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PubMed1986

Terminal inverted repeats of prokaryotic transposable element IS186 which can generate duplications of variable length at an identical target sequence.

The insertion element IS186, which resides in the chromosome of Escherichia coli K-12, is 1338 bp long. Its termini represent 23-bp perfectly inverted repeats, but a variant carries a mismatch at position 23. IS186 transposes preferentially into G + C-rich sequences and generates target duplications of variable length, even at the same integration site.

Base Composition↗
PubMed1987

Characterization of the terminal sequences flanking the transposon that carries the Escherichia coli enterotoxin STII gene.

The Escherichia coli enterotoxin STII gene is flanked by two repeat sequences, approx. 600 bp each and 8 kb apart. This 9-kb DNA fragment has been shown to transpose as a unit and is thus considered a transposon. It is presently designated as Tn4521. In this study, the two terminal sequences of Tn4521 cloned in pPS1 were localized, isolated, and characterized. The two terminal sequences were found to be composed of IS2 sequences and were in an inverted repeat orientation. However, neither repeat contained a complete IS2. The LTR contained bp 1-722, whereas the RTR contained bp 17-536 and 969-1327, all three of the IS2 sequence.

Bacterial Toxins↗
PubMed2002

Cloning and characterization of Acmar1, a mariner-like element in the asiatic honey bee, Apis cerana japonica (Hymenoptera, Apocrita).

A mariner-like element was cloned from the genome of the Asiatic honey bee, Apis cerana japonica (Hymenoptera, Apocrita). The (composite) clone, named Acmar1, was 1,378 bp long, and encoded 336 amino acids corresponding to a transposase-like putative polypeptide in a single open reading frame. The D,D(34)D motif, the catalytic domain of the mariner transposase, was present, although there was a deletion of five amino acid residues within it as compared with the active transposase in Drosophila mauritiana. Nineteen-bp-long imperfect inverted terminal repeat-like sequences flanked by TA dinucleotides, the typical target site for mariner insertion, were observed. Southern blot analysis using a fragment covering two-thirds of the Acmar1 transposase coding sequence as a probe indicated the presence of multiple Acmar1-like elements in the genome. Maximum-parsimony phylogenetic analysis based on the transposase amino acid sequences of insect mariner-like elements revealed that Acmar1 is a member of the mellifera subfamily.

Amino Acid Sequence↗
PubMed2001

Gene rearrangements in radiation-induced thyroid carcinogenesis.

BACKGROUND: Radiation is an accepted risk factor for thyroid carcinogenesis in children. Recent observations in large cohorts of children and young adults who developed papillary thyroid carcinomas (PTC) related to accidental radiation exposure after the Chernobyl reactor accident revealed typical genetic aberrations shedding light on genetic determinants and mechanisms of radiation-induced carcinogenesis. PROCEDURE: A molecular genetic analysis was performed on 191 post-Chernobyl PTC by RT-PCR, multiplex PCR, DNA sequencing, and in some cases 5'RACE. Determination of point mutations was by means of PCR and either allele-specific oligonucleotide hybridization or SSCP and DNA sequencing. RESULTS: In various sporadic thyroid tumor types of adults structural genetic aberrations have been found involving mutations of RAS (codon 12, 13, 61), p53 (exons 5 to 8), Gsalpha (codon 201 and 227), and, at a low prevalence, the receptor tyrosine kinases RET or NTRK1. In contrast, in radiation-induced PTC of children RET rearrangements are by far the most prevalent genetic aberrations. In these RET rearrangements, the transmembrane and extracellular domains of RET are lost, and are replaced by parts of other genes at the 5' end. These genes always contain coiled-coil domains with dimerization potential and lead to constitutive, ligand-independent activation of the ret tyrosine kinase domain at the 3' end of the fusion product. The most frequent radiation-induced RET gene fusions involve the ELE1 (ARA70) gene, a transcription coactivator of the androgen receptor (PTC3), and H4, a gene of unknown function (PTC1). Both rearrangements originate from DNA double strand breaks with repair by intrachromosomal balanced paracentric inversion and recombination by illegitimate DNA endjoining at small stretches of homologous nucleotide sequences and direct or inverted repeats, without significant breakpoint clusters in the involved introns. In addition, five different RET-fused genes, RIalpha, GOLGA5, HTIF, RFG7 and RFG8, have been detected leading to the PTC2, 5, 6, 7 and 8 types of RET rearrangements, respectively. Each fusion leads, in principle, to the same effect: The ret tyrosine kinase is uncoupled from its stringent physiological regulation by replacement of its 5' end and is aberrantly activated by the 5' parts of fused genes in thyrocytes that do not normally express ret tyrosine kinase. Ectopic ret expression, clonal expansion and early invasion are peculiar to the affected cells. The RET-fused gene is obviously decisive for modulating tumor development: ELE1/RET rearrangements lead to most rapid tumor progression and are related to the solid variant of PTC, in contrast to H4/RET rearrangements connected with papillary or follicular variants of PTC. CONCLUSIONS: Typical genetic aberrations are produced by radioiodine uptake in the juvenile thyroid gland. They act as determinants of phenotype, biology, and clinical course of radiation-induced papillary thyroid carcinomas.

Adolescent↗
PubMedDate not supplied

Genetic organization of a linear mitochondrial plasmid (mF) that promotes mitochondrial fusion in Physarum polycephalum.

The mF plasmid which promotes mitochondrial fusion in Physarum polycephalum is a linear molecule with complex terminal inverted repeats (TIRs). Its nucleotide sequence was determined. The mF plasmid is 14,503 bp in size, and contains ten open reading frames (ORFs). All of the ORFs except one are encoded on the same DNA strand (coding strand). The number of amino-acid residues in the putative proteins derived from the nine ORFs on the coding strand are 231, 163, 640, 235, 118, 1130, 366, 309, and 547 from left (5' end) to right (3' end) on the map. The amino-acid sequences of newly-identified ORFs on the mF plasmid did not show significant homology to any amino-acid sequences in the databases. A brief transcriptional map of the mF plasmid was constructed, and the following features were noted. (1) The transcription initiation site was located just inside the end of the left TIRs, but not within the TIRs themselves. (2) Three major transcripts of 1.0, 3.4 and 4.6 knt corresponded to the left region of the mF plasmid, and long, low-abundance (more than 4.6 knt), heterogenous transcripts corresponded to almost the entire mF plasmid. A low-abundance, 3.5-knt transcript corresponding to the coding region of ORF1 130 (a 1 130-amino-acid polypeptide) was also detected, and may be derived from the long transcripts. (3) The quantity of transcripts which included the region near the transcription initiation site was about 500-times more than that which included the region near the inner end of the right TIRs.

Animals↗
PubMed1990

Naphthalene degrading genes on plasmid NAH7 are on a defective transposon.

A 37.5 kb region encompassing a set of the naphthalene degrading genes on the Pseudomonas plasmid NAH7 was found to be transposable only in the presence of the transposase encoded by the Tn1721 subgroup of the class II transposons. This newly identified mobile element, designated Tn4655, contained short (38 bp) terminal inverted repeats which shared extensive sequence homology with those of members of the Tn1721 subgroup. Tn4655 transposed by a two-step process involving formation of the cointegrate followed by its subsequent resolution. In contrast to the defect in the trans-acting factor for the first step, a functional system for the latter step was encoded within a 2.4 kb region in Tn4655. Analysis of deletion and insertion mutants demonstrated that the 2.4 kb region contained the cis-acting (res) site and the gene for a trans-acting factor (resolvase); complementation analysis indicated that Tn4655 resolvase function was not interchangeable with those of other well-studied class II transposons, including the Tn1721 subgroup. Tn4655 had no DNA sequences that were hybridizable with the transposase or resolvase genes of the Tn1721 subgroup.

Base Sequence↗
PubMed1992

Transcripts and translation products of a mitochondrial plasmid of Claviceps purpurea.

Expression of the linear mitochondrial (mt) plasmid pClK1 of strain K of the ascomycete Claviceps purpurea was studied at the RNA and protein level. Using strand-specific probes two major transcripts were detected, corresponding to ORF1 and ORF2 of the plasmid, which most likely code for a DNA-polymerase and an RNA-polymerase, respectively. Primer extension experiments showed that both transcripts start at identical positions from opposite sides within the terminal inverted repeat (TIR). The initiation sequence corresponds to the proposed general mt initiation consensus-sequence. Conserved parts of the putative polymerase ORFs were expressed in an E. coli system and used to prepare antisera. In Western experiments the presence of corresponding proteins was demonstrated in a strain carrying plasmid pClK1, whereas a plasmid-free strain lacked these polypeptides. The sizes of these proteins are in good accordance with the sizes derived from the coding capacity of ORF1 and 2.

Base Sequence↗
PubMed1998

Evidence for circular transposition derivatives from the fungal hAT-transposon Restless.

The structure and function of eukaryotic hAT-transposons has already been elucidated; however, their transposition mechanism is barely understood. We recently have discovered Restless, a fungal member of the hAT-transposon family, which shows transposition in its host Tolypocladium inflatum. Investigations of two strains from T. inflatum, carrying either about 15 copies or only a single copy of the Restless element, indicate the presence of circular transposition intermediates. Using PCR technology, amplicons were identified which carry the joined end of the Restless transposon fused at its inverted repeats. All of eight sequenced PCR fragments contained the intact transposon ends with a short insertion of 1-93 bp of genomic DNA. Remarkably, one of the discovered genomic sequences matches a previously described integration site. Our data are discussed with respect to the transposition mechanism and the horizontal transfer of hAT-transposons.

DNA Transposable Elements↗
PubMed1985

An essential replication gene, repA, of plasmid pSC101 is autoregulated.

Measurements of the rate of replication of a mutant pSC101 plasmid, cloned into a ColE1 vector, showed that insertions of the transposon Tn1000 into the repA gene of pSC101 abolished replication activity, but could be complemented in trans, albeit at a low level. The promoter of the repA gene was mapped by the construction of repA-lacZ gene fusions, and one of the fusions was used to demonstrate that repA protein, provided in trans, could repress expression of beta-galactosidase activity. This repression was primarily due to reduction of transcription of the repA-lacZ fusion. The sequence analysis of mutants of the repA-lacZ fusion gene which were no longer sensitive to the presence of repA protein showed that the site of action of repA was a 22 base-pair sequence, present as an inverted repeat, overlapping the repA promoter. The repA gene is thus autoregulated.

Bacterial Proteins↗
PubMed1986

Functional promoters created by the insertion of transposable element IS1.

We have isolated several insertions of the transposable element IS1 into the proximal promoter (P3) of the beta-lactamase gene of plasmid pBR322, which do not abolish resistance to ampicillin. Using a transcription termination module (omega), we have shown that the gene can be expressed from hybrid promoters, created by the insertion of IS1. The terminal inverted repeats of IS1 carry sequences partially homologous to the "-35" consensus region. Splicing either of these sequences to the existing "-10" region of the beta-lactamase promoter by transposition of IS1 at the proper distance results in the formation of an active hybrid promoter. This interpretation was confirmed by transcription studies in vitro. Gene expression from the hybrid promoters was found to be less efficient than from P3. However, the orientation of IS1 that contributes a "-35" with the greater homology to the known "-35" consensus sequence is significantly more efficient than the other. In addition, we were able to assign a strong determinant of IS1 polarity to a 254 base-pair internal segment of IS1. An examination of the ends of many insertion sequences leads us to expect that the phenomenon described here may occur with several of these transposable elements, and may have an unexpected evolutionary significance.

Base Sequence↗
PubMed1993

The origin of chromosome rearrangements at early stages of AMPD2 gene amplification in Chinese hamster cells.

BACKGROUND: Gene amplification and chromosomal rearrangements are frequent properties of cancer cells, provoking considerable interest in the mechanism of gene amplification and its consequences - particularly its relationship to chromosomal rearrangements. We recently studied the amplification of the gene for adenylate deaminase 2 (AMPD2) in Chinese hamster cells. Using fluorescent in situ hybridization (FISH), we found that early amplification of the AMPD2 gene is based on unequal gene segregation at mitosis, rather than local over-replication. We observed large inverted repeats of the amplified sequences, consistent with an amplification mechanism involving cycles of chromatid breakage, followed by fusion after replication and, in mitosis, the formation of bridges between the fused sister chromatids that leads to further breaks - a process we refer to as chromatid breakage-fusion-bridge (BFB) cycles. Our previous work left open the question of how this mechanism of gene amplification is related, if at all, to the chromosomal rearrangements that generate the dicentric, ring and double-minute (DM) chromosomes observed in some AMPD2-amplified metaphase cells, which are not predicted intermediates of chromatid BFB cycles, although they could be generated by related chromosome BFB cycles. RESULTS: We have addressed this question using FISH with probes for the AMPD2 gene and other markers on the same chromosome. Our results are not consistent with the chromosome BFB cycle mechanism, in which two chromatids break simultaneously and fuse to generate, after replication, a dicentric chromosome. Rather, they suggest that dicentric chromosomes are generated by secondary events that occur during chromatid BFB cycles. Our results also suggest that DM chromosomes are generated by the 'looping-out' of a chromosomal region, generating a circular DNA molecule lacking a centromere; in this case, gene amplification would result from the unequal segregation of DM chromosomes at mitosis. CONCLUSION: We conclude that, at early stages of AMPD2 gene amplification, chromatid BFB cycles are a major source of both 'intrachromosomal' gene amplification and genomic rearrangement, which are first limited to a single chromosome but which can then potentially spread to any additional chromosome. It also seems that, occasionally, a DNA sequence including the AMPD2 gene can be excised, generating a DM chromosome and thus initiating an independent process of 'extrachromosomal' amplification.

Journal Article↗
PubMed2001

PCR-based typing of Mycobacterium avium isolates in an epidemic among farmed lesser white-fronted geese (Anser erythropus).

Mycobacterium avium is an important veterinary pathogen causing avian tuberculosis in birds. The aim of the study was to evaluate the genetic relatedness in M. avium isolates from deep tissues of farmed lesser white-fronted geese with avian tuberculosis and in samples from the farm environment. The strains were analyzed by two PCR-based typing methods, inverted repeat (IR) typing and random amplified polymorphic DNA (RAPD) analysis. The primers for the inverted repeats of the insertion sequences IS1245 and IS1311 were used in IR typing, and the RAPD analysis was performed with six primers. Seven of the nine avian strains yielded an identical pattern in the IR typing, but they could be divided into two groups in the RAPD analysis. The remaining two bird isolates had an identical IR pattern (IR cluster II) which they shared with two environmental isolates. However, the RAPD analysis revealed that these environmental isolates had a RAPD pattern (RAPD cluster VI) distinct and different from either of the bird isolates (RAPD clusters II and IV). In all, four M. avium strains were verified as being inducers of avian tuberculosis in birds, and all were distinct from the three environmental strains identified. Thus, the results did not confirm the preliminary idea that a single strain had caused the epidemic. The polymorphism among M. avium strains highlighted the great biodiversity among an M. avium population even in a limited environmental setting during a short time span, and indicated the high susceptibility to avian tuberculosis of lesser white-fronted geese.

Animal Husbandry↗
PubMed1988

In vivo behavior of genetically engineered herpes simplex viruses R7017 and R7020: construction and evaluation in rodents.

The herpes simplex virus (HSV) recombinant R7017 was constructed from HSV-1 (strain F) by deleting a portion of the thymidine kinase (tk) gene and by replacing the sequences representing the internal inverted repeats and adjacent genes in the L component with a fragment of the HSV-2 genome encoding the glycoproteins G, D, I, and a portion of E. In addition, the R7020 recombinant contains an HSV-1 DNA fragment encoding the tk gene fused to the alpha 4 gene promoter. The results of studies in mice, guinea pigs, and rabbits were as follows: Both recombinants remained unchanged after nine serial, intracerebral passages in mice; the recombinants could not be differentiated with respect to attenuation in mice injected intracerebrally, in vaginally infected guinea pigs, and in rabbits inoculated on the scarified cornea. Given intradermally or intramuscularly, the recombinants prevented severe infections by virulent challenge viruses, and R7020 established latent infections (at a low frequency) in all species tested, whereas latent R7017 virus was detected in rabbits only.

Animals↗
PubMed1988

Multiresistant Staphylococcus aureus: genetics and evolution of epidemic Australian strains.

Molecular and genetic analysis of multiresistant isolates of Staphylococcus aureus from widely separated hospitals in Australia has demonstrated that these are clearly related, and that the predominant strains possess up to three different plasmids, which fall into the following classes: (i) small 1.6 kb plasmids, such as pSK3, which are phenotypically cryptic, (ii) 4.5 kb chloramphenicol resistance plasmids, such as pSK2, and (iii) the pSK1 family of multiresistance plasmids, which range in size from 20 to 42 kb and variously encode resistance to antiseptics and disinfectants, trimethoprim (Tpr), penicillin (Pcr) and the aminoglycosides gentamicin, tobramycin and kanamycin (Gmr Tmr Kmr). Gmr Tmr Kmr is encoded on the pSK1 family plasmids by transposon Tn4001, which was also detected on the chromosomes of some clinical isolates. Tn4001 is composed of inverted repeats of the insertion sequence IS256; these repeats flank a Gmr Tmr Kmr sequence encoding for a 57,000 dalton bifunctional protein with aminoglycoside acetyltransferase [AAC(6')] and phosphotransferase [APH(2")] activities. A Tn4001-like structure, which is defective in transposition but encodes for a Gmr Tmr Kmr determinant homologous with that on Tn4001, occurs on conjugative plasmids from strains isolated in North America. Physical studies indicate that Pcr, via a beta-lactamase, and Tpr, via a trimethoprim-insensitive dihydrofolate reductase (DHFR), are also encoded on the pSK1 family by transposons; these transposons have been designated Tn4002 and Tn4003, respectively. Tn4003 is flanked by direct repeats of the insertion sequence IS257. The evolution of the pSK1 family of multiresistance plasmids is traced through the transposition and genetic rearrangement of resistance determinants. Transposition and genetic rearrangement have also contributed to the evolution of a multiresistant chromosome in Staph. aureus. In the majority of contemporary multiply resistant Staph. aureus strains the determinants for resistance to erythromycin (Emr), fusidic acid, methicillin (Mcr), minocycline, rifampicin, spectinomycin, streptomycin, sulphonamides, tetracycline (Tcr), cadmium (Cdr), and mercury (Hgr) are chromosomally encoded; these strains also possess chromosomally encoded Pcr, via a beta-lactamase. Evidence indicates that some of these determinants, Pcr, Cdr, Hgr, and Tcr, were plasmid encoded in isolates collected from Australian hospitals prior to 1970. Through transposition and site-specific integration, they have since been acquired by the chromosome in more recent Staph. aureus strains.(ABSTRACT TRUNCATED AT 400 WORDS)

Australia↗
PubMed1993

Biological and clinical implications of heat shock protein 27,000 (Hsp27): a review.

Heat shock and other environmental and pathophysiologic stresses stimulate synthesis of heat shock proteins (Hsps). These proteins enable the cell to survive and recover from stressful conditions by as yet uncompletely understood mechanisms. Hsp27 is an important small Hsp (molecular weight, 27,000) found in human cells--both cancer cells and normal cells. This protein, besides its putative role in thermotolerance, is of special clinical interest because of recent data suggesting it may also play a role in drug resistance. In adults, Hsp27 is found particularly in several cell types such as breast, uterus, cervix, placenta, skin, and platelets. Although low-molecular-weight (small) Hsps have been found to be involved in embryogenesis of Xenopus and Drosophila, they have not been detected in human fetal organs. Regulation of expression of the Hsp gene (also known as HSPB1) has been considered a paradigm of gene regulation and is actively being studied in both prokaryotes and eukaryotes. In prokaryotes, the major Hsp genes are transcriptionally regulated by positively and negatively acting transcription factors. In eukaryotes, the genes encoding Hsps contain a regulatory DNA motif (inverted repeats of the pentameric sequence nGAAn) known as the heat shock element. Hsp27 may function as a molecular chaperone and in signal transduction pathways of different cell regulators, and Hsp27 and other Hsps may be active in development of resistance to stressful conditions and agents including cytotoxic drugs. Study findings indicate that some but not all estrogen-positive breast cancers express Hsp27, and overexpression of Hsp27 has been associated with both good and poor prognosis. In endometrial carcinomas, the presence of Hsp27 is correlated with the degree of tumor differentiation as well as with the presence of estrogen and progesterone receptors. Studies suggest, however, that detection of Hsp27 should not be considered to be a method for identifying hormone-responsive tumors or detecting estrogen receptors. Hsp27 seems to be a biochemical marker of estrogenic endometrial response. In patients with cervical cancer, Hsp27 is predominantly expressed in well-differentiated and moderately differentiated squamous cell carcinomas. In addition, expression of Hsp27 seems to be a negative prognostic factor for gastric cancer. Different isoforms of Hsp27 have been found in lymphoid tissue of patients with acute lymphoblastic leukemia, and the protein has also been associated with viral infections. These aspects are summarized and discussed in the present review.

Animals↗
PubMed2005

RNA silencing of mycotoxin production in Aspergillus and Fusarium species.

Mycotoxins are natural fungal products that are defined by their harmful effects on humans and animals. Aflatoxin contamination of maize by Aspergillus species and trichothecene contamination of small grains by Fusarium species are two of the most severe mycotoxin problems in the United States. We are investigating RNA silencing in an effort to identify novel ways to control mycotoxin contamination of crops. Transformation of two Aspergilli (A. flavus and A. parasiticus) and a Fusarium (F. graminearum) with inverted repeat transgenes (IRT) containing sequences of mycotoxin-specific regulatory genes suppressed mycotoxin production in all three plant-pathogenic fungi. This atoxigenic phenotype was stable during infection on corn and wheat, and importantly, F. graminearum IRT strains were less virulent on wheat than were wild type. The IRT did not alter physiological characteristics of the fungi, such as spore production and growth rate on solid media. These results indicate that RNA silencing exists in Aspergillus and Fusarium plant pathogens and suggest that RNA silencing technology may be a useful tool for eliminating mycotoxin contamination of agricultural products.

Aspergillus↗
PubMed1990

The regulatory role of the IS1-encoded InsA protein in transposition.

We show here that the protein InsA, which is encoded by IS1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS1 transposition activity. We demonstrate that it inhibits both IS1-mediated cointegrate formation and transposition of a synthetic IS1-based transposon ('omegon'; omega-on). These results also indicate that the omega-on which does not itself encode IS1 transposition functions can be complemented in trans, presumably by the copies of IS1 resident in the Escherichia coli chromosome. Using insA-lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS1-encoded genes both in cis or in trans. The experiments involving omega-on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS1.

Base Sequence↗
PubMed1989

A human cytomegalovirus early gene has three inducible promoters that are regulated differentially at various times after infection.

The long inverted repeat and the adjacent sequences are major early transcription sites of the human cytomegalovirus genome (M. W. Wathen and M. F. Stinski, J. Virol. 41:462-477, 1982). An early transcription unit which flanks the large terminal repeat was analyzed by RNA mapping at various times after infection. Three unspliced, overlapping RNAs were transcribed from different initiation sites and terminated at the same 3' end. Individual promoters were isolated for all three transcripts. These promoters were activated in trans by viral immediate-early (IE) regulatory proteins after either infection with virus or cotransfection with IE2 alone or IE1 plus IE2 genes. DNA sequence analysis detected TATA and CAAT boxes plus multiple-dyad symmetries in the promoter-regulatory region. Deletion analyses showed that the maximum inducible promoter activity lay in a 230-base-pair region. When in the viral genome, the three promoters were regulated differentially during the course of infection. The upstream promoter was used only at late times after infection. Possible reasons for viral RNAs with multiple 5' ends at different times after infection and the recognition of the upstream promoter at only late times after infection are discussed.

Base Sequence↗