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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↗

PubMed1998

Molecular typing of Staphylococcus aureus based on PCR restriction fragment length polymorphism and DNA sequence analysis of the coagulase gene.

A typing procedure for Staphylococcus aureus was developed based on improved PCR amplification of the coagulase gene and restriction fragment length polymorphism (RFLP) analysis of the product. All coagulase-positive staphylococci produced a single PCR amplification product of either 875, 660, 603, or 547 bp. Those strains of epidemic methicillin-resistant S. aureus 16 (EMRSA-16) studied all gave a product of 547 bp. PCR products were digested with AluI and CfoI, and the fragments were separated by gel electrophoresis. Ten distinct RFLP patterns were found among 85 isolates of methicillin-resistant S. aureus (MRSA) and 10 propagating strains (PS) of methicillin-sensitive S. aureus (MSSA) examined. RFLP patterns 1, 2, and 3 were specific to strains of EMRSA-3, -15, and -16, respectively. By contrast, RFLP patterns 4 and 5 were seen with a heterogeneous collection of strains, together with drug-resistant forms of S. aureus isolated in Europe and four propagating strains used for the international phage set. RFLP pattern 6 was given by the Airedale isolate and PS 95. RFLP pattern 7 encompassed EMRSA-2 (isolate 331), PS 94, and PS 96. An isolate from Germany gave RFLP pattern 8. Eight strains of MSSA gave patterns similar to those of methicillin-resistant strains (RFLP patterns 3, 4, 5, 6, and 7), but two, PS 42E and PS 71, gave unique RFLP patterns 9 and 10, respectively. The coagulase gene PCR products for 24 isolates of MRSA and two isolates of MSSA were sequenced for both strands. The sequences were aligned, and evolutionary lineages were inferred based on pairwise distances between isolates.

Bacterial Typing Techniques↗

PubMed1993

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↗

PubMed1998

Suitability of repetitive-DNA-sequence-based PCR fingerprinting for characterizing epidemic isolates of Salmonella enterica serovar Saintpaul.

Three molecular typing methods, repetitive-sequence-based PCR (rep-PCR) fingerprinting, plasmid profiling, and arbitrarily primed PCR fingerprinting, were used to characterize isolates of Salmonella enterica serovar Saintpaul. Most of the isolates were obtained from epidemic human cases of food-borne salmonellosis, together with some from the food material suspected to be the source of infection, and a few were obtained from other cases apparently not related to the epidemic. All three methods adequately discriminated the epidemic strain from other strains of the serovar. In addition several isolates from human cases which are not identical to the epidemic strain were found. These isolates therefore must have been responsible for some sporadic infections, which were only temporally related to the epidemic. These strains showed a high degree of similarity to a strain isolated from a turkey. rep-PCR fingerprinting with REP-Dt primers and primer ERIC1R, applicable even to crude cell lysates, offers an attractive choice as a primary method for the discrimination of various Salmonella serotypes as well as isolates within serotype Saintpaul.

Bacterial Typing Techniques↗

PubMed1998

Prognostic value of chorionic gonadotropin beta gene transcripts in human breast carcinoma.

The beta subunit of human chorionic gonadotropin is potentially encoded by six genes, which can be categorized into two types based on a sequence change at codon 117: GCC for the type I and GAC for the type II genes. We previously showed that, whereas type I genes were exclusively expressed in normal breast tissues, expression of type II genes was associated with malignant transformation (Bellet, D., et al. Cancer Res., 57: 516-523, 1997). We designed a simple and robust test (the CG117 assay) that measures the percentage of type II over both types of chorionic gonadotropin beta mRNAs. Normal breast tissues consistently had a negative CG117 index, whereas cancer breast tissues showed indexes ranging from 0 to 100%. The prognostic significance of the CG117 index was investigated in a series of 99 unilateral invasive primary breast cancer patients with known long-term outcome (median follow-up, 9 years). The CG117 index was positive in 48 (48.5%) of the 99 tumor mRNA samples. The index was not significantly associated with standard prognostic parameters, including clinical and macroscopic tumor size, histopathological grade, and lymph node status or steroid receptor status. Patients with a positive CG117 index in primary tumor mRNA had significantly shorter metastasis-free survival (P = 0.014) and overall survival (P = 0.038) after surgery, compared to patients with a negative index. The prognostic significance of the CG117 index persisted in Cox multivariate regression analysis, both for metastasis-free survival (P = 0.008) and overall survival (P = 0.016), together with lymph node status (P = 0.027 and P = 0.009, respectively). These findings indicate that the CG117 index may contribute to the identification of high-risk breast cancer patients.

Adult↗

PubMed1998

Rapid method for successful HLA class I and II typing from cadaveric blood for direct matching in cornea transplantation.

BACKGROUND: The aim of the study was to establish fast methods for postmortem HLA class I and II typing of cornea donors using cadaveric blood. METHODS: The commercially available reagents Lymphokwik MN and Dynabeads were evaluated here to provide an enriched living mononuclear cell (MNC) population and B-cell population for HLA class I and II typing of cadaveric blood by serology. Cadaveric blood was obtained 1-80 h post mortem. After isolation of living B-cells and B-cell-depleted living MNC's, cells were serologically typed by double-fluorescence cytotoxicity assay for HLA class I and II antigens. RESULTS: In 373 (81%) of 461 cadaveric blood samples HLA class I typing, and in 36 (62%) of 56 cadaveric blood samples HLA-class II typing, by serology was successful and accomplished within 5 h. Results from the serological HLA class I typing were confirmed by the results of HLA class I typing by RNA-based sequencing in seven cases. To improve the HLA class II typing, DNA typing using PCR with sequence-specific primers was performed in 148 samples and reverse hybridization of PCR-amplified DNA to immobilized HLA class II specific primers in 270 samples. These data were confirmed by DNA-based sequencing in five cases and by sequence-specific oligonucleotide hybridization in all cases. CONCLUSIONS: These results lead to the following typing strategy: HLA class I typing should be performed by serology. HLA class II typing should be performed by DNA technology because of its relative independence of the quality of the blood sample. The strategy we have developed is very successful and fast for tissue typing post mortem, thus expanding the time available for ideal HLA matching, increasing the number of available HLA-matched corneas and therefore reducing the number of graft rejections.

Blood Grouping and Crossmatching↗

PubMed1993

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↗

PubMed1995

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↗

PubMed1994

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↗

PubMed1993

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↗

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↗

PubMed1997

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↗

PubMed1996

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↗

PubMed1994

Structure of a covalent DNA minor groove adduct with a pyrrolobenzodiazepine dimer: evidence for sequence-specific interstrand cross-linking.

The structure of the interstrand cross-linked adduct formed between a C8-C8'-linked pyrrolobenzodiazepine (PBD) dimer (DSB-120; 1,1'-(propane-1,3-diyldioxy)bis[(11aS)-7-methoxy-1,2,3,11a-t etrahydro-5H- pyrrolo[2,1-c][1,4]benzodiazepin-5-one]) and a self-complementary d(CICGATCICG)2 duplex has been determined from high-field 1D- and 2D-NMR data using a simulated annealing procedure. The refined structure supports earlier observations from solution NMR experiments and indicates that the covalently bound molecule spans six DNA base pairs in the minor groove, forming a symmetric cross-link between the spatially separated internal guanines and with active recognition of an embedded 5'-GATC bonding site. This result confirms that template-directed approaches are useful for the design of linked DNA-interactive PBD dimers with viable DNA cross-linking potential. Further, head-to-head connection of the PBD moieties results in an overall retention of 5'-GA bonding site preference for each alkylating PBD subunit. Structural analysis indicates that cross-link formation results in a localized perturbation of the DNA duplex, attributable in part to a mutual reduction in dynamic mobility or "covalent clamping" within the Gua4-Cyt7 base tract. However, ligand-induced distortion is confined to the Cyt7 and Ino8 residues on each strand. The Gua(N2)-Gua(N2) cross-link is stabilized by two directed H-bonds from the formed animal residues to N3 acceptor atoms of adenine bases on the 3'-side of each covalently modified guanine. Evidence for sequence-specific cross-linking with DSB-120 is provided by extended modeling studies which suggest that recognition of the favored d(.GATC.) motif is dominated by van der Waals steric factors, although electrostatic and H-bonded interaction terms also play a key role. This conclusion supports recent covalent footprinting studies revealing that this PBD dimer shows a selectivity for embedded base sequences of the type 5'-(pu/py)GATC(py/pu).

Base 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↗

PubMed1975

Detection of virus-specific DNA and RNA base-sequences in individual cells transformed or infected by adenovirus type 2.

By means of 3H thymidine-labelled adenovirus 2 DNA, adenovirus-specific DNA sequences have been localized in individual nuclei of adenovirus 2- or 12-infected cells, and adenovirus-specific RNA sequences have been detected in the permissive cells as well as in cells transformed by adenovirus type 2. The ability to detect such specific virus RNA base-sequences in particular, by in situ hybridization, should be useful in studying the transcriptional specificities of tumours and neoplasms.

Adenoviridae↗

PubMed1990

Genetic analysis of bacteriophage lambda integrase interactions with arm-type attachment site sequences.

The bacteriophage P22-based challenge phage system was used to study lambda integrase (Int) protein binding to its arm-type recognition sequences in the bacteriophage lambda attachment site. Challenge phages were constructed that carried inserts containing either the contiguous P'123 arm-type sites or the single P'1 site within the P22 phage promoter, Pant, which is required for expression of antirepressor. If Int protein binds to these sequences in vivo, it represses transcription from Pant. We found that Int repressed Pant in phages carrying the P'123 sites more efficiently than those carrying only the P'1 site, suggesting that the protein binds cooperatively at the three adjacent sites. The Int protein from a related lambdoid phage, HK022, also repressed transcription by binding to the same arm-type sites. Mutations in the P'123 or P'1 sites that impair Int binding were isolated by selecting mutant phages that express antirepressor in the presence of Int. DNA sequence analyses showed that most of the mutants in the challenge phages carrying the P'123 sites contained multiple changes and that two mutants contained only single-base-pair changes at positions that are completely conserved among all arm-type sites. Thirty-five mutants were isolated and analyzed from phages containing only the P'1 site. Most mutants contained single-nucleotide changes, and mutations were isolated at 8 of the 10 positions of the site, suggesting that most if not all base pairs in the conserved recognition sequence are involved in Int binding.

Bacteriophage lambda↗

PubMed1996

Use of cleaved amplified polymorphic sequences to distinguish strains of Staphylococcus epidermidis.

We examined the utility of a PCR-based method termed cleaved amplified polymorphic sequences (CAPS) to type 35 well-characterized isolates of Staphylococcus epidermidis. The results were compared with detailed epidemiologic information and typing obtained by using pulsed-field gel electrophoresis (PFGE). To identify CAPS markers for this study, eight pairs of oligonucleotide primers corresponding to five previously sequenced S. epidermidis genes were synthesized and then used to amplify DNA sequences from the S. epidermidis strains by using PCR. Amplified products were reproducibly obtained for seven of eight primer pairs from chromosomal DNA of 33 of the 35 isolates. Seven restriction site polymorphisms were found in five of the amplified products when they were subjected to digestion with a panel of restriction endonucleases. Each fragment-enzyme combination that was polymorphic demonstrated only two alleles in the 33 S. epidermidis isolates analyzed, corresponding to the presence or absence of a single restriction site. Overall, five distinct combinations of alleles were detected and were designated CAPS types A through E. There was a close correlation between the CAPS grouping, the epidemiologic information for the strains, and grouping by PFGE following SmaI digestion of chromosomal DNA. Although PFGE analysis was more discriminatory than typing based on the limited number of CAPS markers used in this study (isolates from the same CAPS group were sometimes distributed into more than one PFGE group), no isolates from the same PFGE group were found in more than one CAPS group. The CAPS procedure was highly reproducible, in contrast to published experience with arbitrarily primed PCR. These preliminary data suggest that CAPS represents a PCR-based technique for strain typing that is highly reproducible, rapid, utilizes widely available technologies, and provides results that are relatively easy to interpret and express.

Bacterial Typing Techniques↗