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Results for “genomic tandem array”

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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↗

PubMed1996

The Ty1-copia group retrotransposons of Allium cepa are distributed throughout the chromosomes but are enriched in the terminal heterochromatin.

The genomic organization and diversity of the Ty1-copia group retrotransposons has been investigated in a monocotyledonous plant, Allium cepa. We used the polymerase chain reaction (PCR) to generate sequences corresponding to a conserved domain of the reverse transcriptase gene of Ty1-copia retrotransposons in this plant. Sequence analysis of 27 of these PCR products shows that they are a highly heterogeneous population, a feature which is common in plants but not in yeast and Drosophila. Slot-blot analysis shows there are 100,000-200,000 copies of Ty1-copia group retrotransposons within the A. cepa genome (2C = 31.7 pg), indicating that they are a significant component of the genome of this plant. In situ hybridization to metaphase chromosomes reveals that Ty1-copia retrotransposons are distributed throughout the euchromatin of all chromosomes of A. cepa but are enriched in the terminal heterochromatic regions, which contain tandem arrays of satellite sequences. This is the first clear evidence for the presence of Ty1-copia retrotransposons in the terminal heterochromatin of plants and contrasts with the distribution of these elements in other plant species.

Allium↗

PubMed2003

Genomic analysis of facioscapulohumeral muscular dystrophy.

The genomic basis of facioscapulohumeral muscular dystrophy (FSHD) is of considerable interest because of the unique nature of the molecular mutation, which is a deletion within a large, complex DNA tandem array (D4Z4). This repeat maps within 30 kb of the 4q telomere. Although D4Z4 repeat units each contain an open reading frame that could encode a homeodomain protein, there is no evidence that the repeat is transcribed, and the underlying disease mechanism probably involves a position effect. A recent study has identified a protein complex bound to D4Z4 that contains YY1 and HMGB2, implicating a role for D4Z4 as a repressor. The 4q telomere has two variants, 4qA and 4qB. Although these alleles are present at almost equal frequencies in the general population, FSHD is associated only with the 4qA allele and never with 4qB. This suggests a functional difference between the telomere variants, either in predisposition to deletions within D4Z4 or in the pathological consequence of the deletion. Comparative mapping studies of the FSHD region in primates, mouse and Fugu rubripes have given insights into the evolutionary history of the D4Z4 repeat and of 4qter, although as yet they have not provided any solutions to the FSHD puzzle.

Animals↗

PubMed1985

Transfection of mouse fibroblast cells with a promoterless herpes simplex virus thymidine kinase gene: number of integrated gene copies and structure of single and amplified gene sequences.

Plasmids carrying the herpes simplex virus thymidine kinase (tk) gene were used to transfect thymidine kinase-deficient cells of the mouse fibroblast cell line LM(tk-). Individual cell clones were cultivated in selective hypoxanthine-aminopterin-thymidine medium to determine the number of integrated plasmid copies which was almost always in the range of one to three copies per genome. In contrast, cells transfected with plasmids carrying a promoterless "truncated" tk gene typically contained between 10 and 25 copies per genome. Surprisingly, when the truncated tk gene was transfected together with a simian virus 40 DNA segment, including its transcriptional enhancer, the number of integrated tk gene copies was always low, between one and three copies per genome. We have analyzed the genomic organization of integrated truncated tk genes by blot hybridization of restricted cellular DNA and concluded that integrated units of plasmid DNA molecules are arranged in tandem arrays which remain stable in most cases for many cell generations. In only 1 of ca. 20 cell clones did we observe a retraction and expansion of the number of integrated promoterless tk genes as a response to the removal or readdition of selective pressure. Surprisingly, the thymidine kinase activity determined in extracts from cells growing in selective hypoxanthine-aminopterin-thymidine medium (high numbers of integrated tk gene copies) was nearly the same as the enzymatic activity in cells growing in nonselective medium (low copy numbers). Moreover, Northern blots of polyadenylated RNA, extracted from cells growing under selective and nonselective conditions, showed that, in both cases, the major species of tk-specific transcripts was ca. 1.5 kilobases in size, as expected for a tk-specific mRNA containing the entire coding region of the gene. Thus, disproportionate DNA replication appeared not to be essential for an active tk gene expression in these cells. We discuss possible pathways leading to the formation of tandem arrays of integrated truncated tk genes and the conditions required for disproportionate DNA replication in the unique case in which we found a retraction and expansion of tk gene copy numbers as a response to selective growth conditions.

Animals↗

PubMed1986

An RNA polymerase I enhancer in Saccharomyces cerevisiae.

By the use of an artificial gene coding for rRNA (rDNA gene), we found that transcription of the major precursor rRNA in Saccharomyces cerevisiae cells is stimulated 15-fold by a positive control element located 2 kilobases upstream of the transcription initiation site. Analysis of in vitro runon transcripts suggests that this promoter element increases the frequency of initiation by RNA polymerase I molecules. A 190-base-pair fragment encompassing the promoter element can stimulate transcription on a centromere plasmid in either orientation, upstream or downstream of the transcription initiation site, suggesting that it is an enhancer element. Integration of artificial rDNA genes into a nonribosomal locus in the genome demonstrates that the rDNA enhancer functions either 5' or 3' to an rRNA transcription unit, suggesting it may operate in both directions within the rDNA tandem array. This is the first observation in S. cerevisiae of the stimulation of transcription by an element placed downstream. Finally, enhancer activity is dependent upon sequences that lie at both boundaries of the 190-base-pair fragment. In particular, a 5-base-pair deletion at the extreme 3' boundary of the 190-base-pair fragment greatly reduces the activation of transcription and implicates a set of inverted repeats.

Base Sequence↗

PubMed1998

Two extended arrays of a satellite DNA sequence at the centromere and at the short-arm telomere of Chinese hamster chromosome 5.

We have cloned a Chinese hamster chromosome-specific repeated sequence (SatCH5). This satellite is composed of a 33-bp unit organized in two extended tandem arrays. It is localized at the centromere and at the short-arm subtelomere of chromosome 5. Altogether, SatCH5 covers about 1-2 Mb per diploid genome and is not present in other species, including the Syrian hamster and mouse. Since it is known in the Chinese hamster and numerous other vertebrate species that telomeric (TTAGGG)n repeats are localized at the centromeres of several chromosomes, we studied the localization of SatCH5 relative to (TTAGGG)n sequences. Using two-color fluorescence in situ hybridization on stretched chromosomes and on DNA fibers, we have shown that at the centromere of chromosome 5 SatCH5 and the (TTAGGG)n arrays are contiguous. SatCH5 is the first chromosome-specific repetitive sequence located at both the pericentromeric and subtelomeric regions of the same chromosome.

Animals↗

PubMed1987

Physical Analysis of the Campoletis sonorensis Virus Multipartite Genome and Identification of a Family of Tandemly Repeated Elements.

This report is an analysis of cross-hybridizing sequences found within the 28 superhelical (SH) DNAs of the multipartite genome of the polydnavirus Campoletis sonorensis virus (CsV). A Southern cross-blot hybridization analysis showed that the majority of CsV EcoRI restriction fragments cross-hybridize to multiple EcoRI fragments. These sequence homologies were analyzed by hybridizing recombinant clones of the CsV SH DNAs B, H, M, and O(1) to Southern blots of undigested CsV DNA, using different hybridization stringencies. The results indicated that homologous regions among the SH DNAs include closely related sequences that are detectable under stringent conditions and related but more diverged sequences which are only detectable under reduced stringencies. A sequence that hybridized to the majority of the CsV SH DNAs was identified and subcloned from the SH DNAs O(1), H, and B. Nucleotide sequence data revealed that these homologous regions contained a family of imperfectly conserved repeated elements. These repeat elements were arranged singly or in direct tandem arrays and had an average length of 540 base pairs. Within the sequenced regions that contained the repeated elements six putative open reading frames were identified. These results show that the CsV genome consists of SH DNAs with complex sequence interrelationships that may have arisen due to multiple recombinational events.

Journal Article↗

PubMed2006

A comprehensive catalog of human KRAB-associated zinc finger genes: insights into the evolutionary history of a large family of transcriptional repressors.

Krüppel-type zinc finger (ZNF) motifs are prevalent components of transcription factor proteins in all eukaryotes. KRAB-ZNF proteins, in which a potent repressor domain is attached to a tandem array of DNA-binding zinc-finger motifs, are specific to tetrapod vertebrates and represent the largest class of ZNF proteins in mammals. To define the full repertoire of human KRAB-ZNF proteins, we searched the genome sequence for key motifs and then constructed and manually curated gene models incorporating those sequences. The resulting gene catalog contains 423 KRAB-ZNF protein-coding loci, yielding alternative transcripts that altogether predict at least 742 structurally distinct proteins. Active rounds of segmental duplication, involving single genes or larger regions and including both tandem and distributed duplication events, have driven the expansion of this mammalian gene family. Comparisons between the human genes and ZNF loci mined from the draft mouse, dog, and chimpanzee genomes not only identified 103 KRAB-ZNF genes that are conserved in mammals but also highlighted a substantial level of lineage-specific change; at least 136 KRAB-ZNF coding genes are primate specific, including many recent duplicates. KRAB-ZNF genes are widely expressed and clustered genes are typically not coregulated, indicating that paralogs have evolved to fill roles in many different biological processes. To facilitate further study, we have developed a Web-based public resource with access to gene models, sequences, and other data, including visualization tools to provide genomic context and interaction with other public data sets.

Computational Biology↗

PubMed1996

FLP-mediated site-specific recombination in microinjected murine zygotes.

The FLP recombinase of yeast catalyses site-specific recombination between repeated FLP recombinase target (FRT) elements in yeast and in heterologous systems (Escherichia coli, Drosophila, mosquito and cultured mammalian cells). In this report, it is shown that transient FLP recombinase expression can recombine and activate an extrachromosomal silent reporter gene following coinjection into fertilized one-cell mouse eggs. Furthermore, it is demonstrated that introduction of a FLP-recombinase expression vector into transgenic one-cell fertilized mouse eggs induces a recombination event at a chromosomal FRT target locus. The resulting event occurred at the one-cell stage and deleted a chromosomal tandem array of a FRT containing lacZ expression cassette down to one or two copies. These results demonstrate that the FLP recombinase can be utilized to manipulate the genome of transgenic animals and suggest that FLP recombinase-mediated plasmid-to-chromosome targeting is feasible in microinjected eggs.

Animals↗

PubMed1992

Recent advances in minisatellite biology.

Highly polymorphic tandemly repeated 'minisatellite' loci are very abundant in the human genome, and of considerable utility in human genetic analysis. This review describes the use of an ordered-array Charomid library in the systematic and efficient cloning of these regions, and in the analysis of the relative overlap between the different probes used to screen for hypervariable loci. Recent work on the process of mutation leading to the generation of new-length alleles is also discussed, including the observation that at least some mutations may be due to unequal exchanges.

Cloning, Molecular↗

PubMed1996

Characterization of a single-locus minisatellite DNA in Xenopus laevis.

We have cloned a minisatellite tandem array (XTA) from Xenopus laevis that contains approximately 200 copies of the 20-bp repeat 5'-CCAACAGCCTGCCCATCCAT-3'. The XTA sequence is present only once per haploid genome and is polymorphic with respect to repeat number and location of flanking restriction endonuclease sites. Although the 20-bp repeat has not previously been described, flanking sequences suggest that it lies proximal to coding regions in the Xenopus genome.

Animals↗

PubMed1995

Simple tandem DNA repeats and human genetic disease.

The human genome contains many repeated DNA sequences that vary in complexity of repeating unit from a single nucleotide to a whole gene. The repeat sequences can be widely dispersed or in simple tandem arrays. Arrays of up to 5 or 6 nt are known as simple tandem repeats, and these are widely dispersed and highly polymorphic. Members of one group of the simple tandem repeats, the trinucleotide repeats, can undergo an increase in copy number by a process of dynamic mutation. Dynamic mutations of the CCG trinucleotide give rise to one group of fragile sites on human chromosomes, the rare folate-sensitive group. One member of this group, the fragile X (FRAXA) is responsible for the most common familial form of mental retardation. Another member of the group FRAXE is responsible for a rarer mild form of mental retardation. Similar mutations of AGC repeats give rise to a number of neurological disorders. The expanded repeats are unstable between generations and somatically. The intergenerational instability gives rise to unusual patterns of inheritance--particularly anticipation, the increasing severity and/or earlier age of onset of the disorder in successive generations. Dynamic mutations have been found only in the human species, and possible reasons for this are considered. The mechanism of dynamic mutation is discussed, and a number of observations of simple tandem repeat mutation that could assist in understanding this phenomenon are commented on.

Chromosome Mapping↗

PubMed2003

Generation of Ci-Brachyury-GFP stable transgenic lines in the ascidian Ciona savignyi.

We report generation of stable transgenic lines of the ascidian Ciona savignyi carrying a Ciona intestinalis-Brachyury-promoter/Green Fluorescent Protein-reporter (Ci-Bra-GFP) construct. The transgenic lines were made using a technique in which the endonuclease I-SceI was coinjected into fertilized eggs with a transgene construct containing flanking recognition sites for I-SceI. Two founder animals, out of 12 F(0) adults tested, were found to transmit the transgene to their offspring (F(1)s) at frequencies of 42% and 23%. The transgene was further inherited by the F(2) in a Mendelian fashion and displayed nonmosaic expression, indicating integration into the genome. The Mendelian inheritance and the absence of mosaicism persisted through the F(3) and F(4) generations. Southern blot analyses showed that the transgene was organized in tandem arrays of no more than 10 copies. Using these Ci-Bra-GFP transgenics, we describe cellular movements and shape changes involved in notochord morphogenesis in both wildtype and mutant embryos.

Animals↗

PubMed1982

Gene transfer into the mouse germ-line.

A cloned rabbit DNA fragment containing the adult beta-globin gene has been introduced into the mouse germ-line by DNA microinjection into the pronuclei of fertilized mouse eggs. Twenty-four adult mice derived from eggs injected with the cloned DNA were screened for the presence of the rabbit gene in their liver DNA by Southern blot hybridization. Nine of these mice contain the rabbit gene in liver and at least six of them transmit it through the germ-line to a fraction of their offspring. Thus, it should be possible to derive permanent strains of mice carrying the foreign gene. The estimated numbers of copies of the rabbit gene per diploid mouse genome range from one or two up to 20 or more. The multiple copies of the rabbit DNA clone appear to be arranged in tandem head-to-tail arrays that are integrated into mouse chromosomal DNA. Chromosomal integration has been directly demonstrated for one mouse by in situ hybridization to metaphase chromosomes using a radioiodinated probe. In this mouse, most if not all the copies of the rabbit DNA fragment are integrated into one homologue of mouse chromosome 1. The possible use of this technique for the study of globin gene regulation is discussed.

Animals↗

PubMed1995

Repetitive sequence in the Epstein-Barr virus EBNA-3C gene is related to a family of minisatellite arrays in the human genome.

A unique feature of the Epstein-Barr virus (EBV) genome is its high content of repetitive sequences. We identified a new human minisatellite element, tentatively designated MEB-1, that is similar to the 10 x "15bp" tandem repeat within the EBV nuclear antigen-3C (EBNA-3C) coding region. Southern blot analysis showed that the human genome has multiple copies of MEB-1-related repeats and that some of them are highly polymorphic. Both MEB-1 and the 10 x "15bp" repeat contain an octamer consensus GC[A/T]GG[A/T]GG, resembling the prokaryotic recombination signal chi. This octamer was also found in another EBV repeat sequence IR3 and the cellular GGA family of repeats that are related to IR3. Since the octamer motif is generally considered to have a role in the generation of a group of minisatellite DNA, these findings suggest that the four viral and cellular repeats have been generated through similar mechanisms promoted by the motif.

Antigens, Viral↗

PubMed2004

Tn5 as an insect gene vector.

The purpose of this study was to explore alternatives to insect-derived transposable elements as insect gene vectors with the intention of improving existing insect transgenesis methods. The mobility properties of the bacterial transposon, Tn5, were tested in mosquitoes using a transient transposable element mobility assay and by attempting to create transgenic insects. Tn5 synaptic complexes were assembled in vitro in the absence of Mg(2+) and co-injected with a target plasmid into developing yellow fever mosquito, Aedes aegypti, embryos. Target plasmids recovered from embryos a day later were screened for the presence of Tn5. Recombinants (transposition events) were found at a frequency of 1.2 x 10(-3). Some transposition events did not appear to be associated with canonical 9 bp direct duplications at the site of insertion and also were associated with either deletions or rearrangements. A Tn5 element containing the brain-specific transgene, 3 x P3DsRed, was assembled into synaptic complexes in vitro and injected into pre-blastoderm embryos of Ae. aegypti. Of the approximately 900 embryos surviving injection and developing into adults, two produced transgenic progeny. Both transgenic events involved the co-integrations of approximately five elements resulting in nested and tandem arrayed Tn5::3 x P3DsRed elements. This study extends the known host range of Tn5 to insects and makes available to insect biologists and others another eukaryotic genome-manipulation tool. The hyperactivity of synaptic complexes may be responsible for the unusual clustering of elements and managing this aspect of the element's behavior will be important in future applications of this technology to insects.

Aedes↗

PubMed1991

A transposon with an unusual LTR arrangement from Chlamydomonas reinhardtii contains an internal tandem array of 76 bp repeats.

TOC1, a transposable element from Chlamydomonas reinhardtii, is 5662 bases long. The 217 and 237 base long terminal repeat sequences of TOC1 are unusually arranged around the 4600 and 123 base unique regions: [217]-4600-[237] [217]-123-[237]. Although TOC1 contains long terminal repeats and most TOC1 elements are complete, features shared with virus-like retroposons, its unique 4600 base region is more similar to the structure of the L1 family of non-virus retroposons: first, 11 3/4 tandemly repeated copies of a 76 base repeat are found 813 bases from the left end of TOC1, and second using the universal genetic code large open reading frames were not found in TOC1. The relationship between TOC1, virus-like retroposons and the L1 family of non-virus retroposons is unclear and may be very distant since only poor similarity was found between the TOC1 encoded ORFs and retrovirus polypeptides. The length of the tandem array of 76 base repeat sequences was conserved in most TOC1 elements and solo 76 base repeat sequences were not found outside TOC1 elements in the C. reinhardtii genome. Nucleotide substitutions allow all copies of the 76 base repeat to be distinguished from one another.

Base Sequence↗

PubMed1992

VH genes in tandem array comprise a repeated germline motif.

In a study of human VH gene heterogeneity, we have previously used sequence-specific oligonucleotide probes to demonstrate polymorphism of 56pl and three highly homologous VH3 germline elements. We now extend these findings with VH nucleotide sequences obtained from a person who possesses restriction fragments corresponding to each of these four VH3 genes. From a lambda-phage library of genomic DNA, distinct phage clones containing putative 56pl, hv3005, 1.9III, and hv3019b9 genes were selected by screening with oligonucleotide probes. PCR amplification, subcloning, and sequencing from the respective clones 3d216, 3d24, 3d28, and 3d277, yielded exact 56p1, hv3005, 1.9III, and hv3019b9 nucleotide sequences. A panel of oligonucleotide probes was shown to hybridize to these cloned VH3 genes with exact specificity, demonstrating the ability of the probes to predict the sequence of detected target DNA. Based on their chromosomal organization and their previously determined distribution in the population, these VH3 genes represent at least three distinct loci. From each of the VH3-containing phage clones, a VH4 element was also identified and sequenced. Linked to 3d24 and 3d28, respectively, were VH4 sequences identical to hv4005 and 1.9II, corroborating previous reports. The VH4 elements linked to 3d216 and 3d277 were distinct from published VH4 sequences. Nucleotide sequence homology was 97 to 99% among the VH3 sequences, and 93 to 99% among the VH4. These findings indicate that the VH3-VH4 gene pairs we have identified are a repeated germline motif, apparently resulting from multiple duplications of tandemly arrayed VH genes.

Amino Acid Sequence↗