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PubMed1989

An unusual wheat insertion sequence (WIS1) lies upstream of an alpha-amylase gene in hexaploid wheat, and carries a "minisatellite" array.

Comparison of the 5' flanking regions of three alpha-amylase genes from chromosome 6B of hexaploid wheat by heteroduplex and sequence analysis revealed the presence of a 1.6 kb stem-loop insertion sequence (WIS1) in one of them. Polymorphism among hexaploid wheat varieties suggests the relatively recent insertion/excision of this sequence from its present position. The complete sequence of the stem-loop insertion shows that it has many of the features found in transposable elements, including target site duplication and terminal inverted repeats. One unusual feature is a tandem array of direct repeats comprising a wheat "minisatellite" sequence. Both the insertion sequence and the minisatellite are found at multiple locations in the wheat genome, but the functional significance of their association in WIS1 is unknown. The minisatellite arrays share a common core structure, and long arrays are polymorphic between different hexaploid varieties.

Base Sequence↗
PubMed2004

Characterization of an EcoRI family of satellite DNA from two species of the genus Eptesicus (Vespertilionidae; Chiroptera).

We have cloned and sequenced a 321 bp band of repetitive DNA from Eptesicus fuscus and E. serotinus observed after gel electrophoresis of EcoRI digested genomic DNA in both species. Southern blot analysis of genomic DNA (from both species) digested with the same enzyme showed the existence of a ladder pattern indicating that the repetitive DNA is arrayed in tandem. The repetitive sequences have a monomer unit of 321 bp which is composed of two subunits of 160 bp, suggested by the existence of a 160 bp band in the ladder of E. fuscus and by the presence of some direct repeats found in the analysis of the consensus sequence. Analysis of the methylation status demonstrated that cytosines in CCGG sequences in this satellite DNA are methylated in E. fuscus but not in the E. serotinus. Alignment of the sequenced clones showed that several nucleotide positions are diagnostic species-specific and consequently the phylogenetic analysis grouped the monomer units from both species in two clearly separated groups.

Animals↗
PubMed1997

The mouse Vcs2 gene is a composite structure which evolved by gene fusion and encodes five distinct salivary mRNA species.

Genes of the VCS (variable coding sequence) family are characterized by an extensive evolutionary divergence in the protein-coding sequence. The VCS family has been characterized by cDNA cloning from submandibular glands in the rat, mouse and humans. At the genomic level, the sequences of two members of this family are known in the rat Rattus norvegicus: the VCSA1 gene, encoding the prohormone-like polypeptide SMR1, and the VCSB1 gene, encoding a salivary Pro-rich polypeptide. No genomic data were available for the VCS genes of other species. To understand the evolution of the VCS gene family better, we have now sequenced 23 kilobases (kb) of the mouse Vcs2 gene. The Vcs2 sequence reveals numerous genomic reorganizations such as an inversion, insertions of short elements and an unusually high number of long interspersed repeated elements (LINEs), which make up 42% of this region. Interestingly, Vcs2 is composed of three different VCS-like regions. The first of these regions contains all the exons necessary to encode the previously described mouse submandibular gland polypeptide MSG2alpha. This region aligns with the entire genomic sequences of rat VCSA1 and VCSB1 genes. The two other regions align with fragments of these rat sequences. The three regions are arrayed in tandem and flanked by LINEs. In particular, the third region also contains exons that were found in mRNA species from the submandibular gland. In total, we have characterized five mRNAs from mouse submandibular glands which have in common their first exon, and are produced by alternative splicing. Vcs2 is thus a single gene that arose by the fusion of three genes (or pseudogenes) of the VCS multigene family.

Alternative Splicing↗
PubMed1998

Arabidopsis thaliana vegetative storage protein (VSP) genes: gene organization and tissue-specific expression.

We have previously identified two cDNAs encoding vegetative storage proteins (VSPs) in Arabidopsis thaliana. Unlike soybean in which VSPs accumulate at high levels in leaves, A. thaliana VSP mRNAs are abundant in flowers. To understand tissue-specific expression and possible roles of VSPs on reproductive organ development, genes corresponding to VSPs (Vsp1 and Vsp2) and their putative promoters were characterized in this study. Genomic sequence analysis revealed that Vsp1 and Vsp2 resemble each other except in their introns, and that these two genes were organized in a tandem array with an interval of 6 kb in a region. The expression patterns of Vsp1 and Vsp2 were examined using transgenic A. thaliana plants carrying a promoter from Vsp1 or Vsp2 fused to a bacterial beta-glucuronidase (GUS) reporter gene. The promoter from Vsp1 expressed its effect in gynoecia, especially in styles, the basal and distal ends of ovaries and in siliques, whereas the promoter from Vsp2 showed its activity in vegetative shoots, petioles, peduncles and receptacles of floral organs. These results suggest that expression of Vsp1 and Vsp2 may be developmentally regulated in A. thaliana. In the transgenic plants, the GUS activity was induced by wounding in an area around the mid-rib of leaves. Therefore, Vsp1 and Vsp2 promoters appear to have elements required for both tissue specificity and wounding.

Arabidopsis↗
PubMed1988

A head-to-tail tandem organization of hsp70 genes in Trypanosoma cruzi.

We describe the isolation and characterization of the T. cruzi hsp 70 DNA coding region which was found to be formed by multigene copies organized in a tandem array in a head-to-tail manner. The restriction pattern of one of the repetition units within the largest clone obtained from the genomic library, clone Tc70.6, shows that the hsp70 coding region should be formed by at least seven identical copies of 2.5 kb. We have found, however, the presence of restriction polymorphisms (Pvu II) within these repeats. Subsequent analysis of the time course of nuclear DNA digestion has revealed that the copy number per haploid genome could be as greater as 10. The analysis of the DNA and amino acid sequence of a fragment (70%) of one of the repetition units has shown the existence of a high homology with all the hsp70 genes of other organisms. The protein sequence homology of the fragment analyzed is as high as 88% when compared with that of the T. brucei hsp70. On the other hand, there are significant restriction site variations between both. The T. cruzi hsp70 contains at the C-terminal end a tetrapeptide repeat of the structure (GMPG)9.

Amino Acid Sequence↗
PubMed1996

Independent and differential expression of two isotypes of human Nm23: analysis of the promoter regions of the nm23-H1 and H2 genes.

We isolated genomic clones of two isotypes of human NDP kinase, nm23-H1 and H2. The nm23-H1 and H2 genes located in a tandem array contained 5 exons and most of the splicing sites in the exon-intron junctions of two isotypes were essentially identical. The regulatory elements of nm23-H1 and H2 genes were also analysed. One major and several minor transcriptional initiation sites were detected in the two isotypes by 5' RACE analysis in HeLa cell. We also identified them by means of an RNase protection assay and primer extension analysis. Promoter activities were found in the 5' flanking sequences of the two genes when placed upstream of the chloramphenicol acetyltransferase gene. Transcriptional activities of nm23-H1 and H2 regulatory regions were measured in a series of human cancer lines. The nm23-H1/nm23-H2 gene transcriptional activity ratio varied depending on the cell line. DNA sequencing of these two genes showed that their promoter regions contain distinct binding sites for known transcriptional factors. These studies suggest that the two isotypes of the nm23 genes might be regulated dissimilarly, and in cell type specific manner.

Base Sequence↗
PubMed2005

Sister chromatids are often incompletely aligned in meristematic and endopolyploid interphase nuclei of Arabidopsis thaliana.

We analyzed whether sister chromatids are continuously aligned in meristematic and endopolyploid Arabidopsis interphase nuclei by studying sister-chromatid alignment at various chromosomal positions. FISH with individual BACs to flow-sorted 4C root and leaf nuclei frequently yielded more than two hybridization signals, indicating incomplete or absent sister-chromatid alignment. Up to 100% of 8C, 16C, and 32C nuclei showed no sister-chromatid alignment at defined positions. Simultaneous FISH with BACs from different chromosomal positions revealed more frequent sister-chromatid alignment in terminal than in midarm positions. Centromeric positions were mainly aligned up to a ploidy level of 16C but became separated or dispersed in 32C nuclei. DNA hypomethylation (of the whole genome) and transcriptional activity (at FWA gene position) did not impair sister-chromatid alignment. Only 6.1% of 4C leaf nuclei showed sister-chromatid separation of the entire chromosome 1 top arm territories. Homozygous transgenic tandem repeat (lac operator) arrays showing somatic homologous pairing more often than average euchromatic loci did not promote an increased frequency of sister-chromatid alignment. The high frequency of separated sister-chromatid arm positions in > or =4C nuclei suggests that sister-chromatid cohesion is variable, dynamic, and not obligatory along the entire chromosome arm in meristematic and differentiated Arabidopsis nuclei.

Arabidopsis↗
PubMed1976

Restriction endonuclease cleavage of satellite DNA in intact bovine nuclei.

We have analyzed the efficiency with which specific nucleotide sequences within nucleosomes are recognized and cleaved by DNA restriction endonucleases. A system amenable to this sort of analysis is the cleavage of the bovine genome with the restriction endonuclease EcoRI. Bovine satellite I comprises 7% of the genome and is tandemly repetitious with an EcoRI site at 1400 base pair (bp) intervals within this sequence. The ease with which this restriction fragment can be measured permits an analysis of the accessibility of this sequence when organized in a nucleosomal array. Initial studies indicated that satellite I sequences are organized in a nucleosomal structure in a manner analogous to that observed for total genomic DNA. We then examined the accessibility of the EcoRI cleavage sites in satellite to endonucleolytic cleavage in intact nuclei. We find that whereas virtually all the satellite I sequences from naked DNA are cleaved into discrete 1400 bp fragments, only 33% of the satellite I DNA is liberated as this fragment from intact nuclei. These data indicate that 57% of the EcoRI sites in nuclei are accessible to cleavage and that cleavage can occur within the core of at least half the nucleosomal subunits. Analysis of the products of digestion suggests a random distribution of nucleosomes about the EcoRI sites of satellite I DNA. Finally, the observation that satellite sequences can be cleaved from nuclei to 1400 bp length fragments with their associated proteins provides a method for the isolation of specific sequences as chromatin. Using sucrose gradient velocity centrifugation, we have isolated a 70% pure fraction of satellite I chromatin. Nuclease digestion of this chromatin fraction reveals the presence of nucleosomal subunits and indicates that specific sequences can be isolated in this manner without gross disorganization of their subunit structure.

Animals↗
PubMed2002

Replication fork block protein, Fob1, acts as an rDNA region specific recombinator in S. cerevisiae.

BACKGROUND: The analysis of homologous recombination in the tandemly repeating rDNA array of Saccharomyces cerevisiae should provide useful information about the stability of not only the rDNA repeat but also the abundant repeated sequences on higher eukaryotic genomes. However, the data obtained so far are not yet conclusive, due to the absence of a reliable assay for detecting products of recombination in the rDNA array. RESULTS: We developed an assay method to detect the products of unequal sister-chromatid recombination (marker-duplication products) in yeast rDNA. This assay, together with the circular rDNA detection assay, was used for the analysis. Marker-duplication occurred throughout the rDNA cluster, preferentially between nearby repeat units. The FOB1 and RAD52 genes were required for both types of recombinant formation. FOB1 showed a gene dosage effect on not only the amounts of both recombinants, but also on the copy number of the repeat. However, unlike the RAD52 gene, the FOB1 gene was not involved in homologous recombination in a non-rDNA locus. In addition, the marker-duplication products were drastically decreased in the mre11 mutant. CONCLUSION: Our data demonstrate that FOB1- and RAD52-dependent homologous recombination cause the gain and loss of a few copies of the rDNA unit, and this must be a basic mechanism responsible for amplification and reduction of the rDNA copy number. In addition, FOB1 may also play a role in the copy number regulation of rDNA tandem repeats.

DNA, Ribosomal↗
PubMed1995

Characterisation of the telomeres at opposite ends of a 3 Mb Theileria parva chromosome.

Bacteriophage lambda clones containing Theileria parva genomic DNA derived from two different telomeres were isolated and the nucleotide sequences of the telomeric repeats and adjacent telomere-associated (TAS) DNA were determined. The T.parva telomeric repeat sequences, a tandem array of TTTTAGGG or TTTAGGG interspersed with a few variant copies, showed a high degree of sequence identity to those of the photosynthetic algae Chlamydomonas reinhardtii (97% identity) and Chlorella vulgaris (87.7% identity) and the angiosperm Arabidopsis thaliana (84.4% identity). Unlike most organisms which have been studied, no significant repetitive sequences were found in the nucleotide sequences of TAS DNA located centromere-proximal to the telomeric repeats. Restriction mapping and hybridisation analysis of lambda EMBL3 clones containing 16 kilobases of TAS DNA derived from one telomere suggested that they did not contain long regions of repetitive DNA. The cloned TAS DNAs were mapped to T.parva Muguga genomic SfiI fragments 8 and 20, which are located at opposite ends of the largest T.parva chromosome. A 126 bp sequence located directly centromere-proximal to the telomeric repeats was 94% identical between the two cloned telomeres. The conserved 126 bp sequence was present on all T.parva Muguga telomeric SfiI fragments.

Animals↗
PubMed1999

Mitochondrial telomere-binding protein from Candida parapsilosis suggests an evolutionary adaptation of a nonspecific single-stranded DNA-binding protein.

The mitochondrial genome in a number of organisms is represented by linear DNA molecules with defined terminal structures. The telomeres of linear mitochondrial DNA (mtDNA) of yeast Candida parapsilosis consist of tandem arrays of large repetitive units possessing single-stranded 5' extension of about 110 nucleotides. Recently we identified the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of C. parapsilosis linear mtDNA and protects it from attack by various DNA-modifying enzymes (Tomáska, L'., Nosek, J., and Fukuhara, H. (1997) J. Biol. Chem. 272, 3049-3059). Here we report the isolation of MTP1, the gene encoding mtTBP of C. parapsilosis. Sequence analysis revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded DNA-binding proteins that nonspecifically bind to single-stranded DNA with high affinity. Recombinant mtTBP displays a preference for the telomeric 5' overhang of C. parapsilosis mtDNA. The heterologous expression of a mtTBP-GFP fusion protein resulted in its localization to the mitochondria but was unable to functionally substitute for the loss of the S. cerevisiae homologue Rimlp. Analysis of the MTP1 gene and its translation product mtTBP may provide an insight into the evolutionary origin of linear mitochondrial genomes and the role it plays in their replication and maintenance.

Amino Acid Sequence↗
PubMed1992

Genomic organization and nucleotide sequence of a long mosaic repetitive DNA in the mouse genome.

A long mosaic repetitive sequence (LMRS) was isolated from a mouse liver genome library using a mouse repetitive DNA as a probe. LMRS exhibits the following features: (1) it is almost 15 kb in length; (2) it is partly organized in tandem array and frequently interrupted by other repeated sequences; and (3) it is located predominantly on the A3 band of the mouse X Chromosome (Chr). One fragment of LMRS (B6) shows restriction fragment length polymorphism (RFLP) between different mouse strains, and is thus potentially useful for mapping studies. The nucleotide sequence confirms a mosaic organization of LMRS which includes three repeats in the 5' part, showing similarity with the 5' end of L1Md-A2, and seven long A + T rich segments in the central part of the element. Our findings suggest that this sequence may have arisen from the duplication of an ancestral motif and has expanded by successive waves of amplification and invasion by foreign sequences.

Animals↗
PubMed1999

Centromeres from telomeres? The centromeric region of the Y chromosome of Drosophila melanogaster contains a tandem array of telomeric HeT-A- and TART-related sequences.

Cytological and cytogenetic studies have previously defined the region needed for centromeric function in the Y chromosome of Drosophila melanogaster. We have identified a YAC clone that originated from this region. Molecular analysis of the YAC and genomic DNAs has allowed the description of a satellite DNA made of telomeric HeT-A- and TART-derived sequences and the construction of a long-range physical map of the heterochromatic region h18. Sequences within the YAC clone are conserved in the centromeric region of the sibling species Drosophila simulans. That telomere-derived DNA now forms part of the centromeric region of the Y chromosome could indicate a telomeric origin of this centromere. The existence of common determinants for the function of both centromeres and telomeres is discussed.

Animals↗
PubMed1984

The two embryonic U1 small nuclear RNAs of Xenopus laevis are encoded by a major family of tandemly repeated genes.

We have identified a large family of U1 RNA genes in Xenopus laevis that encodes two distinct species of U1 RNA. These genes are expressed primarily at the onset of transcription in the 4,000-cell embryo (D. J. Forbes, M. W. Kirschner, D. Caput, J. E. Dahlberg, and E. Lund, Cell 38:681-689, 1984). The two types of embryonic U1 RNA genes are interspersed and are organized in large tandem arrays. The basic 1.9-kilobase repeating unit contains a single copy of each of the embryonic genes and is reiterated ca. 500-fold per haploid genome. This repetitive U1 DNA accounts for more than 90% of all U1 DNA in X. laevis. In addition to this major family, there exist several minor families of dispersed U1 RNA genes, which presumably encode the oocyte and somatic species of X. laevis U1 RNA. Although the embryonic genes are normally inactive in stage VI oocytes, they are expressed when cloned copies are injected into oocyte nuclei.

Animals↗
PubMed1999

Trisomy 20p resulting from inverted duplication and neocentromere formation.

Normal human centromeres contain large tandem arrays of alpha-satellite DNA of varying composition and complexity. However, a new class of mitotically stable marker chromosomes which contain neocentromeres formed from genomic regions previously devoid of centromere activity was described recently. These neocentromeres are fully functional yet lack the repeat sequences traditionally associated with normal centromere function. We report here a supernumerary marker chromosome derived from the short arm of chromosome 20 in a patient with manifestations of dup(20p) syndrome. Detailed cytogenetic, FISH, and polymorphic microsatellite analyses indicate the de novo formation of the marker chromosome during meiosis or early postzygotically, involving an initial chromosome breakage at 20p11.2, followed by an inverted duplication of the distal 20p segment due to rejoining of sister chromatids and the activation of a neocentromere within 20p12. This inv dup(20p) marker chromosome lacks detectable centromeric alpha-satellite and pericentric satellite III sequences, or centromere protein CENP-B. Functional activity of the neocentromere is evidenced by its association with 5 different, functionally critical centromere proteins: CENP-A, CENP-C, CENP-E, CENP-F, and INCENP. Formation of a neocentromere on human chromosome 20 has not been reported previously and in this context represents a new mechanism for the origin of dup(20p) syndrome.

Centromere↗
PubMed1999

Genomic organization of the genes encoding dihydroflavonol 4-reductase for flower pigmentation in the Japanese and common morning glories.

Genomic DNA segments (approximately 17kb) containing three DFR genes in the Japanese and common morning glories were sequenced. The three DFR genes in both plants were found to be arranged in a tandem array, and all of them comprised six exons with identical intron positions. Their DFR-B genes carrying longer introns than the DFR-A and DFR-C genes were expressed extensively in the young buds of pigmented flowers, and the transcription starting site for the DFR-B mRNA of the Japanese morning glory was determined. The DFR-B gene of the common morning glory was expressed considerably in stems, moderately in sepals and leaves, whereas the DFR-A and DFR-C genes of the same plant were expressed scarcely but significantly in the young flower buds and stems. Several novel mobile element-like sequences of around 200bp were found in the genomic DFR regions. A phylogenetic tree indicated that each DFR gene in the Japanese morning glory is most closely related to the corresponding DFR gene in the common morning glory, and that the DFR-B gene is the most diversified gene among the three DFR genes. These structural and functional features of the DFR genes and their evolutionary implications are discussed.

Alcohol Oxidoreductases↗
PubMed1996

The molecular structure, chromosomal organization, and interspecies distribution of a family of tandemly repeated DNA sequences of Antirrhinum majus L.

Monomers of a major family of tandemly repeated DNA sequences of Antirrhinum majus have been cloned and characterized. The repeats are 163-167 bp long, contain on average 60% A+T residues, and are organized in head-to-tail orientation. According to site-specific methylation differences two subsets of repeating units can be distinguished. Fluorescent in situ hybridization revealed that the repeats are localized at centromeric regions of six of the eight chromosome pairs of A. majus with substantial differences in array size. The monomeric unit shows no homologies to other plant satellite DNAs. The repeat exists in a similar copy number and conserved size in the genomes of six European species of the genus Antirrhinum. Tandemly repeated DNA sequences with homology to the cloned monomer were also found in the North American section Saerorhinum, indicating that this satellite DNA might be of ancient origin and was probably already present in the ancestral genome of both sections.

Base Sequence↗