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PubMed1997

Chromosome-specific alpha-satellite DNA from the centromere of chimpanzee chromosome 4.

The centromeric regions of human and primate chromosomes are characterized by diverged subsets of tandemly repeated alpha-satellite DNA. Comparison of the alpha-satellites on known homologous chromosomes in human and chimpanzee provides insight into the very rapid evolution of satellite DNA sequences and the mechanisms that shape complex genomes. By using oligonucleotide primers specific for a conserved region of human alpha-satellite DNA, we have amplified a chromosome-specific alpha-satellite subset from the chimpanzee genome by the polymerase chain reaction. Fluorescence in situ hybridization showed that clones palphaPTR4N and palphaPTR4H are homologous to sequences at the centromere of the chimpanzee chromosome 4. This alpha-satellite subset is organized as a series of pentameric (higher-order) repeats, operationally defined by digestion of genomic DNA with HaeIII, MboI, RsaI, SstI, and XbaI. The lengths of four independent centromeric arrays measured by pulsed-field gel electrophoresis varied between 800 and 3,500 kb (mean = 1,850 kb, SD = 1,000 kb). Nucleotide sequence analysis demonstrated that chimpanzee chromosome 4 alpha-satellite is most closely related to the suprachromosomal subfamily II, which is evolutionarily different from the subfamily I to which the alpha-satellite on the homologous human chromosome 5 belongs. This implies that the human-chimpanzee sequence divergence has not arisen from a common ancestral alpha-satellite repeat(s) but instead represents concerted evolution of distinct repeats on homologous chromosomes.

Animals↗

PubMed1996

A minisatellite polymorphism in intron III of the barramundi (Lates calcarifer) growth hormone gene.

This paper describes the detection of a polymorphism within the growth hormone (GH) gene of the fish barramundi (Lates calcarifer). PCR amplification of barramundi genomic DNA generated three different sized products: A, 409 bp; B, 478 bp; and H, 520 bp. Each barramundi isolate displayed one of four different types of profiles, which contained specific combinations of these PCR products. Sequence analysis confirmed that products A and B are different forms of the barramundi GH gene, and studies showed that product H was an artifact due to heteroduplex formation between the two smaller-sized molecules. The polymorphic nature of these PCR products was due to differences in the number of repeat monomers within the 5' end of the barramundi decaminisatellite, an AT-rich repetitive sequence that was identified within intron III of this gene. The barramundi decaminisatellite consisted of 24 or 28 10-nucleotide imperfect direct repeat monomers in a tandem array. The monomers were grouped into one of three different families and evidence for monomer homogenization by crossover fixation was presented. The barramundi decaminisatellite differed from previously reported AT- or GC-rich minisatellites, although a similar decaminisatellite has been identified in intron III of the tilapia GH gene.

Animals↗

PubMed1999

Related RNAs in lepidopteran cells after in vitro infection with Hyposoter didymator virus define a new polydnavirus gene family.

In the present study, we describe the isolation and the characterization of three different Hyposoter didymator virus (HdV) lepidopteran host-expressed genes, the products of which might interfere with the host physiology during parasitism. In this report, we study the expression of HdV genes in Sf9 cells infected with HdV since results indicate that the Sf9 model mimics to some extent the in vivo model and may be utilized to study expression of HdV genes in lepidopteran host cells. This system allowed us to isolate three HdV-specific cDNAs, termed M24, M27, and M40. cDNA nucleotide sequence analysis demonstrated significant regions of homology. The three cDNAs displayed repeated sequences arranged in tandem array that might have evolved through domain duplication. Similar to other previously described polydnavirus host-expressed genes, two intron positions have been found in the M24 leader region. The cDNAs corresponded to RNAs of 1.5, 1.6, and 2.3 kb that are also detected in parasitized Spodoptera littoralis larvae. They are encoded by different genes likely located on different HdV DNA molecules. Corresponding RNAs are detected early postinfection and remain detectable for at least 10 days postinfection. They encode secreted glycine- and proline-rich proteins. An antiserum raised against a baculovirus recombinant M24-encoded protein detected similar proteins in the culture medium of infected lepidopteran cells and in parasitized host hemolymph. We propose that the three cloned genes belong to an HdV gene family specifically expressed in parasitized lepidopteran hosts.

Amino Acid Sequence↗

PubMed1994

Tandemly repeated transgenes of the human minisatellite MS32 (D1S8), with novel mouse gamma satellite integration.

The human hypervariable minisatellite MS32 has a well characterised internal repeat unit array and high mutation rates have been observed at this locus. Analysis of MS32 mutants has shown that male germline mutations are polarised to one end of the array and frequently involve complex gene conversion-like events, suggesting that tandem repeat instability may be modulated by cis-acting sequences flanking the locus. In order to investigate the processes affecting MS32 mutation rate and mechanism, we have created transgenic mice harbouring an MS32 allele. Here we describe the organisation of eight transgenic insertions. Analysis of these transgenic loci by MVR-PCR and structural analysis of the junctions between mouse flanking DNA and the transgenic loci has shed light on mechanisms of integration and rearrangement of the tandem repeated transgenes. Sequence analysis of the mouse DNA flanking these transgenes has shown that 5 of the 8 insertions have integrated into mouse gamma satellite repeated sequence. This suggests a non-random integration of the MS32 transgene construct into the mouse genome.

Alleles↗

PubMed2006

Characterization of the centromere and peri-centromere retrotransposons in Brassica rapa and their distribution in related Brassica species.

We report the identification and characterization of the major repeats in the centromeric and peri-centromeric heterochromatin of Brassica rapa. The analysis involved the characterization of 88 629 bacterial artificial chromosomes (BAC) end sequences and the complete sequences of two BAC clones. We identified centromere-specific retrotransposons of Brassica (CRB) and various peri-centromere-specific retrotransposons (PCRBr). Three copies of the CRB were identified in one BAC clone as nested insertions within a tandem array of 24 copies of a 176 bp centromeric repeat, CentBr. A complex mosaic structure consisting of nine PCRBr elements and large blocks of 238 bp degenerate tandem repeats (TR238) were found in or near a derivative of 5S-25S rDNA sequences. The chromosomal positions of selected repeats were determined using in situ hybridization. These revealed that CRB is a major component of all centromeres in three diploid Brassica species and their allotetraploid relatives. However, CentBr was not detected in the most distantly related of the diploid species analyzed, B. nigra. PCRBr and TR238 were found to be major components in the peri-centromeric heterochromatin blocks of four chromosomes of B. rapa. These repetitive elements were not identified in B. oleracea or B. nigra, indicating that they are A-genome-specific. GenBank accession numbers: KBrH001P13 (AC 166739); KBrH015B20 (AC 166740); end sequences of KBrH BAC library (CW 978640 - CW 988843); end sequences of KBrS BAC library (DU 826965 - DU 835595); end sequences of KBrB BAC library (DX 010661 - DX 083363).

Brassica↗

PubMed1991

Frequent rearrangements of rRNA-encoding chromosomes in Giardia lamblia.

The ribosomal RNA (rRNA) genes in Giardia lamblia are present as short tandem arrays of a 5.6 Kb repeat unit on at least six telomeres. Four of these telomeres have the same overall organisation comprising a domain ranging in size from 25 to 300 Kb, delineated chromosome internally by a conserved island of restriction enzyme sites. Cloned lines of G. lamblia derived from the WB strain contain polymorphic subsets of chromosomes encoding rRNA genes. However, changes in the size of the rRNA telomere domains of these polymorphic chromosomes alone cannot account for the total size changes in the chromosomes. The rearrangement events are very frequent: 60% of subcloned lines had discrete rearranged karyotypes that differed from each other, suggesting either an estimated rearrangement rate that may be as high as 3% per division or a cloning-induced rearrangement event. The extreme plasticity of the genome has obvious implications for the maintenance of a functional genome and the control of gene expression in Giardia.

Animals↗

PubMed1999

Cis-regulation of inter-allelic exchanges in mutation at human minisatellite MS205 in yeast.

Tandemly repeated DNA is a major component of the human genome, and includes loci contributing to human disease. Minisatellites include the most variable human loci described to date, and the mechanisms by which this variation is generated in humans have been studied in detail. Integration of human minisatellites into yeast not only provides a model for further dissecting the molecular basis of length change mutation at these loci, but also more generally allows the study of complex recombinational events in yeast. We have used human minisatellite MS205 integrated into yeast to study the structural details of length change mutations. Apart from showing that mutation at this locus in yeast has features similar to those observed at some minisatellites in humans, including meiosis-specificity, and polarity, in which exchange events are localised to one extremity of the array, we here, for the first time, directly demonstrate that a flanking element in yeast regulates the mutation process. The results therefore support the hypothesis that flanking initiators are involved in minisatellite mutation in humans. Furthermore, mutant alleles showed more complex rearrangements in one orientation than the other. The data also suggest that the mutational pathway for deletions might be different from the pathway generating inter-allelic exchanges and duplications.

Alleles↗

PubMed1983

Structural proteins of adenovirus-associated virus: subspecies and their relatedness.

Capsids of adenovirus-associated virus (AAV) are known to contain three major structural proteins (A, B, and C). We have further resolved distinct subspecies of two of the major AAV proteins (two forms of protein A and four forms of protein C) which were found in both AAV1 and AAV2 serotypes. All subspecies were accurately synthesized in a cell-free translation system programmed with RNA isolated from infected cells. Analysis of virion proteins from the autonomous parvovirus H1 did not reveal a comparable array of subspecies of its major components. Staphylococcal V8 protease digestion of C proteins from AAV1 and AAV2 yielded very different electrophoretic patterns, indicating a considerable difference between the C proteins of these two serotypes, despite a high degree of genomic homology and an overall similarity in the number and relative proportions of analogous capsid proteins. On the other hand, staphylococcal V8 protease digestion of isolated proteins A, B, and C of AAV2 showed an extensive overlap among these proteins, possibly equivalent to all of protein C. In conjunction with other data, these findings suggest that proteins A, B, and C arise from different in-frame initiation sites contained in mRNA sequences that are transcribed from the right half of the AAV genome. The heterogeneity of subspecies may be explained by a partial read through of several tandem termination codons near the 3' end of AAV mRNA.

Dependovirus↗

PubMed1996

Characterization of a chromosome-specific chimpanzee alpha satellite subset: evolutionary relationship to subsets on human chromosomes.

Alpha satellite DNA is a tandemly repeated DNA family found at the centromeres of all primate chromosomes examined. The fundamental repeat units of alpha satellite DNA are diverged 169- and 172-bp monomers, often found to be organized in chromosome-specific higher-order repeat units. The chromosomes of human (Homo sapiens (HSA)), chimpanzee (Pan troglodytes (PTR) and Pan paniscus), and gorilla (Gorilla gorilla) share a remarkable similarity and synteny. It is of interest to ask if alpha satellite arrays at centromeres of homologous chromosomes between these species are closely related (evolving in an orthologous manner) or if the evolutionary processes that homogenize and spread these arrays within and between chromosomes result in nonorthologous evolution of arrays. By using PCR primers specific for human chromosome 17-specific alpha satellite DNA, we have amplified, cloned, and characterized a chromosome-specific subset from the PTR chimpanzee genome. Hybridization both on Southern blots and in situ as well as sequence analysis show that this subset is most closely related, as expected, to sequences on HSA 17. However, in situ hybridization reveals that this subset is not found on the homologous chromosome in chimpanzee (PTR 19), but instead on PTR 12, which is homologous to HSA 2p.

Animals↗

PubMed1998

Conspiracy of silence among repeated transgenes.

Transgenic experiments in vertebrates often involve the insertion of tandem multiple-copy arrays at single sites. For many transgenes, expression is unpredictable from site to site, a phenomenon usually attributed to a repressive environment caused by nearby sequences. However, an alternative explanation comes from evidence that transgene repeat arrays in flies condense into heterochromatin, suggesting that low levels of expression in vertebrate transgene arrays might result from interactions between repeats within the array. A recent experiment using transgenic mouse lines demonstrates that reduction in copy number of silenced transgenes within an array leads to a striking increase in expression, demonstrating that silencing is intrinsic to the array, and is not attributable to position effects of nearby sequences. This work calls into question functions that have been attributed to vertebrate locus control regions and boundaries, and draws attention to the notion that repeat-induced gene silencing is a system for protection of eukaryotic genomes against threatening sequence elements.

Animals↗

PubMed2006

Mechanisms of human minisatellite mutation in yeast.

Minisatellites are tandem repeat loci, with repeat units ranging in size from 5 bp to 100 bp. The total lengths of repeat arrays vary from about 0.5 kb to 30 kb, and excessive variability in allele length at human minisatellite loci is the result of germline-specific complex recombination events generating new length alleles. Minisatellite alleles also mutate to new lengths in somatic cells, but this occurs at a much lower rate than in the germline. Since recombination is involved in minisatellite mutation, the yeast Saccharomyces cerevisiae is a suitable model organism that has been employed to further dissect the molecular basis of mutation events at human minisatellites. These studies have shown that the mutational behaviour of a minisatellite in meiosis is not determined by the intrinsic properties of the repeat array, but are highly dependent on the position of the minisatellite in the genome. The processes for minisatellite mutation in yeast and humans are identical in the sense that mutation is indeed driven by meiotic recombination, but differ with regard to the types of structural changes that are generated by the recombination events. Tetrad analyses showed that inter-allelic transfers of repeats occur by conversion and not crossing over, and that several chromatids can be involved in successive recombination events in one meiosis, resulting in mutant alleles in several spores. It has been demonstrated that the genes SPO11 and RAD50, involved in the initiation of recombination events, are required for human minisatellite mutation in yeast meiosis. Intrinsic properties of the repeat array appear to determine the stability of human minisatellites in yeast mitosis, since mitotic mutation rates in yeast are highly variable between minisatellites. The repair genes RAD27 and DNA2 stabilise human minisatellites in yeast mitosis, while RAD5 has no effect on mitotic stability. MSH2 depresses human minisatellite frequency in meiotic cells of yeast.

Chromatids↗

PubMed1985

Studies on cellular tandemization of herpes simplex virus thymidine kinase DNA.

The cellular tandemization of the herpes simplex virus (HSV) thymidine kinase (tk) gene was studied in tk- mouse fibroblasts after gene transfer by microinjection into the nucleus or by calcium phosphate-mediated transfection. Three different DNA substrates, designed to yield simple integration patterns, were used: a gel-purified 3.6-kb Bam HI fragment containing the HSV tk gene; the same fragment self-ligated; and the 3.6-kb fragment ligated to a Bam HI-cleaved subset of genomic mouse DNA. The genomic DNA of six independently isolated transformed cell lines was analyzed by Southern blotting and the structure of the tk-specific DNA was studied. The data suggest that modifications (mutations, deletions, recombination events, and recircularization, etc.) of the input DNA fragment occur early after its introduction into the cell. Subsequently these structures are multiplied in a directional manner, generating larger arrays of DNA with distinct and regularly repeated areas. These concatemers can eventually be integrated into the host genome.

Animals↗

PubMed2004

A DNA microarray system for forensic SNP analysis.

Forensic DNA analysis is routinely performed using polymorphic short tandem repeat (STR) markers. However, for degraded or minute DNA samples, analysis of autosomal single nucleotide polymorphisms (SNPs) in short fragments might be more successful. Furthermore, sequencing of mitochondrial DNA (mtDNA) is often performed on highly degraded or scarce samples due to the high copy number of mtDNA in each cell. Due to the increasing number of complete mtDNA genome sequences available, the limited discrimination power of an mtDNA analysis, may be increased by analysis of coding region polymorphisms in addition to the non-coding variation. Since sequence analysis of the coding region would require more material than generally present in forensic samples, an alternative SNP analysis approach is required. We have developed a one-colour microarray-based SNP detection system for limited forensic materials. The method is based on minisequencing in solution prior to hybridisation to universal tag-arrays. In a first outline of a forensic chip, a combination of 12 nuclear and 21 mitochondrial SNP markers are analysed simultaneously. The mitochondrial markers on the chip are polymorphisms within the hypervariable region as well as in the coding region. Even though the number of markers in the current system is limited, it can easily be extended to yield a greater power of discrimination. When fully developed, microarray analysis provides a promising system for efficient sensitive SNP analysis of forensic samples in the future.

Complementarity Determining Regions↗

PubMed2005

The SCAN domain family of zinc finger transcription factors.

Zinc finger transcription factor genes represent a significant portion of the genes in the vertebrate genome. Some Cys2His2 type zinc fingers are associated with conserved protein domains that help to define these regulators. A novel domain of this type, the SCAN domain, is a highly conserved 84-residue motif that is found near the N-terminus of a subfamily of C2H2 zinc finger proteins. The SCAN domain, which is also known as the leucine rich region, functions as a protein interaction domain, mediating self-association or selective association with other proteins. Here we define the mouse SCAN domain and annotate the mouse SCAN family members. In addition to a single SCAN domain, some of the members of the mouse SCAN family members have a conserved N-terminal motif, a KRAB domain, SANT domains and a variable number of C2H2 type zinc fingers (3-14). The genes encoding mouse SCAN domains are clustered, often in tandem arrays, and are capable of generating isoforms that may affect the function of family members. Although the function of most of the family members is not known, an overview of selected members of this group of transcription factors suggests that some of the mouse SCAN domain family members play roles in cell survival and differentiation.

Amino Acid Sequence↗

PubMed2004

Molecular organization of large fragments in the maize B chromosome: indication of a novel repeat.

The supernumerary B chromosome has no apparent effects on plant growth, and its molecular makeup is difficult to unravel, due to its high homology to the normal complement, which prevents conventional cloning. This difficulty was overcome previously by microdissecting the B chromosome under the microscope to result in 19 B clones, one of which is B specific and highly repetitive, dispersing over one-third of the B long arm and most regions of the centromeric knob. To gain insights into the molecular structure of the B chromosome, this sequence was used to screen a genomic library constructed from W22 carrying 16 B's. Five clones (>10 kb each) were isolated, and all were repetitive, showing homology with A chromosomes in Southern and FISH analyses. Two of them were further characterized and sequenced. Each is composed of several restriction fragments with variable degrees of repetitiveness. Some of these are B specific and others have variable degrees of homology with the A chromosomes. The order of each characteristic group is not contiguous; they intersperse within those of other groups. Sequence analysis reveals that their sequences ( approximately 26 kb) have no homology with any published gene other than sequences of transposable elements (retrotransposons and MITEs) and the B as well as the A centromeres. We uncovered a 1.6-kb CL-repeat sequence, seven units of which were present in the two clones in defective forms. Those repeats mostly arrange in tandem array in the B chromosome. Moreover, we detected transposition of a retrotransposon and a MITE element involved in the genesis of these two sequences.

Base Sequence↗

PubMed1998

Selective sweep of a newly evolved sperm-specific gene in Drosophila.

The pattern of genetic variation across the genome of Drosophila melanogaster is consistent with the occurrence of frequent 'selective sweeps', in which new favourable mutations become incorporated into the species so quickly that linked alleles can 'hitchhike' and also become fixed. Because of the hitchhiking of linked genes, it is generally difficult to identify the target of any putative selective sweep. Here, however, we identify a new gene in D. melanogaster that codes for a sperm-specific axonemal dynein subunit. The gene has a new testes-specific promoter derived from a protein-coding region in a gene encoding the cell-adhesion protein annexin X (AnnX), and it contains a new protein-coding exon derived from an intron in a gene encoding a cytoplasmic dynein intermediate chain (Cdic). The new transcription unit, designated Sdic (for sperm-specific dynein intermediate chain), has been duplicated about tenfold in a tandem array. Consistent with the selective sweep of this gene, the level of genetic polymorphism near Sdic is unusually low. The discovery of this gene supports other results that point to the rapid molecular evolution of male reproductive functions.

Amino Acid Sequence↗

PubMed1995

Human gamma X satellite DNA: an X chromosome specific centromeric DNA sequence.

The cosmid clone, CX16-2D12, was previously localized to the centromeric region of the human X chromosome and shown to lack human X-specific alpha satellite DNA. A 1.2 kb EcoRI fragment was subcloned from the CX16-2D12 cosmid and was named 2D12/E2. DNA sequencing revealed that this 1,205 bp fragment consisted of approximately five tandemly repeated DNA monomers of 220 bp. DNA sequence homology between the monomers of 2D12/E2 ranged from 72.8% to 78.6%. Interestingly, DNA sequence analysis of the 2D12/E2 clone displayed a change in monomer unit orientation between nucleotide positions 585-586 from a "tail-to-head" arrangement to a "head-to-tail" configuration. This may reflect the existence of at least one inversion within this repetitive DNA array in the centromeric region of the human X chromosome. The DNA consensus sequence derived from a compilation of these 220 bp monomers had approximately 62% DNA sequence similarity to the previously determined gamma 8 satellite DNA consensus sequence. Comparison of the 2D12/E2 and gamma 8 consensus sequences revealed a 20 bp DNA sequence that was well conserved in both DNA consensus sequences. Slot-blot analysis revealed that this repetitive DNA sequence comprises approximately 0.015% of the human genome, similar to that found with gamma 8 satellite DNA. These observations suggest that this satellite DNA clone is derived from a subfamily of gamma satellite DNA and is thus designated gamma X satellite DNA. When genomic DNA from six unrelated males and two unrelated females was cut with SstI or HpaI and separated by pulsed-field gel electrophoresis, no restriction fragment length polymorphisms were observed for either gamma X (2D12/E2) or gamma 8 (50E4) probes. Fluorescence in situ hybridization localized the 2D12/E2 clone to the lateral sides of the primary constriction specifically on the human X chromosome.

Base Sequence↗

PubMed1990

Structural analysis of the short length ribosomal DNA variant from Pisum sativum L. cv. Alaska.

The genomic clone, RRNpss1, representing the short ribosomal DNA length variant in Pisum sativum L. cv. Alaska, has been isolated and the 2859 bp intergenic spacer, along with the 25S rRNA 3' border and 18S rRNA 5' border, has been sequenced. The intergenic spacer contains nine tandem repeats, approximately 180 bp in length, which show greater than 80% sequence homology to each other. The RNA polymerase I transcription start site and a processing site, located 776 bp and 536 bp upstream of the 5' end of 18S rRNA, respectively, have been determined using S1 analysis. The region surrounding the +1 site shows strong homology between the positions -6 to +10 to the rDNA sites of initiation in radish, maize, and wheat. The sequence CATGCAAA is located 19 bp upstream of the site of initiation, and appears once within each subrepeat and twice more between the end of the subrepeat array and the site of initiation. A previously characterized HpaII site which shows developmental regulation of methylation is located 31 bp downstream of the site of initiation. Using RFLP linkage analysis, the short rDNA length variant of cv. Alaska is assigned to Chromosome 4 where it is genetically independent of the long rDNA length variant which is putatively assigned to Chromosome 7.

Base Sequence↗