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PubMed1995

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↗
PubMed2026

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↗
PubMed1995

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↗
PubMed1994

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↗
PubMed1991

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↗
PubMed1990

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↗
PubMed1987

A human purine nucleoside phosphorylase deficiency caused by a single base change.

Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with a severe defect in T-lymphocyte function. The mutant gene was cloned from one PNP-deficient patient who was the offspring of a consanguineous mating. The exons and intron/exon boundaries of the mutant PNP gene were sequenced and compared with the wild-type cDNA sequence. A single base difference was found in the coding region of the mutant gene, a G to A transition in the third exon. This single base mutation alters the codon at position 89 from Glu to Lys, a result which is consistent with previously published peptide mapping data. The patient was demonstrated to be autozygous for the single base mutation on the basis of hybridization of synthetic oligomers to genomic DNA digests. A mammalian expression vector was constructed containing the entire mutant gene under the transcriptional regulation of its own promoter. In another construction, the single base mutation was reverted to the wild-type sequence by in vitro mutagenesis. An isoelectric focusing gel containing extracts of the cells transfected with the mutant and reverted PNP gene was stained histochemically for PNP activity. The proteins from a similar gel were blotted on a nitrocellulose membrane, and immunoreactive human PNP protein was visualized. Cells transfected with the mutant gene contained no human PNP activity, but expressed immunoreactive PNP which focused at an abnormally alkaline pI. Cells transfected with the reverted gene expressed human PNP activity which co-focused with human PNP from a HeLa cell control, proving that the observed single base change was responsible for the loss of catalytic function.

Amino Acid Sequence↗