Isolation and preliminary characterization of the human and mouse homologues of the bacterial cell cycle gene era.

Era is an essential GTPase that is required for proper cell cycle progression and cell division in Escherichia coli and is found in nearly all bacteria sequenced to date. To determine whether Era is also present in eukaryotic organisms, we searched the dbEST database and found EST clones coding for proteins that were similar to Era. Full sequencing of these ESTs from human and mouse identified a conserved homologue, ERAL1 (Era-like 1). ERAL1 maps to 17q11.2 in human and is located in the syntenic region of mouse chromosome 11. ERAL1 may be an attractive candidate for a tumor suppressor gene since ERAL1 is located in a chromosomal region where loss of heterozygosity is often associated with various types of cancer.

DNA rearrangements on both homologues of chromosome 17 in a mildly delayed individual with a family history of autosomal dominant carpal tunnel syndrome.

Disorders known to be caused by molecular and cytogenetic abnormalities of the proximal short arm of chromosome 17 include Charcot-Marie-Tooth disease type 1A (CMT1A), hereditary neuropathy with liability to pressure palsies (HNPP), Smith-Magenis syndrome (SMS), and mental retardation and congenital anomalies associated with partial duplication of 17p. We identified a patient with multifocal mononeuropathies and mild distal neuropathy, growth hormone deficiency, and mild mental retardation who was found to have a duplication of the SMS region of 17p11.2 and a deletion of the peripheral myelin protein 22 (PMP22) gene within 17p12 on the homologous chromosome. Further molecular analyses reveal that the dup(17)(p11.2p11.2) is a de novo event but that the PMP22 deletion is familial. The family members with deletions of PMP22 have abnormalities indicative of carpal tunnel syndrome, documented by electrophysiological studies prior to molecular analysis. The chromosomal duplication was shown by interphase FISH analysis to be a tandem duplication. These data indicate that familial entrapment neuropathies, such as carpal tunnel syndrome and focal ulnar neuropathy syndrome, can occur because of deletions of the PMP22 gene. The co-occurrence of the 17p11.2 duplication and the PMP22 deletion in this patient likely reflects the relatively high frequency at which these abnormalities arise and the underlying molecular characteristics of the genome in this region.

SERE, a widely dispersed bacterial repetitive DNA element.

The presence of a Salmonella serotype Enteritidis repeat element (SERE) located within the upstream regulatory region of the sefABCD operon encoding fimbrial proteins is reported. DNA dot-blot hybridisation analyses and computerised searches of genetic databases indicate that SERE is well conserved and widely distributed throughout the bacterial and archaeal kingdoms. A SERE-based polymerase chain reaction (SERE-PCR) assay was developed to fingerprint 54 isolates of Enteritidis representing nine distinct phage types and 54 isolates of other Salmonella serotypes. SERE-PCR identified five distinct fingerprint profiles among the 54 Enteritidis isolates; no correlation between phage types and SERE-PCR fingerprint patterns was noticed. SERE-PCR was reproducible, rapid and easy to perform. The results of this investigation suggest that the limited heterogeneity of SERE-PCR fingerprint patterns can be utilised to develop serotype- and serogroup-specific fingerprint patterns for isolates of Enteritidis.

Homologous recombination of a flanking repeat gene cluster is a mechanism for a common contiguous gene deletion syndrome.

Smith-Magenis syndrome (SMS), caused by del(17)p11.2, represents one of the most frequently observed human microdeletion syndromes. We have identified three copies of a low-copy-number repeat (SMS-REPs) located within and flanking the SMS common deletion region and show that SMS-REP represents a repeated gene cluster. We have isolated a corresponding cDNA clone that identifies a novel junction fragment from 29 unrelated SMS patients and a different-sized junction fragment from a patient with dup(17)p11.2. Our results suggest that homologous recombination of a flanking repeat gene cluster is a mechanism for this common microdeletion syndrome.

The human COX10 gene is disrupted during homologous recombination between the 24 kb proximal and distal CMT1A-REPs.

The CMT1A-REPs are two large directly repeating DNA sequences located on chromosome 17p11.2-p12 flanking the region duplicated in patients with Charcot-Marie-Tooth disease type 1A (CMT1A) and deleted in patients with hereditary neuropathy with liability to pressure palsies (HNPP). We have sequenced two cosmids, c74F4 and c15H12, which contain the entire proximal and distal CMT1A-REPs and determined that these repeats are approximately 99% identical across a 24,011 bp region. In addition, both contain an exon of the human heme A:farnesyltransferase gene (COX10). Hybridization studies revealed that COX10 spans the distal CMT1A-REP, while the proximal CMT1A-REP contains an isolated COX10 'pseudo-exon'. There is also a COX10 hybridization signal on chromosome 10 which appears to represent a processed pseudogene. We propose that the distal CMT1A-REP represents the progenitor copy of COX10 exon VI which was duplicated with surrounding intronic sequences during mammalian genome evolution and that the HNPP deletion results in a COX10 null allele.

Detection of the CMT1A/HNPP recombination hotspot in unrelated patients of European descent.

Charcot-Marie-Tooth type 1 disease (CMT1) and hereditary neuropathy with liability to pressure palsies (HNPP) are common inherited disorders of the peripheral nervous system. The majority of CMT1 patients have a 1.5Mb tandem duplication (CMT1A) in chromosome 17p11.2 while most HNPP patients have a deletion of the same 1.5 Mb region. The CMT1A duplication and HNPP deletion are the reciprocal products of an unequal crossing over event between misaligned flanking CMT1A-REP elements. We analysed 162 unrelated CMT1A duplication patients and HNPP deletion patients from 11 different countries for the presence of a recombination hotspot in the CMT1A-REP sequences. A hotspot for unequal crossing over between the misaligned flanking CMT1A-REP elements was observed through the detection of novel junction fragments in 76.9% of 130 unrelated CMT1A patients and in 71.9% of 32 unrelated HNPP patients. This recombination hotspot was also detected in eight out of 10 de novo CMT1A duplication and in two de novo HNPP deletion patients. These data indicate that the hotspot of unequal crossing over occurs in several populations independently of ethnic background and is directly involved in the pathogenesis of CMT1A and HNPP. We conclude that the detection of junction fragments from the CMT1A-REP element on Southern blot analysis is a simple and reliable DNA diagnostic tool for the identification of the CMT1A duplication and HNPP deletion in most patients.

Rapid determination of outbreak-related strains of Neisseria meningitidis by repetitive element-based polymerase chain reaction genotyping.

Outbreaks of invasive disease caused by Neisseria meningitidis have increased in the United States in the 1990s. Repetitive element-based polymerase chain reaction (rep-PCR), a recently developed genotyping method, was used to evaluate a group of 8 outbreak-related and 35 other meningococcal isolates previously typed by multilocus enzyme electrophoresis (MLEE). All were serogroup B or C. Sets of genotypes were generated using primers based on either of two different repetitive sequences. Genotype sets were analyzed in a blinded fashion. Each set correctly identified outbreak-related isolates. Among the other 35 isolates, rep-PCR delineated 14 and 13 strains, respectively, in the two sets of genotypes. Seventeen electrophoretic types had been delineated by MLEE. Rep-PCR holds promise as a rapid, genome-based typing method for delineation of apparent outbreaks of meningococcal disease.

A recombination hotspot responsible for two inherited peripheral neuropathies is located near a mariner transposon-like element.

The Charcot-Marie Tooth disease type 1A (CMT1A) duplication and hereditary neuropathy with liability to pressure palsies (HNPP) deletion are reciprocal products of an unequal crossing-over event between misaligned flanking CMT1A-REP repeats. The molecular aetiology of this apparently homologous recombination event was examined by sequencing the crossover region. Through the detection of novel junction fragments from the recombinant CMT1A-REPs in both CMT1A and HNPP patients, a 1.7-kb recombination hotspot within the approximately 30-kb CMT1A-REPs was identified. This hotspot is 98% identical between CMT1A-REPs indicating that sequence identity is not likely the sole factor involved in promoting crossover events. Sequence analysis revealed a mariner transposon-like element (MITE) near the hotspot which we hypothesize could mediate strand exchange events via cleavage by a transposase at or near the 3' end of the element.

Differential subsequence conservation of interspersed repetitive Streptococcus pneumoniae BOX elements in diverse bacteria.

Evolutionary conservation of an interspersed repetitive DNA sequence, BOX, from Streptococcus pneumoniae was investigated to explore the mosaic nature of these elements. BOX elements consist of various combinations of three subunits, boxA, boxB, and boxC. Eight oligonucleotide probes were designed based on consensus DNA sequences of boxA, boxB, and boxC subunits. DNA hybridization studies and PCR using these probes/primers demonstrate that oligonucleotide sequences within the boxA subunit appear to be conserved among diverse bacterial species. The boxB and boxC subunits show only limited, if any, sequence conservation in bacteria other than S. pneumoniae. Intact BOX elements with boxA, boxB, and boxC subunits were only present in high copy number in pneumococcal strains. This pattern of differential conservation lends support to the modular nature of BOX repetitive elements in that boxA-like subsequences are effectively independent of boxB-like or boxC-like subunits in bacteria other than S. pneumoniae. Furthermore, dendrograms derived from repetitive sequence-based PCR (rep-PCR) fingerprints of S. pneumoniae isolates using the BOXA1R primer yielded clustering patterns that were similar to those obtained previously by other methods, suggesting that these repetitive sequence-based DNA fingerprints represent intrinsic properties of an S. pneumoniae strain's genome. Our results indicate widespread conservation of boxA-like subsequences in the bacterial kingdom, lend support to the mosaic nature of BOX in S. pneumoniae, and demonstrate the utility of boxA-based primers for rep-PCR fingerprinting of many microorganisms.

Vertical transmission of Citrobacter diversus documented by DNA fingerprinting.

OBJECTIVE: To confirm the vertical transmission of Citrobacter diversus from a mother to her infant and to evaluate the epidemiologic usefulness of a new automated procedure for analysis of polymerase chain reaction (PCR)-generated DNA fingerprints. DESIGN: Repetitive element-based PCR (rep-PCR) analysis of C diversus isolates from the blood and amniotic fluid of a mother and the blood of her infant was performed. Unrelated C diversus isolates also were characterized and compared with the isolates from mother and infant. DNA fingerprints were generated by gel electrophoresis of PCR products derived from either unlabeled standard repetitive sequence-based oligonucleotide primers or fluorescent primers. The standard rep-PCR fingerprints were analyzed by visual inspection. The fluorescent primers were used in fluorophore-enhanced rep-PCR (FERP), and the FERP DNA fingerprints were analyzed by an Applied BioSystems (ABI) Model 373A laser scanning unit equipped with Genescan 672 software (Applied Biosystems, Inc, Foster City, CA). SETTING AND PATIENTS: A mother and her newborn infant, both with invasive disease due to C diversus, in an urban tertiary-care hospital. RESULTS: The DNA fingerprints of the maternal blood, amniotic fluid, and infant blood isolates of C diversus were identical by both visual inspection of ethidium bromide-stained agarose gels and computer-aided analysis of FERP patterns. These strains appeared to differ from all but one control isolate, which had been collected 7 years earlier in the same city in which the infant was born. CONCLUSIONS: Vertical transmission of C diversus from mother to infant can occur in utero. Automated analysis of rep-PCR-generated DNA fingerprints derived using fluorescent primers is an objective means for comparing isolates of C diversus and in all likelihood would be useful for other species of bacteria that possess repetitive elements.

Cluster analysis of Helicobacter pylori genomic DNA fingerprints suggests gastroduodenal disease-specific associations.

BACKGROUND: Helicobacter pylori infection is now accepted as the most common cause of chronic active gastritis and peptic ulcer disease. The etiologies of many infectious diseases have been attributed to specific or clonal strains of bacterial pathogens. Polymerase chain reaction (PCR) amplification of DNA between repetitive DNA sequences, REP elements (REP-PCR), has been utilized to generate DNA fingerprints to examine similarity among strains within a bacterial species. METHODS: Genomic DNA from H. pylori isolates obtained from 70 individuals (39 duodenal ulcers and 31 simple gastritis) was PCR-amplified using consensus probes to repetitive DNA elements. The H. pylori DNA fingerprints were analyzed for similarity and correlated with disease presentation using the NTSYS-pc computer program. RESULTS: Each H. pylori strain had a distinct DNA fingerprint except for two pairs. Single-colony DNA fingerprints of H. pylori from the same patient were identical, suggesting that each patient harbors a single strain. Computer-assisted cluster analysis of the REP-PCR DNA fingerprints showed two large clusters of isolates, one associated with simple gastritis and the other with duodenal ulcer disease. CONCLUSIONS: Cluster analysis of REP-PCR DNA fingerprints of H. pylori strains suggests that duodenal ulcer isolates, as a group, are more similar to one another and different from gastritis isolates. These results suggest that disease-specific strains may exist.

DNA fingerprinting of pathogenic bacteria by fluorophore-enhanced repetitive sequence-based polymerase chain reaction.

Fluorophore-labeled oligonucleotide primers complementary to defined interspersed repetitive sequences conserved in diverse bacteria were used in the polymerase chain reaction to generate DNA fingerprint patterns from selected pathogenic bacteria. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction allowed discrimination between unrelated isolates of penicillin-resistant Streptococcus pneumoniae recovered from pediatric patients and Mycobacterium avium cultured from patients with acquired immunodeficiency syndrome. Combinations of oligonucleotide primers labeled with distinct fluorescent dyes enabled simultaneous DNA fingerprinting and Shiga-like toxin gene detection in enterohemorrhagic Escherichia coli isolates. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction was performed with either purified DNA or intact cells that were lysed during the polymerase chain reaction. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction successfully combines polymerase chain reaction amplification and fluorescent label detection for DNA fingerprinting of cultured bacterial pathogens.

Molecular epidemiology of infections due to Enterobacter aerogenes: identification of hospital outbreak-associated strains by molecular techniques.

Molecular techniques were used to study the epidemiology of infections due to Enterobacter aerogenes in a tertiary-care hospital. Sixty-two clinical isolates were collected from 43 patients over 3 months. Restriction endonuclease analysis (REA) of chromosomal DNA and repetitive-element polymerase chain reaction (rep-PCR) with primers based on repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC) bacterial DNA sequences were used for genomic fingerprinting. REA with HindIII or EcoRI yielded complex banding patterns that differentiated community-acquired from some hospital-acquired organisms. Less complex fingerprints were obtained with rep-PCR, which also distinguished between epidemiologically unrelated strains. More discriminatory DNA fingerprints were provided by rep-PCR when REP primers rather than ERIC primers were used. Two clusters of genomically distinct isolates from patients with recent or current exposure to the hospital environment were identified by REA and rep-PCR. Most isolates within each cluster contained characteristic plasmids, and some isolates contained additional plasmids. These results suggest colonization and infection with genotypically related strains of E. aerogenes in a nosocomial setting. Although REA and plasmid profiling are useful techniques for the epidemiological typing of E. aerogenes, genomic fingerprinting with rep-PCR may offer the advantages of ease, speed, and broad species applicability over existing molecular-typing techniques.

Whole-cell repetitive element sequence-based polymerase chain reaction allows rapid assessment of clonal relationships of bacterial isolates.

Repetitive element sequence-based polymerase chain reaction (rep-PCR) enables the generation of DNA fingerprints which discriminate bacterial species and strains. We describe the application of whole-cell methods which allow specimens from broth cultures or colonies from agar plates to be utilized directly in the PCR mixture. The rep-PCR-generated DNA fingerprints obtained with whole-cell samples match results obtained with genomic DNA templates. Examples with different gram-negative bacteria (e.g., Citrobacter diversus, Escherichia coli, and Pseudomonas aeruginosa) and gram-positive bacteria (e.g., Staphylococcus aureus and Streptococcus pneumoniae) are demonstrated. Rapid specimen preparation methods enable rep-PCR-based fingerprinting to be completed in several hours and, therefore, allows the timely analysis of epidemiological relationships.

Penicillin-resistant Streptococcus pneumoniae strains recovered in Houston: identification and molecular characterization of multiple clones.

A sample of 48 penicillin-resistant Streptococcus pneumoniae (PRSP) strains recovered between January 1989 and May 1991, primarily from infected children in Houston, was characterized by multilocus enzyme electrophoresis and repetitive extragenic palindromic-polymerase chain reaction genomic profiling. A heterogeneous array of 22 clonal genotypes was identified, but 64% of the PRSP strains in the sample were assigned to five clones that are closely similar in overall chromosomal character and express serotype 6 capsule. A close genetic association between these five clones and penicillin-resistant 6B clones recovered in Alaska, Iceland, and Spain was identified by multilocus enzyme electrophoresis. Taken together, the results suggest either that the common resistant 6B clones in Alaska, Iceland, Spain, and Houston have a recent ancestor or that isolates of a certain pneumococcal phylogenetic lineage are more likely to develop penicillin resistance.

Conservation and evolution of the rpsU-dnaG-rpoD macromolecular synthesis operon in bacteria.

The macromolecular synthesis (MMS) operon contains three essential genes (rpsU, dnaG, rpoD) whose products (S21, primase, sigma-70) are necessary for the initiation of protein, DNA, and RNA synthesis respectively. PCR amplifications with primers complementary to conserved regions within these three genes, and subsequent DNA sequencing of rpsU-dnaG PCR products, demonstrate that the three genes appear to be contiguous in 11 different Gram-negative species. Within the Gram-negative enteric bacterial lineage, the S21 amino acid sequence is absolutely conserved in 10 species examined. The putative nuteq antiterminator sequence in rpsU consists of two motifs, boxA and boxB, conserved in primary sequence and secondary structure. The terminator sequence, T1, located between rpsU and dnaG is conserved at 31 positions in nine enterobacterial species, suggesting the importance of primary sequence in addition to secondary structure for transcription termination. The intergenic region between rpsU and dnaG varies in size owing to the presence or absence of the Enterobacterial Repetitive Intergenic Consensus (ERIC) DNA element. The rpoD gene contains rearrangements involving a divergent sequence, although two carboxy-terminal regions which encode functional domains are conserved in primary sequence and spacing. Our data suggest that primary sequence divergence and DNA rearrangements in both coding and non-coding sequences account for the interspecies variation in operon structure. However, MMS operon gene organization and cis-acting regulatory sequences appear to be conserved in diverse bacteria.

Analysis of relationships among isolates of Citrobacter diversus by using DNA fingerprints generated by repetitive sequence-based primers in the polymerase chain reaction.

Oligonucleotide probes which match consensus sequences of the repetitive extragenic palindromic (REP) element hybridize to genomic DNA of diverse bacterial species. Primers based on the REP sequence generate complex band patterns with genomic DNA in the polymerase chain reaction (PCR), a technique named REP-PCR. We used REP-PCR with genomic DNA to fingerprint 47 isolates of Citrobacter diversus. Previously, 37 were assigned electrophoretic types (ETs) by multilocus enzyme electrophoresis and 35 were evaluated by using outer membrane protein profiles. Fingerprints were compared by visual inspection and by similarity coefficients (SimCs) based on the number of common bands versus total bands between two given isolates. DNA fingerprints were highly similar visually for patient pairs and outbreak-related sets. SimCs for these were > or = 0.952. Fingerprints of isolates with different ETs generally were distinctive. Among 21 unrelated isolates representing 15 ETs, only 6 of 210 comparisons had SimCs of > or = 0.952. REP-PCR rapidly generated DNA fingerprints which were highly similar for epidemiologically linked isolates of C. diversus and distinct for previously characterized strains within this species. The ability of this method to discriminate between C. diversus isolates with the same biotype was similar to that of multilocus enzyme electrophoresis and outer membrane protein profiles. REP-PCR may be useful in evaluation of apparent outbreaks of this or other bacterial species which possess these extragenic, repetitive elements.

Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes.

Dispersed repetitive DNA sequences have been described recently in eubacteria. To assess the distribution and evolutionary conservation of two distinct prokaryotic repetitive elements, consensus oligonucleotides were used in polymerase chain reaction [PCR] amplification and slot blot hybridization experiments with genomic DNA from diverse eubacterial species. Oligonucleotides matching Repetitive Extragenic Palindromic [REP] elements and Enterobacterial Repetitive Intergenic Consensus [ERIC] sequences were synthesized and tested as opposing PCR primers in the amplification of eubacterial genomic DNA. REP and ERIC consensus oligonucleotides produced clearly resolvable bands by agarose gel electrophoresis following PCR amplification. These band patterns provided unambiguous DNA fingerprints of different eubacterial species and strains. Both REP and ERIC probes hybridized preferentially to genomic DNA from Gram-negative enteric bacteria and related species. Widespread distribution of these repetitive DNA elements in the genomes of various microorganisms should enable rapid identification of bacterial species and strains, and be useful for the analysis of prokaryotic genomes.