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The development of tools for diagnosis of tularemia and typing of Francisella tularensis.

Rapid development of molecular techniques for the diagnosis of infections and typing of microbes has been seen during the last 10 years. The present review exemplifies this development by presenting the work of the authors and others regarding techniques for the diagnosis of tularemia and typing of Francisella tularensis. The lack of rapid and safe methods for the laboratory diagnosis of tularemia was the rationale behind the development of methods for the direct detection of F. tularensis in clinical specimens. Today, detection by polymerase chain reaction has become an important adjunct to clinical decisions for the early diagnosis of tularemia. The elucidation of the epidemiology and epizootology of the disease has been hampered by the lack of suitable methods. During recent years several DNA-based methods that allow rapid identification of the four F. tularensis subspecies, including differentiation of strains of the two clinically important subspecies, the highly virulent type A strains and less virulent type B strains, have been developed. Since F. tularensis strains of any origin exhibit highly conserved genomic sequences, the availability of extensive genome sequence data was a prerequisite for the development of a typing system that allows discrimination of individual isolates. The most discriminatory method is based on multiple-locus variable-number tandem repeat analysis (MLVA) and uses highly variable parts of the F. tularensis genome. The method will be an important tool in future studies of the molecular epidemiology of tularemia.

Bacterial Typing Techniques↗

PubMed2026

Multiomic approaches identify a rare CCG repeat expansion in BCLAF3 in neurodevelopmental disorders.

BACKGROUND: Tandem repeat expansions have been implicated in various neurological conditions. Here, we present a novel hypermethylated CCG repeat expansion on Xp22 in the 5'UTR of BCLAF3 in males with neurodevelopmental disorders. METHODS: We used patient-derived fibroblasts and neuronal models from a family with BCLAF3 repeat expansions to generate multiomic data and investigate downstream molecular consequences of the repeat expansion. To identify additional affected individuals with BCLAF3 repeat expansions, we screened methylation arrays (n = 12,375) and short-read genomes (n = 15,963) from probands with neurodevelopmental presentations. We also characterized BCLAF3 repeat expansions in the general population using long-read sequencing data (n = 793) and population-level short-read sequencing data (n = 410,076). RESULTS: Long-read sequencing validated hypermethylation of expanded repeats. Patient-derived cells showed repressed BCLAF3 RNA and protein expression. We show that the BCLAF3 CCG repeat expansion constitutes a previously uncharacterized fragile site (FRAXG) that shifts the surrounding chromatin compartment from open euchromatin to closed heterochromatin. Using our multiomic screening approaches, we identified three additional unrelated males and one related male cousin with long-read sequencing validated (n = 2) or short-read sequencing predicted (n = 2) repeat expansions. In one family, the BCLAF3 repeats segregate with more severe phenotypes than expected for the primary diagnoses. Long-read sequencing in three carrier mothers showed skewed X-inactivation against the repeat expansion, highlighting the potential deleterious effect of an allele with an expansion. Expansions were absent in long-read sequencing data from control populations. Assessment of the BCLAF3 repeat expansion in the UK Biobank indicates that it may be ~ 20X rarer than FMR1 repeat expansions. CONCLUSIONS: CCG repeat expansions in the 5'UTR of BCLAF3 likely constitute a novel genetic etiology associated with X-linked neurodevelopmental phenotypes in males. Future work will be essential to delineate the phenotypic spectrum and determine a disease pathomechanism.

BCLAF3↗

PubMed1996

Polymorphism and concerted evolution in a tandemly repeated gene family: 5S ribosomal DNA in diploid and allopolyploid cottons.

5S RNA genes and their nontranscribed spacers are tandemly repeated in plant genomes at one or more chromosomal loci. To facilitate an understanding of the forces that govern 5S rDNA evolution, copy-number estimation and DNA sequencing were conducted for a phylogenetically well-characterized set of 16 diploid species of cotton (Gossypium) and 4 species representing allopolyploid derivatives of the diploids. Copy number varies over twentyfold in the genus, from approximately 1,000 to 20,000 copies/2C genome. When superimposed on the organismal phylogeny, these data reveal examples of both array expansion and contraction. Across species, a mean of 12% of nucleotide positions are polymorphic within individual arrays, for both gene and spacer sequences. This shows, in conjunction with phylogenetic evidence for ancestral polymorphisms that survive speciation events, that intralocus concerted evolutionary forces are relatively weak and that the rate of interrepeat homogenization is approximately equal to the rate of speciation. Evidence presented also shows that duplicated 5S rDNA arrays in allopolyploids have retained their subgenomic identity since polyploid formation, thereby indicating that interlocus concerted evolution has not been an important factor in the evolution of these arrays. A descriptive model, one which incorporates the opposing forces of mutation and homogenization within a selective framework, is outlined to account for the empirical data presented. Weak homogenizing forces allow equivalent levels of sequence polymorphism to accumulate in the 5S gene and spacer sequences, but fixation of mutations is nearly prohibited in the 5S gene. As a consequence, fixed interspecific differences are statistically underrepresented for 5S genes. This result explains the apparent paradox that despite similar levels of gene and spacer diversity, phylogenetic analysis of spacer sequences yields highly resolved trees, whereas analyses based on 5S gene sequences do not.

Base Sequence↗

PubMed1994

A middle-repetitive DNA sequence element in the sheep parasitic nematode, Trichostrongylus colubriformis.

A novel repetitive DNA sequence in the sheep parasitic nematode Trichostrongylus colubriformis was cloned and sequenced. A 1.1 kb repetitive sequence (Tc15) which hybridized with DNA from T. colubriformis but not with DNA from two other parasitic nematodes, Haemonchus contortus and Ostertagia circumcincta, or sheep was further characterized. Southern blot analysis showed that the repeat hybridized to a range of fragments in restriction digested T. colubriformis DNA and existed in multiple copy number tandem arrays. However, to define clearly the repetitive monomeric unit further screening of phagemid libraries containing BamH I restriction fragments using a subclone of Tc15 as a probe was carried out. Restriction map and sequence data were compiled for 3 clones containing a 145 bp highly repetitive sequence (designated TcREP) which shared homology with the original pTc15 clone. TcREP hybridized to a tandemly repeating sequence monomer of 145 bp in T. colubriformis DNA which was cloned from various genetic environments in the T. colubriformis genome. TcREP homologous sequences were also found in the genomes of two other species of the same genus (Trichostrongylus axei and Trichostrongylus vitrinus) but not in a fourth species (Trichostrongylus rugatus).

Animals↗

PubMed1999

Rare germinal unequal crossing-over leading to recombinant gene formation and gene duplication in Arabidopsis thaliana.

Small, multigene families organized in a tandem array can facilitate the rapid evolution of the gene cluster by a process of meiotic unequal crossing-over. To study this process in a multicellular organism, we created a synthetic RBCSB gene cluster in Arabidopsis thaliana and used this to measure directly the frequency of meiotic, intergenic unequal crossing-over between sister chromatids. The synthetic RBCSB gene cluster was composed of a silent DeltaRBCS1B::LUC chimeric gene fusion, lacking all 5' transcription and translation signals, followed by RBCS2B and RBC3B genomic DNA. Expression of luciferase activity (luc(+)) required a homologous recombination event between the DeltaRBCS1B::LUC and the RBCS3B genes, yielding a novel recombinant RBCS3B/ 1B::LUC chimeric gene whose expression was driven by RBCS3B 5' transcription and translation signals. Using sensitive, single-photon-imaging equipment, three luc(+) seedlings were identified in more than 1 million F2 seedlings derived from self-fertilized F1 plants hemizygous for the synthetic RBCSB gene cluster. The F2 luc(+) seedlings were isolated, and molecular and genetic analysis indicated that the luc(+) trait was caused by the formation of a recombinant chimeric RBCS3B/1B::LUC gene. A predicted duplication of the RBCS2B gene also was present. The recombination resolution break points mapped adjacent to a region of intron I at which a disjunction in sequence similarity between RBCS1B and RBCS3B occurs; this provided evidence supporting models of gene cluster evolution by exon-shuffling processes. In contrast to most measures of meiotic unequal crossing-over that require the deletion of a gene in a gene cluster, these results directly measured the frequency of meiotic unequal crossing-over (approximately 3 x 10(-6)), leading to the expansion of the gene cluster and the formation of a novel recombinant gene.

Arabidopsis↗

PubMed1994

Conservation of human chromosome 13 polymorphic microsatellite (CA)n repeats in chimpanzees.

Tandemly repeated (dC-dA)n.(dG-dT)n sequences occur abundantly and are found in most eukaryotic genomes. To investigate the level of conservation of these repeat sequences in nonhuman primates, we have analyzed seven human chromosome 13 dinucleotide (CA)n repeat loci in chimpanzees by DNA amplification using primers designed for analysis of human loci. Comparable levels of polymorphism at these loci in the two species, revealed by the number of alleles, heterozygosity, and allele sizes, suggest that the (CA)n repeat arrays and their genomic locations are highly conserved. Even though the proportion of shared alleles between the two species varies enormously and the modal alleles are not the same, allelic lengths at each locus in the chimpanzees are detected within the bounds of the allele size range observed in humans. A similar observation has been noted in a limited number of gorillas and orangutans. Using a new measure of genetic distance that takes into account the size of alleles, we have compared the genetic distance between humans and chimpanzees. The genetic distance between these two species was found to be ninefold smaller than expected assuming there is no selection or mutational bias toward retention of (CA)n repeat arrays. These findings suggest a functional significance for these microsatellite loci.

Alleles↗

PubMed1996

Evidence for a sequence-directed conformation periodicity in the genomic highly repetitive DNA detectable with single-strand-specific chemical probe potassium permanganate.

A single-strand-specific chemical probe, potassium permanganate (KMnO4), was used to study the sequence-dependent conformation periodicity of tandem multicopy repetitive DNA sequences HRS60 and GRS (Nicotiana Species) at the level of single base pair and dinucleotide step. Local DNA structures, sensitive to KMnO4, revealed periodicity of 182 +/- 2 bp, equal to the length of repeat units. Permanganate-sensitive local structures were mapped to both DNA strands of genomic HRS60 sequences and were found to be linked to d(A)n tracts. These adenine tracts are located in the proximity of the intrinsically curved domains. Distamycin A increased reactivity of the DNA but decreased the specificity of DNA cleavage. Similar conformation periodicity has been detected also in the 'canrep' family of repeats (Brassica species). All studied repetitive sequences are predominantly located in the constitutive heterochromatin. We discuss the role of conformation periodicities in relation to a structural code for nucleosome phasing at tandem arrays of DNA repeats.

Base Sequence↗

PubMed1996

Structural analysis of the rDNA intergenic spacer of Brassica nigra: evolutionary divergence of the spacers of the three diploid Brassica species.

EcoRI restriction of the B. nigra rDNA recombinants, isolated from a lambda genomic library, showed that the 3.9-kb fragment corresponded to the Intergenic Spacer (IGS), which was sequenced and found to be 3,928 bp in size. Sequence and dot-matrix analyses showed that the organization of the B. nigra rDNA IGS was typical of most rDNA spacers, consisting of a central repetitive region and flanking unique sequences on either side. The repetitive region was composed of two repeat families-RF 'A' and RF 'B.' The B. nigra RF 'A' consisted of a tandem array of three full-length copies of a 106-bp sequence element. RF 'B' was composed of 66 tandemly repeated elements. Each 'B' element was only 21-bp in size and this is the smallest repeat unit identified in plant rDNA to date. The putative transcription initiation site (TIS) was identified as nucleotide position 3,110. Based on the sequence analysis it was suggested that the present organization of the repeat families was generated by successive cycles of deletions and amplifications and was being maintained by homogenization processes such as gene conversion and crossing-over.A detailed comparison of the rDNA IGS sequences of the three diploid Brassica species-namely, B. nigra, B. campestris, and B. oleracea-was carried out. First, comparisons revealed that B. campestris and B. oleracea were close to each other as the repeat families in both showed high sequence homology between each other. Second, the repeat elements in both the species were organized in an interspersed manner. Third, a 52-bp sequence, present just downstream of the repeats in B. campestris, was found to be identical to the B. oleracea repeats, thereby suggesting a common progenitor. On the other hand, in B. nigra no interspersion pattern of organization of repeats was observed. Further, the B. nigra RF 'A' was identified as distinct from the repeat families of B. campestris and B. oleracea. Based on this analysis, it was suggested that during speciation B. campestris and B. oleracea evolved in one lineage whereas B. nigra diverged into a separate lineage. The comparative analysis of the IGS helped in identifying not only conserved ancestral sequence motifs of possible functional significance such as promoters and enhancers, but also sequences which showed variation between the three diploid species and were therefore identified as species-specific sequences.

Base Sequence↗

PubMed1991

Intrachromosomal location of the telomeric repeat (TTAGGG)n.

Eukaryotic telomeres are specialized DNA-protein structures that are thought to ensure chromosomal stability and complete replication of the chromosome ends. All telomeres which have been studied consist of a tandem array of G-rich repeats which seem to be sufficient for telomere function. Originally, the human telomeric repeat (TTAGGG)n was assumed to be exclusively located at the very end of all human chromosomes. More recent evidence, however, suggests an extension into proterminal regions. Very little is known about the interstitial distribution of telomeric repeats. Here we present evidence for the presence of (TTAGGG)n repeats in internal loci on the long and short arms of different human chromosomes. In addition, we studied the genomic organization of these repeats in more detail and discuss possible functions of interstitial telomeric repeats in the human genome.

Base Sequence↗

PubMed1984

Changes in structure and methylation pattern in a cluster of thymidine kinase genes.

Cell line 101 is a thymidine kinase (TK)-positive derivative of Ltk- which contains ca. 20 copies of the herpes simplex virus TK gene organized in a tandem array. DNA methylation at three sites within the gene and flanking sequences was inversely correlated with expression: the sites were unmethylated in line 101, methylated in each of 4 TK-negative derivatives of 101, and unmethylated in each of 21 TK-positive derivatives derived from them. The same three sites were affected in most of the 20 copies of the TK gene, whereas other sites between them were not affected. Although the entire gene cluster was never lost, indicating that integration into the genome was stable, internal rearrangements occurred at a high frequency. The rearrangements had no obvious correlation with the state of methylation or with the expression of the genes.

Animals↗

PubMed1987

Herpes simplex virus infection generates large tandemly reiterated simian virus 40 DNA molecules in a transformed hamster cell line.

When the simian virus 40 (SV40)-transformed Syrian hamster cell line Elona is infected with herpes simplex virus type 1, an excessive amplification of SV40-specific DNA sequences occurs. Analysis of total DNA from herpes simplex virus-infected cells revealed that amplified DNA sequences were present predominantly in a high-molecular-weight form, consisting of a tandem array of many unit-length SV40 DNA molecules. Repeat units of amplified DNA were found to be very similar to standard SV40 DNA as was shown by restriction analyses, except for a small deletion close to the origin of replication, which could also be detected in the chromosomal DNA of uninfected cells. A procedure, devised for selective enrichment of amplified SV40 DNA molecules from the bulk of cellular and herpesviral DNA, allowed molecular cloning of single repeat units and nucleotide sequence analysis of the relative genomic region.

Animals↗

PubMed2004

ORC, MCM, and histone hyperacetylation at the Kaposi's sarcoma-associated herpesvirus latent replication origin.

The viral genome of Kaposi's sarcoma-associated herpesvirus (KSHV) persists as an extrachromosomal plasmid in latently infected cells. The KSHV latency-associated nuclear antigen (LANA) stimulates plasmid maintenance and DNA replication by binding to an approximately 150-bp region within the viral terminal repeats (TR). We have used chromatin immunoprecipitation assays to demonstrate that LANA binds specifically to the replication origin sequence within the KSHV TR in latently infected cells. The latent replication origin within the TR was also bound by LANA-associated proteins CBP, double-bromodomain-containing protein 2 (BRD2), and the origin recognition complex 2 protein (ORC2) and was enriched in hyperacetylated histones H3 and H4 relative to other regions of the latent genome. Cell cycle analysis indicated that the minichromosome maintenance complex protein, MCM3, bound TR in late-G(1)/S-arrested cells, which coincided with the loss of histone H3 K4 methylation. Micrococcal nuclease studies revealed that TRs are embedded in a highly ordered nucleosome array that becomes disorganized in late G(1)/S phase. ORC binding to TR was LANA dependent when reconstituted in transfected plasmids. DNA affinity purification confirmed that LANA, CBP, BRD2, and ORC2 bound TR specifically and identified the histone acetyltransferase HBO1 (histone acetyltransferase binding to ORC1) as a potential TR binding protein. Disruption of ORC2, MCM5, and HBO1 expression by small interfering RNA reduced LANA-dependent DNA replication of TR-containing plasmids. These findings are the first demonstration that cellular replication and origin licensing factors are required for KSHV latent cycle replication. These results also suggest that the KSHV latent origin of replication is a unique chromatin environment containing histone H3 hyperacetylation within heterochromatic tandem repeats.

Acetylation↗

PubMed1996

Trypanosoma rangeli and Trypanosoma cruzi: molecular characterization of genes encoding putative calcium-binding proteins, highly conserved in trypanosomatids.

Genes encoding a 29-kDa flagellar calcium-binding protein (F29) in Trypanosoma cruzi, strongly homologous to EF-hand calcium-binding protein-encoding genes previously reported in this parasite, were isolated by immunoscreening. F29 is encoded by a number of very similar genes, highly conserved among different T. cruzi isolates. The genes are located on a pair of homologous chromosomes, arranged in one or two clusters of tandem repeats. PCR amplification of Trypanosoma rangeli genomic DNA, using primers derived from the T. cruzi F29 sequence made it possible to isolate the homologous gene in T. rangeli, encoding a 23-kDa protein called TrCaBP. Gene sequence comparisons showed homology to EF-hand calcium-binding proteins from T. cruzi (82.8%), Trypanosoma brucei brucei (60.2%), and Entamoeba histolytica (28.4%). Northern blot analysis revealed that the TrCaBP gene is expressed in T. rangeli as a polyadenylated transcript. The TrCaBP-encoding genes are present in at least 20 copies per cell, organized in tandem arrays, on large T. rangeli chromosomes in some isolates and on two smaller ones in others. This gene, however, seems to be absent from Leishmania.

Amino Acid Sequence↗

PubMed1987

Inversion of 5S ribosomal RNA genes within the genus Coprinus.

In the basidiomycete fungus Coprinus cinereus, the 5S ribosomal RNA (rRNA) genes are found in the rDNA repeat which encodes the other three rRNAs (18S, 5.8S, and 26S), and all genes are transcribed in the same direction (Cassidy et al. 1984). To facilitate examination of the rRNA gene organization in additional species, we developed a method to permit determination of the direction of transcription of the rRNA genes using crude preparations of genomic DNA. We used this method to determine the organization of the rDNA in five other members of the order Agaricales (Flammulina velutipes, Agaricus bisporus, Coprinus micaceus, Coprinus atramentarius, and Coprinus comatus). In four, the organization is the same as it is in Coprinus cinereus. However, in Coprinus comatus, the 5S RNA gene is present in the rDNA repeat, but is transcribed in the opposite direction. This alteration in rDNA organization within the genus Coprinus indicates that inverted 5S genes can arise and become propagated through the tandem array of genes. The presence of orientation-dependent promoter elements within the spacer DNA (Elion and Warner 1984) would be expected to place constraints on such inversions.

Agaricales↗

PubMed2003

Evolution of the cystatin B gene: implications for the origin of its variable dodecamer tandem repeat in humans.

The human cystatin B gene contains a variable number of 12-bp tandem repeats in its promoter region, of which the common alleles contain two or three copies and unusual expansion causes progressive myoclonus epilepsy of the Unverricht-Lundborg type. We undertook a comprehensive analysis of the genomic sequence to address the evolutionary events of this variable repeat. By examination of a contiguous genome sequence spanning 5.0 kb and linkage analysis of detected polymorphic changes, we identified six major intragenic haplotypes in unrelated Japanese subjects. The number of normal repeats was closely correlated with these alleles, indicating that changes in the array should be comparatively rare events during human evolution. To examine the origin of the repeat array further, we also analyzed five primate genomes. Repetitive polymorphism was unlikely in hominoids, and the array originated with the dodecamer itself in the course of primate evolution. The variability conceivably developed after the separation to humans.

Cystatin B↗

PubMed2004

The role of unequal crossover in alpha-satellite DNA evolution: a computational analysis.

Human DNA consists of a large number of tandem repeat sequences. Such sequences are usually called satellites, with the primary example being the centromeric alpha-satellite DNA. The basic repeat unit of the alpha-satellite DNA is a 171 bp monomer. Arbitrary monomer pairs usually have considerable sequence divergence (20-40%). However, with the exception of peripheral alpha-satellite DNA, monomers can be grouped into blocks of k-monomers (4 < or = k < or = 20) between which the divergence rate is much smaller (e.g., 5%). Perhaps the simplest and best understood mechanism for tandem repeat array evolution is unequal crossover. Although it is possible that alpha-satellite sequences developed as a result of subsequent unequal crossovers only, no formal computational framework seems to have been developed to verify this possibility. In this paper, we develop such a framework and report on experiments which imply that pericentromeric alpha-satellite segments (which are devoid of higher order structure) are evolutionarily distinct from the higher order repeat segments. It is likely that the higher order repeats developed independently in distinct regions of the genome and were carried into their current locations through an unknown mechanism of transposition.

Algorithms↗

PubMed1997

What makes Grande1 retrotransposon different?

Grande1 elements constitute a family of Ty3 retrotransposons present in the Zea genus in more than 1000 copies in Zea diploperennis and maize. The sequences of three Grande1 flanking regions, two from Z. diploperennis and one from maize, reveal transposable elements as insertion targets, suggesting a preferential integration of Grande1 elements into other transposable elements. These retrotransposons are remarkable for their large size of around 14 kb, which is a consequence of a very large 3' region of more than 7 kb. Atypical entities within this region are two arrays of unrelated tandem repeats with potential stable stem-loop structures. A large portion of the same region is occupied by ORFs, although only ORF23, whose function is unknown, is presumably transcribed in antisense orientation to the reverse transcriptase ORF. Only ORF23 has a codon usage similar to the one tabulated for highly-expressed maize genes. Correspondingly, the transcript of 900 b that hybridizes with ORF23 probes is found in all the maize tissues explored. This is despite the high level of methylation in the DNA of Grande1. Genomic RNA has not been detected in any tissue or situation studied, probably reflecting a non-functional retrotransposon. The origin of ORF23 and the remainder 3' region might be due to a transduction event.

Base Sequence↗

PubMed2006

Complete cellulase system in the marine bacterium Saccharophagus degradans strain 2-40T.

Saccharophagus degradans strain 2-40 is a representative of an emerging group of marine complex polysaccharide (CP)-degrading bacteria. It is unique in its metabolic versatility, being able to degrade at least 10 distinct CPs from diverse algal, plant and invertebrate sources. The S. degradans genome has been sequenced to completion, and more than 180 open reading frames have been identified that encode carbohydrases. Over half of these are likely to act on plant cell wall polymers. In fact, there appears to be a full array of enzymes that degrade and metabolize plant cell walls. Genomic and proteomic analyses reveal 13 cellulose depolymerases complemented by seven accessory enzymes, including two cellodextrinases, three cellobiases, a cellodextrin phosphorylase, and a cellobiose phosphorylase. Most of these enzymes exhibit modular architecture, and some contain novel combinations of catalytic and/or substrate binding modules. This is exemplified by endoglucanase Cel5A, which has three internal family 6 carbohydrate binding modules (CBM6) and two catalytic modules from family five of glycosyl hydrolases (GH5) and by Cel6A, a nonreducing-end cellobiohydrolase from family GH6 with tandem CBM2s. This is the first report of a complete and functional cellulase system in a marine bacterium with a sequenced genome.

Bacterial Proteins↗