survey of the immune system.
TL;DR: A human T‐lymphoblastoid cell line, TCL‐Fuj, constitutively produced a large amount of human gamma interferon (IFN) in culture fluids and has sustained stable IFN production for more than two years, indicating the requirement of IFN‐mRNA and protein for de novo synthesis.
Abstract: A human T-lymphoblastoid cell line, TCL-Fuj, constitutively produced a large amount of human gamma interferon (IFN) in culture fluids and has sustained stable IFN production for more than two years. When cells were incubated in RPMI-1640 medium with 10% fetal calf serum for three days, IFN activity was detectable at a cell density of 6 X 10(4) cells/ml, whereas 2,000-16,000 units of IFN per ml were produced at 5-10 X 10(5) cells/ml. IFN production was also detected even in serumfree medium and as early as 2 hr after cultivation in fresh medium. IFN was inhibited by treatment of cells with either actinomycin D or cycloheximide, indicating the requirement of IFN-mRNA and protein for de novo synthesis. The molecular weight of the IFN was 45,000-60,000 as determined by Sephacryl S200 gel filtration. Two activity peaks corresponding to molecular weights of 22,000 and 39,000 were obtained by SDS-polyacrylamide gel electrophoresis. Analysis by isoelectric focusing revealed charge heterogeneity with four species at pIs of 6.0, 7.1, 8.6, and 9.3. Conventional IFN-gamma inducers, concanavalin A and 12-O-tetradecanoyl-phorbol-13-acetate, further enhanced the production of IFN in this cell line.
TL;DR: Enterobacteria found in foods may not always indicate direct fecal contamination, but may often originate from environmental sources, and enterobacterial species other than E. coli should also be included in the indicator organisms of microbial standards for processed foods.
Abstract: Taxonomic studies were carried out on 2, 842 strains of gram-negative, facultatively anaerobic, fermentative rods isolated from foods and environmental materials by means of a probabilistic method using a computer. Although coliforms included only a few species in the classification proposed by the Coli-Aerogenes Subcommittee in 1956, the 2, 842 strains studied were divided into 51 species.Strains showing an IMViC pattern on Escherichia coli included authentic E. coli and eight other species that probably originated from the environment. The majority of strains of E. coli were easily differentiated from other coliform organisms by their ability to grow at 44.5°, but 17% of lactose-fermenting and 16% of lactose-nonfermenting strains of E. coli failed to grow at this temperature. In addition, 13.3% of 188 E. coli strains were non- or late lactose-fermenters and 10.4% of 163 lactose-fermenting E. coli produced no gas, so that these strains did not serve as indicator organisms of fecal contamination. Thus, enterobacteria found in foods may not always indicate direct fecal contamination, but may often originate from environmental sources.It is discussed whether enterobacterial species other than E. coli should also be included in the indicator organisms of microbial standards for processed foods. Although the species found in foods in this study would be harmless to healthy persons, they have often been observed as opportunistic pathogens in hospitalized patients with some underlying diseases. It seems desirable, therefore, that separate microbial standards from those for public health should be applied to foods served to hospitalized patients.
TL;DR: A simple and specific method is described, based on the oxidation of PG by propylene glycol dehydrogenase (PGDH) from Microcyclus eburneus, which is accompanied by the reduction of NAD+ to NADH.
Abstract: A simple and specific method is described, for the determination of propylene glycol (PG) in commercial foods. The method is based on the oxidation of PG by propylene glycol dehydrogenase (PGDH) from Microcyclus eburneus, which is accompanied by the reduction of NAD+ to NADH.PG separated from foods by extraction with deionized water and ultrafiltration was readily determined by measuring the absorbance at 340 nm.The use of an enzymatic reaction resulted in almost stoichiometric oxidation of PG, and the method was relatively free from interference. Recoveries of 2 and 10 mg/g of PG from several foods ranged from 85 to 95% for 2 mg/g and 95 to 99% for 10 mg/g with a detection limit of 2 μg.
01 Jan 1984
TL;DR: The major viral protein in the three groups of viruses is the merrorane (M) component, which must play a pivotal role in the assembly of the virus, as it must surround the ribonucleoprotein (RNP) at the same time that it recognizes the site on the plasma membrane where the glycol-proteins have been assembled.
Abstract: Publisher Summary The proteins of the lipid containing viruses such as rhabdo, myxo, and paramyxoviruses assemble at the cell plasma membrane where their close interaction with one another leads to the exclusion of host cell proteins. The viral glycoproteins are synthesized on rough endoplasmic reticulum (ER) and passed on to the smooth ER and the cell plasma membrane, where they remain anchored. The internal viral proteins move through the cytoplasm by some unidentified mechanism and then onto the plasma membrane site, where the glycoproteins have assembled. Virus is produced by budding from the cell. The major viral protein in the three groups of viruses is the merrorane (M) component. This internal protein must play a pivotal role in the assembly of the virus, as it must surround the ribonucleoprotein (RNP) at the same time that it recognizes the site on the plasma membrane where the glycol-proteins have been assembled.