Rice (Oryza sativa) centromeric regions consist of complex DNA

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence in situ hybridization. Six of the elements are middle repetitive, and their copy numbers range from approximately 50 to approximately 300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented approximately 6,000 times in the rice genome and is detected only in Oryza species. Fiber-fluorescence in situ hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and Arabidopsis thaliana chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences.     

Direct cloning of human 10q25 neocentromere DNA using transformation-associated recombination (TAR) in yeast

The transformation-associated recombination (TAR) procedure allows rapid, site-directed cloning of specific human chromosomal regions as yeast artificial chromosomes (YACs). The procedure requires knowledge of only a single, relatively small genomic sequence that resides adjacent to the chromosomal region of interest. We applied this approach to the cloning of the neocentromere DNA of a marker chromosome that we have previously shown to have originated through the activation of a latent centromere at human chromosome 10q25. Using a unique 1.4-kb DNA fragment as a "hook" in TAR experiments, we achieved single-step isolation of the critical neocentromere DNA region as two stable, 110- and 80-kb circular YACs. For obtaining large quantities of highly purified DNA, these YACs were retrofitted with the yeast-bacteria-mammalian-cells shuttle vector BRV1, electroporated into Escherichia coli DH10B, and isolated as bacterial artificial chromosomes (BACs). Extensive characterization of these YACs and BACs by PCR and restriction analyses revealed that they are identical to the corresponding regions of the normal chromosome 10 and provided further support for the formation of the neocentromere within the marker chromosome through epigenetic activation.     

Engineering mammalian chromosomes

Construction of a mammalian artificial chromosome (MAC) will develop our understanding of the requirements for normal chromosome maintenance, replication and segregation while offering the capacity for introducing genes into cells. Construction of MACs with telomere, centromere and replication function has been approached by two methods. The 'top down' strategy uses artificially induced chromosome truncations as a means to define a minimal chromosome that retains the mitotic properties of a normal chromosome. The 'build up' approach has focused on attempts to assemble MAC vectors containing functionally defined telomere repeats together with candidate centromere and replication origin sequences. Here we report on significant advances in both areas, with particular emphasis on two reports showing that stable, low copy number MACs containing a functional centromere can be produced following transfection of naked DNA into the human HT1080 cell line. One approach used a transfection mixture of cloned synthetic alpha-satellite arrays up to 1 Mb in length and unlinked telomeric DNA, in either the presence or absence of random human genomic DNA fragments. In the second approach, MACs were formed from a defined yeast artificial chromosome (YAC) DNA molecule containing 100 kb of highly homo- geneous alphoid DNA retrofitted with human telomere repeats. These results demonstrate for the first time that alpha-satellite DNA can seed de novo centromeres in human cells, indicating that this repetitive sequence family plays an important role in centromere function. The stability of these MACs suggests that they have potential to be developed as gene delivery vectors.     

Structure of the chromosome VII centromere region in Neurospora crassa: degenerate transposons and simple repeats

DNA from the centromere region of linkage group (LG) VII of Neurospora crassa was cloned previously from a yeast artificial chromosome library and was found to be atypical of Neurospora DNA in both composition (AT rich) and complexity (repetitive). We have determined the DNA sequence of a small portion (approximately 16.1 kb) of this region and have identified a cluster of three new retrotransposon-like elements as well as degenerate fragments from the 3' end of Tad, a previously identified LINE-like retrotransposon. This region contains a novel full-length but nonmobile copia-like element, designated Tcen, that is only associated with centromere regions. Adjacent DNA contains portions of a gypsy-like element designated Tgl1. A third new element, Tgl2, shows similarity to the Ty3 transposon of Saccharomyces cerevisiae. All three of these elements appear to be degenerate, containing predominantly transition mutations suggestive of the repeat-induced point mutation (RIP) process. Three new simple DNA repeats have also been identified in the LG VII centromere region. While Tcen elements map exclusively to centromere regions by restriction fragment length polymorphism analysis, the defective Tad elements appear to occur most frequently within centromeres but are also found at other loci including telomeres. The characteristics and arrangement of these elements are similar to those seen in the Drosophila centromere, but the relative abundance of each class of repeats, as well as the sequence degeneracy of the transposon-like elements, is unique to Neurospora. These results suggest that the Neurospora centromere is heterochromatic and regional in character, more similar to centromeres of Drosophila than to those of most single-cell yeasts.     

The centromere enhancer mediates centromere activation in Schizosaccharomyces pombe

The centromere enhancer is a functionally important DNA region within the Schizosaccharomyces pombe centromeric K-type repeat. We have previously shown that addition of the enhancer and cen2 centromeric central core to a circular minichromosome is sufficient to impart appreciable centromere function. A more detailed analysis of the enhancer shows that it is dispensable for centromere function in a cen1-derived minichromosome containing the central core and the remainder of the K-type repeat, indicating that the critical centromeric K-type repeat, like the central core, is characterized by functional redundancy. The centromeric enhancer is required, however, for a central core-carrying minichromosome to exhibit immediate centromere activity when the circular DNA is introduced via transformation into S. pombe. This immediate activation is probably a consequence of a centromere-targeted epigenetic system that governs the chromatin architecture of the region. Moreover, our studies show that two entirely different DNA sequences, consisting of elements derived from two native centromeres, can display centromere function. An S. pombe CENP-B-like protein, Abp1p/Cbp1p, which is required for proper chromosome segregation in vivo, binds in vitro to sites within and adjacent to the modular centromere enhancer, as well as within the centromeric central cores. These results provide direct evidence in fission yeast of a model, similar to one proposed for mammalian systems, whereby no specific sequence is necessary for centromere function but certain classes of sequences are competent to build the appropriate chromatin foundation upon which the centromere/kinetochore can be formed and activated.     

Retrotransposon-related DNA sequences in the centromeres of grass chromosomes

Several distinct DNA fragments were subcloned from a sorghum (Sorghum bicolor) bacterial artificial chromosome clone 13I16 that was derived from a centromere. Three fragments showed significant sequence identity to either Ty3/gypsy- or Ty1/copia-like retrotransposons. Fluorescence in situ hybridization (FISH) analysis revealed that the Ty1/copia-related DNA sequences are not specific to the centromeric regions. However, the Ty3/gypsy-related sequences were present exclusively in the centromeres of all sorghum chromosomes. FISH and gel-blot hybridization showed that these sequences are also conserved in the centromeric regions of all species within Gramineae. Thus, we report a new retrotransposon that is conserved in specific chromosomal regions of distantly related eukaryotic species. We propose that the Ty3/gypsy-like retrotransposons in the grass centromeres may be ancient insertions and are likely to have been amplified during centromere evolution. The possible role of centromeric retrotransposons in plant centromere function is discussed.     

Assay of centromere function using a human artificial chromosome

In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host. When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the alpha21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1-5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the alpha21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that alpha21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes.     

Imaging of acute thoracic aortic injury due to blunt trauma: a review

Special AT-rich sequence-binding protein 1 (SATB1), a DNA-binding protein expressed predominantly in thymocytes, recognizes an ATC sequence context that consists of a cluster of sequence stretches with well-mixed A's, T's, and C's without G's on one strand. Such regions confer a high propensity for stable base unpairing. Using an in vivo cross-linking strategy, specialized genomic sequences (0.1-1. 1 kbp) that bind to SATB1 in human lymphoblastic cell line Jurkat cells were individually isolated and characterized. All in vivo SATB1-binding sequences examined contained typical ATC sequence contexts, with some exhibiting homology to autonomously replicating sequences from the yeast Saccharomyces cerevisiae that function as replication origins in yeast cells. In addition, LINE 1 elements, satellite 2 sequences, and CpG island-containing DNA were identified. To examine the higher-order packaging of these in vivo SATB1-binding sequences, high-resolution in situ fluorescence hybridization was performed with both nuclear "halos" with distended loops and the nuclear matrix after the majority of DNA had been removed by nuclease digestion. In vivo SATB1-binding sequences hybridized to genomic DNA as single spots within the residual nucleus circumscribed by the halo of DNA and remained as single spots in the nuclear matrix, indicating that these sequences are localized at the base of chromatin loops. In human breast cancer SK-BR-3 cells that do not express SATB1, at least one such sequence was found not anchored onto the nuclear matrix. These findings provide the first evidence that a cell type-specific factor such as SATB1 binds to the base of chromatin loops in vivo and suggests that a specific chromatin loop domain structure is involved in T cell-specific gene regulation.     

Early myeloid cell-specific expression of the human cathepsin G gene in transgenic mice

The human cathepsin G (CG) gene is expressed only in promyelocytes and encodes a neutral serine protease that is packaged in the azurophil (primary) granules of myeloid cells. To define the cis-acting DNA elements that are responsible for promyelocyte-specific "targeting," we injected a 6-kb transgene containing the entire human CG gene, including coding sequences contained in a 2.7-kb region, approximately 2.5 kb of 5' flanking sequence, and approximately 0.8 kb of 3' flanking sequence. Seven of seven "transient transgenic" murine embryos revealed human CG expression in the fetal livers at embryonic day 15. Stable transgenic founder lines were created with the same 6-kb fragment; four of five founder lines expressed human CG in the bone marrow. The level of human CG expression was relatively low per gene copy when compared with the endogenous murine CG gene, and expression was integration-site dependent; however, the level of gene expression correlated roughly with gene copy number. The human CG transgene and the endogenous murine CG gene were coordinately expressed in the bone marrow and the spleen. Immunohistochemical analysis of transgenic bone marrow revealed that the human CG protein was expressed exclusively in myeloid cells. Expression of human CG protein was highest in myeloid precursors and declined in mature myeloid cells. These data suggest that the human CG gene was appropriately targeted and developmentally regulated, demonstrating that the cis-acting DNA sequences required for the early myeloid cell-specific expression of human CG are present in this small genomic fragment.     

A neocentromere on human chromosome 3 without detectable alpha-satellite DNA forms morphologically normal kinetochores

A neocentromere at 3q26 was observed in a father and his daughter on a chromosome 3 with deleted centromeric region. No alpha-satellite DNA was detectable at the 3q26 neocentromere, but it was weakly positive with anticentromere (CREST) antibodies. Electron microscopy showed that the neocentromere formed microtubule-associated kinetochores with normal morphology and of the same size as the kinetochores of other large chromosomes. The deleted centromere formed a small linear marker chromosome that reacted strongly with anticentromere antibodies, but showed reduced kinetochore size. The 3q26 neokinetochore was stable under adverse growth conditions, which often caused kinetochore loss in the original 3-centromere on the small marker.     

Formation of de novo centromeres and construction of first-generation human artificial microchromosomes

We have combined long synthetic arrays of alpha satellite DNA with telomeric DNA and genomic DNA to generate artificial chromosomes in human HT1080 cells. The resulting linear microchromosomes contain exogenous alpha satellite DNA, are mitotically and cytogenetically stable in the absence of selection for up to six months in culture, bind centromere proteins specific for active centromeres, and are estimated to be 6-10 megabases in size, approximately one-fifth to one-tenth the size of endogenous human chromosomes. We conclude that this strategy results in the formation of de novo centromere activity and that the microchromosomes so generated contain all of the sequence elements required for stable mitotic chromosome segregation and maintenance. This first-generation system for the construction of human artificial chromosomes should be suitable for dissecting the sequence requirements of human centromeres, as well as developing constructs useful for therapeutic applications.     

A zipper-like duplex in DNA: the crystal structure of d(GCGAAAGCT) at 2.1 A resolution

BACKGROUND: The replication origin of the single-stranded (ss)DNA bacteriophage G4 has been proposed to fold into a hairpin loop containing the sequence GCGAAAGC. This sequence comprises a purine-rich motif (GAAA), which also occurs in conserved repetitive sequences of centromeric DNA. ssDNA analogues of these sequences often show exceptional stability which is associated with hairpin loops or unusual duplexes, and may be important in DNA replication and centromere function. Nuclear magnetic resonance (NMR) studies indicate that the GCGAAAGC sequence forms a hairpin loop in solution, while centromere-like repeats dimerise into unusual duplexes. The factors stabilising these unusual secondary structure elements in ssDNA, however, are poorly understood. RESULTS: The nonamer d(GCGAAAGCT) was crystallised as a bromocytosine derivative in the presence of cobalt hexammine. The crystal structure, solved by the multiple wavelength anomalous dispersion (MAD) method at the bromine K-edge, reveals an unexpected zipper-like motif in the middle of a standard B-DNA duplex. Four central adenines, flanked by two sheared G.A mismatches, are intercalated and stacked on top of each other without any interstrand Watson-Crick base pairing. The cobalt hexammine cation appears to participate only in crystal cohesion. CONCLUSIONS: The GAAA consensus sequence can dimerise into a stable zipper-like duplex as well as forming a hairpin loop. The arrangement closes the minor groove and exposes the intercalated, unpaired, adenines to the solvent and DNA-binding proteins. Such a motif, which can transform into a hairpin, should be considered as a structural option in modelling DNA and as a potential binding site, where it could have a role in DNA replication, nuclease resistance, ssDNA genome packaging and centromere function.     

Nucleotide sequence surrounding the locus marker D21S246 on human chromosome 21

Most of the human Not I linking clones identified to date are considered to be derived from CpG islands because of the recognition sequence of this enzyme, and CpG islands have been reported to be located around the 5' regions of genes. As a pilot study, we determined the complete nucleotide sequence (41,924 bp) of a human cosmid clone (LL21NC02Q7A10) containing the marker D21S246 originating from a Not I linking clone. As a result of sequence analysis, we successfully mapped and revealed the genomic gene structure for KIAA0002 previously reported as a cDNA clone. This gene consists of 15 exons and was shown to exist at the D21S246 locus on human chromosome 21q21.3-q22.1. These results demonstrated that genomic marker-anchored DNA sequencing is a useful approach for the human genome project.     

A stable marker chromosome with a cryptic centromere: evidence for centromeric sequences associated with an inverted duplication

Centromere activation, an important mechanism in karyotype evolution, is occasionally observed in some human chromosome rearrangements. We report a possible occurrence of centromere activation in a marker chromosome containing an atypical centromere associated with an inverted duplication of the region 14q32 --> qter. The marker chromosome's reduced centromere lacks both the alpha and beta satellite sequences usually found at normal centromeres. In an attempt to identify the centromeric sequences, the marker chromosome was flow-sorted and amplified by a degenerate oligonucleotide primer polymerase chain reaction. Reverse chromosome painting experiments showed that the marker chromosome contains sequences that are unique to the distal region of chromosome 14, as well as a low copy number of (centromeric) sequences that are also highly represented in the centromeres of chromosomes 18 and 19. These data suggest the activation of a novel centromere in the 14q32 --> qter region, very likely consequent to the duplication of the region itself.     

Single copy DNA and structural gene sequence relationships among four sea urchin species

Measurements of the divergence of single copy DNA sequences among four sea urichin species are presented. At a standard criterion for reassociation (0.12 M phosphate buffer, 60 degrees C, hydroxyapatite binding) we observe the following extents of reaction and reductions in thermal stability for single copy DNA reassociation between Strongylocentrotus purpuratus tracer and heterologous driver DNA: S. dr?bachiensis 68% and 2.5 degrees C; S. franciscanus 51% and 3.5 degrees C; Lytechinus pictus 12% and 7.5 degrees C. The implied extents of sequence relatedness are consistent with the phylogenetic relationships of these species. The rate of single copy sequence divergence in the evolutionary lines leading to the Strongylocentrotus species is estimated to be 0.06-0.35% per million years. The rate of divergence of total single copy sequence has been compared to that of structural gene sequences represented in S. purpuratus gastrula polysomal messenger RNA. When closely related species, S. purpuratus and S. franciscanus, are compared, these polysomal sequences are found to diverge at a lower rate than does the total single copy sequence. For two very distantly related species, S. purpuratus and L. pictus, a small fraction of the single copy DNA sequence is probably conserved. These conserved sequences are not enriched in their content of structural gene sequences.     

Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution

Virulence genes of pathogenic bacteria, which code for toxins, adhesins, invasins or other virulence factors, may be located on transmissible genetic elements such as transposons, plasmids or bacteriophages. In addition, such genes may be part of particular regions on the bacterial chromosomes, termed 'pathogenicity islands' (Pais). Pathogenicity islands are found in Gram-negative as well as in Gram-positive bacteria. They are present in the genome of pathogenic strains of a given species but absent or only rarely present in those of non-pathogenic variants of the same or related species. They comprise large DNA regions (up to 200 kb of DNA) and often carry more than one virulence gene, the G + C contents of which often differ from those of the remaining bacterial genome. In most cases, Pais are flanked by specific DNA sequences, such as direct repeats or insertion sequence (IS) elements. In addition, Pais of certain bacteria (e,g. uropathogenic Escherichia coli, Yersinia spp., Helicobacter pylori) have the tendency to delete with high frequencies or may undergo duplications and amplifications. Pais are often associated with tRNA loci, which may represent target sites for the chromosomal integration of these elements. Bacteriophage attachment sites and cryptic genes on Pais, which are homologous to phage integrase genes, plasmid origins of replication of IS elements, indicate that these particular genetic elements were previously able to spread among bacterial populations by horizontal gene transfer, a process known to contribute to microbial evolution.