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Results for “genomic tandem array”

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PubMed1992

PCR amplification of tandemly repeated DNA: analysis of intra- and interchromosomal sequence variation and homologous unequal crossing-over in human alpha satellite DNA.

Tandemly repeated DNA can comprise several percent of total genomic DNA in complex organisms and, in some instances, may play a role in chromosome structure or function. Alpha satellite DNA is the major family of tandemly repeated DNA found at the centromeres of all human and primate chromosomes. Each centromere is characterized by a large contiguous array of up to several thousand kb which can contain several thousand highly homogeneous repeat units. By using a novel application of the polymerase chain reaction (repPCR), we are able to amplify a representative sampling of multiple repetitive units simultaneously, allowing rapid analysis of chromosomal subsets. Direct sequence analysis of repPCR amplified alpha satellite from chromosomes 17 and X reveals positions of sequence heterogeneity as two bands at a single nucleotide position on a sequencing ladder. The use of TdT in the sequencing reactions greatly reduces the background associated with polymerase pauses and stops, allowing visualization of heterogeneous bases found in as little as 10% of the repeat units. Confirmation of these heterogeneous positions was obtained by comparison to the sequence of multiple individual cloned copies obtained both by PCR and non-PCR based methods. PCR amplification of alpha satellite can also reveal multiple repeat units which differ in size. Analysis of repPCR products from chromosome 17 and X allows rapid determination of the molecular basis of these repeat unit length variants, which appear to be a result of unequal crossing-over. The application of repPCR to the study of tandemly repeated DNA should allow in-depth analysis of intra- and interchromosomal variation and unequal crossing-over, thus providing insight into the biology and genetics of these large families of DNA.

Base Sequence↗

PubMed1993

Generation of a new adenovirus type 12-inducible fragile site by insertion of an artificial U2 locus in the human genome.

Infection with adenovirus type 12 (Ad12) induces four fragile sites in the human genome (H.F. Stich, G.L. van Hoosier, and J.J. Trentin, Exp. Cell Res. 34:400-403, 1964; H. zur Hausen, J. Virol. 1:1174-1185, 1967). The major site, at 17q21-22, contains the U2 gene cluster, which is specifically disrupted by infection in at least a percentage of the cells (D.M. Durnam, J.C. Menninger, S.H. Chandler, P.P. Smith, and J.K. McDougall, Mol. Cell. Biol. 8:1863-1867, 1988). For direct assessment of whether the U2 locus is the target of the Ad12 effect, an artificial locus, constructed in vitro and consisting of tandem arrays of the U2 6-kbp monomer, was transfected into human cells. We report that integration of this artificial locus on the p arm of chromosome 13 creates a new Ad12-inducible fragile site.

Adenoviruses, Human↗

PubMed1981

Instability and reiteration of DNA sequences within the vaccinia virus genome.

The sequence arrangement within the nontranscribed portion of the inverted terminal repetition of the vaccinia virus genome exists in quasi-stable and unstable forms that are not distinguishable on the basis of viral infectivity. The unstable forms, which composed about 20% of a serially passaged stock of virus, were recognized by terminal heterogeneity on restriction endonuclease analysis. Instead of a single terminal fragment from each end of the genome, an array of eight or more fragments differing in size by 1650-base-pair increments was detected. This feature was not eliminated by repeated plaque purification, indicating that the population of DNA molecules with various numbers of reiterations can rapidly evolve from the DNA of a single virus particle. However, at each successive round of plaque purification, about 20% of the unstable isolates revert back to the more stable form. Stable forms are characterized by the presence of a set of 13-17 tandem 70-base-pair repeats on each side of a 435-base-pair intervening sequence near both ends of the genome. In contrast, the unstable forms possess sets of tandem repeats and intervening sequences that alternate many times in series. The transition between the two genomic forms and the evolution of the unstable form appear to be mediated by recombinational events.

Base Sequence↗

PubMed1998

Putative pheromone receptors related to the Ca2+-sensing receptor in Fugu.

By a combination of PCR with degenerate primers and low stringency probing, we have isolated a large family of genes related to the Ca2+-sensing receptor from the genome of Fugu rubripes. One of the genes (type I) is the Fugu homologue of the Ca2+-sensing receptor. The remaining genes can be divided into five classes (type II-VI) on the bases of gene structure. In several types, the genes occur in clusters as tandem arrays. These genes appear to be the homologues of the vomeronasal pheromone receptors recently described in rodents. The Fugu genes are expressed in the tissues of the nose, suggesting that they may have a similar physiological role.

Animals↗

PubMed2006

[Advance in the study of PPR gene family].

PPR (pentatricopeptide repeats) gene family, one of the largest gene families discovered in plants, is characterized by tandem arrays of pentatricopeptide repeats, which play essential roles in mitochondria or chloroplasts, probably via binding to organellar transcripts. In this review, we summarized the current status in the study of PPR family, including the structure character of PPR gene, the distribution in the chromosome arms and other genomes, as well as the biological function of PPR gene.

Chloroplasts↗

PubMed1997

Variations of the C2H2 zinc finger motif in the yeast genome and classification of yeast zinc finger proteins.

The PROSITE pattern Zinc_Finger_C2H2 was extended to permit the detection of all C2H2 zinc fingers and their parent proteins in the recently completed sequence of the yeast genome. Additionally, a new computer program was written that extracts other zinc binding motifs (non C2H2 'fingers'), overlapping with the classical zinc finger pattern, from the found set of yeast C2H2 fingers. The complete and correct detection of all fingers is a prerequisite for the classification of the yeast zinc finger proteins in functional terms. The detected 53 yeast C2H2 zinc finger proteins do not contain finger clusters with 10 or more repeats, as is frequently found in higher eukaryotes. Only three proteins contain four or more fingers in a cluster. Moreover, nearly all 27 yeast proteins with tandem arrays of two or three finger domains can be classified into nine subgroups with high sequence conservation in their finger clusters, in particular of their DNA recognition helices. These results and application of the recently elaborated finger/DNA recognition rules suggest that the yeast proteins belonging to the same subgroup may recognize identical or very similar DNA sites.

Amino Acid Sequence↗

PubMedDate not supplied

Heterologous and homologous plasmid integration at a spore-pigment locus in Penicillium paxilli generates large deletions.

Mutations in a spore pigmentation locus (brs; brown spore) in Penicillium paxilli were isolated at a relatively-high frequency (0.17%) following integrative transformation of the hygromycin-resistance plasmid pAN7-1. A molecular analysis of four independently-isolated Brs- mutants showed that all contained pAN7-1 integrated at a single-site that was unique for each mutant. A previously-described Brs- mutant, YI-34 (Itoh et al. 1994), was a two-site integration. Three of the mutants had multiple copies of pAN7-1 arranged in head-to-tail tandem arrays. A 9.6-kb BamHI junction fragment was cloned from one of these, YI-33, by plasmid rescue and used to isolate two overlapping lambda clones, lambda WB33-1 and lambda WB33-2, that span about 30 kb in the region of the wild-type locus. When genomic digests of the five Brs- mutants were probed with these lambda clones all of them were found to contain an extensive deletion through a common region of the P. paxilli genome. Subsequent attempts to generate one-step gene replacements within a 4.5-kb EcoRI fragment at the wild-type locus resulted in the isolation of Brs- mutants at a frequency of 1.6%, but all mutants with this phenotype were also found to contain an extensive genomic deletion. Therefore, a common outcome of both heterologous and homologous plasmid integration at this locus is deletion formation.

Base Sequence↗

PubMed2003

Transposition of the Drosophila hydei Minos transposon in the mouse germ line.

We tested the suitability of the fly transposon Minos, a member of the Tc1/mariner superfamily, for insertional mutagenesis in the mouse germ line. We generated a transgenic mouse line expressing Minos transposase in growing oocytes and another carrying a tandem array of nonautonomous transposons. The frequency of transposition in the progeny derived from oocytes carrying both transgenes is 8.2%. Analysis of the new integration sites shows a high frequency of transpositions to a different chromosome. Thus Minos transposition could be an effective system for insertional mutagenesis and functional genomic analysis in the mouse.

Animals↗

PubMed1987

The immunoglobulin heavy chain switch: structural features of gamma 1 recombinant switch regions.

The immunoglobulin heavy chain isotype switch is mediated by a DNA rearrangement involving specific genomic segments referred to as switch regions. Switch regions are composed of tandemly repeated simple sequences. The role of the tandemly repeated structure of switch regions in the switch recombination process is not understood. We mapped eight recombination sites--six in the gamma 1 and two in the gamma 3 tandem arrays. In addition, we obtained molecular clones representing three of the six gamma 1 rearrangements, and determined the nucleotide sequences of the recombination sites in each. In general, the rearrangements are confined to the tandem repeat units, and are not clustered in a particular portion of either the gamma 3 or gamma 1 switch region. Nucleotide sequence analysis of one of the recombinant clones, gamma M35, reveals evidence for a successive switch event wherein a recombination between S mu and S gamma 3 was followed by recombination 57 bp downstream with S gamma 1. gamma 1 sequence data from the molecular clones we obtained, together with similar data from other investigators regarding the gamma 1, gamma 2b, and gamma 2a switch regions, reveals that recombinations tend to occur at homologous positions of the respective gamma-unit repeats, adjacent to the elements AGCT and GGGG found in each. This finding suggests that the cutting and religation step of the recombination process is mediated by a recombinase common to the four gamma-isotypes.

Animals↗

PubMed1985

Organization and chromosomal specificity of autosomal homologs of human Y chromosome repeated DNA.

The human Y chromosome contains a group of repeated DNA elements, identified as 3.4-kilobase pair (kb) fragments in Hae III digests of male genomic DNA, which contain both Y-specific and non-Y-specific sequences. We have used these 3.4-kb Hae III Y fragments to explore the organizational properties and chromosomal distribution of the autosomal homologs of the non-Y-specific (NYS) 3.4-kb Hae III Y elements. Three distinct organizations, termed domains, have been identified and shown to have major concentrations on separate chromosomes. We have established that domain K is located on chromosome 15 and domain D on chromosome 16 and suggested that domain R is on chromosome 1. Our findings suggest that each domain is composed of a tandemly arrayed cluster of a regularly repeating unit containing two sets of repeated sequences: one that is homologous to the NYS 3.4-kb Hae III Y sequences and one that does not cross-react with the 3.4-kb Hae III Y repeats. Thus, these autosomal repeated DNA domains, like their Y chromosome counterparts, consist of a complex mixture of repeated DNA elements interspersed among each other in ways that lead to defined periodicities. Although each of the three identified autosomal domains cross-reacts with 3.4-kb Hae III Y fragments purified from genomic DNA, the length periodicities and sequence content of the autosomal domains are chromosome specific. The organizational properties and chromosomal distribution of these NYS 3.4-kb Hae III homologs seem inconsistent with stochastic mechanisms of sequence diffusion between chromosomes.

Animals↗

PubMed1993

Origin of the main class of repetitive DNA within selected Pennisetum species.

In an attempt to identify relationships among genomes of the allotetraploid Pennisetum purpureum Schumach and closely related Pennisetum species with which it can be successfully hybridized, repetitive DNA sequences were examined. Digestion with KpnI revealed two highly repetitive fragments of 140 bp and 160 bp. The possibility that these sequences could be used as genome markers was investigated. Average sequences were determined for the 140 bp and 160 bp KpnI families from P. purpureum and P. squamulatum Fresen. Average sequences (based upon four or five repeats) were determined for the P. glaucum (L.) R. Br. 140 bp KpnI family and the diploid P. hohenackeri Hochst. ex Steud. 160 bp KpnI family. The average sequences of the 160 bp KpnI families in P. purpureum and P. squamulatum differ by only nine bases. The 140 bp KpnI families of the three related species, P. purpureum, P. squamulantum, and P. glaucum are nearly identical, and thus likely represent a recent divergence from a common progenitor or a common genome. Each repetitive sequence may contain internal duplications, which probably diverged following amplification of the original sequence. The 140 bp KpnI repeat probably evolved from the 160 bp KpnI repeat since the missing 18 bp segment is part of the internal duplication that is otherwise conserved in the subrepeats. Tandemly arrayed repetitive sequences in plants are likely to be composed of subrepeats which have been duplicated and amplified.

Base Sequence↗

PubMed2004

Satellite repeats in the functional centromere and pericentromeric heterochromatin of Medicago truncatula.

Most eukaryotic centromeres contain long arrays of tandem repeats, with unit lengths of 150-300 bp. We searched for such repeats in the functional centromeres of the model legume Medicago truncatula (Medicago) accession Jemalong A17. To this end three repeats, MtR1, MtR2 and MtR3, were identified in 20 Mb of a low-pass, whole genome sequencing data set generated by a random shotgun approach. The nucleotide sequence composition, genomic organization and abundance of these repeats were characterized. Fluorescent in situ hybridization of these repeats on chromosomes at meiosis I showed that only the MtR3 repeat, encompassing stretches of 450 kb to more than 1.0 Mb, is located in the functional portion of all eight centromeres. MtR1 and MtR2 occupy distinct regions in pericentromeric heterochromatin. We also studied the presence and distribution of MtRs in Medicago accession R108-1, a genotype with a genome that is 20% smaller than that of Jemalong A17. We determined that while MtR3 is also centromeric on all pachytene bivalents in R108-1, MtR1 and MtR2 are not present in the R108 genome.

Base Sequence↗

PubMed1997

Mutations at the human minisatellite MS32 integrated in yeast occur with high frequency in meiosis and involve complex recombination events.

Minisatellites are composed of tandem repetitive DNA sequences and are present at many positions in the human genome. They frequently mutate to new length alleles in the germline, by complex and incompletely understood recombination mechanisms which may operate during meiosis. In several minisatellites the mutation events are restricted to one end of the repeat array, indicating a possible association with elements that act in cis. Mutant alleles do not show exchange of flanking regions. To construct a model system suitable for further investigations of the mutation process, we have integrated the human minisatellite MS32, flanked by synthetic markers, in the vicinity of a meiotic recombination hot spot upstream of the LEU2 locus in the yeast Saccharomyces cerevisiae. Here we provide direct evidence for a meiotic origin of MS32 mutations. Mutation events were polarised towards both ends of the minisatellite and varied from simple duplications and deletions to complex intra- and interallelic events. Interallelic events were frequently accompanied by exchange of regions flanking the minisatellite. The results also support the notion that cis-acting elements are involved in the mutational process. The fact that MS32 mutant structures are similar in yeast and human shows that meiotic recombination plays a crucial role in both organisms and emphasises the usefulness of yeast strains harbouring minisatellites as a model system for the study of minisatellite mutation.

Alleles↗

PubMed2001

Detection and isolation of minisatellite Pc-1 binding proteins.

Minisatellites (MNs) are arrays of 5-100 nucleotide repeats that are dispersed throughout the genome of vertebrates. They demonstrate alteration in tumors and in cells exposed to various carcinogens, but the molecular mechanisms underlying the induction of mutations at MNs are largely unknown. Hypervariable MN Pc-1 isolated from the mouse genome consists of tandem repeats of d(GGCAG) flanked with locus-specific sequences at both ends. We have found that MN mutations are induced in NIH3T3 cells by treatment with okadaic acid using a Pc-1 MN fragment as a probe. In order to shed light on the molecular mechanisms, we isolated six MN Pc-1 binding proteins, pA, pB, pD, pE, pF and pG, from nuclear extracts of NIH3T3 cells treated with okadaic acid. While pA and pB bound to the G-rich strand of Pc-1, pD, pE, pF and pG bound to the complementary C-rich strand. Sequence specificities for DNA binding were revealed and one base substitution and insertion into the Pc-1 repeat unit dramatically changed the affinity of each protein, suggesting that they bind to Pc-1 and Pc-1-like MNs in vivo.

3T3 Cells↗

PubMed1979

Cloned pairs of variable region genes for immunoglobulin heavy chains isolated from a clone library of the entire mouse genome.

To investigate the organization of immunoglobulin genes, we have constructed a clone library containing 10(6) randomly generated fragments of mouse embryo DNA, corresponding to eight equivalents of the genome. The cloning method involved methylation of embryo DNA at EcoRI recognition sites, partial digestion by EcoRI* endonclease activity, and direct ligation of the resulting large fragments to the lambda phage vector Charon 4A. The library was searched for sequences homologous to a cloned complementary DNA copy of a mu heavy chain mRNA. Nine clones bearing variable heavy chain (VH) sequences were isolated, representing at least eight distinct VH genes. Thus, multiple related VH genes are available in the genome to contribute to immunoglobulin diversity. Each of the two clones carries a pair of VH genes, one pair separated by 15 +/- 1 kilobase pairs of mouse DNA and the other by 14 +/- 2 kilobase pairs. This indicates that related VH genes are clustered and may occur in a tandem array having a repeating unit of 14--16 kilobase pairs. The large spacer sequences between VH genes cannot, however, be highly conserved.

Animals↗

PubMed1982

Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by homologous recombination.

Animals↗

PubMed2005

Integrating gene and protein expression data: pattern analysis and profile mining.

Proteomics and functional genomics are emerging new research fields devoted to the study of the entire collection of proteins and mRNA transcripts (collectively known as gene products) that define a biological system. DNA microarrays are now a popular platform for measuring changes in messenger RNA transcript levels on a genome-wide scale, while gel-free shotgun profiling methods based on tandem mass spectrometry are increasingly being used to determine the identity, modification states, and relative abundance of large numbers of proteins. By defining the behavior of entire biological pathways and networks under various physiological states, these studies aim to extend traditional reductionist molecular genetic approaches regarding the biological roles of the vast array of uncharacterized gene products. A key goal is to determine how the information encoded by the myriad of expressed gene products is integrated at the molecular, cellular, and even whole organism level to create the dynamic biochemical processes and complex physiological controls that sustain life. While comparison of the complementary information contained in proteomic and mRNA data sets poses considerable analytical challenges, these efforts should provide added insight into the fundamental mechanisms underlying physiology, development, and the emergence of disease. Here, we outline several analytical approaches, methods, and tools that have proven to be helpful in the face of this important challenge.

Algorithms↗

PubMed2000

A polymorphic multigene family encoding an immunodominant protein from Babesia microti.

Human babesiosis in the United States is caused predominantly by Babesia microti, a tick-transmitted blood parasite. Improved testing methods for the detection of infection with this parasite are needed, since asymptomatic B. microti infection represents a potential threat to the blood supply in areas where B. microti is endemic. We performed immunoscreening of an expression library of genomic DNA from a human isolate of B. microti (strain MN1). Among 17 unique immunoreactive clones, we identified 9 which represent a related family of genes with little sequence homology to other known sequences but with an architecture resembling that of several surface proteins of Plasmodium. Within this family, a tandem array of a degenerate six-amino-acid repeat (SEAGGP, SEAGWP, SGTGWP, SGTVGP) was found in various lengths between relatively well conserved segments at the N and C termini. In order to examine within-clone variation, we developed a PCR protocol for direct recovery of a specific bmn1-6 homologue directly from 30 human blood isolates, 4 corresponding hamster isolates, and 5 geographically corresponding Peromyscus leucopus (white-footed mouse) isolates. Isolates from the hamsters had the same sequences as those found in the corresponding human blood, suggesting that genetic variation of bmn1-6 does not occur during passage. However, clones from different patients were often substantially different from each other with regard to the number and location of the degenerate repeats within the bmn1-6 homologue. Moreover, we found that strains that were closely related geographically were also closely related at the sequence level; nine patients, all from Nantucket Island, Mass., harbored clones that were indistinguishable from each other but that were distinct from those found in other northeastern or upper midwestern strains. We conclude that considerable genetic and antigenic diversity exists among isolates of B. microti from the United States and that geographic clustering of subtypes may exist. The nature of the bmn1-6 gene family suggests a mechanism of antigenic variation in B. microti that may occur by recombination, differential expression, or a combination of both mechanisms.

Amino Acid Sequence↗