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PubMed2005

Truncated and RIP-degenerated copies of the LTR retrotransposon Pholy are clustered in a pericentromeric region of the Leptosphaeria maculans genome.

The LMR1 5.2 kb interspersed repeat of Leptosphaeria maculans was described by Taylor and Borgmann [Mol. Plant Microbe Interact. 7 (1994) 181] as an uncharacterized repeated element sharing homologies with both LINEs and SINEs. Here, we used the LMR1 sequence as a template to identify the full-length element within a 184-kb genomic sequence corresponding to the pericentromeric region of the 2.80 Mb chromosome of isolate v23.1.3. This region comprises (i) one 6980-bp full-sized Pholy element bordered by two 275- to 280-bp long terminal repeats (LTRs), (ii) five Pholy-related sequences, usually truncated at their 3' ends, and (iii) five solo-LTRs. Structural features strongly suggested that Pholy corresponds to an ancient copia-like retrotransposon, sharing strong homologies with the Elsa retrotransposon of Stagonospora nodorum. Pholy was also suggested to be specific to pericentromeric regions. Comparative analysis of the structure of the Pholy-like sequences occurring in the 184-kb contig and in other parts of the genome showed that this family of repeats is highly degenerated following extensive repeat induced point mutation (RIP).

Amino Acid Sequence↗
PubMed2004

Excision of the Shigella resistance locus pathogenicity island in Shigella flexneri is stimulated by a member of a new subgroup of recombination directionality factors.

Pathogenicity islands are capable of excision and insertion within bacterial chromosomes. We describe a protein, Rox, that stimulates excision of the Shigella resistance locus pathogenicity island in Shigella flexneri. Sequence analysis suggests that Rox belongs to a new subfamily of recombination directionality factors, which includes proteins from P4, enterohemorrhagic Escherichia coli, and Yersinia pestis.

Amino Acid Sequence↗
PubMed2000

Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites.

Cricket paralysis virus is a member of a group of insect picorna-like viruses. Cloning and sequencing of the single plus-strand RNA genome revealed the presence of two nonoverlapping open reading frames, ORF1 and ORF2, that encode the nonstructural and structural proteins, respectively. We show that each ORF is preceded by one internal ribosome entry site (IRES). The intergenic IRES is located 6,024 nucleotides from the 5' end of the viral RNA and is more active than the IRES located at the 5' end of the RNA, providing a mechanistic explanation for the increased abundance of structural proteins relative to nonstructural proteins in infected cells. Mutational analysis of this intergenic-region IRES revealed that ORF2 begins with a noncognate CCU triplet. Complementarity of this CCU triplet with sequences in the IRES is important for IRES function, pointing to an involvement of RNA-RNA interactions in translation initiation. Thus, the cricket paralysis virus genome is an example of a naturally occurring, functionally dicistronic eukaryotic mRNA whose translation is controlled by two IRES elements located at the 5' end and in the middle of the mRNA. This finding argues that eukaryotic mRNAs can express multiple proteins not only by polyprotein processing, reinitiation and frameshifting but also by using multiple IRES elements.

Animals↗
PubMed1999

Organization and sequence of the gene for the human mitochondrial dicarboxylate carrier: evolution of the carrier family.

The dicarboxylate carrier (DIC) is a nuclear-encoded protein located in the mitochondrial inner membrane. It catalyses the transport of dicarboxylates such as malate and succinate across the mitochondrial membrane in exchange for phosphate, sulphate and thiosulphate. We have determined the sequences of the human cDNA and gene for the DIC. The gene sequence was established from overlapping genomic clones generated by PCRs by use of primers and probes based upon the human cDNA sequence. It is spread over 8.6 kb of human DNA and is divided into 11 exons. Five short interspersed repetitive Alu sequences are found in intron I. The protein encoded by the gene is 287 amino acids long. In common with the rat protein, it does not have a processed presequence to help to target it into mitochondria. It has been demonstrated by Northern- and Western-blot analyses that the DIC is present in high amounts in liver and kidney, and at lower levels in all the other tissues analysed. The positions of introns contribute towards an understanding of the processes involved in the evolution of human genes for carrier proteins.

Alu Elements↗
PubMed2004

Sequencing of the Triticum monococcum hardness locus reveals good microcolinearity with rice.

The Hardness ( Ha) locus on chromosome 5D is the main determinant of grain texture in hexaploid wheat. The related genes Puroindoline-a and -b ( Pina-D1, Pinb-D1) and Grain Softness Protein ( Gsp-D1) are tightly linked at this locus. Mutations in the Pina-D1 and Pinb-D1 genes are associated with increased grain hardness. We report here the complete sequence of a 101-kb BAC clone from Triticum monococcum (A(m ) genome) which includes these three genes, and its comparison with the orthologous region in rice. The genes Gsp-A(m) 1, Pina-A(m) 1 and Pinb-A(m) 1 are separated by 37 kb and 32 kb, respectively, and are organized in the same transcriptional orientation. Four additional genes, including a pair of duplicated genes, were identified upstream of Gsp-A(m) 1 within a high-density gene island. These additional genes were found in the same order and orientation, and the same relative distances apart as similar genes previously annotated on rice chromosome 12. An interesting discovery was a small unannotated putative rice gene that was similar to the Gsp-A(m) 1 gene of T. monococcum (65% similarity at the protein level), and that was disposed in the same orientation, and located in the same position relative to the other orthologous genes. The high gene density observed in this BAC (1 gene per 14 kb) was expected for a distal chromosome region, but the level of microcolinearity with rice was higher than that reported in similar distal regions of other wheat chromosomes. Most of the BAC sequence (40%) was represented by repetitive elements, mainly concentrated in regions adjacent to the genes Pina-A(m) 1 and Pinb-A(m) 1. Rearrangements among these repetitive elements might provide an explanation for the frequent deletions observed at this locus in the genomes of the polyploid wheat species.

Amino Acid Sequence↗
PubMed2001

Association between divergence and interspersed repeats in mammalian noncoding genomic DNA.

The amount of noncoding genomic DNA sequence that aligns between human and mouse varies substantially in different regions of their genomes, and the amount of repetitive DNA also varies. In this report, we show that divergence in noncoding nonrepetitive DNA is strongly correlated with the amount of repetitive DNA in a region. We investigated aligned DNA in four large genomic regions with finished human sequence and almost or completely finished mouse sequence. These regions, totaling 5.89 Mb of DNA, are on different chromosomes and vary in their base composition. An analysis based on sliding windows of 10 kb shows that the fraction of aligned noncoding nonrepetitive DNA and the fraction of repetitive DNA are negatively correlated, both at the level of an entire region and locally within it. This conclusion is strongly supported by a randomization study, in which repetitive elements are removed and randomly relocated along the sequences. Thus, regions of noncoding genomic DNA that accumulated fewer point mutations since the primate-rodent divergence also suffered fewer retrotransposition events. These results indicate that some regions of the genome are more "flexible" over the time scale of mammalian evolution, being able to accommodate many point mutations and insertions, whereas other regions are more "rigid" and accumulate fewer changes. Stronger conservation is generally interpreted as indicating more extensive or more important function. The evidence presented here of correlated variation in the rates of different evolutionary processes across noncoding DNA must be considered in assessing such conservation for evidence of selection.

Animals↗
PubMed2002

A genome-wide screen for normally methylated human CpG islands that can identify novel imprinted genes.

DNA methylation is a covalent modification of the nucleotide cytosine that is stably inherited at the dinucleotide CpG by somatic cells, and 70% of CpG dinucleotides in the genome are methylated. The exception to this pattern of methylation are CpG islands, CpG-rich sequences that are protected from methylation, and generally are thought to be methylated only on the inactive X-chromosome and in tumors, as well as differentially methylated regions (DMRs) in the vicinity of imprinted genes. To identify chromosomal regions that might harbor imprinted genes, we devised a strategy for isolating a library of normally methylated CpG islands. Most of the methylated CpG islands represented high copy number dispersed repeats. However, 62 unique clones in the library were characterized, all of which were methylated and GC-rich, with a GC content >50%. Of these, 43 clones also showed a CpG(obs)/CpG(exp) >0.6, of which 30 were studied in detail. These unique methylated CpG islands mapped to 23 chromosomal regions, and 12 were differentially methylated regions in uniparental tissues of germline origin, i.e., hydatidiform moles (paternal origin) and complete ovarian teratomas (maternal origin), even though many apparently were methylated in somatic tissues. We term these sequences gDMRs, for germline differentially methylated regions. At least two gDMRs mapped near imprinted genes, HYMA1 and a novel homolog of Elongin A and Elongin A2, which we term Elongin A3. Surprisingly, 18 of the methylated CpG islands were methylated in germline tissues of both parental origins, representing a previously uncharacterized class of normally methylated CpG islands in the genome, and which we term similarly methylated regions (SMRs). These SMRs, in contrast to the gDMRs, were significantly associated with telomeric band locations (P =.0008), suggesting a potential role for SMRs in chromosome organization. At least 10 of the methylated CpG islands were on average 85% conserved between mouse and human. These sequences will provide a valuable resource in the search for novel imprinted genes, for defining the molecular substrates of the normal methylome, and for identifying novel targets for mammalian chromatin formation.

Amino Acid Sequence↗
PubMed2006

An analysis of mobile genetic elements in three Plasmodium species and their potential impact on the nucleotide composition of the P. falciparum genome.

BACKGROUND: The completed genome sequences of the malaria parasites P. falciparum, P. y. yoelii and P. vivax have revealed some unusual features. P. falciparum contains the most AT rich genome sequenced so far--over 90% in some regions. In comparison, P. y. yoelii is approximately 77% and P. vivax is approximately 55% AT rich. The evolutionary reasons for these findings are unknown. Mobile genetic elements have a considerable impact on genome evolution but a thorough investigation of these elements in Plasmodium has not been undertaken. We therefore performed a comprehensive genome analysis of these elements and their derivatives in the three Plasmodium species. RESULTS: Whole genome analysis was performed using bioinformatic methods. Forty potential protein encoding sequences with features of transposable elements were identified in P. vivax, eight in P. y. yoelii and only six in P. falciparum. Further investigation of the six open reading frames in P. falciparum revealed that only one is potentially an active mobile genetic element. Most of the open reading frames identified in all three species are hypothetical proteins. Some represent annotated host proteins such as the putative telomerase reverse transcriptase genes in P. y. yoelii and P. falciparum. One of the P. vivax open reading frames identified in this study demonstrates similarity to telomerase reverse transcriptase and we conclude it to be the orthologue of this gene. CONCLUSION: There is a divergence in the frequencies of mobile genetic elements in the three Plasmodium species investigated. Despite the limitations of whole genome analytical methods, it is tempting to speculate that mobile genetic elements might have been a driving force behind the compositional bias of the P. falciparum genome.

Amino Acid Sequence↗
PubMed2000

A contiguous 66-kb barley DNA sequence provides evidence for reversible genome expansion.

Organisms with large genomes contain vast amounts of repetitive DNA sequences, much of which is composed of retrotransposons. Amplification of retrotransposons has been postulated to be a major mechanism increasing genome size and leading to "genomic obesity." To gain insights into the relation between retrotransposons and genome expansion in a large genome, we have studied a 66-kb contiguous sequence at the Rar1 locus of barley in detail. Three genes were identified in the 66-kb contig, clustered within an interval of 18 kb. Inspection of sequences flanking the gene space unveiled four novel retroelements, designated Nikita, Sukkula, Sabrina, and BAGY-2 and several units of the known BARE-1 element. The retroelements identified are responsible for at least 15 integration events, predominantly arranged as multiple nested insertions. Strikingly, most of the retroelements exist as solo LTRs (Long Terminal Repeats), indicating that unequal crossing over and/or intrachromosomal recombination between LTRs is a common feature in barley. Our data suggest that intraelement recombination events deleted most of the original retrotransposon sequences, thereby providing a possible mechanism to counteract retroelement-driven genome expansion.

Base Composition↗
PubMed1996

IRS-Bubble PCR: An Effective Method for Representative Amplification of Human Genomic DNA Sequences from Complex Sources

A rate-limiting step in the analysis of large segments of genomic DNA is the generation of a set of representative short single-copy sequences that can be used for development of fine-structure maps. In direct response to this need, we have developed interspersed repetitive DNA sequence (IRS)-bubble PCR. IRS-bubble PCR was designed to amplify the human DNA content of somatic cell hybrids, yeast artificial chromosomes (YACs), BACs, PACs, cosmids, and lambda phage and to result in greater complexity and representation than standard inter-IRS PCR. Here, we describe the application of IRS-bubble PCR to the generation of complex clone libraries for targeted STS development and the construction of robust hybridization probes for FISH mapping, "chromosome painting," or the assembly of cosmid contigs representing the human DNA content of somatic cell hybrids or YACs.

Journal Article↗
PubMed1996

The var genes of Plasmodium falciparum are located in the subtelomeric region of most chromosomes.

PfEMP1, a Plasmodium falciparum-encoded protein on the surface of infected erythrocytes is a ligand that mediates binding to receptors on endothelial cells. The PfEMP1 protein, which is encoded by the large var gene family, shows antigenic variation and changes in binding phenotype associated with alterations in antigenicity. We have constructed a yeast artificial chromosome contig of chromosome 12 from P. falciparum and show that var genes are arranged in four clusters; two lie amongst repetitive subtelomeric sequences and two occur in the more conserved central region. Analysis of parasite chromosomes by pulsed field gel electrophoresis (PFGE) demonstrates that most contain var genes and two-dimensional PFGE has shown that var genes are located at chromosome ends interspersed amongst repetitive sequences present in the subtelomeric complex. Analysis of a var gene located in the subtelomeric region of chromosome 12 has shown that it has close homologues at the opposite end of the chromosome and in the subtelomeric region of two other chromosomes. This suggests that recombination between heterologous chromosomes has occurred in the subtelomeric regions of these chromosomes. The subtelomeric location of var genes dispersed amongst repetitive sequences has important implications for generation of antigenic variants and novel cytoadherent specificities of this protein.

Animals↗
PubMed1988

Nucleotide sequence and transcription of a rat tRNA(Phe) gene and a neighboring Alu-like element.

A bacteriophage gamma Ch4A clone containing a 22-kb rat DNA insert was isolated and found to contain a solitary tRNA(Phe)GAA gene and, 436 bp downstream of it, an Alu-like element. The nucleotide sequence of a 1141-bp DNA fragment containing these genes was determined. The rat tRNA(Phe)GAA gene, with the exception of an additional A in the extra arm, has a sequence identical to that of a rabbit liver tRNA(Phe). The Alu-like element belongs to the rodent B2 family of short interspersed repetitive nucleotide sequences. This repetitive element, B2Phe, is flanked by 12-bp direct repeats, contains an internal split promoter (block A and block B) for RNA polymerase III and is devoid of an A-rich segment at the 3' end. Like other members of the B2 family, the B2Phe element presents 64% sequence homology with rat serine tRNA and contains a serine (GCT) anticodon. Both tRNA(Phe)GAA gene and B2Phe element were found to be transcriptionally active in HeLa cell and Xenopus oocyte nuclear extracts. The tRNA(Phe) gene transcripts were processed during the course of transcription to form mature-size tRNA(Phe). The transcription efficiency of the B2Phe element was found to be an order of magnitude higher than that of the tRNA(Phe) gene. Competition experiments demonstrate that the B2Phe DNA can form a more stable transcription complex than the tRNA(Phe) gene and compete with it for binding of transcription factors.

Animals↗
PubMed1980

Sequence organization of the nuclear DNA of Schizophyllum commune.

Several methods were used to characterize the organization of repetitive DNA in the fungus Schizophyllum commune. They all failed to show interspersion of repetitive sequences among single copy sequences. Saturation hybridization showed that 2.2% of the double-stranded nuclear DNA coded for rRNA. The size of the ribosomal cistron (11.9.10(6) daltons) was determined by restriction enzyme analysis. From these values it was calculated that about 6% of the nuclear DNA consisted of ribosomal cistrons, which approx. equals the amount of repetitive DNA present. Thus, this simple sequence organization in Schizophyllum commune is fundamentally different from organization patterns in higher eukaryotes.

Agaricales↗
PubMed2004

Species-specific accumulation of interspersed sequences in genus Saccharum.

The genus Saccharum consists of two wild and four cultivated species. Novel interspersed sequences were isolated from cultivated sugar cane S. officinarum. These sequences were accumulated in all four cultivated species and their wild ancestral species S. robustum, but were not detected in the other wild species S. spontaneum and the relative Erianthus arundinaceus. The species-specific accumulation of interspersed sequences would correlate to the domestication of sugar canes.

Agriculture↗
PubMed2002

Genotype-dependent transcriptional activation of novel repetitive elements during cold acclimation of alfalfa (Medicago sativa).

In a search for cold-regulated genes that are differentially expressed in alfalfa genotypes of contrasting freezing tolerance, we screened 1036 arrayed cDNA clones. The screening resulted in isolation of cDNA clones, which demonstrated dramatic differences in expression between hardy and un-hardy alfalfa varieties. Detailed analysis revealed that these cDNAs represent parts of novel non-coding repetitive elements carrying long-terminal repeats (LTR) and other retroelement-like features. Despite strong expression under low temperatures, DNA templates remained highly methylated, and a drug-induced decrease in methylation did not activate transcription under normal temperatures. We identified that these repetitive elements represent a large family and could insert into, or be adjacent to, the unrelated polyprotein sequences of putative retrotransposons. These retrotransposons also showed low temperature-induced transcriptional activation; however, this activation was not genotype-dependent. The retroelements described in this study are the first retroelement characterized in the Medicago genus. Furthermore, they represent the only known example of genotype-specific cold-induced transcriptional activation of multiple copies of a repetitive element whose expression is associated with a genotype difference in cold acclimation.

Acclimatization↗
PubMed1975

Structural organization of the genome of the cellular slime mold Dictyostelium discoideum: interspersion of repetitive and single-copy DNA sequences.

The length and interspersion of reiterated and single-copy DNA sequences in Dictyostelium have been examined. The results indicate that approximately 50-60% of the single-copy sequences in DNA fragments 1500 nucleotides long and 75% of the single-copy sequences in fragments 3000 nucleotides long are linked to short interspersed repeat DNA sequences. The average length of these single-copy sequences is 1500 nucleotides. The length of the reiterated DNA has also been analyzed and shows a bimodal distribution. One half is present in sequences greater than 2000 nucleotides long, while the remainder is present as short fragments 250-450 nucleotides long. These shorter fragments are interspersed with the bulk of the single-copy DNA.

Base Sequence↗