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PubMed1998

Molecular ecology and evolution of Streptococcus thermophilus bacteriophages--a review.

Bacteriophages attacking Streptococcus thermophilus, a lactic acid bacterium used in milk fermentation, are a threat to the dairy industry. These small isometric-headed phages possess double-stranded DNA genomes of 31 to 45 kb. Yoghurt-derived phages exhibit a limited degree of variability, as defined by restriction pattern and host range, while a large diversity of phage types have been isolated from cheese factories. Despite this diversity all S. thermophilus phages, virulent and temperate, belong to a single DNA homology group. Several mechanisms appear to create genetic variability in this phage group. Site-specific deletions, one type possibly mediated by a viral recombinase/integrase, which transformed a temperate into a virulent phage, were observed. Recombination as a result of superinfection of a lysogenic host has been reported. Comparative DNA sequencing identified up to 10% sequence diversity due to point mutations. Genome sequencing of the prototype temperate phage phi Sfi21 revealed many predicted proteins which showed homology with phages from Lactococcus lactis suggesting horizontal gene transfer. Homology with phages from evolutionary unrelated bacteria like E. coli (e.g. lambdoid phage 434 and P1) and Mycobacterium phi L5 was also found. Due to their industrial importance, the existence of large phage collections, and the whole phage genome sequencing projects which are currently underway, the S. thermophilus phages may present an interesting experimental system to study bacteriophage evolution.

Bacteriophages↗
PubMed2006

[Genome conservatism of phiKMV-like bacteriophages (T7 supergroup) active against Pseudomonas aeruginosa].

The T7-like phiKMV bacteriophage active on Pseudomonas aeruginosa was previously isolated by us and shown to have DNA resistant to many endonucleases. A loss of sensitive sites might be a consequence of a long phiKMV evolution on different hosts. To elucidate, whether this trait is shared by other similar phages, several new phiKMV-like phages were isolated from different sources and compared. All studied phiKMV-like phages formed three groups, insignificantly differing in the number and localization of endonuclease-sensitive DNA sites. This confirms that the present-day phages of this species have highly conserved genomes. Mutational "restoration" of the lost sites may be restricted by a lethal effect. The phiKMV-like phages were shown for the first time to increase the rate of in vitro accumulation of giant phiKZ-like phages of P. aeruginosa. This effect is characteristic only of phiKMV-like phages.

Bacteriophage Typing↗
PubMed1997

Parental somatic and germ-line mosaicism for a FBN2 mutation and analysis of FBN2 transcript levels in dermal fibroblasts.

Congenital contractural arachnodactyly (CCA) is an autosomal dominant disorder that is phenotypically related to the Marfan syndrome. CCA has recently been shown to result from mutations in the FBN2 gene, which encodes an elastin-associated microfibrillar protein called fibrillin-2. Two siblings are reported here with classic manifestations of CCA with unaffected parents. Analysis of the FBN2 cDNA from dermal fibroblasts from one of the affected siblings revealed a heterozygous exon splicing error deleting nt 3722-3844 of the FBN2 mRNA. This cDNA deletion resulted in selective removal of one of the 43 calcium-binding EGF-like domains of the fibrillin-2 protein. Analysis of the FBN2 gene in the affected siblings' DNA indicated that the splicing error resulted from an A-to-G transition 15 nt upstream from the 3' splice site of the intron. The genomic mutation resulting in the splicing error alters a putative branch point sequence important for lariat formation, an intermediate structure of normal splicing. The mutation was detectable in DNA from the father's hair bulbs and buccal cells but not his white blood cell DNA, indicating that the father was a somatic mosaic. Analysis of transcript levels by use of dermal fibroblasts from the proband demonstrated that the FBN2 allele containing the exon deletion was expressed at a higher level than the allele inherited from the mother. These results indicate that FBN2 exon splicing errors are a cause of CCA, furthering the understanding of the molecular basis of this disorder. In addition, the demonstration of gonadal mosaicism in the FBN2 gene is important for accurate genetic counseling of families with sporadic cases of CCA. Finally, the preferential expression of the mutated FBN2 allele in dermal fibroblasts may have implications for understanding the pathogenesis and rarity of CCA.

Alleles↗
PubMed2006

Genomic alteration is not associated with fatty and clear cell change in hepatocellular carcinomas and its precursor nodular lesions in cirrhotic liver.

BACKGROUND: The aim of this study was to investigate whether fatty and clear cell areas in large regenerative nodules (LRN), dysplastic nodules (DN), and hepatocellular carcinoma (HCC) show higher degree of genomic mutation compared to non-fatty/clear cell area in the same nodule or non-lesional tissue. METHODS: We examined 22 nodular lesions (9 HCC, 5 DN and 8 LRN) from seven cirrhotic livers removed at transplantation. Frozen sections were used for manual microdissection of areas with fatty/clear cell change. DNA from microdissected tissue was amplified using arbitrarily primed polymerase chain reaction (AP-PCR), and PCR products were run on polyacrilamide gel generating a "fingerprint" band pattern. Autoradiographs were analysed using Adobe Photoshop version 6.0. Fingerprints from lesional tissue were compared to reference tissue and the total number of bands in excess or defect was calculated and divided by the total number of bands identified, obtaining the genomic damage fraction (GDF). RESULTS: Increasing GDF average values were seen from cirrhotic liver (0.13+/-0.04), to LRN (0.16+/-0.1), DN (0.28+/-0.08) and HCC (0.30+/-0.07). A statistically significant difference in GDF values was documented between cirrhotic liver and DN (p=0.008) and HCC (p=0.005) and between HCC and LRN (p=0.02). No significant difference was documented between DN and HCC, and between LRN and cirrhotic liver. Eleven nodules containing fat/clear cell areas were compared to the other 11 nodules without fat/clear cell areas. The GDF was not different between the two groups: 0.29+/-0.11 versus 0.25+/-0.12; p=0.5. The average value of genomic damage fraction between fat/clear cell areas (0.29+/-0.11) and no fat/clear cell areas (0.25+/-0.1) within the same nodules were not significantly different (p=0.11). CONCLUSION: Fatty and clear cell change in nodular lesions in cirrhotic liver may be an epigenetic phenotypic modification caused by microenvironmental factors such as ischaemia rather than indicating areas of increased malignant potential per se.

Journal Article↗
PubMed2001

A large family with hereditary MTC: role of RET genetic analysis in differential diagnosis between MEN 2A and FMTC.

Germline mutations of the RET proto-oncogene cause three different cancer syndromes: multiple endocrine neoplasia type 2A (MEN 2A), multiple endocrine neoplasia type 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). In the absence of biochemical and/or clinical evidence of pheochromocytoma and hyperparathyroidism, patients with MEN 2A disease display the same phenotype of FMTC disease, although prognosis and clinical management in both affected and unaffected familial members are quite different. We studied a family with hereditary MTC, whose proband was referred to us because of enlarged cervical nodes and increased calcitonin serum levels 28 years after the total thyroidectomy for MTC. Cervical node dissection was carried out and subsequently the presence of MTC metastasis was histologically confirmed. A RET genomic mutation at codon 634 (TGC-->TTC) was identified in the proband and in seven out of 19 familial members studied. Accordingly, a hereditary disease was suggested. However, the strong association of RET mutation at codon 634 with the presence of pheochromocytoma in MEN 2 disease suggested a more rigorous management in all gene carriers. Indeed, during the follow-up pheochromocytoma was subsequently identified in the proband. This finding suggests that all families with a pedigree suggestive of FMTC should be regarded at risk from MEN 2A disease, at least when a critical mutation in the RET cysteine domain is detected.

Adolescent↗
PubMed2003

Progressive genomic alterations in intraductal papillary mucinous tumours of the pancreas and morphologically similar lesions of the pancreatic ducts.

Intraductal papillary mucinous tumours (IPMTs) of the pancreas are rare neoplasms characterized by a prominent intraductal component, and by malignant potential. Little data exists concerning numerical chromosome aberrations in IPMTs. The biological significance of mucinous epithelial changes (mucinous hyperplasia) in small branching ducts adjacent to IPMTs also remains unclear. From a series of 12 IPMTs, we investigated by interphase cytogenetics 22 foci with mucinous hyperplasia, 27 foci with borderline lesions, and 11 samples with either intraductal (CIS) or invasive carcinoma. Chromosome 6 loss was detected in areas with mucinous hyperplasia (36.3%), borderline lesions (96.3%), and CIS/invasive carcinoma (100%). Similar losses, indicating clonal progression, were found for chromosome 17 (18.2%, 81.5%, and 100%), and for chromosome 18 (0%, 18.5%, and 100%). Quantitative analysis showed a significant intraductal expansion of cell clones harbouring these numerical aberrations within the spectrum of IPMTs. Mucinous epithelial changes in 11 resection samples with chronic pancreatitis showed monosomy 6 (36%) and monosomy 17 (27%). Conversely, areas with low-grade pancreatic intraepithelial neoplasia (PanIN-1), obtained from eight surgical specimens with ductal adenocarcinoma, showed monosomies for chromosome 6, 17, and 18 (100%, 87%, and 50%, respectively). We conclude that monosomies, as defined by FISH analysis, are frequent in both IPMTs and mucinous hyperplasia of pancreatic ducts adjacent to IPMTs. Monosomy 6 may represent an early event in the stepwise accumulation of genomic mutations necessary for the neoplastic transformation of pancreatic duct epithelia, whereas loss of chromosome 18 may be implicated in the progression of borderline to malignant IPMT. The detection of complex chromosomal aberrations in mucinous epithelial changes, and the quantitative expansion of monosomic cell clones in pancreatic ducts, provide evidence for a continuum between hyperplastic and dysplastic epithelial changes.

Adenocarcinoma, Mucinous↗
PubMed2002

Analysis of mouse polyomavirus mutants with lesions in the minor capsid proteins.

Polyomavirus mutants E, Q and H, expressing non-myristylated VP2, were generated by replacing the N-terminal glycine residue with glutamic acid, glutamine or histidine, respectively. Viruses mutated in either VP2 or VP3 translation initiation codons were also prepared. All mutated genomes, when transfected into murine host cells, gave rise to viral particles. Infectivity of VP2- and VP3- viruses, as measured by the number of cells expressing viral antigens, was dramatically diminished, indicative of defects in the early stages of infection. In contrast, the absence of a myristyl moiety on VP2 did not substantially affect the early steps of virus infection. No differences in numbers of cells expressing early or late viral antigens were observed between wild-type (wt) and E or Q myr- viruses during the course of a life cycle. Furthermore, no delay in virus DNA replication was detected. However, when cells were left for longer in culture, the number of infected cells, measured by typical virus bursts, was much lower when mutant rather than wt genomes were used. In situ, cell fractionation studies revealed differences in the interaction of viral particles with host cell structures. The infectivity of mutants was affected not only by loss of the myristyl group on VP2, but also, and to a greater extent, by alterations of the N-terminal amino acid composition.

Animals↗
PubMed1995

Analysis of cybrids harboring MELAS mutations in the mitochondrial tRNA(Leu(UUR)) gene.

MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes), a maternally inherited mitochondrial disorder, has been associated with an A-->G transition at nucleotide 3243 and a T-->C transition at nucleotide 3271, both in the mitochondrial tRNA(Leu(UUR)) gene. We transferred mitochondria harboring these mutations into human cells lacking endogenous mtDNA (rho o cells), and analyzed the resulting transmitochondrial cytoplasmic hybrid (cybrid) cell lines for the relationship of genotype to phenotype. Cybrids containing high levels of mutated genomes showed decreased rates of synthesis of mitochondrial translation products, reduced respiratory chain function, and increased amounts of a novel unprocessed RNA species (RNA 19). Overall effects on mitochondrial functions were more severe for the MELAS 3243 cybrids as compared to the MELAS 3271 cybrids. These data, combined with our previous observations, suggest that RNA 19 may play an important, but as yet uncharacterized, role in the pathogenesis of this mitochondrial disorder.

Genes↗
PubMed2005

Development of a highly efficient gene targeting system for Fusarium graminearum using the disruption of a polyketide synthase gene as a visible marker.

We cloned a polyketide synthase gene (pks12) from Fusarium graminearum, a devastating fungal pathogen of cereals. Transformation-mediated gene disruption led to an easily detectable albino phenotype of the disruptants. We used the disruption of the pks12 gene as a visible marker for transformation-mediated homologous recombination and optimized the transformation procedure to achieve a high rate of homologous recombination. In combination with the published genomic sequence data and the generation of expressed sequence tags (ESTs) for F. graminearum, this is a useful tool to investigate this important plant pathogen on a molecular level. Optimized transformation of F. graminearum resulted in at least 93% homologous recombination events when the homologous genomic DNA fragment in the vector had a size of approximately 800bp and was linearized in the middle. Using a genomic sequence of approximately 500bp in the transformation vector, 70% of the transformants still exhibited homologous recombination. On the contrary, no more than 10% homologous recombination events were observed when less than 400bp DNA fragments were used. We co-transformed F. graminearum with two different vectors. One vector harboured a DNA insert homologous to the pks12 gene, while the other vector consisted of the same vector backbone carrying the selection marker specific for F. graminearum. About 70% of the transformants had a disrupted pks12 gene, and all of these showed an integration of the second vector into the pks disruption vector. Therefore, the time-consuming construction of a single transformation vector can be avoided; furthermore, it is now easily feasible to express a gene construct at a defined and mutated genomic site.

Base Sequence↗
PubMed1995

A complete plasmid-based complementation system for RNA coliphage Q beta: three proteins of bacteriophages Q beta (group III) and SP (group IV) can be interchanged.

Our laboratory has established a bacteriophage Q beta cDNA-containing plasmid system in which virtually all coding defects present within the 4217 nucleotide Q beta genome can be complemented in trans. In this system, Q beta minus strand RNAs are constitutively transcribed from plasmid cDNA by Escherichia coli RNA polymerase. Replication of these minus strands results in the synthesis of Q beta plus RNA, thereby triggering an infectious cycle in which Q beta phase particles are generated. Genetically engineered Q beta genome mutations that result in defective viral proteins can be complemented in trans by the products of one or more Q beta helper plasmids that express either: (1) Q beta maturation protein, which can complement defects in the Q beta maturation cistron (nucleotides 61 to 1320); (2) Q beta readthrough protein, which can complement defects in the readthrough cistron (nucleotides 1344 to 2330); or (3) Q beta replicase, which can complement defects in the replicase cistron (nucleotides 2352 to 4118). Each plasmid component of this system contains a unique origin of replication and carries a different antibiotic gene, thereby enabling all combinations of these plasmids to coexist in the same host. We have further developed a second series of helper plasmids that generate the corresponding viral proteins of the related group IV RNA phage SP. Each of these SP helper proteins can complement respective defects within the Q beta genome with efficiencies similar to those observed for the Q beta helper proteins. It is now possible to supply functional Q beta or SP proteins in trans to examine Q beta genomes that contain protein coding defects for their ability to synthesize Q beta proteins, replicate Q beta RNA, assemble virions, and/or lyse the host cell.

Allolevivirus↗
PubMed1986

Xenotropic virus-related restriction patterns of non-H-2 histocompatibility mutant mice strains.

The relationship between alteration in the number of xenotropic virus-related sequences and non-H-2 histocompatibility (H) mutations in mice was investigated. Mutant classifications included gain, loss, and loss-gain mutations. Genomic DNA from a panel of non-H-2 H mutant strains on the C57BL/6 and BALB/c backgrounds was digested with a set of restriction enzymes with varying numbers of sites within endogenous xenotropic-related sequences. The digested DNA was then resolved on agarose gels. Southern blots of digested DNA were hybridized with the pXenv probe specific for the env sequence of xenotropic viral sequences. The number of hybridizing bands varied from 7 to 19, depending on the restriction enzyme and inbred background. Most mutant strains were identical in their restriction patterns to the respective background strains. However, two B6 mutant strains, KH84 and HZ54, differed from C57BL/6 at a single band which appeared to be inherited from BALB/c in the derivation of the two congenic strains. The HZ43 strain lacked a male-specific band shared by both C57BL/6 and BALB/c; this loss was evidently independent of the original mutation which was observed to be autosomal. However, the KH148B and KH84 strains on the C57BL/6 background lacked single B6 bands. Both mutants were classified as gain mutants. An examination of previous reciprocal graft rejection patterns and retrovirus linkage to non-H-2 H loci indicated a strong inverse relationship between a linked retroviral sequence and presentation of a non-H-2 H antigen. This inverse correlation is consistent with reports of gene inactivation following retroviral insertion.

Animals↗
PubMed2000

PCR amplification on a microarray of gel-immobilized oligonucleotides: detection of bacterial toxin- and drug-resistant genes and their mutations.

PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency, the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.

Antigens, Bacterial↗
PubMed1995

Identification of clastogenic and/or aneugenic events during the preneoplastic stages of experimental rat hepatocarcinogenesis by fluorescence in situ hybridization.

A growing body of evidence from human and animal cancer cytogenetics studies indicates that aneuploidy is an important chromosome change in carcinogenesis. To understand the role of this genetic phenomenon during the first steps of an experimental cancer model, molecular and cellular techniques were combined. A sequential cytogenetic study of a modified Solt-Farber liver cancer model in the rat was performed to identify the importance of chromosome versus genome mutations. Male Wistar rats were initiated with diethylnitrosamine (DENA), followed by a 2-acetylaminofluorene exposure to select resistant hepatocytes. Chronic phenobarbital (PB) treatment was used to induce promotion. Cell proliferation was induced by a necrogenic dose of CCl4, administered during the selection period (Gerlans protocol) or 3 days before hepatocyte isolation (experimental protocol). In order to discriminate between genetic events causing chromosome breakage (clastogenic) and those that induce chromosome loss (aneugenic), isolated micronucleated hepatocytes (MNH) were analysed for the presence of a centromere in the micronucleus (MN). Non-radioactive in situ hybridization with a rat centromere satellite 1 DNA probe was applied. Our results show that the majority of the observed genetic changes, expressed as MN during different preneoplastic stages, were of clastogenic origin. However, the number of induced aneugenic hepatocytes increased markedly during the promotion period of the Gerlans protocol (approximately 7-fold above control) and during PB exposure in the experimental protocol (approximately 4-fold above control). Additionally, these stages were also characterized by an increased level of MN expression (20.3 < % MNH < 32.8), in comparison with the initiation stage after DENA exposure (13.5 < % MNH < 17.1). Although it is not yet clear if these genetic alterations have a causative nature in neoplastic liver transformation, the use of interphase cytogenetics certainly might lead to a better understanding of the genomic changes which occur during experimental hepatocarcinogenesis.

Aneuploidy↗
PubMed1993

Induction of micronuclei and karyotype aberrations during in vivo mouse skin carcinogenesis.

The induction of chromosome and/or genome mutations during the first steps of skin carcinogenesis was followed in male NMRI mice, treated with a 'two-stage' [9,10-dimethyl-1,2-benzanthracene (DMBA) + phorbol-12-myristate-13-acetate (TPA)], or a 'three-stage' [DMBA+methyl methanesulphonate (MMS) + phorbol-12-retinoate-13-acetate (RPA)] protocol. The scoring of micronuclei (MN) in basal and suprabasal keratinocytes allows a relatively fast in vivo estimation of clastogenic and aneugenic effects of various compounds and treatments. Relevant stages were then further analysed by karyotyping the in vivo treated keratinocytes that were allowed to divide during short in vitro cultivation. DMBA used as initiator in both protocols was able to induce MN. The well-known clastogen MMS had an acute but transient effect on MN induction when used alone or as convertor in the three-stage protocol. Neither the propagator RPA, nor the 'full-promotor' TPA, which can carry out conversion as well as propagation, induced statistically significant numbers of MN when applied on mouse skin. Combined treatments, DMBA+MMS and MMS+RPA, showed higher MN frequencies than when MMS treatments were given alone. The full carcinogenic protocols showed significant frequencies of MN but the time points of appearance differed, indicating that the accumulation of aberrations could be more important than the order of appearance. Karyotypic analysis of those stages where the MN assay detected genome and/or chromosome aberrations revealed no specific loss of chromosomes that might be directly related to the carcinogenic process. When chromosome loss and aberrations were both taken into consideration together, chromosomes 7 and 11 and surely 9, 17 and 18 were more frequently involved than others.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2003

Patterns of transitional mutation biases within and among mammalian genomes.

Significant transition/transversion mutation bias is a well-appreciated aspect of mammalian nuclear genomes; however, patterns of bias among genes within a genome and among species remain largely uncharacterized. Understanding these patterns is important for understanding similarities and differences in mutational patterns among genomes and genomic regions. Therefore, we have conducted an analysis of 7,587 pairs of sequences of 4,347 mammalian protein-coding genes from seven species (human, mouse, rat, cow, sheep, pig, and macaque) and from the introns of 51 gene pairs and multiple intergenic regions (37 kbp, 52 kbp and 65 kbp) from the human, chimpanzee, and baboon genomes. Our analyses show that genes and regions with widely varying base composition exhibit uniformity of transition mutation rate both within and among mammalian lineages, as long as the transitional mutations caused by CpG hypermutability are excluded. The estimates show no relationship to potential intrachromosomal or interchromosomal effects. This uniformity points to similarity in point mutation processes in genomic regions with substantially different GC-content biases.

Animals↗
PubMed1995

Uptake of inorganic carbon in the cyanobacterium Synechocystis PCC6803: physiological and genetic evidence for a high-affinity uptake system.

Synechocystis PCC6803 displays two inorganic carbon-uptake processes, a low-affinity one (apparent Km: 300-400 microM) functional in cells grown under standard or limiting inorganic carbon concentrations, and one with a higher affinity (60 +/- 12 microM), detected only in cells adapted to limiting inorganic carbon conditions. A mutational and screening procedure allowed the isolation of a mutant deficient in the high-affinity system, but only slightly impaired in its growth capacities. The mutated genomic region revealed two open reading frames (ORFs), possibly belonging to an operonic structure. A clone in which the downstream ORF, hatR (high-affinity transport), had been inactivated showed a phenotype close to that of the original mutant. Inactivation of the other ORF, hatA, yielded a clone unable to grow in limiting inorganic carbon conditions. The deduced HatA protein showed no homology with any registered protein. It possessed three hydrophobic domains, including a putative signal peptide. Several hypotheses are considered as to its role. The deduced HatR protein, which possessed the features characteristic of the response regulators of the two-component regulatory systems ubiquitous in bacteria, might be a regulator controlling the activity of the high-affinity transport process. It would belong to the subclass of these molecules lacking the DNA-binding domain.

Amino Acid Sequence↗
PubMed2005

Evidence for widespread reticulate evolution within human duplicons.

Approximately 5% of the human genome consists of segmental duplications that can cause genomic mutations and may play a role in gene innovation. Reticulate evolutionary processes, such as unequal crossing-over and gene conversion, are known to occur within specific duplicon families, but the broader contribution of these processes to the evolution of human duplications remains poorly characterized. Here, we use phylogenetic profiling to analyze multiple alignments of 24 human duplicon families that span >8 Mb of DNA. Our results indicate that none of them are evolving independently, with all alignments showing sharp discontinuities in phylogenetic signal consistent with reticulation. To analyze these results in more detail, we have developed a quartet method that estimates the relative contribution of nucleotide substitution and reticulate processes to sequence evolution. Our data indicate that most of the duplications show a highly significant excess of sites consistent with reticulate evolution, compared with the number expected by nucleotide substitution alone, with 15 of 30 alignments showing a >20-fold excess over that expected. Using permutation tests, we also show that at least 5% of the total sequence shares 100% sequence identity because of reticulation, a figure that includes 74 independent tracts of perfect identity >2 kb in length. Furthermore, analysis of a subset of alignments indicates that the density of reticulation events is as high as 1 every 4 kb. These results indicate that phylogenetic relationships within recently duplicated human DNA can be rapidly disrupted by reticulate evolution. This finding has important implications for efforts to finish the human genome sequence, complicates comparative sequence analysis of duplicon families, and could profoundly influence the tempo of gene-family evolution.

Computational Biology↗
PubMed2000

Quasispecies development of Helicobacter pylori observed in paired isolates obtained years apart from the same host.

Helicobacter pylori isolates show greater genetic diversity than other bacterial species studied, but the basis for this phenomenon is unknown. Whether detectable genomic mutation appears within an H. pylori population during persistent colonization was investigated. Paired H. pylori populations obtained across 7- to 10-year intervals from 13 patients were characterized by use of methods including polymerase chain reaction (PCR) genotyping for cagA, vacA, iceA, recA, and IS605; random arbitrarily primed DNA (RAPD)-PCR and amplified fragment length polymorphism (AFLP) analysis; and ELISA, to determine Lewis phenotypes. Genotyping, including recA sequence analysis, revealed that initial and follow-up populations represented the same population in 11 patients (85%). Nevertheless, distinct dissimilarities were shown within each of these 11 pairs by both RAPD-PCR and AFLP analyses. During follow-up, Lewis-y levels, but not Lewis-x levels, decreased significantly. The changes detected by RAPD-PCR and AFLP indicate that genetic drift occurs within H. pylori populations over the course of years of colonization of a single host.

Adult↗