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Results for “genomic tandem array”

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PubMed2001

Molecular cloning of human squamous cell carcinoma antigen 1 gene and characterization of its promoter.

The squamous cell carcinoma antigen (SCCA) serves as a serological marker for squamous cell carcinomas. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2, which are 95% identical in nucleotide sequence. SCCA1 is a papain-like cysteine proteinase inhibitor, while SCCA2 is a chymotrypsin-like serine proteinase inhibitor. We analyzed here the sequence and the promoter activity of the 5'-flanking region of the SCCA1 gene. Deletion analysis of SCCA1 and SCCA2 promoter identified a 471-bp core promoter region upstream of the transcription start site. The transcriptional activity of SCCA1 promoter was up-regulated in squamous cell carcinoma cells, compared with keratinocyte and adenocarcinoma cells. The ratios of SCCA1 to SCCA2 promoter activity in squamous cell carcinoma, keratinocyte and adenocarcinoma cells were respectively 1.6, 5.3 and 2.8. Position -50 of SCCA1 and SCCA2 promoters played an important role in determining the promoter activities of SCCA1 and SCCA2. These findings suggest that the transcriptional regulation of SCCA1 and SCCA2 might differ among squamous cell carcinoma, keratinocyte and adenocarcinoma cells, and that SCCA1 promoter might be a potential target of gene therapy for squamous cell carcinoma.

Antigens, Neoplasm↗

PubMed2004

SRP-2 is a cross-class inhibitor that participates in postembryonic development of the nematode Caenorhabditis elegans: initial characterization of the clade L serpins.

High molecular weight serpins are members of a large superfamily of structurally conserved proteins that inactivate target proteinases by a suicide substrate-like mechanism. In vertebrates, different clades of serpins distribute predominantly to either the intracellular or extracellular space. Although much is known about the function, structure, and inhibitory mechanism of circulating serpins such as alpha(1)-antitrypsin (SERPINA1) and antithrombin III (SERPINC1), relatively little is known about the function of the vertebrate intracellular (clade B) serpins. To gain a better understanding of the biology of the intracellular serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a model system. A screen of the C. elegans genomic and cDNA databases revealed nine serpin genes, tandemly arrayed on chromosome V. Although the C. elegans serpins represent a unique clade (L), they share significant functional homology with members of the clade B group of intracellular serpins, since they lack typical N-terminal signal peptides and reside intracellularly. To determine whether nematode serpins function as proteinase inhibitors, one family member, srp-2, was chosen for further characterization. Biochemical analysis of recombinant SRP-2 protein revealed SRP-2 to be a dual cross-class inhibitor of the apoptosis-related serine proteinase, granzyme B, and the lysosomal cysteine proteinases, cathepsins K, L, S, and V. Analysis of temporal and spatial expression indicated that SRP-2 was present during early embryonic development and highly expressed in the intestine and hypoderm of larval and adult worms. Transgenic animals engineered to overexpress SRP-2 were slow growing and/or arrested at the first, second, or third larval stages. These data suggest that perturbations of serpin-proteinase balance are critical for correct postembryonic development in C. elegans.

Amino Acid Sequence↗

PubMed1993

Unidirectional dominance of cytoplasmic inheritance in two genetic crosses of Plasmodium falciparum.

Malarial parasites have two highly conserved cytoplasmic DNA molecules: a 6-kb tandemly arrayed DNA that has characteristics of a mitochondrial genome, and a 35-kb circular DNA that encodes functions commonly found in chloroplasts. We examined the inheritance pattern of these elements in two genetic crosses of Plasmodium falciparum clones. Parent-specific oligonucleotide probes and single-strand conformation polymorphism analysis identified single nucleotide changes that distinguished the parental 6- and 35-kb DNA molecules in the progeny. In all 16 independent recombinant progeny of a cross between a Central American clone, HB3, and a Southeast Asian clone, Dd2, the 6- and 35-kb DNAs were inherited from the Dd2 parent. In all nine independent recombinant progeny of a cross between clone HB3 and a likely African clone, 3D7, the 6-kb DNA was inherited from the 3D7 parent. Inheritance of cytoplasmic genomes of the Dd2 and 3D7 parents was, therefore, dominant over that of the HB3 parent. Cytoplasmic DNA molecules were found almost exclusively in the female gametes of malarial parasites; hence, clone HB3 did not appear to have served as a maternal parent for the progeny of two crosses. Defective differentiation into male gametes by clone Dd2 is likely to be a reason for the cytoplasmic inheritance pattern seen in the HB3 x Dd2 cross. However, incompetence of male or female gametes is unlikely to explain the uniparental dominance in recombinant progeny of the HB3 x 3D7 cross, since both parents readily self-fertilized and completed the malaria life cycle on their own. Instead, the data suggest unidirectional parental incompatibility in cross-fertilization of these malarial parasites, where a usually cosexual parental clone can participate only as a male or as a female. Such an incompatibility may be speculated as indicating an early phase of reproductive isolation of P. falciparum clones from different geographical regions.

Animals↗

PubMedDate not supplied

Gene expression of human squamous cell carcinoma antigens 1 and 2 in human cell lines.

The squamous cell carcinoma antigen (SCCA) serves as a serological marker for squamous cell carcinomas. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. SCCA1 is a papain-like cysteine proteinase inhibitor, while SCCA2 is a chymotrypsin-like serine proteinase inhibitor. Little is known concerning how expression of the SCCA1 and SCCA2 genes is regulated in human cell lines. The purpose of this study was to determine whether the SCCA1 gene or SCCA2 gene is more strongly expressed in human cell lines. Squamous cell carcinoma cell lines secreted respectively 4 times and 50 times as much SCCA proteins into medium as normal human keratinocyte and non-squamous cell carcinoma cell lines, as measured by enzyme-linked immunosorbent assay. Quantitative RT-PCR ELISA digoxigenin-labeling assay demonstrated that SCCA1 mRNA expression in squamous cell carcinoma cell lines was respectively 2.8 times and 42 times that in keratinocyte and non-squamous cell carcinoma cell lines. The ratio of SCCA1 to SCCA2 mRNA expression differed distinctly among squamous cell carcinoma, keratinocyte and non-squamous cell carcinoma cell lines (2.8, squamous; 24.1, keratinocyte; 11.0, non-squamous). These findings suggest that SCCA1 is mainly expressed in squamous cell carcinoma, keratinocyte and non-squamous cell carcinoma cell lines and that the ratio of SCCA1 to SCCA2 expression might be a novel marker for the detection of squamous cell carcinoma.

Adenocarcinoma↗

PubMed1995

Structure of genes for sperm-specific nuclear basic protein (SP4) in Xenopus laevis.

Nuclear basic proteins in sperm of Xenopus laevis consist of 6 sperm-specific proteins (SPs1-6) in addition to somatic core histones. Using a cDNA for SP4 as a probe, we cloned genomic DNA containing SP4 genes from a genomic library constructed from recombinant lambda bacteriophage containing 12.0 kbp-EcoRI digests of J-strain X. laevis liver DNA. Construction of restriction maps based on Southern blot analysis revealed the existence of a total of five SP4 genes which are arranged in a tandemly repeated array forming a cluster of simple multigenes per haploid genome, over a range of 18 kbp. Among these genes, the one located at the most upstream position differed from others in possessing a single base substitution which gave rise to a replacement of one out of 78 amino acid residues. The DNA containing the second to the fourth SP4 genes, arranged at about 3 kbp intervals each, was totally sequenced for 10,165 bp. Each gene was found to contain one intron, typical TATA and CCAAT boxes in the 5'-flanking region, and a polyadenylation signal in the 3'-flanking region. Comparative sequence analyses revealed three regions of extensive homology within the upstream non-coding region among three genes, suggesting a possible relevance to their expression at a particular phase of spermatogenesis and/or in testis.

Amino Acid Sequence↗

PubMed1994

Can SINEs: a family of tRNA-derived retroposons specific to the superfamily Canoidea.

A repetitive element of approximately 200 bp was cloned from harbour seal (Phoca vitulina concolour) genomic DNA. The sequence of the element revealed putative RNA polymerase III control boxes, a poly A tail and direct terminal repeats characteristic of SINEs. Sequence and secondary structural similarities suggest that the SINE is derived from a tRNA, possibly tRNA-alanine. Southern blot analysis indicated that the element is predominately dispersed in unique regions of the seal genome, but may also be present in other repetitive sequences, such as tandemly arrayed satellite DNA. Based on slot-blot hybridization analysis, we estimate that 1.3 x 10(6) copies of the SINE are present in the harbour seal genome; SINE copy number based on the number of clones isolated from a size-selected library, however, is an order of magnitude lower (1-3 x 10(5) copies), an estimate consistent with the abundance of SINEs in other mammalian genomes. Database searches found similar sequences have been isolated from dog (Canis familiaris) and mink (Mustela vison). These, and the seal SINE sequences are characterized by an internal CT dinucleotide microsatellite in the tRNA-unrelated region. Hybridization of genomic DNA from representative species of a wide range of mammalian orders to an oligonucleotide (30mer) probe complementary to a conserved region of the SINE confirmed that the element is unique to carnivores of the superfamily Canoidea.

Animals↗

PubMed1992

Sequence organization of variant mouse 4.5 S RNA genes and pseudogenes.

The rodent 4.5 S RNA is an RNA polymerase III product with a sequence related to the Alu family of interspersed repeated DNA. A previous study identified a tandem array of 4.2-kb repeating units that contain the 4.5 S RNA coding sequence as well as many short repetitive sequences. To understand the genomic organization of this gene family, we have isolated and characterized 4.5 S RNA sequences that are part of the tandem array as well as identified members that are not part of the array. One variant 4.5 S RNA gene family member exhibits length polymorphisms in its minisatellite sites relative to the single previously reported gene. The 4.5 S RNA sequences that are not part of the tandem array possess many of the features of processed pseudogenes and are found adjacent to other interspersed repeated elements. These findings suggest that the mouse 4.5 S RNA can behave as a retroposon, resulting in the accumulation of 4.5 S RNA-like elements at many sites in the genome.

Animals↗

PubMed2003

A centromeric tandem repeat family originating from a part of Ty3/gypsy-retroelement in wheat and its relatives.

From a wild diploid species that is a relative of wheat, Aegilops speltoides, a 301-bp repeat containing 16 copies of a CAA microsatellite was isolated. Southern blot and fluorescence in situ hybridization revealed that approximately 250 bp of the sequence is tandemly arrayed at the centromere regions of A- and B-genome chromosomes of common wheat and rye chromosomes. Although the DNA sequence of this 250-bp repeat showed no notable homology in the databases, the flanking or intervening sequences between the repeats showed high homologies (>82%) to two separate sequences of the gag gene and its upstream region in cereba, a Ty3/gypsy-like retroelement of Hordeum vulgare. Since the amino acid sequence deduced from the 250 bp with seven CAAs showed some similarity ( approximately 53%) to that of the gag gene, we concluded that the 250-bp repeats had also originated from the cereba-like retroelements in diploid wheat such as Ae. speltoides and had formed tandem arrays, whereas the 300-bp repeats were dispersed as a part of cereba-like retroelements. This suggests that some tandem repeats localized at the centromeric regions of cereals and other plant species originated from parts of retrotransposons.

Base Sequence↗

PubMed1989

Mutagenesis of phi X174 am3 cs70 incorporated into the genome of mouse L-cells.

The objective of our work with phi X174 has been to develop a shuttle vector that can be used comparatively in bacterial cells, different types of mammalian cells, and possibly in the various tissues of transgenic mice, with a constant mechanism for detection and analysis of mutations independent of any host-cell type. Toward that end, we have efficiently rescued phi X174 am3 cs70 that is host-silent and stably integrated into the genome of mouse L-cells. The particular mouse L-cell line contains tandem arrays, single copies, and fragments of phi X that, upon restriction enzyme excision, can result in 5 potentially active copies per diploid genome. The excised phi X DNA is recovered by column chromatography, ligated, and transfected into highly competent spheroplasts. The Rescue Efficiency, defined as the number of viable phages produced out of the total number of potentially recoverable copies, is approx. 10(-3). The Recovery Ratio, defined as the Rescue Efficiency for chromosomally-integrated phage DNA divided by the Rescue Efficiency for phi X am3 cs70, is close to one. Mouse L-cells containing the integrated phi X174 am3 cs70 were treated with 20 mM ethyl methanesulfonate. The reversion frequency of am3 among progeny phages rescued from treated cells was 1.4 X 10(-5) (193 revertants in 1.4 X 10(7) phages). This is significantly higher than the 5.8 X 10(-7) reversion frequency of am3 (7 revertants in 1.2 X 10(7) phages) among progeny phages rescued from untreated cells.

Animals↗

PubMed2006

Tools for the identification of variable and potentially variable tandem repeats.

BACKGROUND: Tandem repeat arrays showing variation between sequences within a population, between strains or across species may have functional effects. The increasing availability of genomic sequence data makes routine description of observed variation possible, creating a need for tools to describe such variability. RESULTS: We present a set of programs that facilitate the identification of tandem repeats showing variation across multiple sequences or genomes, and the prediction of potentially polymorphic tandem repeats. The VNTRfinder (Variable Number of Tandem Repeats finder) program enables the detection of sequence length variation between arrays of inter-specific or intra-specific tandem repeats. In the absence of comparable sequences to explore observed variation, predictions are provided describing which tandem repeats are more likely to be variable, to help guide and focus further experimental evaluation. CONCLUSION: These tools represent a resource for researchers interested in tandem repeats in nucleotide sequences that are most likely to be of clinical and evolutionary interest. The tools are available at http://bioinformatics.rcsi.ie/vntrfinder/. Downloadable versions for UNIX/LINUX and WINDOWS which permit the consideration of longer and more numerous sequences are also available.

Algorithms↗

PubMed2004

Telomere instability detected in sporadic colon cancers, some showing mutations in a mismatch repair gene.

Human telomeres are essential for genome stability and are composed of long simple tandem repeat arrays (STRs), comprising the consensus TTAGGG repeat interspersed, at the proximal end, with sequence-variant repeats. While the dynamics of telomere attrition through incomplete replication has been studied extensively, the effects on telomeres of error-prone DNA repair processes, known to affect other STRs, are poorly understood. We have compared the TTAGGG and sequence-variant interspersion patterns in the proximal 720 bp of telomeres in colon cancer and normal DNA samples. The frequency of telomere mutations was 5.8% per allele in a randomly collected panel of sporadic colon cancers, showing that telomere mutations occur in vivo. The mutation frequency rose to 18.6% per allele in sporadic tumours that exhibit instability at the polyA tract in the TGFbetaRII gene and to 35% per allele in tumours with somatic mutations in the hMSH2 gene. The majority of the characterized mutations resulted in the loss of one or a few repeats. If the mutation spectrum and frequency described here is reiterated in the rest of the array, there is the potential for extensive telomere destabilization especially in mismatch repair-defective cells. This may in turn lead to a greater requirement for telomere length maintenance earlier in tumourigenesis.

Base Pair Mismatch↗

PubMed1981

Electron microscopy of Achlya deoxyribonucleic acid sequence organization.

Electron microscopic analysis of reassociated deoxyribonucleic acid (DNA) from the aquatic fungus Achlya bisexualis revealed details of the sequence arrangement of the inverted repeats and both the highly and moderately repetitive sequence clusters. We used the gene 32 protein-ethidium bromide technique for visualizing the DNA molecules, a procedure which provides excellent contrast between single- and double-stranded DNA regions. Long (greater than 6-kilobase) DNA fragments were isolated after reannealing to two different repetitive C0t values, and the renatured structures were then visualized in an electron microscope. Our results showed that the inverted repeat sequences were short (0.5 kilobase, number-average) and separated by nonhomologous DNA of various lengths. These pairs of sequences were not clustered within the genome. Both highly repetitive and moderately repetitive DNA sequences were organized as tandem arrays of precisely paired, regularly repeating units. No permuted clusters of repeating sequences were observed, nor was there evidence of interspersion of repetitive with single-copy DNA sequences in the Achlya genome.

Chytridiomycota↗

PubMed1985

The molecular karyotype of Leishmania major and mapping of alpha and beta tubulin gene families to multiple unlinked chromosomal loci.

The arrangement of tubulin genes in the genome of the protozoan parasite Leishmania major was studied by genomic Southern blot analysis and mapping of genes to chromosomes fractionated by pulsed field gradient gel (PFG) electrophoresis. alpha-tubulin genes exist as a tandem array of 2.4 kb PstI fragments. beta-tubulin genes are found as a tandem array of 3.9 kb AvaI or PvuI fragments, but additional genes are also found on other genomic DNA fragments. Chromosome-sized DNA molecules released from promastigotes of L. major were fractionated into at least 17 chromosome bands of approximate size 400-4000 kb by PFG gel electrophoresis. Some bands may be present in non-equimolar amounts suggesting that there may be more than 17 chromosomes. All alpha-tubulin genes were localized to a single band (chromosome 7). beta-tubulin genes were localized to four bands (chromosomes 6, 10, 16 and 17). This shows that the alpha- and beta- tubulin gene families are unlinked in L. major. There is a single chromosomal locus for the alpha-tubulin tandem array whereas beta-tubulin genes exist both as a tandem array and as dispersed genes at four chromosomal loci.

Animals↗

PubMed1995

Molecular characterization of a centromeric satellite DNA in the hemiclonal hybrid frog Rana esculenta and its parental species.

Hybrid water frogs Rana esculenta reproduce by hybridogenesis: one parental genome (of Rana lessonae) is excluded in the germ line, the other (of Rana ridibunda) is clonally transmitted to haploid gametes. The two parental species differ in that the amount of centromeric heterochromatin revealed by differential staining is much higher in Rana ridibunda. An abundant, tandemly arrayed, centromeric satellite DNA, designated RrS1, is revealed in Rana ridibunda genomes by the restriction endonuclease Stul, which generates a major repetitive sequence fragment of 300 and a minor one of 200 bp. This AT-rich (68%) satellite family is located at the centromeres of the five largest chromosomes (1-5) and of a medium to small heterobrachial one (8 or 9); it thus constitutes only part of the centromeric heterochromatin that characterizes all Rana ridibunda chromosomes. RrS1 represents about 2.5% of the genome of Rana ridibunda; it may represent as little as 0.2% of the genome of Rana lessonae, and cannot be detected in Xenopus laevis frogs or Salamandra salamandra and Triturus carnifex salamanders. Segments of the satellite sequence are similar to sequences of yeast centromeric DNA element CDEIII and of the mammalian CENP-B box. A role for RrS1 and other centromeric satellite DNAs in the germ line genome exclusion of the hybridogenetic frog hybrids, although suggested, has not yet been demonstrated.

Animals↗

PubMed1993

A repetitive element in the genome of Atlantic salmon, Salmo salar.

When Atlantic salmon (Salmo salar) genomic DNA is digested with the restriction endonuclease BglI and the fragments separated by agarose-gel electrophoresis, bands corresponding to approximately 430 and 923 bp are visualized after EtdBr staining. The 923-bp band was excised from a preparative gel and used to screen a salmon genomic library for recombinant phage (re-phage) containing the repeat. The BglI repeat element is tandemly arrayed, and an array from one re-phage has been sequenced. The BglI repeats comprise 2.3% of the S. salar genome and have been found in the vicinity of rDNA genes (encoding ribosomal RNA). Southern blot hybridization detects a homologue of the Atlantic salmon BglI repeat in the brown trout (Salmo trutta) genome, but not in other salmonids. However, a DNA fragment with sequence homology to part of the BglI repeat has recently been isolated from Arctic charr (Salvelinus alpinus; S.E. Hartley and W.S.D., unpublished data). In addition, the BglI repeat detects RFLPs in Atlantic salmon.

Animals↗

PubMed2006

Tracking the evolution of alternatively spliced exons within the Dscam family.

BACKGROUND: The Dscam gene in the fruit fly, Drosophila melanogaster, contains twenty-four exons, four of which are composed of tandem arrays that each undergo mutually exclusive alternative splicing (4, 6, 9 and 17), potentially generating 38,016 protein isoforms. This degree of transcript diversity has not been found in mammalian homologs of Dscam. We examined the molecular evolution of exons within this gene family to locate the point of divergence for this alternative splicing pattern. RESULTS: Using the fruit fly Dscam exons 4, 6, 9 and 17 as seed sequences, we iteratively searched sixteen genomes for homologs, and then performed phylogenetic analyses of the resulting sequences to examine their evolutionary history. We found homologs in the nematode, arthropod and vertebrate genomes, including homologs in several vertebrates where Dscam had not been previously annotated. Among these, only the arthropods contain homologs arranged in tandem arrays indicative of mutually exclusive splicing. We found no homologs to these exons within the Arabidopsis, yeast, tunicate or sea urchin genomes but homologs to several constitutive exons from fly Dscam were present within tunicate and sea urchin. Comparing the rate of turnover within the tandem arrays of the insect taxa (fruit fly, mosquito and honeybee), we found the variants within exons 4 and 17 are well conserved in number and spatial arrangement despite 248-283 million years of divergence. In contrast, the variants within exons 6 and 9 have undergone considerable turnover since these taxa diverged, as indicated by deeply branching taxon-specific lineages. CONCLUSION: Our results suggest that at least one Dscam exon array may be an ancient duplication that predates the divergence of deuterostomes from protostomes but that there is no evidence for the presence of arrays in the common ancestor of vertebrates. The different patterns of conservation and turnover among the Dscam exon arrays provide a striking example of how a gene can evolve in a modular fashion rather than as a single unit.

Alternative Splicing↗

PubMed2004

Genomic analysis of Drosophila melanogaster telomeres: full-length copies of HeT-A and TART elements at telomeres.

The repetitive nature of heterochromatin hampers its analysis in general genome-sequencing projects. Specific studies are needed to extend the sequence into telomeric and centromeric heterochromatin. Drosophila telomeres lack the telomerase-generated repeats that are characteristic of other eukaryotic chromosomes. Instead, they consist of tandem arrays of HeT-A and TART elements. Herein, we present the genomic organization of the telomeres in the isogenic strain (y; cn bw sp) that was used for the Drosophila melanogaster sequencing project. The data indicate that the canonical features of telomere organization are widely conserved in evolution. In addition, we have identified full-length elements, likely competent elements, for HeT-A and TART.

Amino Acid Sequence↗

PubMed2004

Expression of the sorghum 10-member kafirin gene cluster in maize endosperm.

Functional analysis of chromosomal segments containing linked genes requires the insertion of contiguous genomic sequences from bacterial artificial chromosomes (BACs) into the genome. Therefore, we introduced a 90-kb large BAC clone carrying a 10-copy tandem array of kafirin storage protein genes from sorghum linkage group J, mixed with a selectable marker gene, directly into maize cells using the particle bombardment method. Transgenic plants were regenerated and seeds from eight different transgenic lines were produced. One such transgenic plant was selected that had the entire kafirin gene cluster on a single continuous DNA fragment spanning more than 45 kb integrated into its genome. When alcohol-soluble proteins from individual T2 and T3 seeds of this event were analyzed, significant levels of kafirin were found in addition to the endogenous zein storage proteins, demonstrating that the large exogenous DNA segment is stably integrated into the maize genome and expressed at high levels in subsequent generations. Therefore, we could provide a new utility of plant transformation by the particle bombardment method for functional genomics of multigene families and the modification of the nutritive quality of cereal grains. Despite a tandem array of highly homologous sequences at the transgenic locus, no gene silencing was observed, probably owing to the effects of co-transformed flanking sequences. The expression studies of the transgenic locus also revealed new features of storage protein gene promoters that differed from previous transient gene expression studies, thereby illustrating the significance of the concentration and configuration of DNA-protein interactions in the regulation of gene expression.

Chromatin↗