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PubMed1998

No influence of age on infection complexity and allelic distribution in Plasmodium falciparum infections in Ndiop, a Senegalese village with seasonal, mesoendemic malaria.

We have shown previously that in Dielmo, a Senegalese village with intense perennial Plasmodium falciparum transmission, the infection complexity and the distribution of some allelic types harbored by asymptomatic carriers was age-dependent. We report here an investigation of these parameters in Ndiop, a village located 5 km from Dielmo, where malaria is mesoendemic and seasonal, and where immunity is acquired at a very low rate, as indicated by the lifelong distribution of P. falciparum clinical attacks. Blood was collected from 143 and 125 inhabitants, including 122 individuals sampled in both surveys, during two cross-sectional surveys at one-month intervals during the 1994 transmission season. Plasmodium falciparum parasites were genotyped for three polymorphic single copy genes. Genetic diversity was very large, with 17, 43, and nine distinct alleles detected for the merozoite surface protein-1 (MSP-1), MSP-2, and glutamate-rich protein loci, respectively. These figures, similar to those previously observed in Dielmo, indicate that the parasite genetic diversity is not directly related to the inoculation rate, at least in the range of transmission intensity studied here. The complexity of the asymptomatic infections (average number of distinct genotypes per isolate) was more than two-fold lower in Ndiop than in Dielmo and importantly, did not decrease with age. Likewise, the allele distribution was not influenced by age, contrasting with the observations made in Dielmo. This indicates that the number of parasite types per isolate and the influence of age on complexity and allele distribution depend on the level of endemicity, consistent with the interpretation that they reflect acquired anti-parasite immunity.

Adolescent↗

PubMed2002

The transposable elements of the Drosophila melanogaster euchromatin: a genomics perspective.

BACKGROUND: Transposable elements are found in the genomes of nearly all eukaryotes. The recent completion of the Release 3 euchromatic genomic sequence of Drosophila melanogaster by the Berkeley Drosophila Genome Project has provided precise sequence for the repetitive elements in the Drosophila euchromatin. We have used this genomic sequence to describe the euchromatic transposable elements in the sequenced strain of this species. RESULTS: We identified 85 known and eight novel families of transposable element varying in copy number from one to 146. A total of 1,572 full and partial transposable elements were identified, comprising 3.86% of the sequence. More than two-thirds of the transposable elements are partial. The density of transposable elements increases an average of 4.7 times in the centromere-proximal regions of each of the major chromosome arms. We found that transposable elements are preferentially found outside genes; only 436 of 1,572 transposable elements are contained within the 61.4 Mb of sequence that is annotated as being transcribed. A large proportion of transposable elements is found nested within other elements of the same or different classes. Lastly, an analysis of structural variation from different families reveals distinct patterns of deletion for elements belonging to different classes. CONCLUSIONS: This analysis represents an initial characterization of the transposable elements in the Release 3 euchromatic genomic sequence of D. melanogaster for which comparison to the transposable elements of other organisms can begin to be made. These data have been made available on the Berkeley Drosophila Genome Project website for future analyses.

Animals↗

PubMed2000

Patterns of ribosomal gene variation in elite commercial chicken pure line populations.

The nucleolus organizer region (NOR) encodes the tandemly repeated 18S, 5.8S and 28S ribosomal (r) RNA genes. The NORs of broiler and layer commercial chicken pure lines were studied to establish the type and extent of genetic variation at this important locus. The parameters studied were gene copy number, repeat size, and diversity of NOR-types. The populations were organized into three groups for analysis including brown-egg broiler (13 lines), brown-egg layer (six lines), and white-egg layer (eight lines). The ribosomal gene copy number average of the white-egg layer populations was significantly lower (329 genes) than that of the brown-egg layers (372 genes); the brown-egg broiler ribosomal gene average was intermediate (350 genes). The white-egg layer populations exhibited a ribosomal repeat unit average size of 36 kb, significantly different from the brown-egg layer and brown-egg broiler average repeat unit size of 32.5 and 33.9 kb, respectively. NOR array size was similar among the three groups (6 mb). The brown-egg broiler populations exhibited polymorphic NOR patterns, intra- and interline, whereas the white-egg layer populations were essentially monomorphic for NOR-type; brown-egg layers exhibited an intermediate level of NOR diversity. Some NOR array characteristics may be a function of breed origin as brown-egg commercial populations, both broilers and layers, have similar breed origins and exhibited similarities for predominant repeat unit size as compared with white-egg layer populations. However, the finding that brown-egg broiler lines typically exhibit a greater number of segregating NOR-types than brown-egg layer lines suggests that the selection schemes of broiler vs. layer pure line populations may also have influenced the degree of variation at this gene complex.

Animals↗

PubMed2005

Influence of reagent formulation on mRNA quantification by RT-PCR using imported external standard curves.

Use of an imported external standard curve is common in real-time quantitative RT-PCR. Two practical strategies for long-term experiments include importing a grand mean standard curve to all accumulated runs or using daily imported standard curves, fixing the slope at the beginning of the experiment and calibrating successive runs with curves generated from this imported slope, adding a single standard that registers the variation in the y-intercept. This study determines the influence that a change in reagent lots has on these two calibration approaches when determining mRNA copy numbers of the ornithine decarboxylase and porphobilinogen deaminase genes. Two sets of determinations were run with the use of lot A and lot B. A marked decrease in the crossing points (Cp) in the standards for both genes at all concentration levels was observed with the change in lots. A grand mean standard curve was generated for each gene and each set and comparisons between the sets were performed. Statistically significant differences were found with respect to the y-intercept but not the slope, suggesting that the change of reagent lot affected the detection sensitivity but not the efficiency of the reaction. The excellent correlation coefficients obtained for these curves for each gene were not achieved when overall data from both sets were combined to generate an overall grand mean standard curve. We conclude that when faced with a change of RT-PCR reagent lot that will affect the detection sensitivity of the method, samples should be calculated with either the daily imported standard curves or with the respective grand mean standard curve for each lot.

Base Sequence↗

PubMed2000

Molecular clocks reduce plasmid loss rates: the R1 case.

Plasmids control their replication so that the replication frequency per plasmid copy responds to the number of plasmid copies per cell. High sensitivity amplification in replication response to copy number deviations generally reduces variation in copy numbers between different single cells, thereby reducing the plasmid loss rate in a cell population. However, experiments show that plasmid R1 has a gradual, insensitive replication control predicting considerable copy number variation between single cells. The critical step in R1 copy number control is regulation of synthesis of a rate-limiting cis-acting replication protein, RepA. De novo synthesis of a large number of RepA molecules is required for replication, suggesting that copy number control is exercised at multiple steps. In this theoretical kinetic study we analyse R1 multistep copy number control and show that it results in the insensitive replication response found experimentally but that it at the same time effectively prohibits the existence of only one plasmid copy in a dividing cell. In combination with the partition system of R1, this can lead to very high segregational stability. The R1 control mechanism is compared to the different multistep copy number control of plasmid ColE1 that is based on conventional sensitivity amplification. This implies that while copy number control for ColE1 efficiently corrects for fluctuations that have already occurred, R1 copy number control prevents their emergence in cells that by chance start their cycle with only one plasmid copy. We also discuss how regular, clock-like, behaviour of single plasmid copies becomes hidden in experiments probing collective properties of a population of plasmid copies because the individual copies are out of phase. The model is formulated using master equations, taking a stochastic approach to regulation, but the mathematical formalism is kept to a minimum and the model is simplified to its bare essence. This simplicity makes it possible to extend the analysis to other replicons with similar design principles.

Bacteriocin Plasmids↗

PubMed1994

The 8993 mtDNA mutation: heteroplasmy and clinical presentation in three families.

The point mutation at bp 8993 of human mtDNA in the ATPase 6 gene is associated with neurogenic weakness, ataxia and retinitis pigmentosa, and with subacute necrotizing encephalomyelopathy (Leigh disease) when present at high copy number. In this study we describe three new multiplex families with the ATPase 8993 mtDNA mutation and demonstrate a correlation between the percentage heteroplasmy of this mutation and the clinical phenotype. By combining this study with previous data we produce a graph of age of onset of symptoms versus percentage heteroplasmy of the mutation. Finally, we determine that ATP synthesis with NAD-linked substrates in cultured lymphoblast mitochondria from three patients with Leigh disease who had a high percentage heteroplasmy was on average 66% of the rate seen in control lymphoblast mitochondria. Similar rates are observed in lymphoblast mitochondria isolated from patients with Leigh disease due to complex I deficiency. This percentage appears to be independent of the rate of electron transport in mitochondria from patient cell lines with the mtDNA 8993 mutation.

Adenosine Triphosphatases↗

PubMed2005

Sequence divergence and conservation in organizationally distinct subfamilies of Donax trunculus satellite DNA.

Characterization of a low-copy number DTF1 satellite DNA detected in the bivalve mollusk Donax trunculus revealed extensive grouping of monomer sequence variants into subfamilies identified by distinctive combinations of diagnostic nucleotides. It can be anticipated that a large number of subfamilies exists in the genome. In addition to the tandem organization of 169 bp long monomers, at least one subfamily was created through amplification of adjacent repeats in a higher order register. This complex satellite unit consists of two distinctive monomer variants that differ both in specific nucleotide changes and in a deleted segment partially substituted with a short unrelated sequence element. Most of the nucleotide substitutions differing between subfamilies are highly homogenized within a corresponding group of monomer variants, and intra-subfamily variability in general is low. Nucleotide diversity analysis of all sequenced variants of DTF1 satellite revealed the presence of two conserved segments, while the rest of the monomer sequence shows uniform and considerably higher level of variability. The persistence of conserved segments stands in contrast to the sequence and organizational divergence of monomer variant groups, and may indicate constraints in the evolution of DTF1 satellite repeats.

Amino Acid Sequence↗

PubMed2000

Mitochondrial DNA segregation in the developing embryo.

Mitochondrial (mt)DNA is strictly maternally inherited in mammals; new mutations thus segregate along maternal lineages without the benefit of homologous recombination with mtDNA of paternal origin. Despite the high mtDNA copy number (approximately 100000 or more) in mature oocytes, and despite the relatively small number of cell divisions during oogenesis, mtDNA sequence variants segregate rapidly between generations. This paradoxical behaviour has been ascribed to the presence of a mtDNA 'bottleneck' in oogenesis or early embryogenesis. The nature and size of this bottleneck have been the subject of much controversy. This review argues that segregation of mtDNA sequence variants in the female germline occurs primarily during mitosis in the oocyte precursor population. Segregation is rapid because the precursor cells (primordial germ cells and oogonia) contain a relatively small number of mtDNA templates (the bottleneck) and because the replication of mtDNA is under relaxed control. For the most part, the process appears similar in mice segregating polymorphic sequence variants and in human pedigrees segregating pathogenic point mutations. In particular, there is no evidence for selection against high levels of pathogenic mtDNA point mutations in oogenesis, in early embryonic development, or in fetal development, thus suggesting that efficient respiratory chain function is not critical until post-natal life. These results have important practical implications for clinical genetics.

Chromosome Segregation↗

PubMed2006

TRIM retrotransposons occur in apple and are polymorphic between varieties but not sports.

Retrotransposon markers have been demonstrated to be powerful tools for investigating linkage, evolution and genetics diversity in plants. In the present study, we identified and cloned three full-size TRIM (terminal-repeat retrotransposon in miniature) group retrotransposon elements from apple (Malus domestica) cv. 'Antonovka', the first from the Rosaceae. To investigate their utility as markers, we designed primers to match the long terminal repeats (LTRs) of the apple TRIM sequences. We found that PCR reactions with even a single primer produced multiple bands, suggesting that the copy number of these TRIM elements is relatively high, and that they may be locally clustered or nested in the genome. Furthermore, the apple TRIM primers employed in IRAP (inter-retrotransposon amplified polymorphism) or REMAP (retrotransposon-microsatellite amplified polymorphism) analyses produced unique, reproducible profiles for 12 standard apple cultivars. On the other hand, all seven of the sport mutations in this study were identical to their mother cultivar. Genetic similarity values calculated from the IRAP/REMAP analyses or the STMS (sequence tagged microsatellite sites) analysis were generally comparable. PAUP cluster analysis based on IRAP and REMAP markers in apple and Japanese quince generated an NJ tree that is in good accordance with both a tree based on SMTS markers and the origin of the studied samples. Our results demonstrate that, although they do not encode the proteins necessary to carry out a life cycle and are thereby non-autonomous, TRIMs are at least as polymorphic in their insertion patterns as conventional complete retrotransposons.

DNA, Plant↗

PubMed1999

Evolution and horizontal transfer of dUTPase-encoding genes in viruses and their hosts.

dUTPase is a ubiquitous and essential enzyme responsible for regulating cellular levels of dUTP. The dut gene exists as single, tandemly duplicated, and tandemly triplicated copies. Crystallized single-copy dUTPases have been shown to assemble as homotrimers. dUTPase is encoded as an auxiliary gene in a number of virus genomes. The origin of viral dut genes has remained unresolved since their initial discovery. A comprehensive analysis of dUTPase amino acid sequence relationships was performed to explore the evolutionary dynamics of dut in viruses and their hosts. Our data set, comprised of 24 host and 51 viral sequences, includes representative sequences from available eukaryotes, archaea, eubacteria cells, and viruses, including herpesviruses. These amino acid sequences were aligned by using a hidden Markov model approach developed to align divergent data. Known secondary structures from single-copy crystals were mapped onto the aligned duplicate and triplicate sequences. We show how duplicated dUTPases might fold into a monomer, and we hypothesize that triplicated dUTPases also assemble as monomers. Phylogenetic analysis revealed at least five viral dUTPase sequence lineages in well-supported monophyletic clusters with eukaryotic, eubacterial, and archaeal hosts. We have identified all five as strong examples of horizontal transfer as well as additional potential transfer of dut genes among eubacteria, between eubacteria and viruses, and between retroviruses. The evidence for horizontal transfers is particularly interesting since eukaryotic dut genes have introns, while DNA virus dut genes do not. This implies that an intermediary retroid agent facilitated the horizontal transfer process between host mRNA and DNA viruses.

Amino Acid Sequence↗

PubMed1991

Heterogeneity of gene expression of the hemagglutinin-esterase (HE) protein of murine coronaviruses.

The hemagglutinin-esterase (HE) membrane glycoprotein is present only in some members of the coronavirus family, including some strains of mouse hepatitis virus (MHV). In the JHM strain of MHV, expression of the HE gene is variable and corresponds to the number of copies of a UCUAA pentanucleotide sequence present at the 3'-end of the leader RNA. This copy number varies among MHV strains, depending on their passage history. The JHM isolates with two copies of UCUAA in their leader RNA showed a high level of HE expression, whereas the JHM isolate with three copies had a low-level expression. In this study, the analysis of HE gene expression was extended to other MHV strains. The synthesis of HE mRNA in these viruses also correlates with the copy number of UCUAA in the leader RNA and the particular intergenic sequence preceding the HE gene. In one MHV strain, MHV-1, no detectable HE mRNA was synthesized, despite the presence of a proper transcription initiation signal. This lack of HE mRNA expression was consistent with a leader RNA containing three UCUAA copies. However, mutations and deletions within the coding region of the MHV-1 HE gene have generated a stretch of sequence which resembled the transcriptional initiation motif, and was shown to initiate the synthesis of a novel smaller mRNA. These findings strengthened the theory that interactions between leader RNA and transcriptional initiation sequences regulate MHV subgenomic mRNA transcription. Sequence analysis revealed that most MHV strains, through extensive mutations, deletions, or insertions, have lost the complete HE open reading frame, thus turning HE into a pseudogene. This high degree of variation is unusual as the other three structural proteins (spike, membrane, and nucleocapsid) are well-maintained. In contrast to bovine coronavirus, which apparently requires HE for viral replication, the HE protein in MHV may be only an accessory protein which is not necessary for viral replication. JHM and MHV-S, however, have preserved the expression of HE protein.

Amino Acid Sequence↗

PubMed2024

Inverted triplications formed by iterative template switches generate structural variant diversity at genomic disorder loci.

The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a complex genomic rearrangement (CGR). Although it has been identified as an important pathogenic DNA mutation signature in genomic disorders and cancer genomes, its architecture remains unresolved. Here, we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the DNA of 24 patients identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted structural variant (SV) haplotypes. Using a combination of short-read genome sequencing (GS), long-read GS, optical genome mapping, and single-cell DNA template strand sequencing (strand-seq), the haplotype structure was resolved in 18 samples. The point of template switching in 4 samples was shown to be a segment of ∼2.2-5.5 kb of 100% nucleotide similarity within inverted repeat pairs. These data provide experimental evidence that inverted low-copy repeats act as recombinant substrates. This type of CGR can result in multiple conformers generating diverse SV haplotypes in susceptible dosage-sensitive loci.

Humans↗

PubMed1990

Somatic diversification of chicken immunoglobulin light chains by point mutations.

The light-chain locus of chicken has 1 functional V lambda 1 gene, 1 J gene, and 25 pseudo-V lambda-genes (where V = variable and J = joining). A major problem is which somatic mechanisms expand this extremely limited germ-line information to generate many different antibodies. Weill's group [Reynaud, C. A., Anquez, V., Grimal, H. & Weill, J. C. (1987) Cell 48, 379-388] has shown that the pseudo-V lambda-genes diversify the rearranged V lambda 1 by gene conversion. Here we demonstrate that chicken light chains are further diversified by somatic point mutations and by V lambda 1-J flexible joining. Somatic point mutations were identified in the J and 3' noncoding DNA of rearranged light-chain genes of chicken. These regions were analyzed because point mutations in V lambda 1 are obscured by gene conversion; the J and 3' noncoding DNA are presented in one copy per haploid genome and are not subject to gene conversion. In rodents point mutations occur as frequently in the V-J coding regions as in the adjacent flanking DNA. Therefore, we conclude that somatic point mutations diversify the V lambda 1 of chicken. The frequency (0-1%) and distribution of the mutations (decreasing in number with increased distance from the V lambda 1 segment) in chicken were as observed in rodents. Sequence variability at the V lambda 1-J junctions could be attributed to imprecise joining of the V lambda 1 and J genes. The modification by gene conversion of rearranged V lambda 1 genes in the bursa was similar in chicken aged 3 months (9.5%) or 3 weeks (9.1%)--i.e., gene conversion that generates the preimmune repertoire in the bursa seems to level off around 3 weeks of age. This preimmune repertoire can be further diversified by somatic point mutations that presumably lead to the formation of antibodies with increased affinity. A segment with structural features of a matrix association region [(A + T)-rich and four topoisomerase II binding sites] was identified in the middle of the J-C lambda intron (where C = constant).

Animals↗

PubMed2001

Antigen 43, the major phase-variable protein of the Escherichia coli outer membrane, can exist as a family of proteins encoded by multiple alleles.

agn43 encodes a major phase-variable outer-membrane protein, antigen 43 (Ag43), involved in autoaggregation of Escherichia coli cells. The gene is present in single copy on the chromosome of E. coli K-12. In contrast, Southern hybridization and gene inactivation studies demonstrate that control producer strain E. coli ML308-225 possesses duplicate copies of agn43 (agn43A and agn43B). Construction and analyses of single and double knockout mutants clearly show that both alleles are capable of expressing antigen in a phase-variable manner, with observed differences in the ON<-->OFF switch frequencies appearing to favour expression of Ag43B under conditions of normal laboratory growth. Comparative analysis of agn43A and agn43B gene sequences revealed 98% identity at the nucleotide and predicted protein levels, with differences in the protein sequence of the surface-expressed alpha(43) subunit altering the surface probability of one of the predicted epitopes. Analysis of a panel of enteropathogenic E. coli strains by Southern hybridization using agn43-specific gene probes provided strong evidence for the presence of varying numbers of agn43 alleles within clinical isolates. Taken together, the results indicate the presence of a family of distinct Ag43 proteins encoded by multiple chromosomal alleles.

Adhesins, Bacterial↗

PubMed1996

Molecular and evolutionary analyses of a variable series of genes in Borrelia burgdorferi that are related to ospE and ospF, constitute a gene family, and share a common upstream homology box.

In this study we report on the molecular characterization of a series of genes that constitute a gene family related to ospE and ospF. Some members of this family appear to represent recombined or variant forms of ospE and ospF. Variant ospE and ospF genes were found in several Borrelia burgdorferi isolates, demonstrating that their occurrence is not a phenomenon relevant to only a single isolate. Hybridization analyses revealed that the upstream sequence originally identified 5' of the full-length ospEF operon exists in multiple copies ranging in number from two to six depending on the isolate. This repeated sequence, which we refer to as the upstream homology box (UHB), carries a putative promoter element. In some isolates, UHB elements were found to flank copies of ospE and ospF that exist independently of each other. We refer to this group of UHB-flanked genes collectively as the UHB gene family. The evolutionary relationships among UHB gene family members were assessed through DNA sequence analysis and gene tree construction. These analyses suggest that some UHB-flanked genes might actually represent divergent forms of other previously described genes. Analysis of the restriction fragment length polymorphism patterns of the UHB-flanked genes among B. burgdorferi isolates demonstrated that these patterns are highly variable among isolates, suggesting that these genes are not phylogenetically conserved. The variable restriction fragment length polymorphism patterns could indicate recombinational activity in these sequences. The presence of numerous copies of the UHB elements and the high degree of homology among UHB-flanked genes could provide the necessary elements to allow for homologous recombination, leading to the generation of recombination variants of UHB gene family members.

Amino Acid Sequence↗

PubMed2006

Diversity of trematode genetic clones within amphipods and the timing of same-clone infections.

The genetic diversity of trematodes within second intermediate hosts has important implications for the evolution of trematode populations as these hosts are utilized after the parasites reproduce asexually within first intermediate hosts and before sexual reproduction within definitive hosts. We characterised the genetic clonal diversity of the marine trematode Maritrema novaezealandensis within amphipod (Paracalliope novizealandiae) second intermediate hosts using four to six microsatellite loci to determine if multiple copies of identical trematode clones existed within naturally infected amphipods. To determine the relative timing of infections by identical clones within hosts, trematode metacercariae were assigned to six developmental stages and the stages of identical clones were compared. The genotypes of 306 trematodes were determined from 44 amphipods each containing more than one trematode. Six pairs of identical trematode clones were recovered in total (representing five amphipods: 11% of amphipods with greater than one trematode) and all pairs of clones belonged to the same developmental stage. This suggests that identical clone infections are effectively synchronous. A general decrease in the number of metacercariae recovered, prevalence, and mean intensity of infection for each subsequent developmental stage coupled with large numbers of metacercariae (>9) only being recovered from recent infections, supports the occurrence of post-infection amphipod mortality and/or within-host trematode mortality. Taken together, our results indicate that natural infections are characterised by high genetic diversity, but that amphipods also periodically encounter "batches" of genetically identical clones, potentially setting the stage for interactions within and between clonal groups inside the host.

Amphipoda↗

PubMed2005

Different gvpC length variants are transcribed within single filaments of the cyanobacterium Planktothrix rubescens.

Transcripts of the gas vesicle genes gvpA and gvpC were detected in single filaments of the cyanobacterium Planktothrix rubescens using reverse transcription and quantitative real-time PCR. Primers were designed to amplify short sequences within gvpA and three length variants of gvpC. With genomic template DNA, and using Sybr Green to monitor product accumulation, similar amplification efficiencies were observed for each of these genes. The relative copy numbers of gvpC length variants in genomic DNA from five Planktothrix gas vesicle genotypes determined by real-time PCR were similar to those indicated by sequencing the gas vesicle gene clusters. The precipitation of gvp cDNA reverse-transcribed from cellular RNA from single filaments was required before amplification of the gene fragments; without this step it was not possible to detect the accumulation of the expected amplicons by dissociation analysis. Precipitation was also necessary to ensure the generation of product curves that allowed linear regression in an early stage of PCR, a prerequisite for the quantification of low-input cDNA amounts without the need for standard curves. This report shows that different gvpC length variants are transcribed within single Planktothrix filaments, both from laboratory cultures and from natural samples taken from Lake Zurich. This has implications for the efficiency of buoyancy provision by the possible production of gas vesicles of different strengths within individual cyanobacterial filaments. The hypothesis that post-transcriptional regulation may influence the type of protein (GvpC) present in gas vesicles is presented.

Bacterial Proteins↗

PubMed2002

Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets.

The lack of a convenient high-resolution strain-typing method has hampered the application of molecular epidemiology to the surveillance of bacteria of the Mycobacterium tuberculosis complex, particularly the monitoring of strains of Mycobacterium bovis. With the recent availability of genome sequences for strains of the M. tuberculosis complex, novel PCR-based M. tuberculosis-typing methods have been developed, which target the variable-number tandem repeats (VNTRs) of minisatellite-like mycobacterial interspersed repetitive units (MIRUs), or exact tandem repeats (ETRs). This paper describes the identification of seven VNTR loci in M. tuberculosis H37Rv, the copy number of which varies in other strains of the M. tuberculosis complex. Six of these VNTRs were applied to a panel of 100 different M. bovis isolates, and their discrimination and correlation with spoligotyping and an established set of ETRs were assessed. The number of alleles varied from three to seven at the novel VNTR loci, which differed markedly in their discrimination index. There was positive correlation between spoligotyping, ETR- and VNTR-typing. VNTR-PCR discriminates well between M. bovis strains. Thirty-three allele profiles were identified by the novel VNTRs, 22 for the ETRs and 29 for spoligotyping. When VNTR- and ETR-typing results were combined, a total of 51 different profiles were identified. Digital nomenclature and databasing were intuitive. VNTRs were located both in intergenic regions and annotated ORFs, including PPE (novel glycine-asparigine-rich) proteins, a proposed source of antigenic variation, where VNTRs potentially code repeating amino acid motifs. VNTR-PCR is a valuable tool for strain typing and for the study of the global molecular epidemiology of the M. tuberculosis complex. The novel VNTR targets identified in this study should additionally increase the power of this approach.

Alleles↗