RESUMO
Large clusters of coexpressed tissue-specific genes are abundant on chromosomes of diverse species. The genes coordinately misexpressed in diverse diseases are also found in similar clusters, suggesting that evolutionarily conserved mechanisms regulate expression of large multigenic regions both in normal development and in its pathological disruptions. Studies on individual loci suggest that silent clusters of coregulated genes are embedded in repressed chromatin domains, often localized to the nuclear periphery. To test this model at the genome-wide scale, we studied transcriptional regulation of large testis-specific gene clusters in somatic tissues of Drosophila. These gene clusters showed a drastic paucity of known expressed transgene insertions, indicating that they indeed are embedded in repressed chromatin. Bioinformatics analysis suggested the major role for the B-type lamin, LamDm(o), in repression of large testis-specific gene clusters, showing that in somatic cells as many as three-quarters of these clusters interact with LamDm(o). Ablation of LamDm(o) by using mutants and RNAi led to detachment of testis-specific clusters from nuclear envelope and to their selective transcriptional up-regulation in somatic cells, thus providing the first direct evidence for involvement of the B-type lamin in tissue-specific gene repression. Finally, we found that transcriptional activation of the lamina-bound testis-specific gene cluster in male germ line is coupled with its translocation away from the nuclear envelope. Our studies, which directly link nuclear architecture with coordinated regulation of tissue-specific genes, advance understanding of the mechanisms underlying both normal cell differentiation and developmental disorders caused by lesions in the B-type lamins and interacting proteins.
Assuntos
Regulação para Baixo/genética , Lamina Tipo B/metabolismo , Família Multigênica/genética , Testículo/metabolismo , Animais , Sítios de Ligação , Ciclo Celular , Linhagem Celular , Cromatina/genética , Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Lamina Tipo B/genética , Masculino , Membrana Nuclear/metabolismo , Transcrição Gênica/genética , Regulação para Cima/genéticaRESUMO
We previously used subtractive hybridization to isolate cDNAs for genes upregulated in chick hypertrophic chondrocytes (Nurminskaya, M. , and T.F. Linsenmayer. 1996. Dev. Dyn. 206:260-271). Certain of these showed homology with the "A" subunit of human plasma transglutaminase (factor XIIIA), a member of a family of enzymes that cross-link a variety of intracellular and matrix molecules. We now have isolated a full-length cDNA for this molecule, and confirmed that it is avian factor XIIIA. Northern and enzymatic analyses confirm that the molecule is upregulated in hypertrophic chondrocytes (as much as eightfold). The enzymatic analyses also show that appreciable transglutaminase activity in the hypertrophic zone becomes externalized into the extracellular matrix. This externalization most likely is effected by cell death and subsequent lysis-effected by the transglutaminase itself. When hypertrophic chondrocytes are transfected with a cDNA construct encoding the zymogen of factor XIIIA, the cells convert the translated protein to a lower molecular weight form, and they initiate cell death, become permeable to macromolecules and eventually undergo lysis. Non-hypertrophic cells transfected with the same construct do not show these degenerative changes. These results suggest that hypertrophic chondrocytes have a novel, tissue-specific cascade of mechanisms that upregulate the synthesis of plasma transglutaminase and activate its zymogen. This produces autocatalytic cell death, externalization of the enzyme, and presumably cross-linking of components within the hypertrophic matrix. These changes may in turn regulate the removal and/or calcification of this hypertrophic matrix, which are its ultimate fates.
Assuntos
Apoptose/fisiologia , Cartilagem/crescimento & desenvolvimento , Tamanho Celular/fisiologia , Condrócitos/enzimologia , Transglutaminases/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião de Galinha , DNA Complementar/genética , Ativação Enzimática/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Lâmina de Crescimento/enzimologia , Microscopia de Fluorescência , Dados de Sequência Molecular , RNA Mensageiro/análise , Alinhamento de Sequência , Transfecção/genética , Transglutaminases/metabolismo , Regulação para Cima/fisiologiaRESUMO
Rapid adaptive fixation of a new favorable mutation is expected to affect neighboring genes along the chromosome. Evolutionary theory predicts that the chromosomal region would show a reduced level of genetic variation and an excess of rare alleles. We have confirmed these predictions in a region of the X chromosome of Drosophila melanogaster that contains a newly evolved gene for a component of the sperm axoneme. In D. simulans, where the novel gene does not exist, the pattern of genetic variation is consistent with selection against recurrent deleterious mutations. These findings imply that the pattern of genetic variation along a chromosome may be useful for inferring its evolutionary history and for revealing regions in which recent adaptive fixations have taken place.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Dineínas/genética , Evolução Molecular , Genes de Insetos , Variação Genética , Cromossomo X/genética , Alelos , Animais , Dineínas do Axonema , Drosophila/genética , Funções Verossimilhança , Modelos Logísticos , Mutação , Polimorfismo Genético , Seleção GenéticaRESUMO
A number of mechanisms have recently been described that might be important in restricting the level of activity of mariner-like transposable elements (MLEs) in natural populations. These mechanisms include overproduction inhibition, in which increasing the dose of transposase decreases net activity. Another mechanism is mediated by certain missense mutations, in which a mutant transposase protein impairs the activity of the wild-type transposase in heterozygous mutant/nonmutant genotypes. A further mechanism is the potential for transposase titration by defective elements that retain transposase binding activity. The issue of regulation is not only of theoretical importance in understanding the molecular and evolutionary genetics of MLEs, but also of practical significance in learning how best to use MLEs in the germline transformation of insect pests and disease vectors.
Assuntos
DNA Nucleotidiltransferases/fisiologia , Elementos de DNA Transponíveis/fisiologia , Drosophila/genética , Animais , Humanos , TransposasesRESUMO
The intermediate chains (ICs) are the subunits of the cytoplasmic dynein that provide binding of the complex to cargo organelles through interaction of their N termini with dynactin. We present evidence that in Drosophila, the IC subunits are represented by at least 10 structural isoforms, created by the alternative splicing of transcripts from a unique Cdic gene. The splicing pattern is tissue specific. A constitutive set of four IC isoforms is expressed in all tissues tested; in addition, tissue-specific isoforms are found in the ovaries and nervous tissue. The structural variations between isoforms are limited to the N terminus of the IC molecule, where the interaction with dynactin takes place. This suggests differences in the dynactin-mediated organelle binding by IC isoforms. Accordingly, when transiently expressed in Drosophila Schneider-3 cells, the IC isoforms differ in their intracellular targeting properties from each other. A mechanism is proposed for the regulation of dynein binding to organelles through the changes in the content of the IC isoform pool.
Assuntos
Proteínas de Drosophila , Drosophila/metabolismo , Dineínas/química , Proteínas de Insetos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Complexo Dinactina , Éxons/genética , Íntrons/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
A laboratory strain of Drosophila virilis was genetically transformed with a hobo vector carrying the miniwhite cassette using a helper plasmid with an hsp70-driven hobo transposase-coding sequence. The rate of transformation was 0.5% per fertile GO animal. Three transgenic insertions were cloned and characterized and found to be authentic hobo insertions. These results, together with the known widespread distribution of hobo in diverse insect species, suggest that hobo and related transposable elements may be of considerable utility in the germline transformation of insects other than D. melanogaster.
Assuntos
Elementos de DNA Transponíveis , Drosophila/genética , Transformação Genética , Animais , Sequência de Bases , Clonagem Molecular , DNA Nucleotidiltransferases/genética , Primers do DNA/genética , Feminino , Vetores Genéticos , Proteínas de Choque Térmico HSP70/genética , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Plasmídeos/genética , Sequências Repetitivas de Ácido Nucleico , TransposasesRESUMO
The annexin X gene was cloned in the P1 recombinant phage carrying a genomic sequence of approximately 70 kb long. This DNA fragment encompasses at least two annexin X copies and several 7.8-kb tandem units represented by an anonymous sequence fused to the 3' truncated part of the annexin X gene. The proteins of annexin family contain a variable amino-terminal domain and a core domain; the latter includes four structurally conserved repeats that presumably arose as a result of duplications. The annexin X gene of Drosophila is about 2 kb long and contains four exons. Exon 1 encodes four amino-terminal amino acids, exon 2 encodes the remaining part of the amino-terminal domain and the three conserved repeats, and exon 3 and exon 4 encode the fourth repeat. The positions of introns 2 and 3 are strictly conserved with respect to both the amino acid position and codon phase as compared to introns 10 and 12 of the fourth repeat in vertebrate annexin genes. We propose the existence of a primordial annexin coding structure comprising at least two introns whose duplications during evolution have been followed by the loss of ancient introns in the first three repeats of Drosophila and vertebrates. Acquisition of new introns in vertebrates is supposed taking into account that exon borders are not found at homologous locations in four repeats of a given vertebrate annexin. Transcription of the annexin gene was detected in embryonic cell cultures. No profound effects of ecdysterone on the annexin X message content in cell cultures were observed.
Assuntos
Anexinas/genética , Drosophila melanogaster/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Biológica , Linhagem Celular , Clonagem Molecular , DNA , Éxons , Expressão Gênica , Genes de Insetos , Humanos , Íntrons , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
A novel method for estimating neutral rates and patterns of DNA evolution in Drosophila takes advantage of the propensity of non-LTR retrotransposable elements to create nonfunctional, transpositionally inactive copies as a product of transposition. For many LINE elements, most copies present in a genome at any one time are nonfunctional "dead-on-arrival" (DOA) copies. Because these are off-shoots of active, transpositionally competent "master" lineages, in a gene tree of a LINE element from multiple samples from related species, the DOA lineages are expected to map to the terminal branches and the active lineages to the internal branches, the primary exceptions being when the sample includes DOA copies that are allelic or orthologous. Analysis of nucleotide substitutions and other changes along the terminal branches therefore allows estimation of the fixation process in the DOA copies, which are unconstrained with respect to protein coding; and under selective neutrality, the fixation process estimates the underlying mutational pattern. We have studied the retroelement Helena in Drosophila. An unexpectedly high rate of DNA loss was observed, yielding a half-life of unconstrained DNA sequences approximately 60-fold faster in Drosophila than in mammals. The high rate of DNA loss suggests a straightforward explanation of the seeming paradox that Drosophila has many fewer pseudogenes than found in mammalian species. Differential rates of deletion in different taxa might also contribute to the celebrated C-value paradox of why some closely related organisms can have very different DNA contents. New data presented here rule out the possibility that the transposition process itself is highly mutagenic, hence the observed linear relation between number of deletions and number of nucleotide substitutions is most easily explained by the hypothesis that both types of changes accumulate in unconstrained sequences over time.
Assuntos
Drosophila/genética , Evolução Molecular , Genoma , Retroelementos , Animais , DNA , Mutagênese , Seleção GenéticaRESUMO
The ß-catenin signaling axis is critical for normal embryonic development and tissue homeostasis in adults. We have previously shown that extracellular enzyme transglutaminase 2 (TG2) activates ß-catenin signaling in vascular smooth muscle cells (VSMCs). In this study, we provide several lines of evidence that TG2 functions as an activating ligand of the LRP5/6 receptors. Specifically, we show that TG2 synergizes with LRP6 in the activation of ß-catenin-dependent gene expression in Cos-7 cells. Interfering with the LRP5/6 receptors attenuates TG2-induced activation of ß-catenin in Cos-7 cells. Further, we show that TG2 binds directly to the extracellular domain of LRP6, which is also able to act as a substrate for TG2-mediated protein cross-linking. Furthermore, inhibitors of TG2 protein cross-linking quench the observed TG2-induced ß-catenin activation, implicating protein cross-linking as a novel regulatory mechanism for this pathway. Together, our findings identify and characterize a new activating ligand of the LRP5/6 receptors and uncover a novel activity of TG2 as an agonist of ß-catenin signaling, contributing to the understanding of diverse developmental events and pathological conditions in which transglutaminase and ß-catenin signaling are implicated.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Transdução de Sinais , Transglutaminases/metabolismo , beta Catenina/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Camundongos , Ligação Proteica , Proteína 2 Glutamina gama-Glutamiltransferase , Estrutura Terciária de Proteína , Peixe-ZebraRESUMO
Two copies of nonmobile retrotransposon localized in D. melanogaster heterochromatin (mdg1het) were sequenced at the 3'-end. The comparison of 2.5-kb mdg1het sequences with the sequence of cognate euchomatic transposable copies (mdg1tr) revealed an intact mdg1 ORF2 encoding the pol gene in mdg1het, and two-thirds of nucleotide substitutions in this ORF were synonymous. All the known mdg1 regulatory elements in the mdg1het LTR also are conserved, in spite of numerous deletions and nucleotide substitutions elsewhere in this region. These data suggest that the mdg1het subfamily lost its mobility more recently than other functions were lost. The G-->A hypermutation known to occur in the reverse transcription cycle of retroviruses was detected in one mdg1het copy. The structure of the enchancer-like region in mdg1het suggests a reduced transcription level and, therefore, transposition frequency, relative to mdg1tr. The number of nucleotide substitutions suggests that the time of mdg1het mobility loss was less than 0.3-0.5 Myr ago.
Assuntos
Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , Animais , Sequência de Bases , Evolução Biológica , Rearranjo Gênico , Heterocromatina/ultraestrutura , Modelos Genéticos , Dados de Sequência Molecular , Mutação Puntual , Recombinação Genética , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
Analysis of DNA variation is a powerful tool for detecting adaptation at the genomic level. The contribution of adaptive evolution is evident from examples of rapidly evolving genes, which represent the likely targets for strong selection. More subtle adaptation is also an integral component of routine maintenance of gene performance, continuously applied to every gene. Adaptive changes in the population are accomplished through selective sweeps, i.e. complete or partial fixation of beneficial alleles. The evidence is accumulating that selective sweeps are quite frequent events which, together with associated genetic hitchhiking, represent dominant forces that influence molecular evolution by shaping the variability pattern in the genome.
Assuntos
DNA/genética , Evolução Molecular , Genes , Seleção Genética , Animais , Códon/genética , Sequência Conservada/genética , Variação Genética/efeitos dos fármacos , Variação Genética/genética , Genoma , Praguicidas/farmacologia , Polimorfismo Genético/genética , Recombinação Genética/genética , Reprodução/genéticaRESUMO
A strategy of "sequence scanning" is proposed for rapid acquisition of sequence from clones such as bacteriophage P1 clones, cosmids, or yeast artificial chromosomes. The approach makes use of a special vector, called LambdaScan, that reliably yields subclones with inserts in the size range 8-12 kb. A number of subclones, typically 96 or 192, are chosen at random, and the ends of the inserts are sequenced using vector-specific primers. Then long-range spectrum PCR is used to order and orient the clones. This combination of shotgun and directed sequencing results in a high-resolution physical map suitable for the identification of coding regions or for comparison of sequence organization among genomes. Computer simulations indicate that, for a target clone of 100 kb, the scanning of 192 subclones with sequencing reads as short as 350 bp results in an approximate ratio of 1:2:1 of regions of double-stranded sequence, single-stranded sequence, and gaps. Longer sequencing reads tip the ratio strongly toward increased double-stranded sequence.
Assuntos
DNA Recombinante/química , Vetores Genéticos , Análise de Sequência de DNA/métodos , Animais , Clonagem Molecular/métodos , Drosophila/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Moldes GenéticosRESUMO
The 60 kb repeats located in the distal heterochromatin of the X chromosome of Drosophila melanogaster were cloned in overlapping cosmids. These regions, designated as SCLRs, comprised the following types of repeated elements: Stellate genes, which are known to be involved in spermatogenesis; copia-like retrotransposons; LINE elements, including amplified Type I rDNA insertions; and rDNA fragments. The following steps in SCLR formation were hypothesized: insertion of mobile elements into the rDNA and Stellate gene clusters; internal tandem duplication events; recombination between the rDNA cluster and Stellate tandem repeat; and amplification of the whole SCLR structure. There are about nine SCLR copies per haploid genome, but there is approximately a twofold variation in copy number between fly stocks. The SCLR copy number differences between closely related stocks are suggested to be the result of unequal sister chromatid exchange (USCE). The restricted variation in SCLR copy number between unrelated stocks and the absence of chromosomes free of SCLRs suggests that natural selection is active in copy number maintenance.
Assuntos
Evolução Biológica , Drosophila melanogaster/genética , Heterocromatina/genética , Cromossomo X , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Elementos de DNA Transponíveis , DNA Ribossômico , Amplificação de Genes , Dados de Sequência MolecularRESUMO
Physical maps showing the relative locations of cloned DNA fragments in the genome are important resources for research in molecular genetics, genome analysis, and evolutionary biology. In addition to affording a common frame of reference for organizing diverse types of genetic data, physical maps also provide ready access to clones containing DNA sequences from any defined region of the genome. In this paper, we present a physical map of the genome of Drosophila melanogaster based on in situ hybridization with 2461 DNA fragments, averaging approximately 80 kilobase pairs each, cloned in bacteriophage P1. The map is a framework map in the sense that most putative overlaps between clones have not yet been demonstrated at the molecular level. Nevertheless, the framework map includes approximately 85% of all genes in the euchromatic genome. A continuous physical map composed of sets of overlapping P1 clones (contigs), which together span most of the euchromatic genome, is currently being assembled by screening a library of 9216 P1 clones with single-copy genetic markers as well as with the ends of the P1 clones already assigned positions in the framework map. Because most P1 clones from D. melanogaster hybridize in situ with chromosomes from related species, the framework map also makes it possible to determine the genome maps of D. pseudoobscura and other species in the subgenus Sophophora. Likewise, a P1 framework map of D. virilis affords potential access to genome organization and evolution in the subgenus Drosophila.
Assuntos
Evolução Biológica , Mapeamento Cromossômico , Drosophila/genética , Animais , Clonagem Molecular , Hibridização In Situ , Reação em Cadeia da PolimeraseRESUMO
Genomic fragments, homologous to chicken A1(1) collagen cDNA encoding triple-helical domain, were revealed by Southern analysis in various fungi. Such a genomic fragment from Candida utilis was cloned and sequenced. Analysis of the obtained DNA sequence revealed the 119 bp segment, which has possibly originated from the 54 bp module common for the fibrillar collagen genes of higher eukaryotes.
Assuntos
Candida/genética , Galinhas/genética , Colágeno/genética , DNA Fúngico/genética , Homologia de Sequência do Ácido Nucleico , Animais , Sequência de Bases , DNA Complementar/genética , Dados de Sequência MolecularRESUMO
Expression of the X-linked repeated Stellate (Ste) genes, which code for a protein with 38% similarity to the beta-subunit of casein kinase II, is suppressed by the Su(Ste) locus on the Y chromosome. The structure and evolution of the Y-linked repeats in the region of the Su(Ste) locus were studied. The 2800 bp repeats consist of three main elements: the region of homology to the Ste genes, an adjacent AT-rich, Y-specific segment, and mobile element 1360 inserted in the Ste sequence. Amplification of repeats was followed by point mutations, deletions, and insertions of mobile elements. DNA sequencing shows that these repeats may be considered as Ste pseudogenes or as damaged variants of a putative gene(s) encoding a protein quite different from the Ste protein as a result of an alternative splicing pattern. A comparison of 5 variants of the Y-Su(Ste) repeats shows a number of recombination events between amplified and diverged sequences that could be due to either multiple unequal mitotic sister-chromatid exchanges or to gene conversion. It is a first demonstration on a molecular level of these processes occurring in heterochromatic non-rDNA tandemly organized sequences in an eukaryotic genome.
Assuntos
Drosophila melanogaster/genética , Família Multigênica/genética , Pseudogenes/genética , Cromossomo Y , Animais , Sequência de Bases , Evolução Biológica , Elementos de DNA Transponíveis/genética , Amplificação de Genes/genética , Dados de Sequência Molecular , Plasmídeos/genética , Sequências Repetitivas de Ácido Nucleico/genética , Homologia de Sequência do Ácido Nucleico , Cromossomo XRESUMO
Drosophila virilis is a prominent reference species for comparison with Drosophila melanogaster in regard to patterns and mechanisms of molecular and genomic evolution. Sequences were determined for 11 Adh genes from 8 species of the D. virilis species group, including species from both the virilis phylad and the montana subphylad. The genome of D. virilis contains a 6-kb duplication that includes the entire Adh coding region. The pattern of sequence identity within the duplication strongly suggests a recent gene-conversion event bordered by 36-bp indels. As in other Drosophila, the amino-acid coding region of Adh is encoded by three exons interrupted by two short introns. The promoter region includes 16 blocks of sequence that are well conserved in D. virilis, Drosophila hydei, and D. melanogaster. The developmental profile of Adh transcription suggests a distal/proximal promoter switch analogous to that in D. melanogaster. Duplicate Adh genes were also found in Drosophila montana and Drosophila lacicola, which apparently originated independently of that in D. virilis. The Adh genes in all species of the D. virilis group have among the lowest codon bias of any Adh genes so far reported in the genus Drosophila. Taking the low codon bias into account, we estimate the time of divergence between the virilis and montana clades as 9.0 +/- 0.7 Mya and the approximate time of divergence of D. virilis from other members of the virilis phylad as 2.6 +/- 0.4 Mya. The region of the D. virilis genome containing Adh, as well as the chromosome as a whole, gives evidence of extensive rearrangements relative to the genome of D. melanogaster.
Assuntos
Álcool Desidrogenase/genética , Drosophila/genética , Evolução Molecular , Sequência de Aminoácidos , Animais , Sequência de Bases , Drosophila/enzimologia , Genoma , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
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.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Dineínas/genética , Evolução Molecular , Proteínas de Insetos/genética , Espermatozoides , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Anexinas/genética , Fusão Gênica Artificial , Dineínas do Axonema , Sequência de Bases , DNA , Regulação da Expressão Gênica , Genes de Insetos , Masculino , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Seleção Genética , Deleção de Sequência , Espermatozoides/metabolismo , Testículo/metabolismoRESUMO
We have used the polymerase chain reaction (PCR) technique to search the Drosophila melanogaster genome for the presence of sequences with homology to mammalian and yeast centromeric DNA. Using primers based on the human CENP-B box present in alpha-satellite DNA and part of the Saccharomyces cerevisiae CDEIII centromeric sequence, a number of specific DNA fragments were amplified from total genomic DNA. In situ hybridization to polytene and mitotic chromosomes showed these fragments to localise to centromeric and pericentromeric regions. Direct cloning of the amplified fragments into conventional plasmids proved unsuccessful. However, a recombinant P1 clone containing D. melanogaster genomic DNA that supports PCR amplification by the primers was identified. Molecular characterisation of this clone revealed a DNA fragment that localises primarily to the centromere of chromosome 2. Sequence analysis indicated that this fragment contains at least four different repeats, including Rsp, transposable elements, Bari-1 and a new AT-rich repeated sequence that we have designated Porto-1. Detailed fluorescence in situ hybridization analysis shows that Porto-1 is localised very close to the primary constriction of chromosome 2. Sequence analysis suggests that this repeat was specifically amplified by our primers, although limited homology to the CENP-B box or CDEIII elements was found. In situ hybridization to a number of Drosophila species shows Porto-1 to be present only in D. melanogaster.