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PubMed1993

Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis.

Five different genetic elements have been found to be associated with genetic rearrangements in Mycobacterium tuberculosis complex strains. Of these elements, the insertion sequence IS6110 is presently the most frequently used genetic marker for strain differentiation of M. tuberculosis. In the present study we compared five genetic elements for their potentials to differentiate a given cluster of M. tuberculosis strains. Because of the presence of only a single copy of IS6110 or two IS6110 copies at the same chromosomal locus, a large number of strains could not be differentiated by IS6110 fingerprinting. Most strains, including the low-copy-number IS6110 strains, could be differentiated by fingerprinting with the 36-bp direct repeat or the polymorphic GC-rich repetitive DNA element. Less discriminative power was obtained with the major polymorphic tandem repeat and the insertion element IS1081. One strain which did not contain IS6110 DNA was encountered. Until now, this element has invariantly been found to be present in all M. tuberculosis complex strains. On the basis of classical taxonomic criteria and sequencing of the 16S rRNA gene, this strain was shown to be a genuine M. tuberculosis strain. Therefore, the use of this element as a target for polymerase chain reaction-facilitated detection of M. tuberculosis should be reconsidered.

Bacterial Typing Techniques↗
PubMed1996

Gestational pyelonephritis--associated Escherichia coli isolates represent a nonrandom, closely related population.

OBJECTIVE: A select group of Escherichia coli strains known as uropathogenic cause pyelonephritis in nonpregnant individuals. We investigated whether Escherichia coli from gestational pyelonephritis represent a random population or possess common uropathogenic characteristics. STUDY DESIGN: Repetitive element sequence-based polymerase chain reaction, plasmid profiles, hemolysin, and O serotypes were assayed from Escherichia coli isolates of 57 pregnant patients with acute pyelonephritis at different gestational ages. RESULTS: The majority of the first trimester isolates fell primarily into repetitive element sequence-based patterns 1 and 3 and O6, O15, and O75 serotypes. Second-trimester isolates had multiple patterns with high-frequency repetitive element sequence-based polymerase chain reaction 1 and 5 and an unknown (OX) serotype. Pattern 3, predominantly O75 serotype, was found primarily among third-trimester isolates. CONCLUSION: It is likely that Escherichia coli associated with acute pyelonephritis during different trimesters of pregnancy represents nonrandom closely related isolates, and some of these strains may be characteristic in pregnant patients only.

Base Sequence↗
PubMed1991

Identification of high mobility group protein I(Y) as potential progression marker for prostate cancer by differential hybridization analysis.

One of the major problems in the diagnosis of localized prostatic tumors is to predict the aggressiveness of an individual tumor, which is presumably associated with chance to progression. In an attempt to find molecular markers that are specific for aggressive prostatic cancer cells, we compared steady-state mRNA levels of progressionally related prostatic tumors. The Dunning R-3327-H subline, a relatively benign rat prostatic tumor, was compared to the therefrom derived highly aggressive MatLyLu tumor by differential hybridization analysis. The differential screening revealed 26 complementary DNA clones that detected transcripts overexpressed in MatLyLu. Upon further screening on the entire panel of Dunning R-3327 sublines, it appeared that three clones (pBUS1, pBUS19, and pBUS30), detected transcripts specifically expressed in metastatic rat prostatic tumors. The expression pattern of pBUS19 and pBUS30 suggested a relation between these complementary DNAs. Nucleotide sequence analysis, however, could not yet substantiate this. Computer-assisted comparison of the DNA sequences revealed the presence of rat long terminal repeat-like repetitive elements in pBUS19. The differential expression of repetitive elements in progressionally related tumors is interesting, yet similar findings have not been reported in human malignancies. Nucleotide sequence analysis of pBUS1 indicated that this clone is identical or related to high mobility group protein I(Y), a non-histone nuclear protein. From recent studies it appeared that this protein might be implicated in replication and/or transcription processes and is induced in fast proliferating/undifferentiated cells. The overexpression of high mobility group protein I(Y) correlates rather with metastatic ability than with growth rate; hence it may serve as a valuable marker to identify progressionally advanced prostate cancer cells.

Animals↗
PubMed1992

Comparative structural analysis of the transcriptionally active proopiomelanocortin genes A and B of Xenopus laevis.

In the intermediate lobe of the pituitary gland, the prohormone proopiomelanocortin (POMC) is processed to, among other peptides, melanocyte-stimulating hormone (alpha-MSH). In the toad Xenopus laevis alpha-MSH controls skin darkening during background adaptation, and the level of POMC gene transcription in the intermediate lobe depends on the color of the background. In the lobe, two structurally different POMC proteins are produced from two mRNAs that are transcribed to approximately the same level from two POMC genes (A and B). We previously reported the entire nucleotide sequence of Xenopus POMC gene B. To identify conserved-- and thus potential regulatory--DNA elements in the Xenopus POMC gene, we here report the determination and analysis of the complete nucleotide sequence of Xenopus POMC gene A and its 5'- and 3'-flanking regions. Comparison of the two Xenopus POMC genes revealed, in addition to the exons, three highly conserved regions. First, the promoter regions are greater than 90% identical. The second region concerns JH12 repetitive elements situated at approximately the same position in both genes. These elements are greater than 86% identical. The third region is a 500-bp sequence just upstream of exon three (63% identity). Besides these three large regions, several small regions with significant identity were found at similar positions in the two POMC genes. The fact that, except for the JH12 element, the repetitive elements are not conserved between the two POMC genes indicates that these repeats are not functionally important.

Animals↗
PubMed2006

Evidence of influence of genomic DNA sequence on human X chromosome inactivation.

A significant number of human X-linked genes escape X chromosome inactivation and are thus expressed from both the active and inactive X chromosomes. The basis for escape from inactivation and the potential role of the X chromosome primary DNA sequence in determining a gene's X inactivation status is unclear. Using a combination of the X chromosome sequence and a comprehensive X inactivation profile of more than 600 genes, two independent yet complementary approaches were used to systematically investigate the relationship between X inactivation and DNA sequence features. First, statistical analyses revealed that a number of repeat features, including long interspersed nuclear element (LINE) and mammalian-wide interspersed repeat repetitive elements, are significantly enriched in regions surrounding transcription start sites of genes that are subject to inactivation, while Alu repetitive elements and short motifs containing ACG/CGT are significantly enriched in those that escape inactivation. Second, linear support vector machine classifiers constructed using primary DNA sequence features were used to correctly predict the X inactivation status for >80% of all X-linked genes. We further identified a small set of features that are important for accurate classification, among which LINE-1 and LINE-2 content show the greatest individual discriminatory power. Finally, as few as 12 features can be used for accurate support vector machine classification. Taken together, these results suggest that features of the underlying primary DNA sequence of the human X chromosome may influence the spreading and/or maintenance of X inactivation.

Base Sequence↗
PubMed1995

How bats achieve a small C-value: frequency of repetitive DNA in Macrotus.

Bats possess a genome approximately 50-87% the size of other eutherian mammals. We document that the events that have achieved or maintained a small genome size in the Mexican leaf-nosed bat Macrotus waterhousii have resulted in a lower copy number of interspersed and tandemly repetitive elements. These conclusions are based on examination of 1726 randomly chosen recombinant cosmids, with an average insert size of 35.7 kb and representing 2.6% of the haploid genome of M. waterhousii. Probes representative of microsatellites [(GT)n, (CT)n, (AT)n, (GC)n] and a tandem repeat (rDNA) were used to estimate frequency of repetitive elements in the M. waterhousii genome. Of the four dinucleotides, (GT)n was present in 33.5% of the clones, (CT)n was present in 31.0% of the clones, and (AT)n and (GC)n were not represented in any of the clones examined. The 28S rDNA and a repetitive element from M. californicus were found in three and four clones, respectively. The dinucleotides (GT)n and (CT)n occurred together in the same clone more frequently than expected from chance. Although our data do not allow us to empirically test which mechanisms are maintaining copy number of repetitive DNA in the bat genome, the nonrandom association of these different families of repetitive DNA may provide insight into a mechanism that proportionately reduces diverse families of repetitive DNA that are known to be amplified by very different mechanisms.

Animals↗
PubMed1999

Characterization of a Highly Repetitive Sequence Conserved Among the North American Morone Species.

: A highly repetitive DNA sequence family from the genome of the North American Morone has been cloned and characterized. This family, first identified as a HindIII repetitive element, is composed of repeat units that range from 285 to 288 bp in length and comprise approximately 5.5% of the genome. The copy number of the repeat was estimated to be 1.85 x 10(5) per haploid genome set. Data from Southern blot analyses demonstrated that the HindIII repetitive element was tandemly organized. Sequence analysis of six cloned repeat monomers from each of the four North American Morone species, M. saxatilis, M. chrysops, M. americana, and M. mississippiensis, revealed a high degree of conservation of the monomeric unit. The intraspecific sequence variation ranged from 3.2% to 5.4%. A similar level of variation was detected between cloned monomers from the same individual, suggesting that most of the intraspecies variation may be due to variation among copies of the repeat. The interspecific sequence variation ranged from less than 4.6% between M. americana and M. mississippiensis to approximately 16% between the other Morone species pairs. Phylogenetic analysis of the repetitive element nucleotide sequences indicated that M. americana and M. mississippiensis were more closely related to each other than to any other pairs of Morone species. In addition, we reconstructed the Morone phylogeny using 22 previously described morphologic characters. Congruent relationships were obtained between both sets of data. The data suggest that the genus Morone is composed of two sets of sister taxa, M. saxatilis:M. chrysops and M. americana:M. mississippiensis.

Journal Article↗
PubMed1988

Nucleotide sequence and characterization of a repetitive DNA element from the genome of Bordetella pertussis with characteristics of an insertion sequence.

A repeating element of DNA has been isolated and sequenced from the genome of Bordetella pertussis. Restriction map analysis of this element shows single internal ClaI, SphI, BstEII and SalI sites. Over 40 DNA fragments are seen in ClaI digests of B. pertussis genomic DNA to which the repetitive DNA sequence hybridizes. Sequence analysis of the repeat reveals that it has properties consistent with bacterial insertion sequence (IS) elements. These properties include its length of 1053 bp, multiple copy number and presence of 28 bp of near-perfect inverted repeats at its termini. Unlike most IS elements, the presence of this element in the B. pertussis genome is not associated with a short duplication in the target DNA sequence. This repeating element is not found in the genomes of B. parapertussis or B. bronchiseptica. Analysis of a DNA fragment adjacent to one copy of the repetitive DNA sequence has identified a different repeating element which is found in nine copies in B. parapertussis and four copies in B. pertussis, suggesting that there may be other repeating DNA elements in the different Bordetella species. Computer analysis of the B. pertussis repetitive DNA element has revealed no significant nucleotide homology between it and any other bacterial transposable elements, suggesting that this repetitive sequence is specific for B. pertussis.

Base Sequence↗
PubMed2003

Composition-sensitive analysis of the human genome for regulatory signals.

Known transcription regulatory signals which generally act as transcription factor binding sites (TFs) differ significantly in their base composition. Therefore, their occurrence in a genome largely depends on the local base composition. In an attempt to initiate an all human genome analysis for the occurrence of potential TFs, we systematically analyzed the GC-content of distinct functional regions (e. g., upstream and downstream gene regions, exons, long and short introns, repetitive elements) and correlated the frequencies of potential binding sites of a representative set of TFs in these regions. For these analyses, we used the pattern collection of the TRANSFAC database on transcriptional regulation, the information about functionally relevant combinations of them from the database TRANSCompel, and our new resource, TRANSGenomeTM, which provides an overall annotation of the human genome with emphasis on its regulatory characteristics. We show that the occurrence of sequence patterns with regulatory potential may be supported by, but cannot be fully explained by either the GC content of a whole chromosome or its putative promoter regions, nor by the information content of the patterns. Several patterns, HNF-3, NFAT, and GC box, show a clear overrepresentation in all promoter groups as well as in all chromosomes. Other patterns, like E2F and CRE-BP1, are underrepresented in all promoter groups as well as in all chromosomes in comparison with random sequences. Simultaneously, both patterns are over-represented in promoters in comparison with repetitive elements. We define several structural characteristics of the proximal promoters that differentiate them from other functional genomic regions. Two well-known promoter elements, GC- and TATA-boxes, are statistically enriched in promoters in comparison with random sequences, repetitive elements and exons. Altogether, our findings provide insights into the macroheterogeneity amongst the individual chromosomes, into the microheterogeneity among different functional regions of individual chromosomes, contribute to further understanding of structural organization of gene regulatory regions, and give first hints on the development of regulatory features during evolution.

Animals↗
PubMed1990

Prevalence of novel repeat sequences in and around the P1 operon in the genome of Mycoplasma pneumoniae.

The presence of numerous different repetitive elements in the genome of Mycoplasma pneumoniae has been documented by several laboratories. One which we previously identified, denoted as SDC1, has now been further characterized, verified to be distinct from those discussed in previous publications and shown to lack homology to several other species of Mycoplasma when tested under our stringency conditions. As many as eight versions of the SDC1-type repeat, which is more than 400 bp long, are scattered throughout the genome of M. pneumoniae. The prototype for SDC1 is found within a gene encoding a putative 130-kDa membrane-binding protein lying just downstream from the gene encoding the cytadhesin protein P1. In fact, all of the reported M. pneumoniae repetitive elements have at least one representative either within or adjacent to the P1 operon; many if not all of these lie within open reading frames. The function of these repetitive elements is still unclear.

Base Sequence↗
PubMed1990

Alpha-galactosidase A gene rearrangements causing Fabry disease. Identification of short direct repeats at breakpoints in an Alu-rich gene.

Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-linked gene encoding the lysosomal enzyme, alpha-galactosidase A (EC 3.2.1.22). Six alpha-galactosidase A gene rearrangements that cause Fabry disease were investigated to assess the role of Alu repetitive elements and short direct and/or inverted repeats in the generation of these germinal mutations. The breakpoints of five partial gene deletions and one partial gene duplication were determined by either cloning and sequencing the mutant gene from an affected hemizygote, or by polymerase chain reaction amplifying and sequencing the genomic region containing the novel junction. Although the alpha-galactosidase A gene contains 12 Alu repetitive elements (representing approximately 30% of the 12-kilobase (kb) gene or approximately 1 Alu/1.0 kb), only one deletion resulted from an Alu-Alu recombination. The remaining five rearrangements involved illegitimate recombinational events between short direct repeats of 2 to 6 base pairs (bp) at the deletion or duplication breakpoints. Of these rearrangements, one had a 3' short direct repeat within an Alu element, while another was unusual having two deletions of 1.7 kb and 14 bp separated by a 151-bp inverted sequence. These findings suggested that slipped mispairing or intrachromosomal exchanges involving short direct repeats were responsible for the generation of most of these gene rearrangements. There were no inverted repeat sequences or alternating purine-pyrimidine regions which may have predisposed the gene to these rearrangements. Intriguingly, the tetranucleotide CCAG and the trinucleotide CAG (or their respective complements, CTGG and CTG) occurred within or adjacent to the direct repeats at the 5' breakpoints in three and four of the five alpha-galactosidase A gene rearrangements, respectively, suggesting a possible functional role in these illegitimate recombinational events. These studies indicate that short direct repeats are important in the formation of gene rearrangements, even in human genes like alpha-galactosidase A that are rich in Alu repetitive elements.

Base Sequence↗
PubMed1995

Characterization of a repetitive DNA element in a brown algal virus.

We describe a family of repetitive sequences found in viruses infecting the brown alga Feldmannia sp. Previously we have demonstrated that the dsDNA genomes of viruses infecting one Feldmannia sp. isolate exist as two size classes of 160 and 179 kb. Repetitive sequences within these genomes were first demonstrated based on the anomalous hybridization among five BamHI fragments from digested virus DNA. Sequence analysis of one of those fragments, B2.4, revealed the presence of 173-bp direct repeats. The restriction maps of the cross-hybridizing BamHI fragments in the two FsV (Feldmannia sp. virus) genome size classes show that these repeats are not widely dispersed, rather they are confined to a small region of each virus genome. We estimate the number of these repeats in the 179-kb genome to be about 109 and in the 169-kb genome to be about 41. In the 179-kb genome, the repeats are contained within a 22-kb region, about 12% of that virus genome, and in the 160-kb genome the repeats are contained within a 10-kb region, about 6% of the genome. The difference in repeat numbers can account for 62% of the size difference between the two size classes of the FsV genome.

Base Sequence↗
PubMed2005

Distortion of quantitative genomic and expression hybridization by Cot-1 DNA: mitigation of this effect.

Cross-hybridization of repetitive sequences in genomic and expression arrays is reported to be suppressed with repeat-blocking nucleic acids (C(o)t-1 DNA). Contrary to expectation, we demonstrated that C(o)t-1 also enhanced non-specific hybridization between probes and genomic targets. When added to target DNA, C(o)t-1 enhanced hybridization (2.2- to 3-fold) to genomic probes containing conserved repetitive elements. In addition to repetitive sequences, C(o)t-1 was found to be enriched for linked single copy (sc) sequences. Adventitious association between these sequences and probes distort quantitative measurements of the probes hybridized to desired genomic targets. Quantitative microarray hybridization studies using C(o)t-1 DNA are also susceptible to these effects, especially for probes that map to genomic regions containing conserved repetitive sequences. Hybridization measurements with such probes are less reproducible in the presence of C(o)t-1 than for probes derived from sc regions or regions containing divergent repeat elements, a finding with significant ramifications for genomic and expression microarray studies. We mitigated the requirement for C(o)t-1 either by hybridizing with computationally defined sc probes lacking repeats or by substituting synthetic repetitive elements complementary to sequences in genomic probes.

DNA↗
PubMed1993

Gene sequence, cDNA construction, expression in Escherichia coli and genetically approached purification of porcine interleukin-1 beta.

A genomic clone (PIL3) covering the 8.8-kb prointerleukin-1 beta ('catabolin') gene of the domesticated swine (Sus scrofa domestica) was isolated from a genomic library and characterized by nucleotide sequencing. Typical features of the gene include a seven-exon structure, with the highest degree of nucleotide and amino acid conservation among human and porcine genes being found in the receptor-binding portion encoded by exons six and seven. Three 250-bp repetitive elements with a > 75% similarity to the pig repetitive element-1 family sequence are located in untranslated gene segments. Southern-hybridization experiments disclosed extensive genomic heterogeneity of the porcine interleukin-1 beta (IL-1 beta) gene region, suggesting a duplication of at least the 3' half of the gene in the porcine genome. Since similar hybridization patterns were observed for wild boar (Sus scrofa) genomic DNA, it was concluded that this gene rearrangement had preceded domestication of the wild swine. In addition, the cDNA for processed porcine IL-1 beta was constructed through polymerase-chain-reaction-mediated exon fusion by overlap extension starting from the genomic template. Recombinant IL-1 beta was expressed in Escherichia coli as a fusion protein containing an N-terminal hexahistidine tag followed by a factor-Xa-cleavage site. The protein was efficiently purified through adoption of a scheme that consisted of four alternating cycles of immobilized metal-ion-affinity chromatography and size-exclusion chromatography. 13.8 mg highly purified recombinant porcine IL-1 beta was obtained starting from a 900-ml thermo-induced E. coli culture (final endotoxin concentration < 0.22 ng/ml). The protein behaved homogeneously as a monomeric species, which was reactive in Western-blot experiments with an anti-(human-IL-1 beta) serum and which appeared to induce gelatinase B in MDBK cells in a dose-dependent fashion.

Amino Acid Sequence↗
PubMed1984

DNA and chromatin structure of the human alpha 1 (I) collagen gene.

The human alpha 1 (I) collagen gene and 48 kilobase pairs of flanking DNA have been isolated on two overlapping cosmids. The alpha 1 (I) gene is 18 kilobase pairs long and contains a single repetitive element of the Alu family; at least 15 repetitive elements are present in the flanking DNA. Analysis of chromatin structure in nuclei isolated from cultured fibroblasts demonstrated a single chromatin domain greater than 65 kilobase pairs in length that contained 9 DNase I-hypersensitive sites. The pattern of hypersensitive sites was also determined in nuclei derived from placental tissue. Five of the DNase I-hypersensitive sites were observed in both placental and fibroblast chromatin including one site near the 5' end and another near the 3' end of alpha 1 (I). An additional two sites located near the 3' end of the alpha 1 (I) gene in fibroblast chromatin are associated with the tissue-specific use of different polyadenylation sites. Two DNase I-hypersensitive sites found only in fibroblast chromatin and one site found only in placental chromatin were located more than 10 kilobase pairs away from the alpha 1 (I) gene and may be related to tissue-specific expression of other genes in the domain. However, the only abundant placental mRNAs from the 65-kilobase pair domain were those transcribed from the alpha 1 (I) gene. These findings suggest that physical linkage does not play a predominant role in controlling coordinate expression of collagen genes.

Base Composition↗
PubMed1995

An outbreak of Burkholderia (formerly Pseudomonas) cepacia respiratory tract colonization and infection associated with nebulized albuterol therapy.

OBJECTIVE: To investigate an outbreak of Burkholderia (formerly Pseudomonas) cepacia respiratory tract colonization and infection in mechanically ventilated patients. DESIGN: A retrospective case-control and bacteriologic study. SETTING: Veterans Affairs medical center. PATIENTS: 42 mechanically ventilated patients who developed respiratory tract colonization or infection with B. cepacia and 135 ventilator-dependent controls who were not colonized and did not develop infections. MEASUREMENTS: Clinical and demographic data; benzalkonium chloride concentrations and pH levels in albuterol sulfate solutions; repetitive-element polymerase chain reaction (PCR)-mediated molecular fingerprinting on eight patient isolates and three environmental B. cepacia isolates that were available for study. RESULTS: 42 patients had B. cepacia respiratory tract colonization or infection. Observation of intensive care unit and respiratory care personnel showed faulty infection control procedures (for example, the same multiple-dose bottle of albuterol was used for many mechanically ventilated patients). More case patients (39 [92.9%]) than controls (95 [70.4%]; P = 0.006) received nebulized albuterol, and case patients (67.5 treatments) received more treatments than controls (18 treatments; P < 0.001). In-use albuterol solutions had pH values that were unstable, and benzalkonium chloride concentrations declined over time to levels capable of supporting bacterial growth. Medication nebulizers and in-use bottles of albuterol harbored B. cepacia. Molecular fingerprints of patient isolates and environmental B. cepacia isolates were identical using repetitive-element PCR. No further isolates of B. cepacia were identified after institution of appropriate infection control procedures. CONCLUSIONS: Multiple-dose medications and reliance on benzalkonium chloride as a medication preservative provide a mechanism for nosocomial spread of microorganisms, particularly if infection control procedures are not carefully followed. Repetitive-element PCR is a useful fingerprinting technique for molecular epidemiologic studies of B. cepacia.

Aged↗
PubMed1994

Characterization of a human locus in transition.

The spermatid-specific nucleoprotamine genes PRM1 and PRM2 and the transition protein gene TNP2 are clustered at a single site on human chromosome 16p13.2. To begin to understand the mechanism governing their genesis and coordinate regulation the primary sequence of this approximately 40.6 kilobase region was determined. This cluster of genes is embedded within a series of repetitive elements, including numerous Alu elements distributed at a frequency of > 1 Alu element/kilobase. Multiple Alu elements have integrated into separate truncated L1 sequences within this region. Many of these Alu elements are tandemly inserted or clustered. The role of repetitive elements in the genomic organization and evolution of this gene cluster is discussed. Computer-assisted sequence analysis revealed the presence of structural sequence elements often associated with the boundary regions of active transcriptional domains. Further analysis identified a CpG island at the 3' end of this segment of chromosome 16 and other candidate coding segments within this region indicative of an additional linked gene. These sequence landmarks are commensurate with the complexity of the region.

Base Sequence↗
PubMed1997

Characterization of an interspersed repetitive DNA element in the genome of Trypanosoma cruzi.

We report the molecular characterization of a middle repetitive DNA sequence, named C6, isolated from the Trypanosoma cruzi genome. C6 appears to be a composite repeated element since 3 subregions may be defined within it on the basis of sequence similarities with other T. cruzi genomic sequences. Sequences homologous to C6 are interspersed in the genome and can be mapped out on most chromosomal bands of different T. cruzi. strains. The copy number of the C6 element is about 1000 per haploid genome. Given the species specificity and different genomic distribution of C6 homologous sequences among the T. cruzi strains the C6 element could be a useful probe for diagnosis and typing of parasites. C6 is a polymorphic marker with potential as a tool for physical mapping of the T. cruzi genome.

Animals↗