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Repetitive sequence-based PCR versus pulsed-field gel electrophoresis for typing of Enterococcus faecalis at the subspecies level.

Repetitive sequence-based PCR was compared to pulsed-field gel electrophoresis (PFGE) for the ability to discriminate Enterococcus faecalis isolates at the subspecies level. The BOXA2R primer, derived from repetitive sequences in Streptococcus pneumoniae, was applied to 41 isolates of E. faecalis collected from various sources. The REP1R-Dt and REP2-Dt primers, derived from the gram-negative repetitive extragenic palindromic element, were also applied to 18 selected isolates. Of the 41 isolates examined, 7 were beta-lactamase producing and 8 were vancomycin resistant. By PFGE, 17 isolates had distinct patterns; the other 24 were classified into eight different clonal groups. By PCR using the BOXA2R primer, 16 isolates generated distinct patterns; the other 25 were classified into nine different clonal groups. There were only minor differences in the PCR results obtained by using the BOXA2R primer and the REP1R-Dt and REP2-Dt primers. Two isolates among vancomycin-resistant enterococci from the greater Houston, Tex., area were related by PFGE, distinct by PCR with the BOXA2R primer, and related by PCR with the REP1R-Dt and REP2-Dt primers. Clonal relationships among the remaining 39 isolates were similar by both PFGE and PCR. PCR reliably discriminated all epidemiologically unrelated isolates. Although PCR is less time consuming than PFGE, PCR results were more difficult to interpret than PFGE results, perhaps because fewer bands were generated by PCR than by PFGE and some PCR products were inconsistently seen.

Bacterial Typing Techniques

Characterisation of group A streptococcal isolates from tropical Australia with high prevalence of rheumatic fever: probing for signature sequences to identify members of the family of serotype 5.

The prevalence of rheumatic heart disease (RHD) in Northern Territory Aboriginal communities is high, but there is a low isolation rate of historically rheumatic fever associated M types (such as M5) of group A streptococci (GAS). Many isolates are M non-typable (MNT). Serology suggests that the population is exposed to M5-like isolates; some RHD patients having high IgM or IgG titres to two M5 B-repeat region peptide epitopes, B1 (KQQESK) and B4 (EQKSKQ). To identify relatives of M5 in our collection of GAS, oligonucleotide probes to the B1 and B4-repeat regions shared by M5 and a local M5-like isolate, were used to screen 101 isolates for the presence of signature sequences. In all, 28% of the tropical Australian isolates contained the signature sequences, identifying members of the M5 family. The 5' region of the genes for M proteins from three members of the M5 family fell into two sequence types. Hybridisation to probes based on these sequences suggested that among tropical Australian isolates there are at least three distinct sequence types that contained the M5 signature sequences. These results suggest that a considerable number of M5 family GAS are circulating in tropical Australia.

Amino Acid Sequence

Base-pair resolution conservation data improves cell type specific sequence-to-expression prediction.

MOTIVATION: Genomic sequence-to-activity models can decipher gene regulatory mechanisms and predict the functional impact of regulatory variants. However, current models struggle to integrate information from sequences outside promoters, especially information from cell type specific regulatory elements. RESULTS: Here, we propose incorporating base-pair resolution evolutionary conservation data into genomic sequence-to-expression predictors. We explore two training strategies-training from scratch or fine-tuning an existing sequence-only model with additional conservation input. We find that in both cases, base-pair resolution conservation data improves cell type specific sequence-to-expression prediction, with training from scratch yielding the greatest benefit. The improvement in cell type specific expression prediction can be attributed in part to the fact that models trained on sequence and conservation data learn to better recognize cell type specific regulatory elements than models trained on sequence alone. AVAILABILITY: Code is available at https://github.com/ni-lab/basenji-phyloP.

Conserved Sequence

Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells.

The adenovirus penton base protein has a cell rounding activity and may lyse endosomes during virus entry into the cytoplasm. We found that penton base that was expressed in Escherichia coli also caused cell rounding and that cells adhered to polystyrene wells that were coated with the protein. Mutant analysis showed that both properties required an Arg-Gly-Asp (RGD) sequence at residues 340 to 342 of penton base. In flat adherent cells, virus mutants with amino acid substitutions in the RGD sequence were delayed in virus reproduction and in the onset of viral DNA synthesis. In nonadherent or poorly spread cells, the kinetics of mutant virus reproduction were similar to those of wild-type adenovirus type 2. Expression of the mutant phenotype exclusively in the flat cells that we tested supports a model in which penton base interacts with an RGD-directed cell adhesion molecule during adenovirus uptake or uncoating.

Adenoviruses, Human

High-resolution HLA-DPB typing based upon computerized analysis of data obtained by fluorescent sequencing of the amplified polymorphic exon 2.

To differentiate 32 HLA-DPB alleles, conventional techniques such as serology and cellular typing are inadequate for high-resolution DPB typing. The most refined DNA typing until now is SSO typing and new selected oligonucleotides can be added to this system to distinguish new allele sequences. DNA sequencing, however, reveals directly the sequence information of all polymorphic HVRs and has the advantage of being independent from exon polymorphisms. We have developed a new DNA-based typing approach that is rapid, fully automated, and therefore suitable for routine typing. The system is based upon direct sequencing of amplified DNA with fluorescent-labeled primers. The designation of alleles is obtained by a comparison of all polymorphic positions in the determined sequence with all known allele sequences retained in a database along with their heterozygous combinations. Sequence data at both constant and polymorphic positions are used for quality control. In this study, the typing results of a panel of 91 previous SSO-typed DNA samples are described. After comparison with the SSO-typing results, we conclude that with this SBT system allele assignment is reliable. The method is easy to perform since both sequencing and assignment are automated. Furthermore, the system is easily applicable to other gene systems.

Base Sequence

In vitro evolution used to define a protein recognition site within a large RNA domain.

A minimum of 460 nucleotides of 16S ribosomal RNA are needed to fold the target site for E. coli ribosomal protein S4, although a much smaller region within this large domain is protected from chemical reagents by the protein. Starting with a 531-nucleotide tRNA fragment, cycles of mutagenesis, selection with S4, and amplification ('in vitro evolution') were used to obtain a pool of 30 RNA sequences selected for S4 recognition but approximately 30% different from wild type. Numerous compensatory base pair changes have largely preserved the same secondary structure among these RNAs as found in wild-type sequences. A 20-base deletion and a single nucleotide insertion are among several unusual features found in most of the selected sequences and also prevalent among other prokaryotic rRNAs. Most of the compensatory base changes and selected features are located outside of the region protected by S4 from chemical reagents. It was unexpected that S4 would select for RNA structures throughout such a large domain; the selected features are probably contributing indirectly to S4 recognition by promoting correct tertiary folding of the region actually contacted by S4. The role of S4 may be to stabilize this domain (nearly one-third of the 16S rRNA) in its proper conformation for ribosome function.

Base Sequence

Typing of Mycoplasma pneumoniae by nucleic acid sequence-based amplification, NASBA.

Nucleic acid sequence-based amplification, NASBA, is an isothermal amplification technique for nucleic acids and was used for typing a collection of 24 Mycoplasma pneumoniae strains. A set of primers was chosen from the 16S rRNA sequence alignment of Mycoplasma species. The nucleotide sequences of the (-)RNA amplicons were determined for M. pneumoniae strains M15/83 (type 1) and FH (type 2), and revealed a one-point difference at the 16S rRNA level between the two types. Based on this result, two type-specific probes were constructed. The probes were hybridized in solution with the amplified nucleic acids of 24 M. pneumoniae strains in an enzyme-linked gel assay (ELGA). The results obtained by NASBA-based typing are in agreement with the classification of the 24 M. pneumoniae strains into two types by other typing methods, confirming the reliability of this technique.

Bacterial Typing Techniques

Analysis of clonal relationships among isolates of Shigella sonnei by different molecular typing methods.

Shigella sonnei is a major cause of diarrheal disease in developed as well as in developing countries. Epidemiologic studies of this organism have been limited by the lack of a simple and effective method for comparing strains. In this study, we have compared different molecular typing methods, i.e., plasmid profile analysis, restriction endonuclease analysis of plasmids, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis (PFGE), and enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR (ERIC-PCR) for typing 20 clinical isolates of S. sonnei collected from six incidents of infection. PFGE and ERIC-PCR fingerprintings had the highest discriminatory power for discrimination of epidemiologically related isolates from epidemiologically unrelated strains of S. sonnei, and both gave seven distinct strain types among these isolates and the type strain of the species. Plasmid study and ribotyping produced only six and typing techniques demonstrated two distinct patterns, respectively, among these strains. All of these molecular an identical fingerprint for eight temporally related sporadic isolates. It is possible that these temporally related isolates belonged to a single bacterial clone and circulated obscurely through the community. Our results indicate that the ERIC-PCR technique represents a rapid and simple means for typing S. sonnei with a level of discrimination equivalent to that of PFGE but greater than those of plasmid profile analysis, restriction endonuclease analysis of plasmids, and ribotyping.

Bacterial Typing Techniques

Measurement of mRNA for E-selectin, VCAM-1 and ICAM-1 by reverse transcription and the polymerase chain reaction.

Stimulation of cultured human umbilical vein endothelial cells by cytokines such as interleukin-1 and tumour necrosis factor induces de novo synthesis and expression of the adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In general, alterations in cell surface expression of these molecules are known to be related to increased gene transcription and altered levels of mRNA. The extension of these observations to the study of inflammatory processes in different human organs necessitates the development of techniques for the quantification of mRNA in small tissue samples. Here we present a method for the quantification of mRNA for E-selectin, VCAM-1 and ICAM-1 using reverse transcription and the polymerase chain reaction (RT-PCR). For each molecule of interest a mutant RNA was synthesised consisting of the wild-type sequence deleted of 15-20 bases. The mutant and wild-type RNA sequences are recognised by the same primers, and can therefore be amplified competitively in the same tube by RT-PCR. As the mutant and wild-type RNAs compete for the primers, the amount of wild-type RNA can be determined by the size of the dominant product that results after addition of known quantities of mutant RNA. Using this detection and quantification method we have examined the dose dependency and time course of mRNA accumulation following TNF-alpha stimulation of HUVEC. Similar time-courses of E-selectin, ICAM-1 and VCAM-1 mRNA accumulation were observed by competitive RT-PCR as by laser densitometry of Northern blots. Finally we were able to show that the technique could measure changes in levels of mRNA for these three molecules in human skin biopsies taken at different times during the development of a delayed hypersensitivity response to tuberculin purified protein derivative. This technique should be useful for the study of adhesion molecule mRNA in small tissue culture samples and in biopsies.

Base Sequence

A computer method for finding common base paired helices in aligned sequences: application to the analysis of random sequences.

We describe a new computer program that identifies conserved secondary structures in aligned nucleotide sequences of related single-stranded RNAs. The program employs a series of hash tables to identify and sort common base paired helices that are located in identical positions in more than one sequence. The program gives information on the total number of base paired helices that are conserved between related sequences and provides detailed information about common helices that have a minimum of one or more compensating base changes. The program is useful in the analysis of large biological sequences. We have used it to examine the number and type of complementary segments (potential base paired helices) that can be found in common among related random sequences similar in base composition to 16S rRNA from Escherichia coli. Two types of random sequences were analyzed. One set consisted of sequences that were independent but they had the same mononucleotide composition as the 16S rRNA. The second set contained sequences that were 80% similar to one another. Different results were obtained in the analysis of these two types of random sequences. When 5 sequences that were 80% similar to one another were analyzed, significant numbers of potential helices with two or more independent base changes were observed. When 5 independent sequences were analyzed, no potential helices were found in common. The results of the analyses with random sequences were compared with the number and type of helices found in the phylogenetic model of the secondary structure of 16S ribosomal RNA. Many more helices are conserved among the ribosomal sequences than are found in common among similar random sequences. In addition, conserved helices in the 16S rRNAs are, on the average, longer than the complementary segments that are found in comparable random sequences. The significance of these results and their application in the analysis of long non-ribosomal nucleotide sequences is discussed.

Base Composition

Assignment of HLA-DPB alleles by computerized matching based upon sequence data.

Routine HLA typing by serology has been supported by DNA or protein analysis of the respective molecules in cases when serologic typing was inconclusive or difficult to perform. DNA analysis by RFLP, SSO, or PCR amplification with SSP is a reliable tool for the identification of alleles. Because DNA sequences may now be determined routinely, we developed software based on sequence data from known sequences for allele assignment of polymorphic gene systems. We describe the assignment of HLA-DPB alleles based upon sequence data obtained after PCR amplification and subsequent sequencing of exon 2. Our software includes a database containing all known HLA-DPB sequences, which offers the possibility of analyzing heterozygous individuals by combinatorial comparison through all alleles and thus identifying the one or two alleles involved. Quality control of the sequence has been included by the alignment of constant regions of the sequence combined with related polymorphism of known polymorphic nucleotides and the identification of the positions crucial for the allele assignment. The ability to type for HLA-DPB alleles routinely by automatic sequence determination and subsequent automated analysis offers new perspectives for HLA-DNA typing.

Alleles

Development of conditioning programs for dressage horses based on time-motion analysis of competitions.

The time-motion characteristics of Canadian basic- and medium-level dressage competitions are described, and the results are applied in formulating sport-specific conditioning programs. One competition was analyzed at the six levels from basic 1 to medium 3. Each test was divided into a series of sequences based on the type and speed of activity. The durations of the sequences were measured from videotapes. The basic-level tests had fewer sequences, and they were shorter in distance and duration than the medium tests (P < 0.10), but the average speed did not differ between the two levels. It is recommended that horses competing at the basic levels be conditioned using 5-min exercise periods, with short (10-s) bursts of lengthened trot and canter included at basic 2 and above. In preparation for medium-level competitions, the duration of the work periods increases to 7 min, 10- to 12-s bursts of medium or extended trot and canter are included, and transitions are performed frequently to simulate the energy expenditure in overcoming inertia.

Animals

Molecular cloning and sequence analysis of the penton base genes of type II avian adenoviruses.

We describe here the identification of the penton base gene of hemorrhagic enteritis virus (HEV), a type II avian adenovirus, in a 2477-base pair (bp)-EcoRI fragment of the viral DNA by sequence analysis. Identification is based on an extensive amino acid homology between the HEV-open reading frame and the penton base of a fowl adenovirus (FAV-10) and various human adenoviruses. The 1344 bp-penton base gene of HEV encodes a 448-amino acid polypeptide of molecular weight of 50,843 Da. The nucleotide sequences of penton base genes of HEV and marble spleen disease virus (MSDV) are identical. The HEV penton base lacks the RGD motif, present in most human adenoviruses (Ad2, Ad3, Ad4, and Ad 12) suggesting that HEV may not use alpha v integrins to gain entry into host cells. Further sequence analysis revealed the presence of a Leu-Asp-Val (LDV) motif in the HEV penton base amino acid sequence similar to most of the human adenoviruses. LDV motif on the fibronectin has been shown to interact with the alpha 4 beta 1 integrins on cells, which includes lymphocytes and monocytes. The presence of LDV motif in the penton base of HEV implicates the involvement of alpha 4 beta 1 integrins in the viral internalization into host cells.

Amino Acid Sequence

IS6770, an enterococcal insertion-like sequence useful for determining the clonal relationship of clinical enterococcal isolates.

Enterococci expressing resistance to antimicrobial agents are increasingly important nosocomial pathogens. Effective strategies to prevent or abort outbreaks of resistant enterococcal infection will rely on an accurate understanding of the mechanisms by which these organisms spread. A 1065-bp insertion-like sequence (IS6770) is present in varying copy numbers in > 90% of enterococcal strains thus far examined. Hybridization patterns resulting from hybridization of enterococcal genomic DNA with an internal IS6770 probe vary considerably between unrelated strains and correlate well with results of pulsed-field gel electrophoresis and field-inversion gel electrophoresis in identifying clonal relationships among enterococcal isolates. IS6770 analysis of several outbreaks of resistant enterococci has confirmed the spread of single resistant clones rather than the emergence of resistance within the resident flora. These results suggest that IS6770 hybridization will be a useful tool for tracing the epidemiology of nosocomial enterococcal infections.

Amino Acid Sequence

Typing of Listeria spp. by random amplified polymorphic DNA (RAPD) analysis.

Random amplified polymorphic DNA (RAPD) analysis, a variation of the polymerase chain reaction (PCR) in which a single primer is used, was evaluated for use as a simple and reliable method with which to type Listeria spp. Representatives of six species of Listeria were studied. Five isolates of L. innocua and four isolates of L. seeligeri were all distinguishable from one another, but the four isolates of L. ivanovii tested, although distinguishable from other Listeria spp., were not differentiated. Among L. monocytogenes serovars 1/2a (eight isolates), 1/2b (eight isolates) and 4b (10 isolates), at least six, three and six RAPD patterns were observed, respectively. Fourteen neonatal cross-infection sets of L. monocytogenes isolates, shown to be indistinguishable by serotyping and phage typing, were examined with three different primers. With one primer, three of the sets were shown to consist of closely related, but distinguishable, strains. In the other 11 cases, each set of strains was indistinguishable with all three primers. These preliminary data indicate that RAPD analysis has promise as a method for typing Listeria spp.

Amino Acid Sequence

Ureaplasma urealyticum urease genes; use of a UGA tryptophan codon.

Nucleotide sequence analysis of a Ureaplasma urealyticum DNA fragment, homologous to cloned urease genes of other prokaryotes, revealed three consecutive open reading frames. The molecular weights of the three deduced polypeptides are 11.2 kD, 13.6 kD and 66.6 kD. These values are consistent with the size of the three subunits previously reported for purified native urease. A significant sequence homology was found between the three polypeptides of the ureaplasmal urease and the single polypeptide of jack bean (Canavalia ensiformis) urease. Codon usage indicates that UGA is a tryptophan codon in this mollicute. Use of polymerase chain reactions has disclosed the existence of genetic polymorphism among the urease genes of different serotypes of U. urealyticum.

Amino Acid Sequence

Molecular detection of streptomycin-producing streptomycetes in Brazilian soils.

Actinomycetes were isolated from soybean rhizosphere soil collected as two field sites in Brazil. All the isolates were identified as Streptomyces species and were screened for streptomycin production and the presence of two genes, strA and strB1, known to be involved in streptomycin biosynthesis in Streptomyces griseus. Antibiotic resistance profiles were determined for 53 isolates from cultivated and uncultivated sites, and approximately half the strains were streptomycin resistance. Clustering by the unweighted pair group method with averages indicated the presence of two major clusters, with the majority of resistant strains from cultivated sites being placed in cluster 1. Only representatives from this cluster contained strA. Streptomycetes containing strA and strB1 were phenotypically diverse, and only half could be assigned to known species. Sequence comparison of 16S rRNA and trpBA (tryptophan synthetase) genes revealed that streptomycin- producing streptomycetes were phylogenetically diverse. It appeared that a population of streptomycetes had colonized the rhizosphere and that a proportion of these were capable of streptomycin production.

Amino Acid Sequence

DNA fingerprinting of Vibrio cholerae strains with a novel insertion sequence element: a tool to identify epidemic strains.

A novel Vibrio cholerae insertion sequence element, designated IS1004, was characterized and used for DNA fingerprinting of Vibrio spp. IS1004 comprises 628 bp and contains an open reading frame whose product shows a large degree of sequence identity with the IS200-encoded transposase. IS1004 was present in one to eight copies in most of the V. cholerae strains analyzed. The IS1004-generated fingerprints of epidemic V. cholerae strains with serotype O1 were closely related, although it was possible to distinguish between the two biotypes, classical and El Tor. Non-O1 serotype strains generally showed heterogeneous patterns unrelated to those of the epidemic O1 strains. Several strains were observed with identical or related fingerprint patterns but expressed different serotypes. Conversely, strains with different fingerprint patterns but identical serotypes were also found. These observations indicate that the gene clusters coding for distinct O antigens may be transferred horizontally between V. cholerae strains. Two examples of non-O1 strains with a fingerprint resembling that of epidemic O1 strains were found; they were the O139 Bengal strain and an O37 strain. The O139 Bengal strain is closely related to the El Tor biotype. The O37 strain was responsible for a large cholera outbreak in Sudan in 1968 and was classified as a noncholera vibrio. Our study, however, shows that the O37 Sudan strain is genetically closely related to classical O1 strains. Similar to O139 Bengal, O37 Sudan lacked most of the O1 antigen cluster but did contain flanking genes. Thus, O37 Sudan represents a second example of an epidemic V. cholerae strain carrying non-O1 antigens. This study underlines the importance of genotypic methods for the differentiation of V. cholerae strains and for recognition of strains with epidemic potential.

Amino Acid Sequence