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PubMed1983

New suppressors of frameshift mutations in Salmonella typhimurium.

Several new types of suppressor mutants have been isolated. These were identified among revertants of mutants originally generated by mutagens other than the acridine-derived ICR191. The new suppressors correct mutations other than those with runs of C or G which are recognized by the previously described suppressors. Several frameshift mutations are corrected by more than one suppressor type. Apparently, the DNA base sequence near these mutant sites includes sites of action for several distinct suppressor types.

Base Sequence↗
PubMed1976

Studies on the poky mutant of eurospora crassa. Fingerprint analysis of mitochondrial ribosomal RNA.

Base sequence and methylation of mitochondrial ribosomal RNAs from wild type and poky strains of Neurospora crassa were compared to determine whether a mutational lesion exists in poky 19 S RNA. At the outset, new procedures were developed for the isolation of intact nucleic acids from Neurospora mitochondria based on the substitution of Ca2+ for Mg2+ in the isolation media to inhibit mitochondrial nuclease activity. Using these procedures, intact and highly purified 32P-labeled ribosomal RNAs were extracted from purified mitochondrial ribosomal subunits of wild type and poky and compared using three complementary fingerprinting systems: two-dimensional electrophoresis of T1 plus phosphatase digests and homochromatography of T1 and pancreatic RNase digests. In supplementary experiments, 32P-labeled wild type RNA was co-fingerprinted with 32P-labeled poky and ratios of 32P/33P radioactivity were determined in each fragment to detect possible differences in stoichiometry. In addition, levels and patterns of methylated nucleotides were compared using procedures based on in vivo labeling with [methyl-3H]methionine and [32P]orthophosphate. In all these experiments, no difference was detected between wild type and poky in base sequence or methylation of either 19 S or 25 S RNA. Levels of methylation of Neurospora mitochondrial ribosomal RNAs were extremely low (less than 0.1% of the nucleotides), and results based on fingerprint analysis and DEAE-cellulose chromatography of alkaline hydrolysates of the [3H]methyl-labeled RNA suggested that 25 S RNA contains two ribose methylations, while 19 S RNA contains no methylated nucleotides.

Calcium↗
PubMed1990

Characterization of the African HIV-1 isolate CBL-4 (RUT) by partial sequence analysis and virus neutralization with peptide antibody and antisense phosphate-methylated DNA.

The HIV-1 isolate CBL-4 (RUT), originating from Tanzania, was characterized using a comprehensive virus-typing system. This system included sequence analysis of the region coding for the neutralization domain in the third variable region (V3) of the external envelope and of the tat responsive (TAR) region after polymerase chain reaction (PCR) amplification of these sequences from cellular DNA in the CBL-4 (RUT) producer line. Based on independent cluster analysis of TAR and V3 sequences the CBL-4 (RUT) virus was positioned closest to the Z6 (and ELI) African virus family. The V3 amino acid sequence on the surface of the virus particle was confirmed by the inhibition of neutralization of CBL-4 (RUT) by a synthetic peptide derived from the nucleic acid sequence. Using antisense phosphate-methylated DNA covering the TAR loop region of LAV-1/HTLV-IIIB, inhibition of HTLV-IIIB and HTLV-IIIRF infection was seen, whereas no inhibition was observed for CBL-4 (RUT), indicating two or more mismatches in the TAR loop region, a characteristic shared with Z6 virus, but not with ELI. We propose a virus-typing system based on sequence analysis confirmed by virus neutralization with a peptide binding antibody and inhibition by antisense phosphate-methylated DNA to group viruses for laboratory use and vaccine design.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Identification of sequence types among the M-nontypeable group A streptococci.

Streptococcal diseases, namely, acute glomerulonephritis and acute rheumatic fever, are common features in the aboriginal population of the Northern Territory of Australia. We examined the group A streptococcal M types identified during various surveys conducted since 1987. Streptococci were predominantly isolated from skin infections. A high proportion of the isolates could not be serotyped by conventional means and were designated M nontypeable (MNT). M-specific DNA sequences from the MNT isolates were examined, and sequence types were proposed for the classification of MNTs. This allowed a more precise estimate of the M types present in a population study.

Amino Acid Sequence↗
PubMed1995

Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis.

A total of 53 field and reference strains, including the type strains of the seven named species (nomenspecies) and belonging to the 18 described genomic species (DNA groups) of the genus Acinetobacter, were studied by amplified ribosomal DNA restriction analysis (ARDRA). Restriction analysis with the enzymes AluI, CfoI, MboI, RsaI, and MspI of the enzymatically amplified 16S rRNA genes allowed us to identify all species except the genomic species 4 (Acinetobacter haemolyticus) and 7 (A. johnsonii), 5 (A. junii) and 17, and 10 and 11, which clustered pairwise in three respective groups. Further analysis with the enzyme HaeIII, HinfI, NciI, ScrFI, or TaqI did not allow us to differentiate the species within these three clusters. However, use of a few additional simple phenotypic tests (hemolysis, growth at 37 degrees C, production of acid from glucose, and gelatin hydrolysis) can be used to differentiate between the species within these clusters. ARDRA proved to be a rapid and reliable method for the identification of most of the Acinetobacter genomic species, including the closely related DNA groups 1 (A. calcoaceticus), 2 (A. baumannii), 3, and 13. The results of this study suggest that ARDRA can be used for the identification of Acinetobacter species and as such may help to elucidate the ecology and clinical significance of the different species of this genus. Since ARDRA uses universal 16S rRNA gene primers, it is expected to be applicable to the identification of most bacterial species. Furthermore, ARDRA is less prone to contamination problems than PCR for detection, since the use of cultured organisms results in a large initial quantity of target DNA.

Acinetobacter↗
PubMed2025

Molecular epidemiological surveillance for non-tuberculous mycobacterial pulmonary disease: a single-center prospective cohort study.

UNLABELLED: Bacterial species cultured from sputum change during treatment or observation for non-tuberculous mycobacterial pulmonary disease; however, strain-level changes remain unrecognized. Variable number tandem repeat typing is a standard technique for strain identification; nonetheless, its labor-intensive and time-consuming nature limits routine clinical use. Therefore, we aimed to elucidate species-subspecies and strain dynamics in non-tuberculous mycobacteria and develop a simple sequence-based strain-level determination method. We performed a single-center prospective cohort study of 112 patients with non-tuberculous mycobacterial pulmonary disease. Whole-genome sequencing was performed on two sputum samples collected at enrollment and at the end of follow-up, followed by variable number tandem repeat (VNTR) typing. We also developed a simple long-read sequencing-based digital VNTR (dVNTR) typing method and evaluated its efficacy. Our results demonstrate that core genome multi-locus sequencing typing revealed species/subspecies changes in 13 patients (11.6%); VNTR typing detected strain changes in 16 patients (14.3%) without species/subspecies changes. Overall, pathogen shifts occurred in 29 patients (shift [+] group, 25.9%), whereas 83 had no detectable pathogen shift (shift [-] group, 74.1%). Interestingly, macrolide and amikacin susceptibility changed in both groups, but resistance remained higher in shift (-) patients. dVNTR results aligned with those of conventional VNTR typing. In conclusion, since susceptibility factors remain unclear, routine species/subspecies identification and molecular typing, such as VNTR, are optimal for patient care. Core genome multi-locus sequencing typing with a dVNTR identified pathogen shifts, innovating non-tuberculous mycobacterial pulmonary disease management.Clinical TrialsThis study is registered with UMIN as UMIN 000056067. IMPORTANCE: Pulmonary non-tuberculous mycobacterial disease is a chronic infection in which the causative pathogens may change at the species, subspecies, or strain level over time. Accurate tracking of these changes is essential for optimizing treatment; however, conventional clinical practice lacks efficient methods for monitoring such dynamics. Our study revealed pathogen changes in approximately one-quarter of patients over 1.5 years, prompting the development of a novel surveillance system that integrates next-generation sequencing for both species-subspecies identification and strain-level molecular epidemiology. This innovation enables real-time monitoring of pathogen dynamics, allowing clinicians to promptly adjust treatment strategies and improve patient care through more informed decision-making.

Humans↗
PubMed1987

Restriction endonucleases recognizing DNA sequences of four base pairs facilitate differentiation of herpes simplex virus type 1 strains.

Variation of restriction endonuclease cleavage patterns between herpes simplex virus type 1 (HSV-1) strains was investigated using restriction endonucleases recognizing DNA sequences of four base pairs (4-bp enzymes) (HaeIII, HhaI, Sau3AI). The analysis made feasible identification of variations not heretofore detectable, using enzymes recognizing DNA sequences of six base pairs (BamHI, KpnI, SalI), and proved to be most useful for distinction and classification of HSV-1 strains. Ten of the fifteen strains analysed using 4-bp enzymes were closely related, and were put into group 1, and the other five not so related strains were classed into the non-group 1. The observations suggested a possible common ancestor from which group 1 strains had derived.

Animals↗
PubMed1991

Protection of macaques with a simian immunodeficiency virus envelope peptide vaccine based on conserved human immunodeficiency virus type 1 sequences.

This report describes the vaccination of rhesus macaques with peptides selected from regions of the simian immunodeficiency virus (SIV) envelope that are hydrophilic, immunoreactive, and highly homologous with corresponding conserved envelope sequences of the human immunodeficiency virus (HIV). The peptides, produced as beta-galactosidase fusion proteins, induced virus-neutralizing and peptide-specific antibodies. After challenge with virulent virus, controls became virus positive and developed gradually rising antibody titers to SIV over 63 weeks. Immunized macaques developed a postchallenge anamnestic response to SIVenv antigens within 3-6 weeks followed by a gradual, fluctuating decline in SIV antibody titers and partial or total suppression of detectable SIV. Virus suppression correlated with prechallenge neutralizing antibody titers. Although the average CD4+ cell count in the blood of immunized macaques remained constant, the control macaques exhibited a progressive decrease developing about week 55 after challenge. The conserved nature of the HIV and SIV peptides and the similar humoral immunoreactivity in the respective hosts suggest that homologous HIV peptides may be important components of a successful immunization strategy.

Amino Acid Sequence↗
PubMed1989

Role of the extra G-C pair at the end of the acceptor stem of tRNA(His) in aminoacylation.

All sequenced histidine tRNAs have one additional nucleotide at the 5' end compared with other tRNA species. To investigate the role of this unique structure in aminoacylation, we constructed in vitro transcripts corresponding to the E. coli histidine tRNA sequence and its variants at the G-1-C73 base pair, by using T7 RNA polymerase transcription system. A transcript having a wild-type sequence with no modified bases was a good substrate for histidyl-tRNA synthetase (HisRS), and aminoacylation activity was affected by introduction of a triphosphate at the 5' terminus. Base replacements at position 73 caused a marked decrease of Vmax, and deletion and substitution of the G-1 had a remarkable effect on the aminoacylation. A mutant having an A-1-U73 pair was also not a good substrate for HisRS. Comparison among G-1-deficient mutants showed that A was preferable rather than C as the base at position 73. These data demonstrate that the set of the G-1-C73 pair at the end of the acceptor stem of histidine tRNA is crucial for the catalytic process of aminoacylation.

Acylation↗
PubMed1980

Structure of genes for virus-associated RNAI and RNAII of adenovirus type 2.

A DNA sequence, 552 base pairs in length, encoding the two "virus-associated" (VA) RNAs of adenovirus type 2 is presented. Comparison of the oligonucleotide maps of VA RNAI and VA RNII with the established sequence permits identification of the genes for these RNAs. VA RNAI is 157-160 nucleotides long and VA RNAII 158-163 nucleotides long, depending on the exact length of their heterogeneous 3' end. The genes are separated by a spacer of about 98 nucleotides. The RNAs exhibit scattered regions of primary sequence homology and can adopt secondary structures which resemble each other closely in their configuration and stability. VA RNAII is also capable of assuming a different configuration that is energetically more favorable. The data suggest that the two RNA genes may have arisen by duplication of an ancestral gene and that the folding of the RNA chain may be of importance for the function of VA RNAs. Hypothetical RNA polymerase III recognition sequences and the coding potential of the region are discussed.

Adenoviruses, Human↗
PubMed1987

A single base change in the Shine-Dalgarno region of 16S rRNA of Escherichia coli affects translation of many proteins.

A single base mutation was constructed at position 1538 of Escherichia coli 16S rRNA, changing a cytidine to a uridine. This position is in the Shine-Dalgarno region, thought to be involved in base-pairing to mRNA during initiation of protein synthesis. The mutation was constructed by using a synthetic oligodeoxynucleotide that differs in sequence by one base from the wild-type sequence of 16S rRNA. This oligonucleotide was used as a primer on single-stranded DNA of phage M13, into which was cloned a specific region of DNA encoding 16S rRNA. The mutation is lethal when expressed from the normal promoters of rRNA operons, P1 and P2, in a high-copy-number plasmid. Expression can be repressed by a temperature-sensitive repressor, cI857, in combination with the bacteriophage lambda PL promoter. Induction of transcription by temperature shift yields mutant 16S rRNA that is processed and assembled into functional ribosomal subunits. The presence of mutant ribosomes retards cell growth and dramatically alters incorporation of [35S]methionine into a large proportion of the cellular proteins. The change in level of synthesis of individual proteins correlates with the change in base-pairing between mutant rRNA and the Shine-Dalgarno region of the mRNA.

Bacterial Proteins↗
PubMed1995

Identification of Campylobacter jejuni and C. coli using the rpoB gene and a cryptic DNA fragment from C. jejuni.

Campylobacter jejuni (Cj) and C. coli (Cc) clinical isolates, obtained from three different sources, were characterized using two Cj DNA probes, CJ01 and CJ02. These probes were selected at random by virtue of their stability in Escherichia coli (Ec). CJ01 hybridized specifically with DNA from Cj reference strains, but not with DNA from Cc, C. lari (Cl) nor C. fetus (Cf) reference strains. Using clinical isolates characterized by genome-genome hybridization and biotype, CJ01 hybridized with DNA derived from all Cj strains. However, DNA from four out of ten Cc strains, from three different sources, also hybridized with CJ01, suggestive of this region being heterogeneous between clinical isolates of both species. The nucleotide sequence analysis of CJ01 reveals two incomplete open reading frames (ORFs) that did not show significant homology with any other known sequences. CJ02 hybridized specifically with DNA from Cj and Cc reference strains, but not with DNA from Cl and Cf reference strains. The specificity and sensitivity were maintained upon hybridization with DNA from 31 clinical isolates. CJ02 has an uninterrupted ORF whose deduced amino-acid sequence showed extensive homology with the central region of the Ec and Salmonella typhimurium (St) RNA polymerase beta subunits (52 and 66% similarity, respectively). The most conserved segments correspond to putative functional domains.

Amino Acid Sequence↗
PubMed1995

Differentiation of strains in Mycobacterium tuberculosis complex by DNA sequence polymorphisms, including rapid identification of M. bovis BCG.

The Mycobacterium tuberculosis complex includes M. tuberculosis, M. bovis, M. microti, and M. africanum. Seven strains of the M. tuberculosis complex were sequenced in a region of about 300 bp which contains multiple 15-bp tandem repeats and which is part of a 1,551-bp open reading frame. Four distinct sequences were obtained, each defining a sequevar. A sequevar includes the strain or strains with a given sequence. The type strain M. tuberculosis TMC 102 (H37Rv) was designated sequevar MED-G. When compared to MED-G, sequevar LONG had an insertion of one 15-bp tandem repeat and sequevar SHORT had a deletion of one tandem repeat. Sequevar MED-C had a G-->C substitution, coding for the conservative change Ser-->Thr. BanI cuts only sequevar MED-C at the site of the substitution. PCR-restriction enzyme analysis was used to determine the sequevars of 92 M. tuberculosis complex strains. All 23 M. bovis BCG strains belonged to sequevar MED-C. The M. africanum type strain was sequevar SHORT. The remaining 68 strains of M. tuberculosis, M. bovis (not BCG), and M. microti were sequevars LONG (3 strains) or MED-G (65 strains). PCR-restriction enzyme analysis was applied to reference strains and clinical isolates with a worldwide distribution. This method provides rapid, sensitive, and specific identification of the important vaccine strain M. bovis BCG.

Amino Acid Sequence↗
PubMed1987

Regulation of the herpes simplex virus type 1 late (gamma 2) glycoprotein C gene: sequences between base pairs -34 to +29 control transient expression and responsiveness to transactivation by the products of the immediate early (alpha) 4 and 0 genes.

The glycoprotein C (gC) gene of herpes simplex virus type 1 is a true late gene, in that its expression occurs late in infection with a strict requirement for viral DNA replication. Recently, we reported on gC expression during infection with mutant viruses carrying deletions in the gC gene promoter. Analysis of RNA extracted from cells infected with individual mutants showed that the DNA sequences required for regulated expression of this late gene lie within bases -34 to +124 relative to the 5' end of the mRNA. In the present study, the deleted gC promoter sequences were fused to the bacterial chlorampheniol acetyltransferase (CAT) gene and expression was measured in short-term transfection assays after transactivation by infection with HSV or cotransfection with a second plasmid carrying the immediate early genes of HSV-1. The 63 base pair sequence located between -34 to +29 on the gC promoter was sufficient to give induction of CAT activity following infection and on cotransfection with plasmids which code for the immediate early gene products ICP4 and ICPO. This 63 base pair region contains the TATA homology and the transcriptional start site of the gC gene, and apparently contains specific promoter elements not found in a similar region of the HSV TK promoter. This was shown by removing the distal upstream region of the TK promoter, 5' to -37, and found that the TK gene was no longer activated by infection or cotransfection with an alpha 4-alpha 0 gene containing plasmid.

Animals↗
PubMed1986

Intron mutations affect splicing of Saccharomyces cerevisiae SUP53 precursor tRNA.

The Saccharomyces cerevisiae amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the role of intron structure and sequence on precursor tRNA splicing in vivo and in vitro. This gene encodes a pre-tRNA which contains a 32-base intervening sequence. Two types of SUP53 intron mutants were constructed: ones with an internal deletion of the natural SUP53 intron and ones with a novel intron. These mutant genes were transcribed in vitro, and the end-processed transcripts were analyzed for their ability to serve as substrates for the partially purified S. cerevisiae tRNA endonuclease and ligase. The in vitro phenotype of these mutant RNAs was correlated with the in vivo suppressor tRNA function of these SUP53 alleles after integration of the genes into the yeast genome. Analysis of these mutant pre-tRNAs, which exhibited no perturbation of the mature domain, clearly showed that intron structure and sequence can have profound effects on pre-tRNA splicing. All of the mutant RNAs, which were inefficiently spliced or unspliced, evidenced cleavage only at the 5' splice junction. Base changes in the intron proximal to the 3' splice junction could partially rescue the splicing defect. The implications of these data for tRNA endonuclease-substrate interactions are discussed.

Computer Simulation↗
PubMed1986

Immunochemistry of the Lewis blood-group system: isolation and structures of Lewis-c active and related glycosphingolipids from the plasma of blood-group O Le(a-b-) nonsecretors.

Five different glycosphingolipid fractions (GL-3, 285 micrograms; GL-5, 1090 micrograms; GL-6, 615 micrograms; GL-7, 555 micrograms; and GL-8, 155 micrograms) have been isolated from 25 liters of plasma of O Le(a-b-) nonsecretors by means of ethanol extraction, several steps of Folch distribution, and reversed-phase, silicic acid, and ion-exchange column chromatography of native or peracetylated substances. Final purification, accomplished by preparative silica gel high-performance thin-layer chromatography, led to chromatographic homogeneity of GL-3 and GL-6. In the hemagglutination inhibition as well as quantitative passive hemagglutination techniques two of these substances (GL-3, GL-5) exhibited distinct, and the other three (GL-6-GL-8) very strong, Lec blood-group activities when tested against two different Lec antisera of human or goat origin. The fragments' structures were elucidated by fast atom bombardment and electron impact mass spectrometry of permethylated derivatives in order to determine molecular weight, sugar sequence, position of branching points, and type of oligosaccharide chains, as well as fatty acid and sphingosine patterns of the ceramide residue. Combined gas-liquid chromatography and mass spectrometry of partially methylated alditol acetates identified sugar composition and glycosidic linkages. Thus, the following structures could be established: (formula; see text) In contrast to the structurally homogeneous GL-3, minor amounts of 4-O-substituted GlcNAc pointed to a small contamination of GL-6 by branched type 2 ceramide nonasaccharide analogs. Glycolipids containing hepta- or nonasaccharides as in GL-3 or GL-6 could also be identified in fractions GL-5 (ceramide heptasaccharide) and GL-7 and GL-8 (ceramide nonasaccharide). These latter fractions revealed, however, distinct heterogeneity due to the presence of a small amount of either a type 2 analog of GL-3 (GL-5) or linear, mainly type 2, ceramide hexa- (GL-5, GL-7) or octasaccharides (GL-8). In addition to previous immunochemical communications the presented Lec active structures of GL-3 and GL-6 provide evidence that 3-fucosyl-N-acetyllactosamine in combination with a type 1 based oligosaccharide sequence and a 3,6-galactosyl branching point are essential parts of the Lec antigenic determinant (as marked in the formula of GL-6).

ABO Blood-Group System↗
PubMed2026

Potential dissemination and persistence of Clostridium perfringens along the slaughtering process in French cattle, pig or poultry slaughterhouses.

Clostridium perfringens is a major foodborne pathogen associated with meat products, yet its dissemination routes and persistence within slaughterhouses remain poorly understood. In this study, whole-genome sequencing combined with multilocus sequence typing (MLST), core genome MLST (cgMLST), and core single nucleotide polymorphism (SNP) analysis was applied to 286 C. perfringens isolates collected from cattle, pig, and poultry slaughterhouses in France. MLST analysis revealed extensive genetic diversity, with most isolates assigned to novel allelic profiles rather than previously described sequence types. Phylogenetic analyses based on cgMLST and SNP data revealed frequent recovery of closely related isolates from feces, meat, surfaces, and air, highlighting widespread dissemination of strains within slaughterhouses during processing. Notably, close genetic related isolates recovered from air and other sample types are consistent with air-associated dissemination within slaughterhouse environments. In addition, the detection of closely related strains across different sampling campaigns suggests the potential persistence of C. perfringens within slaughterhouse environments over time. Most isolates were classified as toxinotype A (97.9%), with a few belonging to toxinotypes D (1.0%) and G (1.0%), and in silico analyses revealed a broad distribution of virulence-associated genes. Antimicrobial resistance genes (ARGs) were commonly detected, particularly those conferring resistance to tetracyclines, although isolates carrying multiple ARGs remained infrequent. Overall, this study provides new insights into the genomic diversity, dissemination pathways, and persistence of C. perfringens in multi-species slaughterhouses. These findings highlight the potential role of air-associated dissemination in contamination dynamics and underscore the importance of improved hygiene control strategies to mitigate food safety risks along the meat production chain.

Antimicrobial resistance gene (ARG)↗