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PubMed1995

The murine AIDS defective provirus acts as an insertional mutagen in its infected target B cells.

In susceptible mice, the murine AIDS (MAIDS) defective virus can induce marked expansion of its target cells, the majority of which belong to the B-cell lineage. This expansion, which appears to be critical for the development of the immunodeficiency syndrome, is initially polyclonal but becomes oligoclonal late in the disease, suggesting the involvement of a secondary genetic event(s) during this proliferation. To determine whether integration of the MAIDS defective provirus into particular regions of the cellular genome contributes to this oligoclonal expansion, we searched for common provirus integration sites in enlarged lymphoid organs of MAIDS mice. We identified two common proviral integration sites, Dis-1 and Dis-2, which were occupied by a defective provirus at frequencies of 20 and 13%, respectively. Our analysis revealed that the Dis-1 region corresponds to the Sfpil1 (Spi-1, PU.1) locus, which maps on chromosome 2, and encodes a transcription factor. Insertion of the MAIDS defective provirus into this region led to a two- to threefold increase in the expression of Sfpi1 RNA. The Dis-2 locus was found to map to mouse chromosome 11, between Hox2 and Scya. It appears to be a novel locus probably harboring a gene involved in B-cell proliferation. The present study indicates that the MAIDS defective provirus can act as an insertional mutagen, thus contributing to the oligoclonal expansion of infected cells. The detection of two common proviral integration sites, each of which targetted at a low frequency in diseased organs, suggests that the deregulation of a unique gene through provirus insertion is essential for neither proliferation of infected B cells nor development of the immunodeficiency syndrome.

Animals↗
PubMed1995

The distribution of the transposable element Bari-1 in the Drosophila melanogaster and Drosophila simulans genomes.

The distribution of the transposable element Bari-1 in D. melanogaster and D. simulans was examined by Southern blot analysis and by in situ hybridization in a large number of strains of different geographical origins and established at different times. Bari-1 copies mostly homogeneous in size and physical map are detected in all strains tested. Both in D. melanogaster and in D. simulans a relatively high level of intraspecific insertion site polymorphism is detectable, suggesting that in both species Bari-1 is or has been actively transposing. The main difference between the two sibling species is the presence of a large tandem array of the element in a well-defined heterochromatic location of the D. melanogaster genome, whereas such a cluster is absent in D. simulans. The presence of Bari-1 elements with apparently identical physical maps in all D. melanogaster and D. simulans strains examined suggests that Bari-1 is not a recent introduction in the genome of the melanogaster complex. Structural analysis reveals unusual features that distinguish it from other inverted repeat transposons, whereas many aspects are similar to the widely distributed Tc1 element of C. elegans.

Animals↗
PubMed1991

Site-specific integration of genes into hot spots for recombination flanking aadA in Tn21 transposons.

Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA- background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5' CTAAAACAAAGTTA 3' (hs2). The double-site-specific recombination occurred with a frequency of 10(-5)-10(-6). The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a transacting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations.

Base Sequence↗
PubMed1986

Regulation of exoprotein gene expression in Staphylococcus aureus by agar.

Insertion of the erythromycin-resistance transposon Tn551 into the Staphylococcus aureus chromosome at a site which maps between the purB and ilv loci has a pleiotrophic effect on the production of a number of extracellular proteins. Production of alpha, beta and delta hemolysin, toxic shock syndrome toxin (TSST-1) and staphylokinase was depressed about fifty-fold while protein A production was elevated twenty-fold. Hybridization analysis showed that the defect in expression of TSST-1 and alpha hemolysin was at the transcriptional level. Inability of the mutant strain to express either a cloned TSST-1 gene or the chromosomal gene indicates that the transposon has inactivated a trans-active positive control element. This element has been designated agr for accessory gene regulator.

Bacterial Proteins↗
PubMed1981

DNA sequence of a plasmid-encoded dihydrofolate reductase.

The sequence of the methotrexate-resistant dihydrofolate reductase (DHFR) gene borne by the plasmid R-388 was determined. The gene was subcloned and mapped by an in vitro mutagenesis method involving insertion of synthetic oligonucleotide decamers encoding the BamHI recognition site. Sites of insertion that destroyed the methotrexate resistance fell in two regions separated by 300 bp within a 1.2 kb fragment. One of these regions encodes a 78 amino acid polypeptide homologous to another drug-resistant DHFR. The second region essential for DHFR expression appears to be the promoter of the DHFR gene.

Base Sequence↗
PubMed2003

Reduction in visceral slide is a good sign of underlying postoperative viscero-parietal adhesions in children.

BACKGROUND/PURPOSE: Viscera stuck to the anterior abdominal wall from previous surgery risk injury during laparoscopic surgery. A prospective study was conducted to determine if these adhesions are detectable on ultrasound scan by showing a reduction in the normal visceral slide. METHODS: Patients undergoing laparoscopic procedure after a previous laparotomy underwent preoperative real-time ultrasound scan to observe if viscera slides freely under the abdominal wall. A reduction in slide was considered a positive sign of underlying adhesions. These findings were correlated with the operative findings. RESULTS: Anterior abdominal wall scans were performed on 17 children. Reduced visceral slide was seen in 10. Viscero-parietal adhesions were found in 9 of 10 patients. Visceral slide was reduced in a very localized area in 6 patients, and, in these, a loop of bowel (n = 3), liver and bowel (n = 2), or liver (n = 1) was adherent. In 4, reduced visceral slide was seen over a wide area. Extensive adhesions were found in 3 of 4. One renal transplant patient with peritonitis had a false-positive ultrasound scan. At laparotomy there were no adhesions. The peritonitis is thought to have prevented an adequate examination. Seven patients had normal visceral slide. Of these, 4 had no adhesions, but 3 children had flimsy omental adhesions. The sensitivity and specificity of visceral slide in predicting adhesions were 75% and 80%, respectively. CONCLUSIONS: Reduction in visceral slide is a good sign of underlying postoperative viscero-parietal adhesions. Ultrasonographic mapping of the abdominal wall may be useful in selecting an adhesion-free site for trocar insertion in children with previous operations requiring laparoscopic procedures.

Abdominal Wall↗
PubMed2000

DNA sequencing with transposons.

The use of transposons offers the possibility of a directed approach to DNA sequencing, where a target DNA up to about 6kb in length can be sequenced quickly and with minimal redundancy. Transposons are mobile DNA elements which can be inserted in a reasonably random fashion into the target DNA. An important part of this process is the location of the transposon insertions (known as mapping) and the selection of a sensible subset of transposons to use as priming sites for sequencing reactions. This paper presents a probabilistic method of scoring selected subsets of transposons and a graph-theoretic algorithm for selection of a subset of maximal score.

Algorithms↗
PubMed1994

Genetic characterization of a Rhizobium meliloti lactose utilization locus.

We identified several linked genes of a lactose regulon in Rhizobium meliloti. These were lacZ, the structural gene for beta-galactosidase; lacR, the lactose repressor gene; and two genes encoding proteins of unknown function, lacW and lacX. Insertion mutants in lacW and lacZ belonged to a single genetic complementation group, and lacW appeared to lie upstream of lacZ in an operon. Expression of lacZ, lacW and lacX was repressed by lacR, and expression of lacZ and lacW was derepressed by lactose. lacZ was not required for induction of lacW by lactose, suggesting that lactose itself, rather than a processed form of lactose, may be the actual inducer molecule. Expression of all three genes was repressed by succinate, and the lacR independence of this repression showed that inducer exclusion could not be the sole mechanism. This pattern of lac gene organization and regulation differs in several ways from that observed in enteric bacteria.

DNA Transposable Elements↗
PubMed1992

Cloning of a probe for a previously undescribed enterobacterial tetracycline resistance gene.

An 8.35 kb BamHI fragment was cloned from the plasmid R714a. It encoded resistances to chloramphenicol, streptomycin, spectinomycin and tetracycline. Tetracycline resistance was determined by a locus without homology to the known enterobacterial gene classes, TetA-TetE. Further subcloning of the fragment located an unusual tetracycline resistance gene on a 4.7 kb BamHI-BglII fragment. A physical-genetic map of this fragment indicated that the gene was bisected by a PstI site. Deletion analysis and insertion mutagenesis were used to define a suitable probe. An intragenic PstI-AvaI fragment of 1.2 kb was identified, and used as a non-radioactive probe, being specific for this previously undescribed enterobacterial Tet gene.

Cloning, Molecular↗
PubMed1974

Apparent mutagenic effect of induction of lambda prophage inserted between lysA and thyA.

Induction of a heat-inducible abnormal lambda prophage inserted between lysA and thyA in Escherichia coli resulted in a number of auxotrophic mutants in the surviving cured-cell populations. These mutants could not be accounted for by deletions arising on formation of lambda hybrid particles carrying regions adjacent to the insertion site. The properties of these mutants, which were almost all spontaneously revertable, have been described and mapped by F' episome complementation. Tentatively, it was suggested that induction of the lambda lysogen leads to a mutagenic state.

Arginine↗
PubMed1998

Interactions between the structural domains of the RNA replication proteins of plant-infecting RNA viruses.

Brome mosaic virus (BMV), a positive-strand RNA virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with N-terminal putative capping and C-terminal helicase-like sequences. These two proteins are part of a multisubunit complex which is necessary for viral RNA replication. We have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo RNA replication assays and biochemical assays of protein-protein interaction, thus permitting the identification of additional interacting domains. We now map an interaction found to take place between two 1a proteins. Using previously characterized 1a mutants, a perfect correlation was found between the in vivo phenotypes of these mutants and their abilities to interact with wild-type 1a (wt1a) and each other. Western blot analysis revealed that the stabilities of many of the noninteracting mutant proteins were similar to that of wt1a. Deletion analysis of 1a revealed that the N-terminal 515 residues of the 1a protein are required and sufficient for 1a-1a interaction. This intermolecular interaction between the putative capping domain and itself was detected in another tripartite RNA virus, cucumber mosaic virus (CMV), suggesting that the 1a-1a interaction is a feature necessary for the replication of tripartite RNA viruses. The boundaries for various activities are placed in the context of the predicted secondary structures of several 1a-like proteins of members of the alphavirus-like superfamily. Additionally, we found a novel interaction between the putative capping and helicase-like portions of the BMV and CMV 1a proteins. Our cumulative data suggest a working model for the assembly of the BMV RNA replicase.

Animals↗
PubMed1994

Genetic cholesteryl ester transfer protein deficiency caused by two prevalent mutations as a major determinant of increased levels of high density lipoprotein cholesterol.

Genetic determinants of HDL cholesterol (HDL-C) levels in the general population are poorly understood. We previously described plasma cholesteryl ester transfer protein (CETP) deficiency due to an intron 14 G(+1)-to-A mutation(Int14 A) in several families with very high HDL-C levels in Japan. Subjects with HDL-C > or = 100 mg/dl (n = 130) were screened by PCR single strand conformational polymorphism analysis of the CETP gene. Two other mutations were identified by DNA sequencing or primer-mediated restriction map modification of PCR products: a novel intron 14 splice donor site mutation caused by a T insertion at position +3 from the exon14/intron14 boundary (Int14 T) and a missense mutation (Asp442 to Gly) within exon 15 (D442G). The Int14 T mutation was only found in one family. However, the D442G and Int14 A mutations were highly prevalent in subjects with HDL-C > or = 60 mg/dl, with combined allele frequencies of 9%, 12%, 21% and 43% for HDL-C 60-79, 80-99, 100-119, and > or = 120 mg/dl, respectively. Furthermore, prevalences of the D442G and Int14 A mutations were extremely high in a general sample of Japanese men (n = 236), with heterozygote frequencies of 7% and 2%, respectively. These two mutations accounted for about 10% of the total variance of HDL-C in this population. The phenotype in a genetic compound heterozygote (Int14 T and Int14 A) was similar to that of Int14 A homozygotes (no detectable CETP and markedly increased HDL-C), indicating that the Int14 T produces a null allele. In four D442G homozygotes, mean HDL-C levels (86 +/- 26 mg/dl) were lower than in Int14 A homozygotes (158 +/- 35 mg/dl), reflecting residual CETP activity in plasma. In 47 D442G heterozygotes, mean HDL-C levels were 91 +/- 23 mg/dl, similar to the level in D442G homozygotes, and significantly greater than mean HDL-C levels in Int14 A heterozygotes (69 +/- 15 mg/dl). Thus, the D442G mutation acts differently to the null mutations with weaker effects on HDL in the homozygous state and stronger effects in the heterozygotes, suggesting dominant expression of a partially defective allele. CETP deficiency, reflecting two prevalent mutations (D442G and Int14 A), is the first example of a genetic deficiency state which is sufficiently common to explain a significant fraction of the variation in HDL-C in the general population.

Adult↗
PubMed1997

Genomic distribution of the retrovirus-like element ZAM in Drosophila.

The mobile element ZAM, recently identified in Drosophila melanogaster, is similar in structure and coding potential to vertebrate retroviruses. In this paper, we analyze the insertional and structural polymorphism of this element and show that members of this family appear to have a long evolutionary history in the genome of Drosophila. It is present in all the species of the D. melanogaster subgroup and in more distantly related species like D. takahashii, D. ananassae, or D. virilis but in a lower copy number or with a lower homology. Two categories of strains have been previously identified in D. melanogaster: strains with a high copy number of ZAM and strains with a low copy number. Here, we show that ZAM is at least in a low copy number in each tested strain of the species analyzed. The study of ZAM's genomic distribution by FISH mapping analysis to salivary gland polytene chromosomes or on mitotic chromosomes indicates that most of the insertion sites of ZAM elements are associated with the constitutive heterochromatin regardless of the ZAM copy number. In addition, our results suggest that multiple ZAM elements are present at the insertion sites visualized by in situ experiments.

Animals↗
PubMed1991

Epicardial mapping in patients with "nodoventricular" accessory pathways.

Some patients with electrophysiologic features suggesting nodoventricular fibers have been shown to have right parietal atrioventricular (AV) accessory pathways with decremental conduction properties intraoperatively. The experience with 11 patients (7 women and 4 men, mean age +/- standard deviation 25 +/- 5 years) who had electrophysiologic features consistent with a nodoventricular pathway and who underwent operative correction was reviewed. At electrophysiologic study, all patients had absent or minimal preexcitation in sinus rhythm. During atrial pacing and extrastimulus testing, maximal preexcitation with left bundle branch block morphology developed and the AH and AV intervals progressively prolonged. Preexcited tachycardia was initiated in all patients (AV reentrant tachycardia in 10 patients and AV node reentrant tachycardia in 1 patient). At operation all patients had a right parietal accessory pathway demonstrated. Intraoperative mapping demonstrated the earliest site of ventricular activation during anterograde preexcitation to be at the midanterior right ventricle, consistent with insertion of these pathways into the right bundle branch system, in 7 patients. The ventricular insertion was at the AV groove in 4 patients, in keeping with the typical Wolff-Parkinson-White syndrome. Retrograde conduction over the pathway was not demonstrated in any patient. Two patients had evidence of a second accessory AV pathway in the left paraseptal region. Operative AV node ablation was electively performed in 2 patients without affecting preexcitation in either case. In 1 of these patients, accessory pathway conduction was temporarily abolished by ice mapping in the right anterolateral AV groove.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1996

Characterization and mapping of Ds-GUS-T-DNA lines for targeted insertional mutagenesis.

The transposition patterns of the Ds-GUS transposon T-DNA in 23 independent single-copy lines have been characterized and the map positions of 10 of them on three of the five Arabidopsis chromosomes are reported. Using overexpressed Activator (Ac) elements as a transposase source, it was found that the primary determinant of transposition frequency is the insertion site of the Ac-T-DNA. Neither the structure of the transposon T-DNA nor, in most cases, its insertion site have a significant effect on transposition frequency. Both the frequency and timing of transposition are influenced by the parent through which the transposon and transposase T-DNAs are transmitted. Overall, nearly 75% of plants in which excision has occurred bear a reinserted element and very short-range transpositions predominate, underlining the advantage of using mapped transposons for insertional mutagenesis.

Arabidopsis↗
PubMed1985

Baculovirus (MNPV) genomic variants: characterization of Spodoptera exempta MNPV DNAs and comparison with other Autographa californica MNPV DNAs.

A nuclear polyhedrosis virus (NPV) strain from Spodoptera exempta (SeMNPV-25 baculovirus) is a restriction endonuclease DNA map variant similar to Autographa californica NPV (AcMNPV baculovirus). Fourteen restriction endonuclease variants were identified and isolated from a SeMNPV baculovirus stock with 12 of the variants found at low frequency (less than 3%). The DNA from each variant was compared to the prototype SeMNPV-25 for insertions, deletions and new restriction sites. Regions of variation were defined on the prototype SeMNPV-25 genome, and the nature of the variation within these regions was determined. These data are discussed and compared with the existing data on other variants of AcMNPV. A comparison of the physical maps revealed that all the SeMNPV variants were different from those reported for AcMNPV. Although the SeMNPV variants were distinctive, they were clearly genomic AcMNPV variants. The regions of the baculovirus genomic variation were identified, and three separate mechanisms are suggested for their generation. Five regions (hr1 to hr5) were associated with intragenic homologous viral sequences, five regions (vI to vIII) may be associated with the insertion of DNA sequences of cellular origin, and two regions (pI and pII) were associated with mutations resulting in the addition or loss of a PstI site. Physical maps were generated for SeMNPV variant regions vI, hr2, vII and vIII.

Animals↗
PubMedDate not supplied

Physical genome analysis of bacteria.

Pulsed-field gel electrophoresis (PFGE) is a general analytical tool to separate large DNA molecules and may therefore be applied to problems from all areas of bacteriology. The genome size of bacteria covers the range of 0.6 to 10 megabase pairs. For genome fingerprinting, the bacterial chromosome is cleaved with a restriction endonuclease that gives a resolvable and informative number of five to one hundred fragments on the PFGE gel. Restriction enzymes are chosen according to GC content, degree of methylation, and codon usage of the respective bacterial genus. Macrorestriction fingerprinting allows the identification of bacterial strains and the distinction between related and unrelated strains. If fragment patterns of several restriction digestions are quantitatively evaluated, strains can be classified according to genetic relatedness at the level of genus, species, and biovar. In particular, members of a clonal lineage can be uncovered. Hence, any problem from applied, environmental, and clinical microbiology may be addressed by PFGE restriction analysis where the spatiotemporal spread of a bacterial clone is of interest. In bacterial genomics, PFGE is employed for the top-down construction of macrorestriction maps of the chromosome which yields data about genome organization, mobile genetic elements, and the arrangement of gene loci and gene families. The genomic diversity of a bacterial species is elucidated by comparative chromosome mapping. Map positions of restriction sites and gene loci of interest serve as landmarks to assess the extent of gross chromosomal modification, namely insertions, deletions and inversions. Intra- and interspecies comparisons of genome organization provide insights into the structure and diversity of bacterial populations and the phylogeny of bacterial taxa.

Bacteria↗