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PubMed2001

Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.

The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements.

Bromodeoxyuridine↗
PubMed1986

Foreign DNA introduced by calcium phosphate is integrated into repetitive DNA elements of the mouse L cell genome.

We investigated the sites of integration of exogenous DNA fragments introduced by DNA-mediated gene transfer. Mouse Ltk- cells were transformed with the herpes simplex virus thymidine kinase gene and pBR322 DNA by the calcium phosphate precipitation method. Some of the integrated exogenous DNA sequences were recovered from the stable tk+ transformants in the form of plasmids that were capable of propagation in bacteria. Four plasmids derived from two cloned cell lines were analyzed in detail by nucleotide sequencing and hybridization techniques. These plasmids contained a total of seven cellular-exogenous DNA junctions. In all cases, there was no sequence homology between the exogenous and cellular DNA sequences adjacent to the joining sites, and no specific exogenous or cellular sequences occurred at the junctions. Rearrangement or deletion of Ltk- DNA was always associated with the integration of exogenous DNA. All of the assignable cellular sequences at the junctions were repetitive sequences. Two of these sequences were from the MIF-1 repetitive sequence family, and a third consisted of a 40-base pair simple copolymer of alternating deoxyadenosine-deoxythymidine. Our results suggest that repetitive sequences are relatively favorable sites for the integration of exogenous DNA.

Animals↗
PubMed2006

Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey.

BACKGROUND: Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. AIMS: In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC) library for the nurse shark, Ginglymostoma cirratum. RESULTS: The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 x 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6-28 primary positive clones per probe of which 50-90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. CONCLUSION: We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.

Animals↗
PubMed1995

Virulence-associated fyuA/irp2 gene cluster of Yersinia enterocolitica biotype 1B carries a novel insertion sequence IS1328.

The fyuA/irp2 gene cluster, which is part of the Yersinia pestis pigmentation (pgm) locus encoding genes involved in iron uptake and virulence, is present in all pesticin-sensitive bacteria. In Y. enterocolitica biotype 1B strains (serotypes O8, O20, O21), the fyuA/irp2 gene cluster carries an insertion of a novel repetitive sequence, IS1328. It was also found in the genome of Y. enterocolitica O5 (biotype 1A) and O13 (biotype 1B), but not in pesticin-sensitive Y. pseudotuberculosis O1 and Escherichia coli Phi. The 1353-bp repetitive element has 12-bp perfect inverted terminal repeats. A single open reading frame is capable of encoding a 334-amino acid polypeptide. IS1328 DNA has high homology with the DNA sequences located downstream of the aggR gene from the enteroaggregative E. coli (EAggEC), to the region of the R751 plasmid flanking Tn501, to the sequence following the merR gene of S. marcescens pDU1358 plasmid, and to the sequences of K. pneumoniae plasmid pCFF04. The putative polypeptide has 36.4% identity with the transposase encoded by the Coxiella burnetii IS1111a insertion sequence. The IS1328 insertion sequence could be responsible for the deletions of the fyuA/irp2 gene cluster observed in Y. enterocolitica O8 and could represent a member of a new group of widely distributed repetitive elements.

Amino Acid Sequence↗
PubMed2001

Specificity of insertion sequence-based PCR assays for mycobacterium tuberculosis complex.

OBJECTIVE: To determine the specificity of different insertion sequence-targeted polymerase chain reaction (PCR) tests for Mycobacterium tuberculosis complex. DESIGN: One M. bovis BCG strain, two M. tuberculosis strains and ten species of mycobacteria other than tuberculosis (MOTT) were tested by three PCR assays based on the repetitive elements IS6110, IS1081 and IS990 under variable amplification conditions (different temperatures of primer annealing and numbers of reaction cycles). RESULTS: DNA amplifications based on the three insertion sequences yielded fragments of expected sizes only in DNA from M. tuberculosis complex strains when the tests were conducted at high stringency (65 degrees C). At the annealing temperature of 60 degrees C the PCR assay with IS6110-specific primers yielded a 245 bp fragment also in nine MOTT strains tested. This could result from previously reported homology between non-tuberculous mycobacteria and a central region of IS6110. Amplification assays based on IS1081 and IS990 gave false-positive results in some MOTT isolates only under very low stringency (55 degrees C), which could be due to non-specific priming of the target DNA at that temperature. CONCLUSION: Repetitive elements IS1081 and IS990 may represent a more reliable alternative to the more widely used IS6110 PCR target for tuberculosis diagnosis.

DNA Transposable Elements↗
PubMed2006

Prevalence and typing of Listeria monocytogenes in raw catfish fillets.

Raw channel catfish fillets collected from three processing plants during four time periods were tested for the presence of Listeria species. Listeria monocytogenes was the predominant Listeria species found in these catfish fillets, with 25 to 47% prevalence. Other Listeria species, such as L. welshimeri, L. innocua, L. ivanovii, L. grayi, and L. seeligeri, were also found. L. monocytogenes isolates were further fingerprinted by a repetitive element PCR. Forty distinctive electrophoretic types (ETs) and three genetic clusters were determined by Dice coefficient analysis and UPGMA (unweighted pair group method using arithmetic averages). Twenty of 40 ETs were represented by a single isolate, and the other 20 ETs were represented by 2 to 11 isolates. Thirty-five ETs, represented by 76 isolates, were found in processing plant A, B, or C and designated plant-specific types. The remaining five ETs, represented by 21 isolates, were found in multiple plants and designated nonplant-specific types. In addition, 10 ETs from 52 isolates were found repeatedly during different seasons. Plant-specific and nonplant-specific L. monocytogenes coexisted in processed catfish fillets. Some isolates were persistently found in processed fillets, suggesting that either the current sanitation procedures used by these plants are inadequate or that these isolates originated from the natural habitats of the catfish. The results also suggest that the repetitive element PCR is a useful tool for differentiating L. monocytogenes subtypes and can be used for tracing the source of a contamination.

Animals↗
PubMed2006

The repetitive DNA elements called CRISPRs and their associated genes: evidence of horizontal transfer among prokaryotes.

We have found direct DNA repeats 21-47 bp in length interspersed with nonrepetitive sequences of similar length, or clustered regularly interspaced short palindromic repeats (CRISPRs) in a wide range of diverse prokaryotes, including many Archaeal and Eubacterial species. A number of cas, CRISPR-associated genes have also been characterized in many of the same organisms. Phylogenetic analysis of these cas genes suggests that the CRISPR loci have been propagated via HGT, horizontal gene transfer. We suggest a mechanism by which this HGT has occurred, namely, that the CRISPR loci can be carried between cells on megaplasmids > or = 40 kb in length.

Base Sequence↗
PubMed1995

Characterization of a DNA sequence that detects repetitive DNA elements in the Asian rice gall midge (Orseolia oryzae) genome: potential use in DNA fingerprinting of biotypes.

We have isolated based on reverse genome hybridization, and sequenced a DNA clone, pNZE25, from a partial genomic library of the Asian rice gall midge Orseolia oryzae (Wood-Mason) (O.o.). Clone pNZE25 is highly A+T rich (67%), lacks any open reading frame and does not display homology to sequences in GenBank. Clone pNZE25 detects a 120-bp repeat in the O.o. genome, as seen from the generation of a 120-bp ladder after Southern analysis of HinfI-digested genomic DNA. When used to probe O.o. genomic DNA digested with DraI, HaeIII or AluI, pNZE25 generates individual specific DNA fingerprints on target DNA isolated from gall midge biotypes collected from different parts of India.

Animals↗
PubMed1991

The rodent B2 sequence can affect expression when present in the transcribed region of a reporter gene.

The mouse B2 element is a moderately repetitive nt sequence of 180 bp transcribed by RNA polymerase III (Pol III) at high levels in embryonic and transformed cells. The B2 sequence is present in either orientation within the noncoding regions of a number of genes transcribed by RNA polymerase II (Pol II). We sought to determine if the small B2 transcripts generated by Pol III are natural antisense RNA molecules which might hybridize to complementary sequences present within Pol II transcripts. Chimaeric reporter genes encoding Escherichia coli gpt were constructed containing a B2 repeat in either orientation within the 5'- or 3'-untranslated regions. These constructs were transfected into embryonal carcinoma (EC) cells and expression of the reporter gene was analysed in EC cells and retinoic acid-treated EC cells, which contain high and low levels of small B2 RNAs, respectively. Although the B2 sequences affected expression of the reporter gene, these effects did not appear to be due to hybridization of the small B2 RNA to the reporter transcripts. The presence of B2 sequences near a Pol II-transcribed gene can alter expression of that gene in a position- and orientation-dependent manner, suggesting these repetitive elements may be cis-acting regulators of gene expression.

Animals↗
PubMed1998

SERE, a widely dispersed bacterial repetitive DNA element.

The presence of a Salmonella serotype Enteritidis repeat element (SERE) located within the upstream regulatory region of the sefABCD operon encoding fimbrial proteins is reported. DNA dot-blot hybridisation analyses and computerised searches of genetic databases indicate that SERE is well conserved and widely distributed throughout the bacterial and archaeal kingdoms. A SERE-based polymerase chain reaction (SERE-PCR) assay was developed to fingerprint 54 isolates of Enteritidis representing nine distinct phage types and 54 isolates of other Salmonella serotypes. SERE-PCR identified five distinct fingerprint profiles among the 54 Enteritidis isolates; no correlation between phage types and SERE-PCR fingerprint patterns was noticed. SERE-PCR was reproducible, rapid and easy to perform. The results of this investigation suggest that the limited heterogeneity of SERE-PCR fingerprint patterns can be utilised to develop serotype- and serogroup-specific fingerprint patterns for isolates of Enteritidis.

Animals↗
PubMed1999

Tumoral environment triggers transcript anomalies in established tumors: induction of altered gene expression and of aberrant, truncated and B2 repeat-containing gene transcripts.

In addition to eugenetic changes, cancerous cells exhibit extensive modifications in the expression levels of a variety of genes. The phenotypic switch observed after inoculation of T lymphoma cells into syngenic mice illustrates the active participation of tumoral environment in the induction of an aberrant gene expression pattern. To further substantiate this contribution, we performed polymerase chain reaction (PCR)-based subtraction suppression hybridization (SSH) to identify genes that are differentially expressed in tumor-derived EL4/13.3 cells compared to the same cells isolated from cultures. Besides a number of unknown genes, the subtracted library contained several known genes that have been reported to be expressed at increased levels in tumors and/or to contribute to carcinogenesis. Apart from clones representing translated transcripts, the subtracted library also contained a high number of clones representing B2 repeat elements, viz. short interspersed repetitive elements that are transcribed by RNA polymerase III. Northern blotting confirmed the induction of B2 transcripts in tumor tissue and also revealed induction of chimeric, B2 repeat-containing mRNA. The appearance of chimeric transcripts was accompanied by aberrant, shorter-than-full-length transcripts, specifically from upregulated genes. Accordingly, in addition to altered gene expression, tumoral environmental triggers constitute a potent mechanism to create an epigenetic diversity in cancers by inducing extensive transcript anomalies.

Animals↗
PubMed2001

Demonstration of reinfection and reactivation in HIV-negative autopsied cases of secondary tuberculosis: multilesional genotyping of Mycobacterium tuberculosis utilizing IS 6110 and other repetitive element-based DNA fingerprinting.

SETTING: Secondary tuberculosis may follow reinfection or endogenous reactivation. The design of effective preventive and treatment protocols requires knowledge about the relative importance of these mechanisms in specific communities. Molecular typing of Mycobacterium tuberculosis has permitted linkage of cases and demonstration of patterns of inter- or intrapatient strain diversity correlating with reinfection and reactivation. OBJECTIVE: The use of DNA fingerprinting to examine intrapatient strain diversity in autopsied, HIV-negative individuals resident in a high incidence community. DESIGN: Autopsy (12 cases) and pneumonectomy (one case) permitted multilesional sampling for bacterial culture and comparative genotyping. Mycobacterium tuberculosis strains were typed using probes directed against IS 6110, the direct repeat sequence (DR) and MTB484(1). RESULTS: In two patients, the demonstration of pulmonary infection by two distinct strains suggested dual infection of these individuals. In one other case, the strain isolated from a Ghon-focus was identical to that obtained from all secondary cavitating lesions, which suggested reactivation of the primary infection. In the remaining cases, all isolates were identical, but primary lesions could not be identified with certainty or were culture negative. One of these cases showed evidence of strain evolution. CONCLUSIONS: Multilesional strain genotyping suggested both reinfection and reactivation in a series of HIV-negative autopsied patients with secondary tuberculosis. In most (11/13) cases, the same strain (or clonal variant) was present in all lesions, pulmonary and extrapulmonary.

AIDS-Related Opportunistic Infections↗
PubMed1991

Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes.

Dispersed repetitive DNA sequences have been described recently in eubacteria. To assess the distribution and evolutionary conservation of two distinct prokaryotic repetitive elements, consensus oligonucleotides were used in polymerase chain reaction [PCR] amplification and slot blot hybridization experiments with genomic DNA from diverse eubacterial species. Oligonucleotides matching Repetitive Extragenic Palindromic [REP] elements and Enterobacterial Repetitive Intergenic Consensus [ERIC] sequences were synthesized and tested as opposing PCR primers in the amplification of eubacterial genomic DNA. REP and ERIC consensus oligonucleotides produced clearly resolvable bands by agarose gel electrophoresis following PCR amplification. These band patterns provided unambiguous DNA fingerprints of different eubacterial species and strains. Both REP and ERIC probes hybridized preferentially to genomic DNA from Gram-negative enteric bacteria and related species. Widespread distribution of these repetitive DNA elements in the genomes of various microorganisms should enable rapid identification of bacterial species and strains, and be useful for the analysis of prokaryotic genomes.

Bacteria↗
PubMed2001

Transcription attenuation associated with bacterial repetitive extragenic BIME elements.

Transcription attenuation comprises several processes that affect transcript elongation and transcription termination, and has an important role in regulating gene expression. In most cases, transcription attenuation is used as a regulatory mechanism that allows the cell to adjust protein synthesis levels in response to a specific signal. Here, by using a tRNA gene as a transcriptional reporter, we characterize a new type of transcription attenuation mechanism in Escherichia coli that involves bacterial interspersed mosaic elements (BIMEs), the main family of repetitive extragenic elements. The transcription termination factor Rho is required for attenuation in association with BIMEs, thus revealing a new role for Rho as a BIMEs-dependent global regulator of gene expression. By mutational analyses, we identified nucleotide determinants of BIMEs that are required for attenuation and showed that this process relies on a sequence-specific mechanism. Our data are consistent with a model in which BIMEs provoke a pause in RNA polymerase movement and Rho acts ultimately to terminate transcription. BIME-dependent transcription attenuation may be used as a means to differentially regulate expression of adjacent genes belonging to a single operon. BIMEs are dispersed in more than 250 operons such that attenuation can simultaneously affect expression of a large number of genes encoding unrelated proteins. This attenuation phenomenon, together with the known ability of BIMEs to stabilize upstream mRNA, reveals how dispersion of these abundant repetitive elements may affect gene regulation at the genome level.

Base Sequence↗
PubMed1983

Organization and evolution of repeated DNA sequences in closely related plant genomes.

In common with many other eukaryotic species, the genomes of species in the genus Allium contain a high proportion of repeated DNA sequences, which may be implicated in the considerable differences in genome size that are seen between even very closely related species. The gross organization of repetitive sequences within the genome of Allium sativum and of some other related species has been investigated using DNA/DNA hybridization studies. Such studies show that there has been much modulation in the amounts of different repeated DNA families during the evolution of the genus Allium; these repetitive elements are interspersed in all species with sequences of low repetition. The organization and distribution of one particular repetitive family within the genus has been examined using a cloned hybridization probe. Hybridization of this probe to DNA from related genomes reveals that this element is present in all Allium species examined, but with large-scale modulation of its abundance, and some considerable changes in its sequence environment. The evolution of such genome-specific arrangements of common repetitive elements and the possible mechanisms by which they might be maintained are discussed.

Autoradiography↗
PubMed1987

Structure and evolution of a family of interspersed repetitive DNA sequences in Caenorhabditis elegans.

The structure of three members of a repetitive DNA family from the genome of the nematode Caenorhabditis elegans has been studied. The three repetitive elements have a similar unitary structure consisting of two 451-bp sequences in inverted orientation separated by 491 bp, 1.5 kb, and 2.5 kb, respectively. The 491-bp sequence separating the inverted 451-bp sequences of the shortest element is found adjacent to one of the repeats in the other two elements as well. The combination of the three sequences we define as the basic repetitive unit. Comparison of the nucleotide sequences of the three elements has allowed the identification of the one most closely resembling the primordial repetitive element. Additionally, a process of co-evolution is evident that results in the introduction of identical sequence changes into both copies of the inverted sequence within a single unit. Possible mechanisms are discussed for the homogenization of these sequences. A direct test of one possible homogenization mechanism, namely homologous recombination between the inverted sequences accompanied by gene conversion, shows that recombination between the inverted repeats does not occur at high frequency.

Animals↗