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PubMed2005

Genotyping Mycobacterium ulcerans and Mycobacterium marinum by using mycobacterial interspersed repetitive units.

A novel category of variable tandem repeats (VNTR) called mycobacterial interspersed repetitive units (MIRUs) has been identified for Mycobacterium ulcerans (n = 39), M. marinum (n = 27), and one related organism. Fifteen MIRU loci were identified in the genome of M. marinum and were used to genotype M. ulcerans, M. marinum, and an M. marinum-like organism that is considered a possible missing link between M. marinum and M. ulcerans. Seven MIRU loci were polymorphic, and locus-specific PCRs for four of these loci differentiated seven M. ulcerans genotypes, four M. marinum genotypes, and a unique genotype for the missing link organism. The seven M. ulcerans genotypes were related to six different geographic origins of isolates. All isolates from West and Central Africa, including old and recent isolates, belonged to the same genotype, emphasizing the great spatiotemporal homogeneity among African isolates. Unlike the M. ulcerans genotypes, the four M. marinum genotypes could not be clearly related to the geographic origins of the isolates. According to MIRU-VNTR typing, all M. ulcerans and M. marinum isolates of American origin were closely related, suggesting a common American ancestor for these two pathogenic species on the American continents. MIRU typing has significant potential value for discriminating between reoccurrence and reinfection for M. ulcerans disease.

Bacterial Typing Techniques↗

PubMed1983

Interspersed sequence organization and developmental representation of cloned poly(A) RNAs from sea urchin eggs.

A random primed complementary DNA (cDNA) clone library constructed from total maternal poly(A) RNA of sea urchin eggs was screened with two cloned genomic repetitive sequence probes. Sets of cDNA clones reacting with each of these repetitive sequences were recovered. Most of the cloned transcripts included both single copy and repeat sequence elements. Except for the shared repeat sequence element, both the repetitive and single copy regions of the members of each set of clones failed to crossreact. Single copy probes linked to the repeats on the cloned maternal RNAs are represented in an asymmetric manner. It follows that many different genomic members of a given dispersed repeat sequence family are represented in the maternal RNA. RNA gel blots carried out with several repeat probes display about 10 to 20 prominent maternal poly(A) RNAs containing transcripts of each repetitive sequence family. The interspersed maternal transcripts are 3000 to 15,000 bases in length. Maternal transcripts reacting with single copy probes derived from the cloned cDNAs persist during embryonic development, and in some cases appear to be augmented by similar, newly synthesized embryo transcripts. Two examples were found in which additional transcripts of different length appear at specific developmental stages. The transcribed single copy regions are highly polymorphic in the genomes of different individual sea urchins, and comparisons of closely related sea urchin species showed that both the prevalence and length of specific maternal transcripts change rapidly during evolution. Nucleotide sequences of two homologous repeat elements occurring on different cloned transcripts displayed translation stop codons in every possible reading frame. These repeat sequences display structural features suggesting that there has been evolutionary transposition into transcription units active during oogenesis. The repeat elements and their flanking single copy regions reside either in very long 3' or 5'-terminal sequences, or in unprocessed intervening sequences in the maternal poly(A) RNA. These findings lead us to the proposal that the majority of the cytoplasmic poly(A) RNA in echinoderm eggs and early embryos is similar in form to RNAs that occur in the nucleus rather than to the messenger RNA of later cells.

Animals↗

PubMed2006

A new mechanism for generating TCR diversity: A TCR Jalpha-like gene that inserts partial nucleotide sequences in a D-gene manner.

delta-TCR genes of two gammadelta-T cell hybridomas were found to contain an identical 19-nt sequence in their non-germline N-regions. To determine if this sequence represented a third murine TCR Ddelta gene, genomic PCR was performed by using it as a primer together with primers for interspersed repetitive elements (IRE). Sequencing revealed that the 19-nt segment is part of a 61-nt gene with flanking 5' and 3' recombination signaling sequences (RSS). Southern blot analysis confirmed the presence of this 61-nt gene in the genome of several mouse strains. The gene is unusual in that the distal 24 nucleotides of its 3' RSS region are contributed by the 5' portion of a B2 IRE sequence that includes an apparently non-functional RNA splice site within the 3' nonamer sequence. It has sequence similarities with the Ddelta1 gene (81%) at its 5' end and with Jalpha genes (73%) overall. Tyramide-FISH analysis identified the gene to exist within or adjacent to the TCR alpha/delta locus on chromosome 14. Surveys of available TCR sequences reveal possible partial insertions of the 61-nt gene in other delta-TCR and in alpha-TCR gene sequences. Thus, the unique 61-nt gene is Jalpha gene-like in structure but D gene-like in function.

Amino Acid Sequence↗

PubMed1984

Non-Alu family interspersed repeats in human DNA and their transcriptional activity.

Randomly selected human genomic clones have been surveyed for the presence of non-Alu family interspersed repeats. Four such families of repeats have been isolated and characterized with respect to repetition frequency, interspersion, base sequence, sequence divergence, in vitro RNA polymerase III transcription, elongation of transcripts in isolated nuclei, and in vivo transcription. The two most abundant of the four families of repeats correspond to previously reported families of repeats, namely the kpn I family and poly (CA). We conclude that most of the highly repetitive (greater than 50,000 copies) human interspersed repeats have already been identified. Two lower abundance repeats families are also described here. The abundance with which each of these families is represented in nuclear RNA qualitatively corresponds to their genomic reiteration frequencies. Further, the complementary strands of each repeat family are approximately symmetrically transcribed. The abundance of these repeats in cytoplasmic RNA is qualitatively less than in nuclear RNA. The bulk of the in vivo transcriptional activity of these repeats thus appears to be nonspecific read through from other promoters.

Animals↗

PubMed1981

DNA sequence organization in the flax genome.

The complexity of the flax genome has been determined by reassociation kinetics. The total complexity of one constituent genome was 3.5 . 10(8) nucleotide pairs. The single copy sequences comprised 44% of the genome and showed a long period interspersion pattern with the repetitive sequences. The repetitive sequences occurred in clusters which stretched for at least 10 000 base pairs. Within these clusters the individual repetitive elements were about 650 base pairs. These elements themselves showed little interspersion of different frequency classes in lengths less than 3000 base pairs. The repetitive sequence duplexes formed on reassociation, except for the satellite DNA, showed a high thermal stability. The fold-back DNA comprised 1% of the total genome, and was itself clustered in a small fraction of the genome.

Base Sequence↗

PubMed1996

Genomic repetitive DNA elements of Trypanosoma cruzi.

Repetitive DNA sequences are interspersed throughout the genomes of mammals and other higher eukaryotes, and represent a substantial portion of the genome. Although it has been generally assumed that the redundant DNA is present only in the complex genomes of high order organisms, over the past few years a number of repetitive DNA sequences have been also detected in the protozoan parasite Trypanosoma cruzi. A compilation of the repetitive DNA sequences found in the T. cruzi genome is here presented by Jose Maria Requena, Manuel Carlos López and Carlos Alonso, who also speculate on their possible origin and functional implications regarding retrotransposition and gene regulation.

Journal Article↗

PubMed1992

Molecular analysis of the lethal(1)B214 region at the base of the X chromosome of Drosophila melanogaster.

Approximately 50 kb of genomic DNA was isolated from polytene chromosome bands 19F1 and 2 of Drosophila melanogaster. Bands 19F1 and 2 are in the immediate vicinity of the beta-heterochromatin at the base of the X chromosome and encompass the little fly-like and lethal(1)B214 complementation groups. The cloned DNA consists of an approximately 21 kb stretch of unique or low copy number sequence that is bounded by repetitive elements interspersed with further unique sequences. The presence of repeated sequences is characteristic of regions within and adjacent to beta-heterochromatin. At least part of a tRNA gene cluster is present within the 50 kb of cloned DNA. The cloned region also produces at least 18 discrete size classes of developmentally regulated poly(A)+ RNA species. A 2 kb EcoRI fragment (E10), which lies in the 21 kb stretch of unique sequence, generates seven of these transcripts (of sizes 3.5, 3.35, 2.1, 2.0, 1.5, 1.2 and 1.0 kb) in wild-type flies. However, a small deletion of approximately 75 bp in E10 in a lethal(1)B214 mutant allele is associated with alterations in the production or processing of all seven of these transcripts. These data identify E10 sequences as belonging to the lethal(1)B214 gene and suggest that the wild-type lethal(1)B214 gene encodes multiple transcripts. Furthermore, no transcripts of the same size and having the same developmental profile as those generated by the wild-type E10 fragment were identified by probes covering the remainder of the cloned region. This suggests that at least the larger transcripts hybridizing to E10 are partly transcribed from sequences located outside the cloned region, which indicates that the lethal(1)B214 gene extends for more than 20 kb and contains other transcriptionally active sequences within it.

Animals↗

PubMed1997

Differences and similarities between various tandem repeat sequences: minisatellites and microsatellites.

Tandemly repetitive DNA sequences are abundantly interspersed in the genome of practically all eukaryotic species studied. The relative occurrence of one type of repetitive sequence and its location in the genome appear to be species specific. A common property of repetitive sequences within the living world is their ability to give rise to variants with increased or reduced number of repeats. This instability depends upon numerous parameters whose exact role is unclear: the number of repeats, their sequence content, their chromosomal location, the mismatch repair capability of the cell, the developmental stage of the cell (mitotic or meiotic) and/or the sex of the transmitting parent. It is now apparent that mutations in repetitive sequences are a common cause of human disease, including cancer and disorders which may exhibit a dominant mode of inheritance. Two mechanisms have been proposed to explain the instability of repetitive sequences: DNA polymerase slippage, which may account for the instability of short repeats and unequal recombination which reshuffles repeat variants and maintains repeat heterogeneity in minisatellites. The purpose of this review is to show that no general rule can explain the instability of repetitive sequence. Each sequence of repeats is under the influence of local and general biological activities that determine its level of instability.

Animals↗

PubMed1975

Genome complexity and in vivo transcription in human leukemic leukocytes.

Human DNA contains 35 percent repetitive and 65 percent nonrepetitive sequences. Eight satellite DNAs have now been identified in human DNA. The highly repetitive non-satellite sequences are arranged in blocks 1,100 nucleotides long and interspersed with intermediate repetitive and nonrepetitive sequences greater than 2,500 nucleotides long. Approximately 2.5-3.9 percent of the nonrepetitive sequences are represented in total cellular RNA of normal and leukemic leukocytes.

Base Sequence↗

PubMed1984

Nature and origin of the RNA associated with simian virus 40 large tumor antigen.

Simian virus 40 (SV40) large tumor (T) antigen isolated from mammalian cells undergoing lytic or transforming infection is associated with small RNA fragments ("T-antigen RNA") that are protected from nuclease digestion. The rather high complexity of the ribonuclease T1 fingerprints of T-antigen RNA suggested that it is mainly derived from cellular heterogeneous nuclear RNAs. In the present study, 5'-32P-labeled T-antigen RNA was hybridized to monkey, mouse, and human Alu and SV40 DNA, and the nucleotide sequence of 37 T1 oligonucleotides was determined. The results suggest that the bulk of T-antigen RNA is derived from noncoding, double-stranded, ordered regions of cellular heterogeneous nuclear RNAs that exhibit sequence homologies with interspersed repetitive elements of the cellular genome. The possible biological implications of these results are discussed.

Animals↗

PubMed1997

A newly isolated family of short interspersed repetitive elements (SINEs) in coregonid fishes (whitefish) with sequences that are almost identical to those of the SmaI family of repeats: possible evidence for the horizontal transfer of SINEs.

The SmaI family of repeats is present only in the chum salmon and the pink salmon, and it is not present in five other species in the same genus or in other species in closely related genera. In the present study, we showed that another short interspersed repetitive elements (SINEs) family, which is almost identical to the SmaI family, is present in all fishes in the subfamily Coregoninae, being regarded as the most primitive salmonids. This new family of SINEs was designated the SmaI-cor family (SmaI family of repeats in coregonids). The consensus sequence of the SmaI-cor family was found to be 98.6% homologous to that of the SmaI family. Accordingly, it is difficult to explain the high degree of homology between these two families of SINEs by any mechanism other than the horizontal transfer of SINEs. The estimates of the rate of neutral mutation of nuclear genes, comparing chum salmon and European whitefish, confirmed this possibility. Our results strongly suggest that a member(s) of the SmaI-cor family might have been transferred horizontally from one coregonid species to a common ancestor of chum and pink salmon or to these two species independently, to allow subsequent amplification of the SmaI family in their respective genomes.

Animals↗

PubMed1991

Mitochondrial D-loop sequences are integrated in the rat nuclear genome.

We have cloned two fragments of rat nuclear DNA (nucDNA), 3.3 x 10(3) nucleotide-pairs (knp) and 9.1 knp, that contain a 0.5 knp section sharing 80% sequence identity with the mitochondrial DNA (mtDNA) heavy strand origin of replication (D-loop) nascent strand and 88% identity with each other. The light and heavy strand promoters of the D-loop region are not present in either clone, thus they likely do not function as replication origins in the nuclear genome. The nucDNA sequences surrounding the mtDNA-like sequences are not mitochondrial, thus the mtDNA-like sequences are demonstrably covalently linked in the nuclear genome. Indeed, the surrounding nuclear sequences of each clone also share 88% identity. This sequence arrangement strongly suggests an initial insertion of mtDNA into nucDNA with subsequent amplification of an encompassing region of nucDNA. Divergence calculations suggest that the mtDNA insertion occurred around 13.6 million years ago (MYA) with the subsequent separation occurring around 6.5 MYA. The mtDNA-like sequences of the nuclear clones hybridize strongly to a number of different BamHI-PstI restriction fragments, suggesting either repeated integration and/or frequent mutational events producing new restriction enzyme sites. It is not yet known if one or more of the uncloned D-loop-like sequences are associated with promoters, which would suggest possible function. The 3.3 knp nucDNA fragment is present in low copy number. In contrast, the 9.1 knp nucDNA fragment appears to be moderately repeated. The elements do not appear to be tandemly repeated. The nucDNA clones contain remnants of rat long interspersed repetitive element (LINE) sequences; in addition the 9.1 knp fragment contains sequences with similarity to portions of viral reverse transcriptase and RNaseH genes. Until now, all mtDNA-like sequences found in the nuclear genome have been coding sequences. This is the first confirmation by sequence analysis of a portion of the mtDNA control region in the nuclear genome.

Amino Acid Sequence↗

PubMed1998

REP-PCR fragments as biomarkers for differentiating gastroduodenal disease-specific Helicobacter pylori strains.

We previously identified four potential putative gastroduodenal disease fragments by using the interspersed repetitive extragenic palindromic DNA sequence based PCR (REP-PCR) technique. We investigated these fragments with regard to their disease specificity. The putative disease-specific REP-PCR fragments were cloned, mapped by restriction enzymes, cross-hybridized, and confirmed by Southern hybridization. The four fragments were also used as probes against REP-PCR amplicons from H. pylori isolates obtained from gastritis (N = 20), duodenal ulcer (N = 30), and gastric cancer patients (N = 30). Three of these fragments (1.4- and 0.76-kb for gastritis; 1.35 kb for duodenal ulcer) were amplified without any discrimination between any disease-specific H. pylori isolates. However, amplification following hybridization with the fourth 0.81-kb fragment was observed only from gastritis (60%) and duodenal ulcer (52%) but with none (0%) of gastric cancer patients. Nucleotide sequence analysis of the 0.81-kb fragment revealed that it was an open reading frame of the hypothetical protein HP0373 matched to the position of 380,966 to 383,068 nucleotides of the H. pylori complete genome sequence. Hence, the REP-PCR sequence was not a extragenic palindromic DNA sequence. The hypothetical protein was also present in all the tested isolates. The REP-PCR fingerprinting technique is useful to differentiate disease-specific H. pylori strains based on the interspersed repetitive extragenic palindromic DNA sequences; however, it may not be useful to identify disease-specific virulence determinant(s) without being confirmed by DNA sequence analysis and functional studies.

Amino Acid Sequence↗

PubMed1992

Towards a physical map of the fertility genes on the heterochromatic Y chromosome of Drosophila hydei: families of repetitive sequences transcribed on the lampbrush loops Nooses and Threads are organized in extended clusters of several hundred kilobases.

The understanding of structure and function of the so-called fertility genes of Drosophila is very limited due to their unusual size--several megabases--and their location on the heterochromatic Y chromosome. Since mapping of these genes has mainly been done by classical cytogenetic analyses using a small number of cytologically visible lampbrush loops as the sole markers for particular fertility genes, the resolution of the genetic map of the Y chromosome is restricted to 3-5 Mb. Here we demonstrate that a substantially finer subdivision of the megabase-sized fertility genes in the subtelomeric regions of the Y chromosome of Drosophila hydei can be achieved by a combination of digestion with restriction enzymes having 6 bp recognition sequences, and pulsed field gel electrophoresis. The physical subdivision is based upon large conserved fragments of repetitive DNA in the size range from 50 up to 1600 kb and refers to the long-range organization of several families of repetitive DNA involved in Y chromosomal transcription processes in primary spermatocytes. We conclude from our results that at least five different families of repetitive DNA specifically transcribed on the lampbrush loops nooses and threads are organized as extended clusters of several hundred kb, essentially free of interspersed non-repetitive sequences.

Animals↗

PubMed1976

Evolutionary divergence and length of repetitive sequences in sea urchin DNA.

The organization of repetitive and single copy DNA sequences in sea urchin DNA has been examined with the single strand specific nuclease S1 from Aspergillus. Conditions and levels of enzyme were established so that single strand DNA was effectively digested while reassociated divergent repetitive duplexes remained enzyme resistant. About 25% of sea urchin DNA reassociates with repetitive kinetics to form S1 resistant duplexes of two distinct size classes derived from long and short repetitive sequences in the sea urchin genome. Fragments 2,000 nucleotides long were reassociated to Cot 20 and subjected to controlled digestion with S1 nuclease. About half of the resistant duplexes (13% of the DNA) are short, with a mode size of about 300 nucleotide pairs. This class exhibits significant sequence divergence, and principally consists of repetitive sequences which were interspersed with single copy sequences. About one-third of the long duplexes (4% of the DNA) are reduced in size after extensive S1 nuclease digestion to about 300 nucleotide pairs. About two-thirds of the long resistant duplexes (8% of the DNA) remains long after extensive SI nuclease digestion. These long reassociated duplexes are precisely base paired. The short duplexes are imprecisely paired with a melting temperature about 9 degrees C below that of precisely paired duplexes of the same length. The relationship between length of repetitive duplex and precision of repetition is confirmed by an independent method and has been observed in the DNA of a number of species over a wide phylogenetic area.

Animals↗

PubMed1979

Characterization of the nuclear genome of pearl millet.

The nuclear genome of pearl millet has been characterized with respect to its size, buoyant density in CsCl equilibrium density gradients, melting temperature, reassociation kinetics and sequence organization. The genome size is 0.22 pg. The mol percent G + C of the DNA is calculated from the buoyant density and the melting temperature to be 44.9 and 49.7%, respectively. The reassociation kinetics of fragments of DNA 300 nucleotides long reveals three components: a rapidly renaturing fraction composed of highly repeated and/or foldback DNA, middle repetitive DNA and single copy DNA. The single copy DNA consists of 17% of the genome. 80% of the repetitive sequences are at least 5000 nucleotide pairs in length. Thermal denaturation profiles of the repetitive DNA sequences show high Tm values implying a high degree of sequence homogeneity. About half of the single copy DNA is short (750--1400 nucleotide paris) and interspersed with long repetitive DNA sequences. The remainder of the single copy sequences vary in size from 1400 to 8600 nucleotide pairs.

DNA↗

PubMed1979

Genomic organization in the flesh fly Sarcophaga bullata.

The genome of the flesh fly Sarcophaga bullata has been characterized both cytologically and biochemically. S. bullata has a haploid DNA level of 0.61 picograms which is five times larger than the haploid genome size of Drosophila melanogaster. Reassociation kinetics of Sarcophaga DNA shows that its sequence organization is very similar to that of D. melanogaster in having a very large proportion of single copy DNA (81%) and only small amounts of highly and moderately repetitive DNA (9% and 6%, respectively). cRNAs from all three sequence classes were prepared and their cytological distributions on biploid and polytene cells determined by in situ hybridization. The cytological distribution of the highly repetitive probe was found to be restricted to the centromeric heterochromatin of two of the five autosomes and this sequence class was also found to be markedly underreplicated in polytene foot-pad cells. No highly repetitive DNA was localized on either of the sex chromosomes, but only on the two large centromeric regions of chromosomes C and E. Moderately repetitive DNA was found uniformly distributed on all of the autosomes in both testis and polytene foot-pad squashes. As in the case of the highly repetitive sequence probe, no moderately repetitive DNA was detected on either the X or Y chromosomes. Moderately repetitive DNA in Sarcophaga was also shown to have the "Drosophila type" pattern of sequence interspersion with a moderately repetitive element of congruent to 5,000 nucleotides adjacent to a unique element of greater than 10,000 nucleotides. The Sarcophaga genome is the largest for which this type of interspersion has so far been demonstrated.

Animals↗

PubMed1983

The nucleotide sequence of a new human repetitive DNA consists of eight tandem repeats of 66 base pairs.

Three cloned human DNA fragments obtained from a fibroblast genomic DNA were sequenced and identified as containing members of the well-known 300-bp Alu family of interspersed, middle-repetitive DNA sequences. One of these cloned DNA fragments, p16, also contains members of a new repetitive DNA family, which repeats several thousand times in the human genome. Each member of the new 528-bp family consists of eight tandem repeats of a 66-bp sequence. An AluI recognition site is present at the same location in each repeat, and a 25-bp sequence occurs twice (as a tandem repeat) in each of the eight repeats. There is no sequence homology between the new 528-bp family and the 300-bp Alu family, and the new family lacks the flanking 7- to 20-bp direct repeats as well as the dAMP-rich sequences characteristic of the 300-bp Alu family. Construction of a putative evolutionary tree indicates that six duplication events are needed to give rise to the eight tandemly repeated 66-bp units in the new 528-bp family.

Base Sequence↗