Evolution of the major pilus gene cluster of Haemophilus influenzae

Haemophilus influenzae is a ubiquitous colonizer of the human respiratory tract and causes diseases ranging from otitis media to meningitis. Many H. influenzae isolates express pili (fimbriae), which mediate adherence to epithelial cells and facilitate colonization. The pilus gene (hif) cluster of H. influenzae type b maps between purE and pepN and resembles a pathogenicity island: it is present in invasive strains, absent from the nonpathogenic Rd strain, and flanked by direct repeats of sequence at the insertion site. To investigate the evolution and role in pathogenesis of the hif cluster, we compared the purE-pepN regions of various H. influenzae laboratory strains and clinical isolates. Unlike Rd, most strains had an insert at this site, which usually was the only chromosomal locus of hif DNA. The inserts are diverse in length and organization: among 20 strains, nine different arrangements were found. Several nontypeable isolates lack hif genes but have two conserved open reading frames (hicA and hicB) upstream of purE; their inferred products are small proteins with no data bank homologs. Other isolates have hif genes but lack hic DNA or have combinations of hif and hic genes. By comparing these arrangements, we have reconstructed a hypothetical ancestral genotype, the extended hif cluster. The hif region of INT1, an invasive nontypeable isolate, resembles the hypothetical ancestor. We propose that a progenitor strain acquired the extended cluster by horizontal transfer and that other variants arose as deletions. The structure of the hif cluster may correlate with colonization site or pathogenicity.     

Generation of depth-perception information in stereoscopic nuclear magnetic resonance imaging by non-linear magnetic field gradients

This study was undertaken to characterize serial Haemophilus parainfluenzae strains from epidemiologically unrelated chronic obstructive pulmonary disease (COPD) patients and from healthy carriers. A comprehensive approach was used including different phenotypical and molecular typing methods: biotyping, antibiotyping, conventional ribotyping, pulsed field gel electrophoresis (PFGE) assay, and PCR-ribotyping. Conventional ribotyping and PFGE analysis were confirmed as excellent procedures to differentiate isolates of the same species and biotype. Conversely, in our study, PCR-ribotyping proved to be suitable for taxonomic purposes, unambiguously identifying H. parainfluenzae from H. influenzae but not discriminating strains at the intraspecific level for epidemiological typing. Phylogenetic analysis of restriction fragment length polymorphism (RFLP) data of sequences related to the rrn operon demonstrated that H. parainfluenzae strains associated to COPD are spread among many diverging lineages.     

Randomized study of n-of-1 trials versus standard practice

Fate of 32P-labelled pJ1-8N19 DNA was followed in the mutant strain N19 and wild type H. influenzae Rd, during post-uptake incubation. Integration of the insert fragment carrying novr gene into the host genome was measured at various time intervals during post-uptake incubation. Negligible amount of label transfer and no detectable transfer of biological activity (novr) was observed in mutant strain N19 compared to wild type strain Rd. These observations correlated poor extra chromosomal establishments of the donor plasmid in the mutant strain N19.     

Integration of 101 DNA markers across human Xp11 using a panel of somatic cell hybrids

One hundred and one DNA markers previously assigned to the short arm of the human X chromosome were localized on a hybrid mapping panel consisting of ten radiation-reduced, and four classical somatic cell hybrids. Of the 101 DNA markers, 16 are genes, two are pseudogenes, 13 are expressed sequence tags, 32 are simple tandem repeats (STRs), four are restriction fragment length polymorphisms, one is a variable number of tandem repeats, and 33 are sequence tagged sites (STSs). Three of these markers, two STSs and one STR, were generated from the products of an inter-Alu PCR library of a radiation-reduced hybrid containing Xp11.4-->p11.22 as its only human DNA content. A second STR was isolated from a region-specific cosmid containing the gene ZNF21. The 101 DNA markers fell into 22 bins based on their retention on the hybrids of this panel, which, in combination with YAC contig data, could be further resolved into 24 bins. This hybrid map of Xp11 has an average resolution of approximately 0.8 Mb.     

A new transformation-deficient mutant of Haemophilus influenzae Rd with normal DNA uptake

Haemophilus influenzae Rd is a gram-negative natural transformer. A mutant strain, RJ248, that has normal DNA uptake and translocation but whose transformation frequency is 300 times lower than that of wild-type H. influenzae and whose phage recombination is 8 times lower was isolated. The affected gene, comM, is induced during competence development in wild-type H. influenzae but not in RJ248.     

Flanking region sequences and internal repeat structure of the pYNH24 (D2S44) 2 kbp insert analyzed by polymerase chain reaction and partial digestion with RsaI

The 2 kbp YNH24 pUC18 insert was partially sequenced, amplified with fluorescence-labeled primers, and characterized by incomplete digestion with the restriction enzyme RsaI. A characteristic RsaI profile with one restriction site in each repeat unit was obtained when the fragments were analyzed on a DNA sequencer. We developed this procedure with the specific aim of analyzing the internal repeat structure in recombinant alleles often observed in the YNH24 variable number of tandem repeat system (D2S44). Nonetheless, it should also be possible to apply the method when analyzing other tandem repeats, even when little information regarding their sequences is available. In addition, when the repeat array is amplified, either a 5' or a 3' fluorescence-labeled primer can be used to enable analysis from both directions of fairly long (> 3.6 kbp) alleles. The RsaI fragments obtained by partial digestion of the amplified pYNH24 insert corresponded well to the RsaI sites found in the sequenced regions. The 5' flanking region contained eight 30-34 bp sequences similar to the repeat unit and also several RsaI sites, whereas only a few RsaI sites and no repeat units were found in the 3' flanking region. Twenty-five consecutive RsaI sites were revealed, in the center of the pYNH24 insert, indicating a region with strictly repeated core sequences; this repetitive block comprised less than half of the total insert. Two approximately 2.24 kbp HinfI alleles from two different heterozygous individuals were found to have identical internal structure, which was very similar to that of the insert although it exhibited only 15 RsaI repeats.     

Sexual differences in branched chain amino acid metabolism into fatty acids and cholesterol in Harderian gland of golden hamster

PURPOSE: To investigate the telomere hypothesis of cellular aging as the mechanism for cell cycle arrest in normal human corneal endothelium. METHODS: The corneal endothelium and epithelium from 21 human corneas from 13 donors 5 weeks to 84 years of age were dissected and frozen at -70 degrees C. Purified DNA, digested with the restriction enzyme, HinfI, was run on 0.7% agarose gels, probed with radiolabeled (AATCCC)4, and exposed to a phosphor screen. The length of the terminal restriction fragment (TRF) was determined by densitometry. RESULTS: The cells of the corneal endothelium had TRF lengths ranging from 11.0 to 14.0 kbp (mean, 12.2 +/- 0.9). Corneal epithelial specimens showed TRF lengths that were always less than (mean, 10.4 +/- 1.0; range 9.0-12.0) the corresponding endothelial TRF lengths. Human corneal endothelial cells, transformed with human papillomavirus type 16 oncogenes E6 and E7, showed decreasing TRF lengths from 11 kbp at population doubling level (PDL) 15 to 9.5 kbp at PDL 73. Neither the endothelial and epithelial cells from human donors nor the transformed pre-immortalized human endothelial cells showed evidence of telomerase activity. CONCLUSIONS: Human corneal endothelial cells have long telomeres throughout life. Their limited replicative ability does not appear to result from critically short telomere lengths.     

Sequence-specific DNA cleavage by Fe2+-mediated fenton reactions has possible biological implications

Preferential cleavage sites have been determined for Fe2+/H2O2-mediated oxidations of DNA. In 50 mM H2O2, preferential cleavages occurred at the nucleoside 5' to each of the dG moieties in the sequence RGGG, a sequence found in a majority of telomere repeats. Within a plasmid containing a (TTAGGG)81 human telomere insert, 7-fold more strand breakage occurred in the restriction fragment with the insert than in a similar-sized control fragment. This result implies that telomeric DNA could protect coding DNA from oxidative damage and might also link oxidative damage and iron load to telomere shortening and aging. In micromolar H2O2, preferential cleavage occurred at the thymidine within the sequence RTGR, a sequence frequently found to be required in promoters for normal responses of many procaryotic and eucaryotic genes to iron or oxygen stress. Computer modeling of the interaction of Fe2+ with RTGR in B-DNA suggests that due to steric hindrance with the thymine methyl, Fe2+ associates in a specific manner with the thymine flipped out from the base stack so as to allow an octahedrally-oriented coordination of the Fe2+ with the three purine N7 residues. Fe2+-dependent changes in NMR spectra of duplex oligonucleotides containing ATGA versus those containing AUGA or A5mCGA were consistent with this model.     

Sequence similarities between the genes encoding the S.NgoI and HaeII restriction/modification systems

The DNA sequence encoding the S.NgoI restriction/modification (R/M) system was identified from a gene bank made from Neisseria gonorrhoeae strain WR302 by identifying recombinant plasmids that induced the reporter system in a methylase detection strain AP1-200-9 (Piekarowicz et al., 1991) and were resistant to digestion with NgoI. The DNA sequence was determined from one of these (pUCP30). M.NgoI is a protein of 315 aa with a predicted MW of 35296 Da and R.NgoI is a protein of 350 aa with a predicted MW of 40650 Da. The termination codon of M.NgoI overlapped the start codon of R.NgoI. The same strategy was used to clone the R/M system encoding HaeII from Haemophilus aegyptius strain ATCC 11116. The DNA sequence from one clone representing this class (pAP704) was determined. HaeII methylase is a protein of 318 aa with a predicted MW of 35669 Da and R.HaeII contains 352 aa with a predicted MW of 40800 Da. aa alignments between the two methylases indicated that they were 74.3% identical and 79% similar. DNA sequence alignments revealed 68% identity. An aa alignment between the two restriction enzymes indicated that they were 60% identical and 68% similar. DNA sequence alignments revealed 61% identity. The DNA sequences flanking these two systems were identified and used to determine the genomic organization of the two systems. The S.NgoI genes were found between two genes, one with high homology to GTP binding proteins of unknown function and one with homology to genes involved in tRNA synthetase synthesis. The HaeII R/M genes were located between two genes, mucF and mucE. The DNA sequence of the HaeII R/M system was compared to the genomic DNA sequence of H. influenzae Rd. Although the DNA sequences flanking the HaeII system were > 99% identical to contiguous DNA fragments found in the genome of H. influenzae Rd, no homology was seen with the DNA sequences encoding the HaeII R/M system, indicating that it is not found in this strain. Given the vast difference in the GC content of S.NgoI and HaeII, their apparent insertion into polycistronic operons, and their difference in codon usage when compared to the species from which they were isolated, the data suggest that these R/M systems originated in an organism other than Neisseria or Haemophilus.     

Stereology of nerve cross sections

Among strains of Haemophilus influenzae, the ability to catabolize tryptophan (as detected by indole production) varies and is correlated with pathogenicity. Tryptophan catabolism is widespread (70 to 75%) among harmless respiratory isolates but is nearly universal (94 to 100%) among strains causing serious disease, including meningitis. As a first step in investigating the relationship between tryptophan catabolism and virulence, we have identified genes in pathogenic H. influenzae which are homologous to the tryptophanase (tna) operon of Escherichia coli. The tna genes are located on a 3.1-kb fragment between nlpD and mutS in the H. influenzae type b (Eagan) genome, are flanked by 43-bp direct repeats of an uptake signal sequence downstream from nlpD, and appear to have been inserted as a mobile unit within this sequence. The organization of this insertion is reminiscent of pathogenicity islands. The tna cluster is found at the same map location in all indole-positive strains of H. influenzae surveyed and is absent from reference type d and e genomes. In contrast to H. influenzae, most other Haemophilus species lack tna genes. Phylogenetic comparisons suggest that the tna cluster was acquired by intergeneric lateral transfer, either by H. influenzae or a recent ancestor, and that E. coli may have acquired its tnaA gene from a related source. Genomes of virulent H. influenzae resemble those of pathogenic enterics in having an island of laterally transferred DNA next to mutS.     

In situ detection of tandem DNA repeat length

A simple method for scoring short tandem DNA repeats is presented. An oligonucleotide target, containing tandem repeats embedded in a unique sequence, was hybridized to a set of complementary probes, containing tandem repeats known lengths. Single-stranded loops structures formed on duplexes containing a mismatched (different) number of tandem repeats. No loop structure formed on duplexes containing a matched (identical) number of tandem repeats. The matched and mismatched loop structures were enzymatically distinguished and differentially labeled by treatment with S1 nuclease and the Klenow fragment of DNA polymerase.     

Rice (Oryza sativa) centromeric regions consist of complex DNA

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence in situ hybridization. Six of the elements are middle repetitive, and their copy numbers range from approximately 50 to approximately 300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented approximately 6,000 times in the rice genome and is detected only in Oryza species. Fiber-fluorescence in situ hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and Arabidopsis thaliana chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences.     

PCR amplification of ribosomal DNA for species identification in the plant pathogen genus Phytophthora

We have developed a PCR procedure to amplify DNA for quick identification of the economically important species from each of the six taxonomic groups in the plant pathogen genus Phytophthora. This procedure involves amplification of the 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) with the ITS primers ITS 5 and ITS 4. Restriction digests of the amplified DNA products were conducted with the restriction enzymes RsaI, MspI, and HaeIII. Restriction fragment patterns were similar after digestions with RsaI for the following species: P. capsici and P. citricola; P. infestans, P. cactorum, and P. mirabilis; P. fragariae, P. cinnamomi, and P. megasperma from peach; P. palmivora, P. citrophthora, P. erythroseptica, and P. cryptogea; and P. megasperma from raspberry and P. sojae. Restriction digests with MspI separated P. capsici from P. citricola and separated P. cactorum from P. infestans and P. mirabilis. Restriction digests with HaeIII separated P. citrophthora from P. cryptogea, P. cinnamomi from P. fragariae and P. megasperma on peach, P. palmivora from P. citrophthora, and P. megasperma on raspberry from P. sojae. P. infestans and P. mirabilis digests were identical and P. cryptogea and P. erythroseptica digests were identical with all restriction enzymes tested. A unique DNA sequence from the ITS region I in P. capsici was used to develop a primer called PCAP. The PCAP primer was used in PCRs with ITS 1 and amplified only isolates of P. capsici, P. citricola, and P. citrophthora and not 13 other species in the genus. Restriction digests with MspI separated P. capsici from the other two species. PCR was superior to traditional isolation methods for detection of P. capsici in infected bell pepper tissue in field samples. The techniques described will provide a powerful tool for identification of the major species in the genus Phytophthora.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Differential display of genome subsets containing specific interspersed repeats

A genomic differential display method was developed that analyzes many restriction fragment length polymorphisms simultaneously. Interspersed repeat sequences were used to reduce DNA sample complexity and to target genomic subsets of interest. This work focused on trinucleotide repeats because of their importance in human inherited diseases. Immobilized repeat-containing oligonucleotides were used to capture genomic DNA fragments containing sequences complementary to the oligonucleotide. Captured fragments were amplified by PCR and fluorescently labeled using primers complementary to the repeat sequence and/or to the known sequences ligated to the ends of the restriction fragments. The labeled PCR fragments were displayed by size on a high-resolution automated fluorescent DNA sequencing instrument. Although there was a conservation in the overall pattern of displayed genome subsets, many clear and reproducible differences were detected when genomes from different individuals were compared. Fewer differences were detected within, than between, monozygotic twin pair genomes. In control experiments, the method distinguished between Huntington disease alleles with normal and expanded CAG repeat lengths.     

Microsatellite instability in yeast: dependence on repeat unit size and DNA mismatch repair genes

We examined the stability of microsatellites of different repeat unit lengths in Saccharomyces cerevisiae strains deficient in DNA mismatch repair. The msh2 and msh3 mutations destabilized microsatellites with repeat units of 1, 2, 4, 5, and 8 bp; a poly(G) tract of 18 bp was destabilized several thousand-fold by the msh2 mutation and about 100-fold by msh3. The msh6 mutations destabilized microsatellites with repeat units of 1 and 2 bp but had no effect on microsatellites with larger repeats. These results argue that coding sequences containing repetitive DNA tracts will be preferred target sites for mutations in human tumors with mismatch repair defects. We find that the DNA mismatch repair genes destabilize microsatellites with repeat units from 1 to 13 bp but have no effect on the stability of minisatellites with repeat units of 16 or 20 bp. Our data also suggest that displaced loops on the nascent strand, resulting from DNA polymerase slippage, are repaired differently than loops on the template strand.     

Telomere analysis by fluorescence in situ hybridization and flow cytometry

Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.