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PubMed2006

Use of oligoarrays for characterization of community-onset methicillin-resistant Staphylococcus aureus.

Until recently, methicillin-resistant Staphylococcus aureus (MRSA) was considered the prototype of a hospital-acquired bacterial pathogen. However, recent reports have shown that MRSA has now emerged in the community. Characterization of specific markers for distinguishing the origin of isolates could contribute to improved knowledge of MRSA epidemiology. The release of whole-genome sequences of hospital- and community-acquired S. aureus strains allowed the development of whole-genome content analysis techniques, including microarrays. We developed a microarray composed of 8,191 open reading frame-specific oligonucleotides covering >99% of the four sequenced S. aureus genomes (N315, Mu50, MW2, and COL) to evaluate gene contents of hospital- and community-onset S. aureus strains. In parallel, pulsed-field gel electrophoresis, variable number of tandem repeats, antibiogram, staphylococcal cassette chromosome-mec element typing, and presence of the Panton-Valentine leukocidin gene were evaluated in a collection of 15 clinical isolates. Clusters obtained with microarrays showed a high degree of similarity with those obtained by pulsed-field gel electrophoresis or variable number of tandem repeats. Clusters clearly segregated hospital-onset strains from community-onset strains. Moreover, the microarray approach allowed definition of novel marker genes and chromosomal regions specific for given groups of isolates, thus providing better discrimination and additional information compared to pulsed-field gel electrophoresis and variable number of tandem repeats. Finally, the comparative genome hybridization approach unraveled the occurrence of multiple horizontal transfer events leading to community-onset MRSA as well as the need for a specific genetic background in recipient strains for both the acquisition and the stability of the mec element.

Adult↗

PubMed2006

Homozygosity mapping with SNP arrays identifies TRIM32, an E3 ubiquitin ligase, as a Bardet-Biedl syndrome gene (BBS11).

The identification of mutations in genes that cause human diseases has largely been accomplished through the use of positional cloning, which relies on linkage mapping. In studies of rare diseases, the resolution of linkage mapping is limited by the number of available meioses and informative marker density. One recent advance is the development of high-density SNP microarrays for genotyping. The SNP arrays overcome low marker informativity by using a large number of markers to achieve greater coverage at finer resolution. We used SNP microarray genotyping for homozygosity mapping in a small consanguineous Israeli Bedouin family with autosomal recessive Bardet-Biedl syndrome (BBS; obesity, pigmentary retinopathy, polydactyly, hypogonadism, renal and cardiac abnormalities, and cognitive impairment) in which previous linkage studies using short tandem repeat polymorphisms failed to identify a disease locus. SNP genotyping revealed a homozygous candidate region. Mutation analysis in the region of homozygosity identified a conserved homozygous missense mutation in the TRIM32 gene, a gene coding for an E3 ubiquitin ligase. Functional analysis of this gene in zebrafish and expression correlation analyses among other BBS genes in an expression quantitative trait loci data set demonstrate that TRIM32 is a BBS gene. This study shows the value of high-density SNP genotyping for homozygosity mapping and the use of expression correlation data for evaluation of candidate genes and identifies the proteasome degradation pathway as a pathway involved in BBS.

Animals↗

PubMed1991

Identification and characterization of "zinc-finger" domains by the polymerase chain reaction.

We have developed a method for amplifying DNA fragments containing tandem arrays of "zinc-finger" sequences of the transcription factor IIIA (Cys2-His2) type by using the polymerase chain reaction. Because these sequences occur as tandem arrays, a ladder of bands is produced upon amplification using primers derived from the amino- and carboxyl-terminal sequences of a zinc-finger domain. The "rungs" of this ladder correspond to DNA fragments encoding one zinc-finger domain, two adjacent zinc-finger domains, and so on. This is demonstrated by isolating individual bands corresponding to n zinc-finger domains and reamplifying them with the same primers. This yields a band of the original size as well as bands corresponding to 1 through (n - 1) zinc-finger domains. Direct evidence that these bands encode zinc-finger domains was obtained by cloning and sequencing a collection of the amplification products. Due to the lack of redundancy in the sequences obtained, we conclude that each band corresponds to a large number of unique zinc-finger-encoding sequences. The results from amplification reaction mixtures using genomic DNA from a variety of sources as template provide further evidence that zinc-finger domains occur widely and frequently in eukaryotic genomes. We believe that this method is a powerful technique for the isolation and characterization of zinc-finger-encoding genes.

Amino Acid Sequence↗

PubMed2000

Genomic organization of the human cholesterol-responsive ABC transporter ABCA7: tandem linkage with the minor histocompatibility antigen HA-1 gene.

We have recently cloned a novel cholesterol-responsive ABC transporter, designated ABCA7, which is predominantly expressed in human leukocytes. Here we report the structure of the human ABCA7 gene. The ABCA7 gene spans a region of approximately 32 kb and comprises 46 exons. Its putative promoter sequence contains potential binding sites for transcription factors with roles in hematopoiesis and cholesterol metabolism. Surprisingly, sequence analysis of the ABCA7 3' gene flanking region revealed that the terminal exon of ABCA7 borders immediately on the 5' end of the coding region of the recently identified human minor histocompatibility antigen HA-1. We demonstrate that the coding regions of ABCA7 and HA-1 are physically separated by a 1.7-kb intergene region. Subsequent genomic structure analysis showed that the HA-1 gene consists of 23 exons which extend across a 16-kb genomic region. Our results provide evidence that the genes for the human minor histocompatibility antigen HA-1 and the ABC transporter ABCA7 are arranged in a head-to-tail array and that both genes localize to a common locus of approximately 48 kb size on chromosome 19p13.3.

ATP-Binding Cassette Transporters↗

PubMed2006

Environment arrays: a possible approach for predicting changes in waterborne bacterial disease potential.

Current molecular techniques for identifying bacteria in water have proven useful, but they are not reliably predictive of impending disease outbreaks. Genomics-based approaches will help to detect the presence of pathogens quickly and well before they grow into a population that poses a risk to public health. We suggest that genomics is only one component of the toolbox that will be needed to identify emerging waterborne threats. We propose a methodology beyond genomics, based on activity in the mobile genome. This approach makes use of a new device called an environment array. The array will depend upon the same research necessary for genomics-based detection, but will not require an a priori knowledge of virulence genes. Environment arrays are assembled from molecular profiles of the infectious elements that transfer between bacteria. The advantage of the array is that it monitors the activity of the mobile genome, rather than the presence of particular DNA sequences. Environmental arrays should thus be many times more sensitive than traditional hybridization or PCR-based techniques that target already-known DNA sequences. Mobile elements are known to respond to new environmental conditions that may correlate with a chemical contamination or the bloom of bacterial pathogens, potentially allowing for a much broader application in detecting unknown or unanticipated biological and chemical contaminants.

Bacteria↗

PubMed2001

A tandemly repeated DNA sequence is associated with both knob-like heterochromatin and a highly decondensed structure in the meiotic pachytene chromosomes of rice.

Highly repetitive tandem DNA sequence repeats are often associated with centromeric and telomeric regions of eukaryotic chromosomes. The rice tandem repeat Os48 is organized as long arrays of a 355 bp monomer and is mainly located in the telomeric regions. The chromosomal locations of the Os48 sequence were determined by fluorescence in situ hybridization (FISH) on rice pachytene chromosomes. The majority of the Os48 loci are associated with brightly 4',6-diamidino-2-phenylindole (DAPI)-stained and knob-like heterochromatin in rice pachytene chromosomes. As with other DNA sequences located in the heterochromatic regions, the cytosines of the CG and C(A/T)G sites within the Os48 repeat are heavily methylated. Surprisingly, a proportion of the FISH signals are highly decondensed and deviate significantly from the DAPI-stained periphery of the pachytene chromosomes. This highly decondensed chromatin structure has not been reported in pachytene chromosomes prepared from alcohol/acid-fixed meiotic samples in any other eukaryotic species. The condensation of the Os48 sequences is dynamic during prophase I of meiosis. The FISH signals derived from the Os48 repeat progress from a condensed configuration between leptonema and early pachynema into a decondensed structure from middle pachynema to diakinesis, and then return to a condensed form at metaphase I.

Base Sequence↗

PubMed2006

The clustering of four subfamilies of satellite DNA at individual chromosome ends in Silene latifolia.

The satellite DNA (satDNA) on the ends of chromosomes has been isolated and characterized in the dioecious plant Silene latifolia. BAC clones containing large numbers of repeat units of satDNA in a tandem array were isolated to examine the clustering of the repeat units. satDNA repeat units were purified from each isolated BAC clone and sequenced. To investigate pairwise similarities among the repeat units, a phylogenetic tree was constructed using the neighbor-joining algorithm. The repeat units derived from 7 BAC clones were grouped into SacI, KpnI, #11F02, and #16E07 subfamilies. The SacI and KpnI subfamilies have been reported previously. Multicolored fluorescence in situ hybridization (FISH) using SacI or KpnI subfamily probes resulted in different signal intensities and locations at the chromosomal ends, indicating that each chromosomal end has a unique composition of subfamilies of satDNA. For example, the p arm of the X chromosome exhibited signal composition similar to that on the pseudo autosomal region (PAR) of the Y chromosome, but not to that on the q arm of the X chromosome. The satDNA has not been completely homogenized in the S. latifolia genome. Each subfamily is available for a probe of FISH karyotyping.

Base Sequence↗

PubMed1990

Heterogeneity and organization of the ribosomal RNA genes of Cucurbita maxima.

Thirty-six clones were recovered from Cucurbita maxima genomic DNA which had been enriched for rDNA and cleaved at the unique repeat unit Hind III site. Twenty-nine of these, which contain complete rDNA units, were compared to a standard whose intergenic spacer (IGS) nucleotide sequence has been determined. Twenty-one are identical in length and restriction site pattern. Eight which differ from the standard in length do so because of addition or deletion of varying numbers of IGS subrepetitive units of two different classes, with four of the length variants being different in both of these classes. Seven clones were isolated which contain incomplete repeat units, six of which are composites of rDNA and non-rDNA material. They have been cleaved at the unique rDNA Hind III site at one end and at a non-rDNA Hind III site at the other. We consider it most likely that these are derived from the termini of repeat unit tandem arrays, although other explanations are possible. Twelve individual plants of two different cultivars were examined for heterogeneity of IGS length distribution. They all appear to be identical in this regard.

Cloning, Molecular↗

PubMed2002

Rice genome organization: the centromere and genome interactions.

Over the last decade, many varied resources have become available for genome studies in rice. These resources include over 4000 DNA markers, several bacterial artificial chromosome (BAC) libraries, P-1 derived artificial chromosome (PAC) libraries and yeast artificial chromosome (YAC) libraries (genomic DNA clones, filters and end-sequences), retrotransposon tagged lines, and many chemical and irradiated mutant lines. Based on these, high-density genetic maps, cereal comparative maps, YAC and BAC physical maps, and quantitative trait loci (QTL) maps have been constructed, and 93 % of the genome has also been sequenced. These data have revealed key features of the genetic and physical structure of the rice genome and of the evolution of cereal chromosomes. This Botanical Briefing examines aspects of how the rice genome is organized structurally, functionally and evolutionarily. Emphasis is placed on the rice centromere, which is composed of long arrays of centromere-specific repetitive sequences. Differences and similarities amongst various cereal centromeres are detailed. These indicate essential features of centromere function. Another view of various kinds of interactive relationships within and between genomes, which could play crucial roles in genome organization and evolution, is also introduced. Constructed genetic and physical maps indicate duplication of chromosomal segments and spatial association between specific chromosome regions. A genome-wide survey of interactive genetic loci has identified various reproductive barriers that may drive speciation of the rice genome. The significance of these findings in genome organization and evolution is discussed.

Centromere↗

PubMed1984

A 3' co-terminal family of mRNAs from the herpes simplex virus type 1 short region: two overlapping reading frames encode unrelated polypeptide one of which has highly reiterated amino acid sequence.

We have used DNA sequencing, mRNA mapping and in vitro translation to characterise three partially overlapping genes in the genome of herpes simplex virus (HSV) type 1. These genes specify three mRNAs with distinct 5' termini but a common 3' terminus, the longest of which is immediate-early (IE) mRNA-5. The 12,000 MW (12K) IE polypeptide encoded by IEmRNA-5 is translated from an 88 codon open reading frame, leaving a 1200 base 3' non-translated region. The second mRNA (mRNA-B) is initiated within the coding sequence of IEmRNA-5, and encodes a 21K polypeptide. The 12K and 21K polypeptide coding regions do not overlap. The third mRNA (mRNA-C) is initiated within the coding region of mRNA-B, and encodes a 33K polypeptide. The reading frame for 33K has a 110 codon out-of-frame overlap with the 21K reading frame. This is the first instance of overlapping genes described for HSV. The 21K polypeptide is thought to be a DNA binding protein and is remarkable for an array of 24 tandem repeats of the sequence X/Pro/Arg (where X represents predominantly Glu, Asp, Thr, Ser or Val) in its C-terminal portion. This array, which occupies most of the region of overlap with 33K, can vary in repeat number between virus strains.

Amino Acid Sequence↗

PubMed2002

Molecular evolution of the nontandemly repeated genes of the histone 3 multigene family.

In some species, histone gene clusters consist of tandem arrays of each type of histone gene, whereas in other species the genes may be clustered but not arranged in tandem. In certain species, however, histone genes are found scattered across several different chromosomes. This study examines the evolution of histone 3 (H3) genes that are not arranged in large clusters of tandem repeats. Although H3 amino acid sequences are highly conserved both within and between species, we found that the nucleotide sequence divergence at synonymous sites is high, indicating that purifying selection is the major force for maintaining H3 amino acid sequence homogeneity over long-term evolution. In cases where synonymous-site divergence was low, recent gene duplication appeared to be a better explanation than gene conversion. These results, and other observations on gene inactivation, organization, and phylogeny, indicated that these H3 genes evolve according to a birth-and-death process under strong purifying selection. Thus, we found little evidence to support previous claims that all H3 proteins, regardless of their genome organization, undergo concerted evolution. Further analyses of the structure of H3 proteins revealed that the histones of higher eukaryotes might have evolved from a replication-independent-like H3 gene.

Amino Acid Sequence↗

PubMed1998

Influence of intron length on alternative splicing of CD44.

Although the splicing of transcripts from most eukaryotic genes occurs in a constitutive fashion, some genes can undergo a process of alternative splicing. This is a genetically economical process which allows a single gene to give rise to several protein isoforms by the inclusion or exclusion of sequences into or from the mature mRNA. CD44 provides a unique example; more than 1,000 possible isoforms can be produced by the inclusion or exclusion of a central tandem array of 10 alternatively spliced exons. Certain alternatively spliced exons have been ascribed specific functions; however, independent regulation of the inclusion or skipping of each of these exons would clearly demand an extremely complex regulatory network. Such a network would involve the interaction of many exon-specific trans-acting factors with the pre-mRNA. Therefore, to assess whether the exons are indeed independently regulated, we have examined the alternative exon content of a large number of individual CD44 cDNA isoforms. This analysis shows that the downstream alternatively spliced exons are favored over those lying upstream and that alternative exons are often included in blocks rather than singly. Using a novel in vivo alternative splicing assay, we show that intron length has a major influence upon the alternative splicing of CD44. We propose a kinetic model in which short introns may overcome the poor recognition of alternatively spliced exons. These observations suggest that for CD44, intron length has been exploited in the evolution of the genomic structure to enable tissue-specific patterns of splicing to be maintained.

Alternative Splicing↗

PubMed2001

Microarray-based survey of repetitive genomic sequences in Vicia spp.

A modified DNA microarray-based technique was devised for preliminary screening of short fragment genomic DNA libraries from three Vicia species (V. melanops, V. narbonensis, and V. sativa) to isolate representative highly abundant DNA sequences that show different distribution patterns among related legume species. The microarrays were sequentially hybridized with labeled genomic DNAs of thirteen Vicia and seven other Fabaceae species and scored for hybridization signals of individual clones. The clones were then assigned to one of the following groups characterized by hybridization to: (1) all tested species, (2) most of the Vicia and Pisum species, (3) only a few Vicia species, and (4) preferentially a single Vicia species. Several clones from each group, 65 in total, were sequenced. All Group I clones were identified as rDNA genes or fragments of chloroplast genome, whereas the majority of Group II clones showed significant homologies to retroelement sequences. Clones in Groups III and IV contained novel dispersed repeats with copy numbers 10(2)-10(6)/1C and two genus-specific tandem repeats. One of these belongs to the VicTR-B repeat family, and the other clone (S12) contains an amplified portion of the rDNA intergenic spacer. In situ hybridization using V. sativa metaphase chromosomes revealed the presence of the S12 sequences not only within rDNA genes, but also at several additional loci. The newly identified repeats, as well as the retroelement-like sequences, were characterized with respect to their abundance within individual genomes. Correlations between the repeat distributions and the current taxonomic classification of these species are discussed.

Blotting, Southern↗

PubMed2000

The core histone N termini function independently of linker histones during chromatin condensation.

The relationships between the core histone N termini and linker histones during chromatin assembly and salt-dependent chromatin condensation were investigated using defined chromatin model systems reconstituted from tandemly repeated 5 S rDNA, histone H5, and either native "intact" core histone octamers or "tailless" histone octamers lacking their N-terminal domains. Nuclease digestion and sedimentation studies indicate that H5 binding and the resulting constraint of entering and exiting nucleosomal DNA occur to the same extent in both tailless and intact chromatin arrays. However, despite possessing a normal chromatosomal structure, tailless chromatin arrays can neither condense into extensively folded structures nor cooperatively oligomerize in MgCl(2). Tailless nucleosomal arrays lacking linker histones also are unable to either fold extensively or oligomerize, demonstrating that the core histone N termini perform the same functions during salt-dependent condensation regardless of whether linker histones are components of the array. Our results further indicate that disruption of core histone N termini function in vitro allows a linker histone-containing chromatin fiber to exist in a decondensed state under conditions that normally would promote extensive fiber condensation. These findings have key implications for both the mechanism of chromatin condensation, and the regulation of genomic function by chromatin.

Chromatin↗

PubMedDate not supplied

Cryptic simplicity in DNA is a major source of genetic variation.

DNA regions which are composed of a single or relatively few short sequence motifs usually in tandem ('pure simple sequences') have been reported in the genomes of diverse species, and have been implicated in a range of functions including gene regulation, signals for gene conversion and recombination, and the replication of telomeres. They are thought to accumulate by DNA slippage and mispairing during replication and recombination or extension of single-strand ends. In order to systematize the range of DNA simplicity and the genetic nature of the regions that are simple, we have undertaken an extensive computer search of the DNA sequence library of the European Molecular Biology Laboratory (EMBL). We show here that nearly all possible simple motifs occur 5-10 times more frequently than equivalent random motifs. Furthermore, a new computer algorithm reveals the widespread occurrence of significantly high levels of a new type of 'cryptic simplicity' in both coding and noncoding DNA. Cryptically simple regions are biased in nucleotide composition and consist of scrambled arrangements of repetitive motifs which differ within and between species. The universal existence of DNA simplicity from monotonous arrays of single motifs to variable permutations of relatively short-lived motifs suggests that ubiquitous slippage-like mechanisms are a major source of genetic variation in all regions of the genome, not predictable by the classical mutation process.

Animals↗

PubMed1994

Developmental gene expression in Leishmania donovani: differential cloning and analysis of an amastigote-stage-specific gene.

Leishmania protozoans are the causative agents of leishmaniasis, a major parasitic disease in humans. During their life cycle, Leishmania protozoans exist as flagellated promastigotes in the sand fly vector and as nonmotile amastigotes in the mammalian hosts. The promastigote-to-amastigote transformation occurs in the phagolysosomal compartment of the macrophage cell and is a critical step for the establishment of the infection. To study this cytodifferentiation process, we differentially screened an amastigote cDNA library with life cycle stage-specific cDNA probes and isolated seven cDNAs representing amastigote-specific transcripts. Five of these were closely related (A2 series) and recognized, by Northern (RNA) blot analyses, a 3.5-kb transcript in amastigotes and in amastigote-infected macrophages. Expression of the amastigote-specific A2 gene was induced in promastigotes when they were transferred from culture medium at 26 degrees C and pH 7.4 to medium at 37 degrees C and pH 4.5, conditions which mimic the macrophage phagolysosomal environment. A2 genes are clustered in tandem arrays, and a 6-kb fragment corresponding to a unit of the cluster was cloned and partially sequenced. An open reading frame found within the A2-transcribed region potentially encoded a 22-kDa protein containing repetitive sequences. The recombinant A2 protein produced in Escherichia coli cells was specifically recognized by immune serum from a patient with visceral leishmaniasis. The A2 protein repetitive element has strong homology with an S antigen of Plasmodium falciparum, the protozoan parasite responsible for malaria. Both the A2 protein of Leishmania donovani and the S antigen of P. falciparum are stage specific and developmentally expressed in mammalian hosts.

Amino Acid Sequence↗

PubMed2001

A cluster of three novel Ca2+ channel gamma subunit genes on chromosome 19q13.4: evolution and expression profile of the gamma subunit gene family.

The CACNG1 gene on chromosome 17q24 encodes an integral membrane protein that was originally isolated as the regulatory gamma subunit of voltage-dependent Ca2+ channels from skeletal muscle. The existence of an extended family of gamma subunits was subsequently demonstrated upon identification of CACNG2 (22q13), CACNG3 (16p12-p13), and CACNG4 and CACNG5 (17q24). In this study, we describe a cluster of three novel gamma subunit genes, CACNG6, CACNG7, and CACNG8, located in a tandem array on 19q13.4. Phylogenetic analysis indicates that this array is paralogous to the cluster containing CACNG1, CACNG5, and CACNG4, respectively, on chromosome 17q24. We developed sensitive RT-PCR assays and examined the expression profile of each member of the gamma subunit gene family, CACNG1-CACNG8. Analysis of 24 human tissues plus 3 dissected brain regions revealed that CACNG1 through CACNG8 are all coexpressed in fetal and adult brain and differentially transcribed among a wide variety of other tissues. The expression of distinct complements of gamma subunit isoforms in different cell types may be an important mechanism for regulating Ca2+ channel function.

Amino Acid Sequence↗

PubMed2002

An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22.

The cytidine (C) to uridine (U) editing of apolipoprotein (apo) B mRNA is mediated by tissue-specific, RNA-binding cytidine deaminase APOBEC1. APOBEC1 is structurally homologous to Escherichia coli cytidine deaminase (ECCDA), but has evolved specific features required for RNA substrate binding and editing. A signature sequence for APOBEC1 has been used to identify other members of this family. One of these genes, designated APOBEC2, is found on chromosome 6. Another gene corresponds to the activation-induced deaminase (AID) gene, which is located adjacent to APOBEC1 on chromosome 12. Seven additional genes, or pseudogenes (designated APOBEC3A to 3G), are arrayed in tandem on chromosome 22. Not present in rodents, this locus is apparently an anthropoid-specific expansion of the APOBEC family. The conclusion that these new genes encode orphan C to U RNA-editing enzymes of the APOBEC family comes from similarity in amino acid sequence with APOBEC1, conserved intron/exon organization, tissue-specific expression, homodimerization, and zinc and RNA binding similar to APOBEC1. Tissue-specific expression of these genes in a variety of cell lines, along with other evidence, suggests a role for these enzymes in growth or cell cycle control.

APOBEC-1 Deaminase↗