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Tandem genes and clustered genes.

Two patterns of gene repetition are described: tandem arraying and clustering. Tandemly arrayed genes reside within segments of DNA that are repeated head-to-tail a number of times. Clustered genes are linked but irregularly spaced, are often mutually inverted in an unpredictable pattern and are connected by non-conserved DNA. Tandem arrays are homogenized by both unequal recombination and gene conversion, are necessary for the maintenance of large gene families, can expand and contract rapidly in response to changing demand, can keep functionally related genes equal in number, and do not engender increased genetic complexity. Gene clusters are homogenized by conversion only, seldom if ever contain more than 50 members, are stable in number, and often engender increased genetic complexity. The interrelationships among these properties are discussed. Tandem gene arrays can evolve into gene clusters. It is suggested that this occurs when some change in the array inhibits unequal recombination but not gene conversion. The most common such change is inversion of part of the tandem array with respect to the rest; however, arrays can evolve into clusters without inversion. Clustered genes are sometimes re-amplified into new tandem arrays. Clustered genes are probably more durable than tandemly arrayed genes during periods of relaxed selection, and in the case of fish antifreeze protein genes, seem to behave as a genetic memory.

Animals↗

Genomic Identification and Comparative Characterization of Chemosensory Genes in Two Walnut Pests.

Conogethes punctiferalis (generalist) and Atrijuglans aristata (specialist) are important pests of walnut fruits. Chemosensory genes play critical roles in host location and mating, making them promising candidates for pest management research. However, genome-wide identification of these gene families has not yet been performed for either species, and cross-species comparisons are often confounded by differences in annotation quality and methodology. Here, we employed a unified pipeline for genome annotation and gene family identification to systematically characterize the odorant receptor (OR), gustatory receptor (GR), odorant-binding protein (OBP), and chemosensory protein (CSP) genes of both species and further analyzed their physicochemical properties, chromosomal distribution, and phylogeny. Genome annotation identified 13,236 and 14,083 protein-coding genes in C. punctiferalis and A. aristata, respectively, with BUSCO completeness of 94.6% and 94.5%. We identified 121 candidate chemosensory genes in C. punctiferalis (46 ORs, 23 GRs, 32 OBPs, and 20 CSPs) and 137 in A. aristata (65 ORs, 26 GRs, 28 OBPs, and 18 CSPs). Notably, A. aristata possesses more OR genes (65) than C. punctiferalis (46). Both species possess a single conserved ORco, with three and four pheromone receptor (PR) genes in C. punctiferalis and A. aristata, respectively. Chemosensory genes were either dispersed or tandemly arrayed on chromosomes, with each family clustering into conserved functional branches. This study provides a reliable foundation for comparative chemosensory evolution studies and a catalog of candidate genes for future functional validation.

Atrijuglans aristata↗

Subrepeats within the BR1 beta repeat unit in Chironomus pallidivittatus can be classified into different types depending on codon usage.

A new type of repeat unit was isolated from Balbiani ring 1 of Chironomus pallidivittatus and designated BR1 beta repeat. It consists of a constant and a subrepeated part, like previously described units belonging to the core blocks of the BR genes. The subrepeated part contains 10-codon subrepeats with an arrangement similar to the subrepeats of the previously described BR2 beta gene. The present unit differs from earlier reported core units firstly in a much lower number of copies (about 15) per genome, which are tandemly arranged. Secondly, the number of subrepeats per BR1 beta repeat unit can show great variations. On the basis of the pattern of codon usage, three types of subrepeats can be distinguished. One type lies 5'-proximal in the subrepeat array and consists of variable numbers of subrepeats almost identical at the nucleotide level. The last complete subrepeat represents another type, with consistent differences in codon usage as compared to subrepeats of the proximal type. Finally, there is an intermediate type represented by the subrepeat preceding the distal one. Here, codon characteristics from proximal and distal subrepeats are mixed in a patchy and irregular way. The evolution of the arrays can be understood either as being the result of subrepeat formation in two steps (occurring before and after amplification of whole repeat units) or as the result of a continuous process in which there is evidence for participation of gene conversion.

Amino Acid Sequence↗

Cloning and characterization of a novel zinc-finger protein-encoding cDNA from the mouse eye lens.

Zinc fingers (Zf) are a common structural motif found in many nucleic acid-binding proteins. In an effort to identify potential transcription factors in the mouse eye lens, we have isolated a Zf-containing clone from a newborn mouse lens cDNA library. The clone, named pMLZ-4, is 4.5 kb in length and contains an open reading frame of 1073 bp. The putative pMLZ-4 protein consists of a short, N-terminal acidic domain followed by twelve tandemly arrayed Zf of the C2H2 variety. The remaining 3.2 kb of the cDNA comprises the 3'-untranslated region. PCR analysis detected the presence of pMLZ-4 RNA in liver, heart, kidney, spleen and brain of newborn mice. Hybridization of pMLZ-4 to genomic DNA from a number of species of vertebrates revealed the presence of homologous sequences only in mouse and rat. Unexpectedly, the probe also hybridized to a single band in yeast DNA digested with EcoRI. NIH3T3 cells were stably transformed with a construct that over-expresses the pMLZ-4 mRNA. The stably transformed cells did not differ in appearance from untransformed cells, and an analysis of proteins from transformed and untransformed cells failed to detect any differences resulting from over-expression of the pMLZ-4 mRNA.

3T3 Cells↗

Isolation and characterization of simple sequence repeat loci in the gray tree frog, Hyla chrysoscelis.

A gray tree frog (Hyla chrysoscelis) genomic library was constructed and characterized with regard to the incidence and complexity of simple sequence repeat (SSR) loci. The partial genomic library, containing approximately 10,000 clones with an average-sized insert of 350 bp, was screened with six SSR repeat oligonucleotides (AC, AG, ACG, AGC, AAC, and AAG). Screening identified 31 unique positive clones containing 41 SSR loci. Sequences of tandemly arrayed dinucleotide repeats were more common (36 of 41) than trinucleotide repeats. Twenty-six loci were identified using the AC dinucleotide probe, while 7 loci were identified using the AG dinucleotide probe. An additional 3 AT dinucleotide loci were serendipitously identified. The AT repeats generally comprised the longest dinucleotide repeat loci. The SSR repeat loci reported here should provide potent markers for identity, parentage, and short-lineage determinations in large-scale experiments using gray tree frogs.

Animals↗

Transposable elements and the evolution of heterochromatin.

Drosophila P elements were shown to insert frequently into telomeric and centromeric heterochromatin, and to prefer a region associated with efficient copy number regulation. Upon excision, P elements frequently altered the number of repeats in a tandem array of heterochromatic sequences, by inducing unequal gene conversion. These studies suggest that a flux of transposable element insertions and excisions has the capacity to rapidly and nonrandomly modify heterochromatic sequences dispersed at multiple chromosomal sites. We propose that transposable elements maintain genomic heterochromatin in a state of dynamic equilibrium and drive its rapid evolution.

Animals↗

Genesis and evolution of the Evx and Mox genes and the extended Hox and ParaHox gene clusters.

BACKGROUND: Hox and ParaHox gene clusters are thought to have resulted from the duplication of a ProtoHox gene cluster early in metazoan evolution. However, the origin and evolution of the other genes belonging to the extended Hox group of homeobox-containing genes, that is, Mox and Evx, remains obscure. We constructed phylogenetic trees with mouse, amphioxus and Drosophila extended Hox and other related Antennapedia-type homeobox gene sequences and analyzed the linkage data available for such genes. RESULTS: We claim that neither Mox nor Evx is a Hox or ParaHox gene. We propose a scenario that reconciles phylogeny with linkage data, in which an Evx/Mox ancestor gene linked to a ProtoHox cluster was involved in a segmental tandem duplication event that generated an array of all Hox-like genes, referred to as the 'coupled' cluster. A chromosomal breakage within this cluster explains the current composition of the extended Hox cluster (with Evx, Hox and Mox genes) and the ParaHox cluster. CONCLUSIONS: Most studies dealing with the origin and evolution of Hox and ParaHox clusters have not included the Hox-related genes Mox and Evx. Our phylogenetic analyses and the available linkage data in mammalian genomes support an evolutionary scenario in which an ancestor of Evx and Mox was linked to the ProtoHox cluster, and that a tandem duplication of a large genomic region early in metazoan evolution generated the Hox and ParaHox clusters, plus the cluster-neighbors Evx and Mox. The large 'coupled' Hox-like cluster EvxHox/MoxParaHox was subsequently broken, thus grouping the Mox and Evx genes to the Hox clusters, and isolating the ParaHox cluster.

Animals↗

Linkage of the calmodulin and ubiquitin loci in Trypanosoma cruzi.

We describe here the organization of the calmodulin genes of Trypanosoma cruzi and their linkage to the ubiquitin gene family. The nucleotide sequence of the CalA2 gene has been determined and is 85% homologous to the protein coding sequence of the calmodulin genes of the African trypanosome, Trypanosoma brucei. The proteins encoded by CalA2 and the T. brucei genes contain a single mismatch out of a total of 149 amino acids. The genome of T. cruzi contains eight calmodulin genes present at two distinct loci (2.8 and 2.65) each of which is linked to downstream ubiquitin genes. Within each locus two calmodulin gene families have been defined, CalA and CalB. Each calmodulin locus consist of alternating tandem arrays of the CalA and CalB genes. Both 2.8 and 2.65 calmodulin loci and their respective downstream ubiquitin genes share the same DNA coding strand. Transcription of the calmodulin genes in the epimastigote stage of T. cruzi results in the generation of two stable mRNAs of 1.6Kb and 1.1Kb.

Amino Acid Sequence↗

Detection of deleted genomic DNA using a semiautomated computational analysis of GeneChip data.

Genomic diversity within and between populations is caused by single nucleotide mutations, changes in repetitive DNA systems, recombination mechanisms, and insertion and deletion events. The contribution of these sources to diversity, whether purely genetic or of phenotypic consequence, can only be investigated if we have the means to quantitate and characterize diversity in many samples. With the advent of complete sequence characterization of representative genomes of different species, the possibility of developing protocols to screen for genetic polymorphism across entire genomes is actively being pursued. The large numbers of measurements such approaches yield demand that we pay careful attention to the numerical analysis of data. In this paper we present a novel application of an Affymetrix GeneChip to perform genome-wide screens for deletion polymorphism. A high-density oligonucleotide array formatted for mRNA expression and targeted at a fully sequenced 4.4-million-base pair Mycobacterium tuberculosis standard strain genome was adapted to compare genomic DNA. Hybridization intensities to 111,000 probe pairs (perfect complement and mismatch complement) were measured for genomic DNA from a clinical strain and from a vaccine organism. Because individual probe-pair hybridization intensities exhibit limited sensitivity/specificity characteristics to detect deletions, data-analytical methodology to exploit measurements from multiple probes in tandem locations across the genome was developed. The TSTEP (Tandem Set Terminal Extreme Probability) algorithm designed specifically to analyze the tandem hybridization measurements data was applied and shown to discover genomic deletions with high sensitivity. The TSTEP algorithm provides a foundation for similar efforts to characterize deletions in many hybridization measures in similar-sized and larger genomes. Issues relating to the design of genome content screening experiments and the implications of these methods for studying population genomics and the evolution of genomes are discussed.

Algorithms↗

The tubulin genes of Trypanosoma cruzi.

The organization of the alpha- and beta-tubulin genes in the genome of Trypanosoma cruzi have been analysed by Southern blotting using tubulin probes derived from Trypanosoma brucei. The tubulin array appears to be more complex in this organism than in other members of the same family. Some tubulin genes are tightly clustered in an alternating (alpha-beta)n array with a basic repeat unit length of 4.3 kb. However, other pairs of alternating alpha- and beta-tubulin sequences appear to be physically separated from the basic group. This finding indicates that the tubulin gene cluster present in T. cruzi is less perfectly conserved than in T. brucei. T. (Herpetosoma) rangeli is similar to T. (Schizotrypanum) cruzi in its tubulin gene organization whereas most of these genes are tandemly clustered in the genome of T. (Trypanozoon) evansi, with a basic repeat unit length of 3.6 kb as previously described for T. (Trypanozoon) brucei. Two overlapping recombinant clones containing T. cruzi tubulin sequences have been isolated from a genomic cosmid library of T. cruzi epimastigotes using the T. brucei tubulin probes. Partial sequencing of the T. cruzi beta-tubulin gene has confirmed its identity and shows more than 70% homology with the sea urchin, chicken and T. b. rhodesiense beta-tubulin reported gene sequences. Analysis of tubulin gene organization through the parasite life cycle does not show evidence of major rearrangements within the repeat unit. Several T. cruzi strains and cloned lines whilst sharing the 4.3-kb tubulin repeat unit, exhibited very variable tubulin gene organization with tubulin probes. These striking differences in the organization of this structural gene among T. cruzi strains and cloned lines suggest that the heterogeneity previously reported in parasite populations may be related to a very dynamic, diploid genome.

Animals↗

Repetitive DNA and chromosome evolution in plants.

Most higher plant genomes contain a high proportion of repeated sequences. Thus repetitive DNA is a major contributor to plant chromosome structure. The variation in total DNA content between species is due mostly to variation in repeated DNA content. Some repeats of the same family are arranged in tandem arrays, at the sites of heterochromatin. Examples from the Secale genus are described. Arrays of the same sequence are often present at many chromosomal sites. Heterochromatin often contains arrays of several unrelated sequences. The evolution of such arrays in populations is discussed. Other repeats are dispersed at many locations in the chromosomes. Many are likely to be or have evolved from transposable elements. The structures of some plant transposable elements, in particular the sequences of the terminal inverted repeats, are described. Some elements in soybean, antirrhinum and maize have the same inverted terminal repeat sequences. Other elements of maize and wheat share terminal homology with elements from yeast, Drosophila, man and mouse. The evolution of transposable elements in plant populations is discussed. The amplification, deletion and transposition of different repeated DNA sequences and the spread of the mutations in populations produces a turnover of repetitive DNA during evolution. This turnover process and the molecular mechanisms involved are discussed and shown to be responsible for divergence of chromosome structure between species. Turnover of repeated genes also occurs. The molecular processes affecting repeats imply that the older a repetitive DNA family the more likely it is to exist in different forms and in many locations within a species. Examples to support this hypothesis are provided from the Secale genus.

Animals↗

(CA/GT)(n) microsatellites affect homologous recombination during yeast meiosis.

One of the most common microsatellites in eukaryotes consists of tandem arrays of the dinucleotide GT. Although the study of the instability of such repetitive DNA has been extremely fruitful over the last decade, no biological function has been demonstrated for these sequences. We investigated the genetic behavior of a region of the yeast Saccharomyces cerevisiae genome containing a 39-CA/GT dinucleotide repeat sequence. When the microsatellite sequence was present at the ARG4 locus on homologous chromosomes, diploid cells undergoing meiosis generated an excess of tetrads containing a conversion of the region restricted to the region of the microsatellite close to the recombination-initiation double-strand break. Moreover, whereas the repetitive sequence had no effect on the frequency of single crossover, its presence strongly stimulated the formation of multiple crossovers. The combined data strongly suggest that numerous recombination events are restricted to the initiation side of the microsatellite as though progression of the strand exchange initiated at the ARG4 promoter locus was impaired by the repetitive sequence. This observation corroborates in vitro experiments that demonstrated that RecA-promoted strand exchange is inhibited by CA/GT dinucleotide tracts. Surprisingly, meiotic instability of the microsatellite was very high (>0.1 alterations per tetrad) in all the spores with parental and recombinant chromosomes.

Argininosuccinate Lyase↗

Macromolecular organization and genetic mapping of a rapidly evolving chromosome-specific tandem repeat family (B77) in cotton (Gossypium).

Isolation and characterization of the most prominent repetitive element families in the genome of tetraploid cotton (Gossypium barbadense L; [39]) revealed a small subset of families that showed very different properties in tetraploids than in their diploid progenitors, separated by 1-2 million years. One element, B77, was characterized in detail, and compared to the well-conserved 5S and 45S rRNA genes. The 572 bp B77 repeat was found to be concentrated in several discontinuous tandem arrays confined to a single 550 kb SalI fragment in tetraploid cotton. Genetic mapping based on the absence of the pentameric 'rung' in the G. barbadense 'ladder' showed that B77 maps to a D-subgenome chromosome. In situ hybridization supports the contention that the array is confined largely to a single chromosomal site in the D-subgenome. The B77 repeat has undergone a substantial increase in copy number since formation of tetraploid cotton from its diploid relatives. RFLPs observed among tetraploid cotton species suggest that amplification and/or rearrangement of the repeat may have continued after divergence of the five tetraploid cotton species. B77 contains many short direct repeats and shares significant DNA sequence homology with a Nicotiana alata retrotransposon Tna1-2 integrase motif. The recent amplification of B77 on linkage group D04 suggests that the D-subgenome of tetraploid cotton may be subject to different evolutionary constraints than the D-genome diploid chromosomes, which exhibit few genome-specific elements. Further, the abundance of B77 in G. gossypioides supports independent evidence that it may be the closest extant relative of the D-genome ancestor of cotton.

Base Sequence↗

Characterization of VSG gene expression site promoters and promoter-associated DNA rearrangement events.

The expressed variant cell surface glycoprotein (VSG) gene of Trypanosoma brucei is located at the 3' end of a large, telomeric, polycistronic transcription unit or expression site. We show that the region 45 kb upstream of the VSG gene, in the expression site on a 1.5-Mb chromosome, contains at least two promoters that are arranged in tandem, directing the transcription of the expression site. DNA rearrangement events occur specifically, at inactivation of the expression site, and these events delete the most upstream transcribed region and replace it with a large array of simple-sequence DNA, leaving the downstream promoter intact. Because of the placement of simple-sequence DNA, the remaining downstream promoter now becomes structurally identical to previously described VSG promoters. The downstream promoter is repetitive in the genome, since it is present at several different expression sites. Restriction fragment length polymorphism mapping allows grouping of the expression sites into two families, those with and those without an upstream transcription unit, and the DNA rearrangement events convert the expression sites from one type to the other. Deletion of the upstream transcription unit also leads to the loss of several steady-state RNAs. The findings may indicate a role for promoter-associated DNA rearrangement events, and/or interactions between tandemly arranged promoters, in expression site transcriptional control.

Animals↗

Repetitive DNA sequences in Crocus vernus Hill (Iridaceae): the genomic organization and distribution of dispersed elements in the genus Crocus and its allies.

Eight clones of repetitive DNA were isolated from Crocus vernus Hill. The genomic organization of the clones was analyzed by in situ hybridization to C. vernus and Southern hybridization to a range of Crocus and other species. Seven clones were used for in situ hybridization. Sequence analysis showed that all eight clones were nonhomologous, and thus represented eight different sequence-families. In situ hybridization showed that six were dispersed in high copy numbers on all chromosomes of the C. vernus genome, whereas one was localized proximal to the secondary constriction, at the NOR (nucleolar organizer region) and was not further analyzed, as it was considered part of the 18S-25S rDNA repeat. Except for short palindromes, none of the sequences showed notable internal structures. Clone pCvKB4 showed homology to the reverse transcriptase gene of Ty1-copia-like retrotransposons; the others showed no homology to known sequences. When used as probes for Southern hybridization, four showed a ladder of 3-4 bands superimposed by irregular patterns, indicating organization in short tandem arrays. Each clone had a unique distribution among Crocus species (12-16 species analyzed with each clone) and six species of Iridaceae, Liliaceae, and Amaryllidaceae; all seven investigated sequences were Iridaceae specific and four were Crocus specific. The species distribution of these seven clones showed notable discrepancies with the taxonomic subdivision of the genus at the subgenus, section, and series levels. The results suggest that the phylogeny and taxonomic structure of the genus Crocus might need reconsideration. The analysis of repetitive DNA as a major and rapidly evolving part of the genome could contribute to the study of species relationships and evolution.

Base Sequence↗

Microsatellite evolution inferred from human-chimpanzee genomic sequence alignments.

Most studies of microsatellite evolution utilize long, highly mutable loci, which are unrepresentative of the majority of simple repeats in the human genome. Here we use an unbiased sample of 2,467 microsatellite loci derived from alignments of 5.1 Mb of genomic sequence from human and chimpanzee to investigate the mutation process of tandemly repetitive DNA. The results indicate that the process of microsatellite evolution is highly heterogeneous, exhibiting differences between loci of different lengths and motif sizes and between species. We find a highly significant tendency for human dinucleotide repeats to be longer than their orthologues in chimpanzees, whereas the opposite trend is observed in mononucleotide repeat arrays. Furthermore, the rate of divergence between orthologues is significantly higher at longer loci, which also show significantly greater mutability per repeat number. These observations have important consequences for understanding the molecular mechanisms of microsatellite mutation and for the development of improved measures of genetic distance.

Animals↗

Active microeletronic chip devices which utilize controlled electrophoretic fields for multiplex DNA hybridization and other genomic applications.

Microelectronic DNA chip devices that contain planar arrays of microelectrodes have been developed for multiplex DNA hybridization and a variety of genomic research and DNA diagnostic applications. These devices are able to produce almost any desired electric field configuration on their surface. This ability to produce well-defined electric fields allows charged molecules (DNA, RNA, proteins, enzymes, antibodies, nanobeads, and even micron scale semiconductor devices) to be electrophoretically transported to or from any microlocation on the planar surface of the device. Of key importance to the device function is the permeation layer which overcoats the microelectrodes. The permeation layer is generally a porous hydrogel material that allows water molecules and small ions (Na+, CI-, etc.) to freely contact the microelectrode surface, but impedes the transport of the larger analytes (oligonucleotides, DNA, RNA, proteins, etc.). The permeation layer prevents the destruction of DNA at the active microelectrode surface, ameliorates the adverse effects of electrolysis products on the sensitive hybridization reactions, and serves as a porous support structure for attaching DNA probes and other molecules to the array. In order to maintain rapid transport of DNA molecules, facilitate hybridization, and work within constrained current and voltage ranges, low conductance buffers and various electronic pulsing scenarios have also been developed. These active microelectronic array devices allow electrophoretic fields to be used to carry out accelerated DNA hybridization reactions and to improve selectivity for single nucleotide polymorphism (SNP), short tandem repeat (STR), and point mutation analysis.

Animals↗

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↗