Isolation and characterization of a human variable copy number tandem repeat at Xcen-p11.22

A subclone (M27B) has been isolated from a cosmid randomly selected from a library enriched for human X-chromosomal material. The subclone is extensively single-copy sequence, but also contains three complete copies and one partial copy of a 26-bp repeat, within which exists an inverted repeat having the potential to form a cruciform loop structure. Genomic sequences in this repeat region are apparently refractory to cloning, and rearrangements occurring during this process result in deletions. M27B detects multiple X-linked restriction fragments for a wide range of enzymes including MspI and HpaII. We have assigned the locus recognized by the probe (DXS255) to Xcen-Xp11.4 by mapping with a somatic cell hybrid panel and further refined its localization to Xp11.22 by in situ hybridization. The repeat sequence that is presumed to be responsible for the hypervariability observed does not show close similarity to other variable copy number tandem repeats described.     

Effect of repeat copy number on variable-number tandem repeat mutations in Escherichia coli O157:H7

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 x 10(-4) mutations/generation and a combined 28-locus rate of 6.4 x 10(-4) mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r2= 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r2= 0.833, P < 0.0001) or excluded (r2= 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data.     

The contribution of tandem repeat number to the O-glycosylation of mucins

The serine- and threonine-rich tandem repeat (TR) units that make up the characteristic feature of mucin glycoproteins are often polymorphic with substantial genetic variation in TR number. The precise effect of TR number on O-glycosylation is not fully understood, although the TR number of several mucins may be associated with apparent susceptibility to certain human diseases. To evaluate the contribution of TR number to O-glycosylation, we generated a series of chimeric mucins carrying increasing numbers of TR units from the MUC5B mucin in the context of an epitope-tagged MUC1 mucin backbone. These mucins were expressed in Caco2 colon carcinoma cell clones and purified by immunoprecipitation. O-Glycosylation was investigated by western blotting with antibodies to known carbohydrate structures and by fast atom bombardment-mass spectrometry. Additional carbohydrate epitopes were detected with antibodies on chimeric mucins with a higher TR number in comparison to those with fewer TRs. Using mass spectrometry, higher-molecular-weight glycans were detected more frequently on the mucins with extended TRs compared to those with fewer TRs. However no novel carbohydrate structures were seen, suggesting that TR number does not affect the specificity of O-glycosylation.     

Influence of temperature on the number of Legionella pneumophila in hot water systems

The occurrence of legionella in the hot water systems of two buildings (A and B) was investigated in relation to the water temperature. The peripheral parts of both hot water systems were found to be colonized by these organisms. A temperature of 60 degrees C in the hot water mains returning from the building eliminated legionellas from the mains as well as from the peripheral taps and showers. Legionellas could be isolated from taps, showers and the mains when the temperature in the return mains was kept at 54 degrees C. The hot water systems could not be completely decontaminated by raising the hot water temperature in the return mains to 70 degrees C combined with flushing all the taps and showers. It is suggested that failure to decontaminate the systems is due to dead ends in the pipeline network, which are not reached by the hot water and that these dead ends are the source for recolonization of the systems.     

Influence of temperature on the number of Legionella pneumophila in hot water systems

The occurrence of legionella in the hot water systems of two buildings (A and B) was investigated in relation to the water temperature. The peripheral parts of both hot water systems were found to be colonized by these organisms. A temperature of 60C in the hot water mains returning from the building eliminated legionellas from the mains as well as from the peripheral taps and showers. Legionellas could be isolated from taps, showers and the mains when the temperature in the return mains was kept at 54C. The hot water systems could not be completely decontaminated by raising the hot water temperature in the return mains to 70C combined with flushing all the taps and showers. It is suggested that failure to decontaminate the systems is due to dead ends in the pipeline network, which are not reached by the hot water and that these dead ends are the source for recolonization of the systems.     

Identification and characterization of novel ColE1-type, high-copy number plasmid mutants in Legionella pneumophila

ColE1-type plasmids are commonly used in bacterial genetics research, and replication of these plasmids is regulated by interaction of RNA I and RNA II. Although these plasmids are narrow-host-range, they can be maintained in Legionella pneumophila under antibiotic selection, with low-copy number and instability. Here, we have described the isolation of two novel spontaneous mutants of pBC(gfp)Pmip, pBG307 and pBG309, which are able to mark the L. pneumophila with strong green fluorescence when exposed to visible light. One of the mutants, pBG307, has a single CG-->TA mutation in RNA II promoter located 2-bases upstream the - 10 region. Another one, pBG309, has the same mutation, as well as an additional CG-->AT mutation in the 76th nucleotide of RNA I, or in the 6th nucleotide of RNA II. A plasmid with the single mutation in RNA I, pBG308, was also constructed. Characterization of these plasmids carrying the enhanced green fluorescent protein (gfpmut2) gene revealed that the green fluorescence intensities of these plasmids were 2- to 30-fold higher than that of the wild type and both of the mutations contribute to increase the plasmid copy number and/or plasmid stability. The mutation located in RNA II promoter played a more dominant role in elevating the copy number, compared to the mutation in RNA I. We also tested the mutant plasmids for replication in Escherichia coli, and found that their copy number and stability were dramatically decreased, except pBG307. Our data suggest that these plasmids might be useful and convenient in genetic studies in L. pneumophila.     

Genomic copy number variation and its potential role in lipoprotein and metabolic phenotypes

PURPOSE OF REVIEW: This review examines the role of copy number variation in the human genome as a newly recognized determinant of lipoprotein and metabolic phenotypes. RECENT FINDINGS: Much of the recent progress defining the molecular basis of lipoprotein disorders has been the result of studying genomic DNA at the single nucleotide level, for instance with nucleotide sequence analysis or genotyping to detect single nucleotide polymorphisms. Focus on single nucleotides, however, fails to capture the complete spectrum of potential genetic variability. Recent genome-wide mapping studies have demonstrated the surprising ubiquity of large-scale copy number variations in apparently healthy people, adding to the complexity of the 'normal' genome, but also emphasizing this form of genetic variation as a potential disease mechanism. The application of this understanding to the genetics of lipoprotein disorders has been rapid. For instance, the use of novel techniques to detect copy number variations, such as multiplex ligation-dependent probe amplification, has revealed many additional causative mutations in the low-density lipoprotein receptor gene in patients with familial hypercholesterolemia. SUMMARY: Copy number variations thus represent a new level of genomic variation that is both an important mechanism of monogenic lipoprotein disorders and a potential contributor to common complex lipoprotein and metabolic phenotypes in the general population.     

Physiology and morphology of Legionella pneumophila in continuous culture at low oxygen concentration.

Two strains of Legionella pneumophila serogroup 1 monoclonal subgroup Pontiac were grown for the first time in continuous culture using a chemically defined medium. The influence of temperature on physiology and morphology was investigated by fixing the growth rate (equal to the dilution rate, D) at 0.08 h-1 and controlling the pH and dissolved oxygen concentration of the culture. Serine provided the principal source of carbon and energy but growth was limited by tyrosine. The bacterium behaved as a microaerophile in this medium, with maximal growth occurring at 0.31 (mg O2)I-1 (equivalent to a dissolved oxygen tension of 4% (v/v) air saturation at 30 degrees C). The cultures consisted of flagellated, short rods at 24 degrees C, but exhibited an increased level of pleomorphism and the loss of flagella as the temperature was increased to 37 degrees C. The presence of intracellular granules was noted, and their abundance was temperature-dependent. Polyhydroxybutyrate was present in L. pneumophila, and the proportion of the cell dry weight that it accounted for varied with temperature, being maximal at 24 degrees C. The ratio of saturated to unsaturated fatty acids in the cells decreased as the temperature was reduced towards 24 degrees C, so as to maintain membrane fluidity at low growth temperature.     

The role of RhoA in the regulation of cell morphology and motility

Members of the Rho family of small GTPases are key regulators of the actin cytoskeleton, particularly in relation to the cell shape changes and the adhesion dynamic that drive cell migration. Here, we report the effect of activation or inhibition of the function of RhoA on cell motility and morphology. Both in the presence and the absence of serum, expression of constitutively active RhoA dramatically inhibited L929 fibroblasts' cell motility, and induced a rounding of the cells and a decrease in the number of processes per cell. In contrast, expression of a dominant negative mutant of RhoA had no effect on cell motility or morphology in steady-state conditions with or without serum in the medium. Inhibition of p160ROCK, a kinase effector of RhoA, only partially inhibited cell migration. Conversely, when cells were submitted to a period of serum deprivation followed by addition of serum, inhibition of endogenous RhoA by expression of the dominant negative mutant of RhoA impeded cell motility after serum stimulation. Thus, RhoA activity is required for stimulation of cell locomotion by serum factors. It was also observed that the addition of serum factors to quiescent L929 and NR6wtEGFR fibroblasts resulted in a delayed motility response of several hours compared to the immediately induced morphological changes, indicating the absence of a previously assumed direct correlation between changes in cell motility and cell morphology in response to serum addition. The motility response of L929 and NR6wtEGFR fibroblasts to serum stimulation required protein synthesis.     

Intracellular parasitism,the driving force of evolution of Legionella pneumophila and the genus Legionella

Legionella pneumophila is an intracellular pathogen that causes a severe pneumonia called Legionnaires' disease that is often fatal when not promptly diagnosed and treated. Legionella parasitize aquatic protozoa with which it co-evolved over an evolutionary long time. The close relationship between hosts and pathogens, their co-evolution, led to molecular interactions such as the exchange of genetic material through horizontal gene transfer (HGT). Genome sequencing of L. pneumophila and of the entire genus Legionella that comprises over 60 species revealed that Legionellae have co-opted genes and thus cellular functions from their eukaryotic hosts to a surprisingly high extent. Acquisition and loss of these eukaryotic-like genes and domains is an on-going process underlining the highly dynamic nature of the Legionella genomes. Although the large amount and diversity of HGT in Legionella seems to be unique in the prokaryotic world the analyses of more and more genomes from environmental organisms and symbionts of amoeba revealed that such genetic exchanges occur among all amoeba associated bacteria and also among the different microorganisms that infect amoeba. This dynamic reshuffling and gene-acquisition has led to the emergence of Legionella as human pathogen and may lead to the emergence of new human pathogens from the environment.     

lbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin

Under iron stress, Legionella pneumophila secretes legiobactin, a nonclassical siderophore that is reactive in the chrome azurol S (CAS) assay. Here, we have optimized conditions for legiobactin expression, shown its biological activity, and identified two genes, lbtA and lbtB, which are involved in legiobactin production. lbtA appears to be iron repressed and encodes a protein that has significant homology with siderophore synthetases, and FrgA, a previously described iron-regulated protein of L. pneumophila. lbtB encodes a protein homologous with members of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtA or lbtB were defective for legiobactin, producing 40 to 70% less CAS reactivity in deferrated chemically defined medium (CDM). In bioassays, mutant CDM culture supernatants, unlike those of the wild type, did not support growth of iron-limited wild-type bacteria in 2',2'-dipyridyl-containing buffered charcoal yeast extract (BCYE) agar and a ferrous iron transport mutant on BCYE agar without added iron. The lbtA mutant was modestly defective for growth in deferrated CDM containing the iron chelator citrate, indicating that legiobactin is required in conditions of severe iron limitation. Complementation of the lbt mutants restored both siderophore expression, as measured by the CAS assay and bioassays, and bacterial growth in deferrated, citrate-containing media. The lbtA mutant replicated as the wild type did in macrophages, amoebae, and the lungs of mice. However, L. pneumophila expresses lbtA in the macrophage, suggesting that legiobactin, though not required, may play a dispensable role in intracellular growth. The discovery of lbtAB represents the first identification of genes required for L. pneumophila siderophore expression.     

Exploring the role of copy number variants in human adaptation

Over the past decade, the ubiquity of copy number variants (CNVs, the gain or loss of genomic material) in the genomes of healthy humans has become apparent. Although some of these variants are associated with disorders, a handful of studies documented an adaptive advantage conferred by CNVs. In this review, we propose that CNVs are substrates for human evolution and adaptation. We discuss the possible mechanisms and evolutionary processes in which CNVs are selected, outline the current challenges in identifying these loci, and highlight that copy number variable regions allow for the creation of novel genes that may diversify the repertoire of such genes in response to rapidly changing environments. We expect that many more adaptive CNVs will be discovered in the coming years, and we believe that these new findings will contribute to our understanding of human-specific phenotypes.     

Characterization of the uup locus and its role in transposon excisions and tandem repeat deletions in Escherichia coli

Null mutations in the Escherichia coli uup locus (at 21.8 min) serve to increase the frequency of RecA-independent precise excision of transposable elements such as Tn10 and to reduce the plaque size of bacteriophage Mu (Uup(-) phenotype). By the combined approaches of physical mapping of the mutations, complementation analyses, and protein overexpression from cloned gene fragments, we have demonstrated in this study that the Uup(-) phenotype is the consequence of the absence of expression of the downstream gene (uup) of a two-gene operon, caused either directly by insertions in uup or indirectly by the polar effect of insertions in the upstream gene (ycbY). The promoter for uup was mapped upstream of ycbY by primer extension analysis on cellular RNA, and assays of reporter gene expression indicated that it is a moderately active, constitutive promoter. The uup mutations were also shown to increase, in a RecA-independent manner, the frequencies of nearly precise excision of Tn10 derivatives and of the deletion of one copy of a chromosomal tandem repeat, suggesting the existence of a shared step or intermediate in the pathways of these latter events and that of precise excision. Finally, we found that mutations that increase the frequency of precise excision of Tn10 are divisible into two categories, depending upon whether they did (uup, ssb, polA, and topA) or did not (mutHLS, dam, and uvrD) also increase precise excision frequency of the mini-Tn10 derivatives. It is suggested that the differential response of mini-Tn10 and Tn10 to the second category of mutations is related to the presence, respectively, of perfect and of imperfect terminal inverted repeats in them.     

Intrinsic polymorphism of variable number tandem repeat loci in the human genome.

In the human genome, short tandem repetitive (STR) DNA sequences often show restriction fragment length polymorphisms (RFLPs) due to variation in the number of copies of the repeat unit. For a subset of these sequences known as minisatellites or variable number tandem repeat loci (VNTR), it has been proposed that a homologous "core" sequence of 10-12 nucleotides is involved in the mechanism(s) generating the polymorphism. In our present study we have prepared oligonucleotide probes complementary to one or two repeat units of several VNTR loci. Under stringent hybridization and wash conditions these probes hybridize locus specifically thus allowing the evaluation of the intrinsic polymorphism of individual loci. Our results indicate that not all of the loci having STR DNA sequences are polymorphic despite the fact that they share the "core" sequence. This suggests that more than the DNA sequence of the locus is involved in the mechanism(s) generating the polymorphism.     

The human genome puzzle - the role of copy number variation in somatic mosaicism

The discovery of copy number variations (CNV) in the human genome opened new perspectives in the study of the genetic causes of inherited disorders and the etiology of common diseases. Differently patterned instances of somatic mosaicism in CNV regions have been shown to be present in monozygotic twins and throughout different tissues within an individual. A single-cell-level investigation of CNV in different human cell types led us to uncover mitotically derived genomic mosaicism, which is stable in different cell types of one individual. A unique study of immortalized B-lymphoblastoid cell lines obtained with 20 year interval from the same two subjects shows that mitotic changes in CNV regions may happen early during embryonic development and seem to occur only once, as levels of mosaicism remained stable. This finding has the potential to change our concept of dynamic human genome variation. We propose that further genomic studies should focus on the single-cell level, to understand better the etiology and physiology of aging and diseases mediated by somatic variations.     

Microevolution of pandemic Vibrio parahaemolyticus assessed by the number of repeat units in short sequence tandem repeat regions

The emergence of the pandemic strain Vibrio parahaemolyticus O3:K6 in 1996 caused a large increase of diarrhea outbreaks related to seafood consumption in Southeast Asia, and later worldwide. Isolates of this strain constitutes a clonal complex, and their effectual differentiation is possible by comparison of their variable number tandem repeats (VNTRs). The differentiation of the isolates by the differences in VNTRs will allow inferring the population dynamics and microevolution of this strain but this requires knowing the rate and mechanism of VNTRs' variation. Our study of mutants obtained after serial cultivation of clones showed that mutation rates of the six VNTRs examined are on the order of 10(-4) mutant per generation and that difference increases by stepwise addition of single mutations. The single stepwise mutation (SSM) was deduced because mutants with 1, 2, 3, or more repeat unit deletions or insertions follow a geometric distribution. Plausible phylogenetic trees are obtained when, according to SSM, the genetic distance between clusters with different number of repeats is assessed by the absolute differences in repeats. Using this approach, mutants originated from different isolates of pandemic V. parahaemolyticus after serial cultivation are clustered with their parental isolates. Additionally, isolates of pandemic V. parahaemolyticus from Southeast Asia, Tokyo, and northern and southern Chile are clustered according their geographical origin. The deepest split in these four populations is observed between the Tokyo and southern Chile populations. We conclude that proper phylogenetic relations and successful tracing of pandemic V. parahaemolyticus requires measuring the differences between isolates by the absolute number of repeats in the VNTRs considered.