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PubMed1978

Regional preference of insertion of Tn501 and Tn802 into RP1 and its derivatives.

The sites of insertion of Tn501 into RP1 and into derivatives of this plasmid that either lack the Tn801 (TnA) element or contain it in a different location have been determined. Similarly, the sites of insertion of Tn802 into a derivative of RP1 that lacks the Tn801 element and into recombinants of this plasmid with Tn501 were determined. 'Hot spots' for insertion were observed with both transposons; but it is clear that a particular DNA sequence is not sufficient to define a 'hot spot', since a particular region does contain many insertions when present in one plasmid but does not do so when part of another.

Base Sequence↗
PubMed1978

Studies on the control region of the bipolar argECBH operon of Escherichia coli. I. Effect of regulatory mutations and IS2 insertions.

Several mutations affecting the control or the potential of gene expression in the argECBH bipolar operon have been characterized by enzyme assays, genetic mapping, dominance tests and pulse labelled RNA determinations. None of the mutations involves DNA rearrangements detectable by heteroduplex analysis (Charlier et al., 1978). Partially constitutive transcription of both argE and argCBH has been observed in mutant L10 while constitutive argE transcription and normal argCBH control characterize mutants L9, LL13 and LL2. The control region thus appears to contain two overlapping operators, as suggested previously (Elseviers et al., 1972). Two mutants (L2, LL1) and strain 6-8 from Bretscher and Baumberg (1976) display an increase in acetylornithinase specific activity (argE product) without concommittant increased argE transcription. In addition, they exhibit a decreased argCBH transcription. It is suggested that in these organisms, argE translation and argCBH transcription may be affected by the same genetic event; this explanation is compatible with present working hypothesis for the structure of the control region. An interpretation in terms of messenger attenuation also appears possible. From the properties of two strains harbouring an IS2 insertion in the control region (Charlier et al., 1978) the following conclusion may be drawn: 1. When inserted in orientation I close to the proximal end of a silent gene IS2 appears to promote a low but detectable transcription readthrough into that gene. 2. Insertion of an IS2 element in orientation II close to a neighbouring gene is not a sufficient condition to express that gene at a high rate. The properties of the two insertions appear compatible with the structure proposed for the control region.

Acetyltransferases↗
PubMed1978

Evidence for a common mechanism for the insertion of the Tn10 transposon and for the generation of Tn10-stimulated deletions.

Mutations in and near the Salmonella typhimurium histidine transport operon were generated by insertion of the translocatable tetracycline-resistance element Tn10. Deletion mutants affecting histidine transport genes were subsequently isolated in several of the Tn10-containing strains. Tn10 insertions in hisJ occurred preferentially at one site, designated site A. This same site was also the preferential endpoint of deletions originating from Tn10 insertions at two neighboring sites. Thus, Tn10 insertion and Tn10-stimulated deletion formation appear to involve a common DNA-recogition step.

Genes↗
PubMed1979

The occurrence of long transcription units among the X and Y ribosomal genes of Drosophila melanogaster: transcription of insertion sequences.

Most of the ribosomal transcription units (rTUs) in Drosophila melanogaster observed by electron microscopy measure about 8 kb; a length which corresponds to the size of the 38S precursor to ribosomal RNA in D. melanogaster. However, interspersed among these rTUs are transcription units that are much longer (up to 14.6 kb) than the 8 kb expected for rTUs. Some of these larger length estimates can be attributed to stretching but an important fraction is significantly larger and has up to 60 more fibers per gene.--The following evidence suggests that these larger transcription units are ribosomal genes consisting of insertion sequences. The long transcription units are within the sizes expected for rTUs containing insertion sequences as reported by other workers. Their RNP fibers cross-react with antibodies raised against ribosomal proteins in a manner similar to that observed for ribosomal RNP. They are interspersed among rTUs in the X chromosome.--These putative ribosomal genes carrying insertions are present both in the X and, although to a lesser extent, in the Y ribosomal chromatin as is indicated by their existence in nurse cells of both Oregon R females and females of the genotype sc4sc8/sc4sc8/y+ Y. Analysis of the fiber patterns of "long TUs" supports the hypothesis that the insertion region is being transcribed.--"Long TUs" are found in tandem with non-transcribed spacer regions which are heterogeneous in length with a mean of 1.53+/-0.61 micrometers (or 8.5+/-3.4 kb).

Animals↗
PubMed1977

Transcription of insertion elements IS1 and IS2 in vitro.

Insertion elements IS1 and IS2 integrated within the gal operator-promoter region, an IS1 element in gene galT and insertions IS1 and IS2 integrated in the xycIIOP region of phage lambda were transcribed in vitro with E. coli RNA-polymerase. The insertion elements are transcribed exclusively by polymerase molecules started at the gal promoter and the lambdaPR promoter respectively. No promoter exists on IS1 or IS2 which can be recognized by RNA-polymerase in the pure in vitro transcription system used. Both insertions apparently are transcribed with a lower elongation rate than gal operon DNA or lambdaDNA. RNAs transcribed from the termini of IS1 and IS2 respectively were analysed by hybridization experiments. They are different in sequence.

Base Sequence↗
PubMed1978

Analysis of transposable elements inserted in the genomes of bacteriophages Mu and P1.

We have examined the genomes of the temperate bacteriophages Mu and P1 and some of their insertion mutants for hybridization with the prokaryotic transposable elements IS1 and IS2. We used the DNA blotting-hybridization technique in which denatured DNA fragments are transferred to nitrocellulose paper directly from agarose gels and hybridized to 32P-labeled probe DNA. The 800 base pair insertion in an X mutant of Mu was found to hybridize with IS1. The chloramphenicol resistance transposon, Tn9, in Mu X cam mutants was found to be located at or close to the sites of IS1 insertion in X mutants; Tn9 also hybridized with IS1. The restriction endonuclease BalI cleaved IS1 once; it cleaved Tn9 in all Mu X cam mutants twice to release a fragment of about 1700 base pairs. These results support the conclusion that Tn9 contains one copy of IS1 at each end. In the P1cam isolate, from which Tn9 was transposed to Mu, BalI made a third cut in Tn9 giving rise to fragments of about 850 base pairs. The data further suggested that Tn9 is present in tandem copies in the P1cam isolate we examined. P1 itself was found to harbor IS1. The two P1 strains tested had a common fragment containing IS1; one strain had an additional copy of IS1. The IS1 element common to the P1 strains was shown to be the site of the Tn9 insertion in the P1cam isolate examined. No hybridization between IS2 and any of the Mu and P1 strains could be detected.

Chloramphenicol↗
PubMed1978

Control of expression of a cloned yeast (Saccharomyces cerevisiae) gene (trp5) by a bacterial insertion element (IS2).

A hybrid ColE1 plasmid [pYe(trp5)1], containing a yeast DNA segment that complements auxotrophic point mutations and deletions in the Escherichia coli tryptophan synthetase gene (trpAB), has been isolated. Expression of the yeast tryptophan synthetase activity from the cloned yeast gene (trp5) is relatively inefficient in E. coli, as measured by growth rates of trpAB/pYe(trp5)1 strains on minimal media lacking tryptophan and by enzyme assays. Faster growing variants occur spontaneously at a frequency of one in 10(4)--10(5) cells plated and produce higher levels of the yeast enzyme. Plasmid DNA [pYe(trp5)2] from one of these variants was shown to contain a DNA insertion (1.3 kilobase pairs) in the cloned yeast DNA segment in relatively close proximity to the trp5 gene. This DNA insert was identified as a bacterial IS2 element, which carries a promoter for RNA transcription when inserted in the proper orientation. The spontaneous integration of a bacterial DNA insertion element into cloned eukaryotic DNA can result in more efficient expression of the foreign gene.

Bacteriocin Plasmids↗
PubMed1979

Transposition of the Escherichia coli insertion element gamma generates a five-base-pair repeat.

We have determined DNA sequences surrounding the termini of the Escherichia coli insertion element gamma delta, both at its normal locus on the F (fertility) factor and at three different sites of insertion into the plasmid pBR322. After transposition, a five-base-pair pBR322 sequence is duplicated and appears in direct orientation adjacent to each end of the element. No such duplication flanks the ends of gamma delta in F, and there is no apparent homology between the sequences surrounding gamma delta in F and the five-base-pair duplications generated by insertion. These findings suggest that the duplications are not essential for transposition and that they do not act to direct gamma delta to a homologous site in the target chromosome. In addition, we find that the 35-base-pair inverted repeat that comprises the termini of gamma delta is strikingly similar in sequence to the ends of both the ampicillin-resistance transposon Tn3 and a 200-nucleotide-long sequence on the plasmid pSC101 which has been shown to mediate recombination with phage f1 replicative form. Within the terminal region, there is a specific heptanucleotide sequence common to each of the above elements and to bacteriophage Mu, all of which generate five-base-pair repeats upon insertion.

Base Sequence↗
PubMed1979

Enzymatic insertion of purine bases into depurinated DNA in vitro.

An enzymatic activity that inserts purines into depurinated DNA was found in a soluble enzyme extract of Escherichia coli. This activity brings about the insertion of adenine and guanine into the appropriate apurinic sites in double-stranded DNA by using the corresponding deoxyribonucleoside triphosphates as the purine donors. Magnesium ions are required for this activity, it is inhibited by caffeine, and it does not act on depurinated single-stranded DNA. The insertion activity described here may represent a step in a repair mechanism, "base-insertion repair," whereby apurinic sites (which may occur in double-stranded DNA either due to the removal of damaged purines with specific glycosylases or by spontaneous depurination) are directly filled with the correct missing purine base.

Caffeine↗
PubMed1978

Rat alpha-crystallin A chain with an insertion of 22 residues.

Rat lens alpha-crystallin contains, besides the usual alphaA and alphaB subunits, an additional minor chain. This subunit was purified by ion-exchange chromatography and its primary structure studied. It appeared to be an elongated alphaA-like chain, having an insertion of 22 residues between position 63 and 64 of an otherwise normal alphaA2 chain. Therefore this subunit was called alphaAIns, i.e. an alphaA chain with an inserted sequence. This inserted region, which contains three methionyl, five basic and no acidic residues, apparently results in an adequately functioning alphaAIns chain. The alphaAIns chain may be the product of a gene which has originated, after duplication of the alphaA gene, by insertion in one of the copies of a stretch of 66 nucleotides of unknown origin, or alternatively be the result of unusual transcription or processing of precursor mRNA (pre-mRNA), leaving an extra 66 nucleotides internally in the mRNA to be translated.

Amino Acid Sequence↗
PubMed1976

Suppression of polarity of insertion mutations in the gal operon and N mutations in bacteriophage lambda.

Bacterial mutations (psuA and psu) known for their ability to suppress the polarity on nonsense mutations are shown to suppress the polarity of certain insertion mutations in the gal operon. The short insertion, IS1 (800 nucleotide pairs), is about 15 to 50% suppressed, whereas longer insertions, IS2 (1,400 nucleotide pairs), and IS3 (1,200 nucleotide pairs), are not. Some of the polarity suppressor mutations (psu-1, psu-2, and psu-3) are at least partially permissive for N-gene mutations (N7 and N53) of bacteriophage lambda, suggesting a relationship between natural and mutational polar signals. That this relationship may be complex is indicated by the fact that other suppressor mutations, effective in suppressing nonsense or insertion polarity, fail entirely to permit the growth of lambda N mutants.

Cell-Free System↗
PubMed1977

Isolation, mapping, and examination of effects of TnA insertions in ColE1 plasmids.

Twelve TnA insertions of ColE1 deoxyribonucleic acid have been isolated and mapped by electron microscopic studies of heteroduplex molecules. Insertions only blocking the production of active colicin clustered in one region of the map, whereas insertions only blocking the expression of deoxyribonucleic acid nicking activity associated with the plasmid "relaxation complex" clustered in another region of the map. The location of one insertion that blocks both the expression of colicin immunity and the production of active colicin suggests that the expression of both characteristics are coordinately controlled.

Ampicillin↗
PubMed1979

Insertional inactivation of trpC in cloned Bacillus trp segments: evidence for a polar effect on trpF.

Plasmid pUB110 was previously used as a vector to clone fragments of deoxyribonucleic acid that complement the trpC2 mutation in Bacillus subtilis from endonuclease EcoRI digested B. licheniformis, B. pumilus, and B. subtilis cellular deoxyribonucleic acid. Each of several such trp plasmids was subsequently shown to contain a segment of the trp gene cluster on the basis of genetic complementing activity. In the present study, analysis of the Trp enzyme levels in B. subtilis harboring the constructed trp plasmids confirms the genetic constitution of the plasmids. Thus, plasmids that complement mutations in specific trp genes specify the corresponding enzyme activities. The levels of the plasmid-specified Trp enzymes in B. subtilis were generally above the repressed level of the chromosomally specified Trp enzymes and equal to or below the derepressed levels of the chromosomally specified Trp enzymes. Certain cloned trp segments contain a single HindIII-sensitive site. Insertion of HindIII-generated deoxyribonucleic acid fragments into these trp plasmids resulted in inactivation of trpC complementing activity, loss of the trpC-specified enzyme activity, and a 10-fold reduction in the specific activity of the plasmid-specified trpF product. The HindIII insertions had no detectable effect on the level of the trpD product, nor did the insertions detectably alter plasmid-specified complementing activity other than to abolish trpC complementation. Removal of the HindIII insertions was accompanied by recovery of trpC complementing activity and restoration of the trpC-and trpF-determined enzymes to the levels specified by the parent plasmids.

Bacillus↗
PubMed1979

Sites of insertion of TnA and TnM in RP1 and its derivatives.

The sites of insertion of TnM into the plasmid RP1, into derivatives of RP1 lacking TnA, and into derivatives in which the location of TnA within RP1 has been altered, were determined. Similarly, the sites of insertion of TnA into derivatives of RP1 from which TnA had been deleted, both with and without copies of TnM, have been examined. These studies show the presence in the plasmids of 'hot-spots' for TnA and TnM insertion. It is clear from the observations, however, that a particular DNA sequence in the recipient replicon is not sufficient to definite a 'hot-spot' since particular sequences sometimes do, and sometimes do not, contain many sites of insertion for a given transposon.

Base Sequence↗
PubMed1979

Timing of the IUD insertion.

The relationship between the time in the menstrual cycle when a TCu-200 or Cu-7-200 is inserted and subsequent IUD-related events was evaluated. For women who had either TCus or Cu-7s inserted, the pregnancy, expulsion and medical removal rates were similar for insertions performed at any time of the menstrual cycle.

Aging↗
PubMed2026

LINE-1 insertion intermediates recombine with one another or with DNA breaks to form genome rearrangements.

LINE-1 (L1) retrotransposition is common in human cancers and rearrangements at insertion sites can contribute to cancer-driving oncogene amplifications and promote genome instability. However, the mechanisms underlying rearrangements of L1 retrotransposition intermediates are poorly understood. To address this gap, we developed GFP-based recombination reporter assays to study the formation of L1 retrotransposition-mediated rearrangements. Using these reporters combined with long-read sequencing, we find that L1 retrotransposition cDNA intermediates can recombine with distal DNA breaks to generate chromosomal rearrangements. We also find that two independent L1 insertion cDNA intermediates on distinct genomic loci can recombine with each other to generate chromosomal rearrangements. Both types of rearrangements depend on L1-encoded ORF2p endonuclease and reverse transcriptase activities. Using these reporters, we discover that L1 retrotransposition-mediated rearrangements are robustly induced when the recombining sequences share extensive homology and that their formation requires the homologous recombination factor BRCA1. In contrast, we find L1 retrotransposition-mediated rearrangements are suppressed by the mismatch repair factor MSH2 when the recombining sequences contain mismatches. Given the repetitive nature of our genome, these findings highlight the risk of L1 insertion intermediates becoming substrates for aberrant recombination and promoting genome instability.

Long Interspersed Nucleotide Elements↗
PubMed2024

Identifying transgene insertions in Caenorhabditis elegans genomes with Oxford Nanopore sequencing.

Genetically modified organisms are commonly used in disease research and agriculture but the precise genomic alterations underlying transgenic mutations are often unknown. The position and characteristics of transgenes, including the number of independent insertions, influences the expression of both transgenic and wild-type sequences. We used long-read, Oxford Nanopore Technologies (ONT) to sequence and assemble two transgenic strains of Caenorhabditis elegans commonly used in the research of neurodegenerative diseases: BY250 (pPdat-1::GFP) and UA44 (GFP and human α-synuclein), a model for Parkinson's research. After scaffolding to the reference, the final assembled sequences were ∼102 Mb with N50s of 17.9 Mb and 18.0 Mb, respectively, and L90s of six contiguous sequences, representing chromosome-level assemblies. Each of the assembled sequences contained more than 99.2% of the Nematoda BUSCO genes found in the C. elegans reference and 99.5% of the annotated C. elegans reference protein-coding genes. We identified the locations of the transgene insertions and confirmed that all transgene sequences were inserted in intergenic regions, leaving the organismal gene content intact. The transgenic C. elegans genomes presented here will be a valuable resource for Parkinson's research as well as other neurodegenerative diseases. Our work demonstrates that long-read sequencing is a fast, cost-effective way to assemble genome sequences and characterize mutant lines and strains.

Caenorhabditis elegans↗
PubMed1978

Soluble gentamicin ophthalmic inserts as a drug delivery system.

A comparison was made of soluble 14C-gentamicin ophthalmic inserts with drop, ointment, and the subconjunctival routes of administration. The insert is a solid, solubilizable collagen polymer containing 14C-gentamicin. We compared the levels of 14C-gentamicin in the rabbit tear film and in multiple corneal and scleral biopsies to determine which route of administration gave the best results. The wafer route of administration gave the highest tear film and tissue concentration of drug. The tear film concentration by subconjunctival injection was surprisingly low. Soluble collagen inserts offer a new method of delivering high doses of gentamicin in infected corneal tissue in a convenient and atraumatic fashion.

Administration, Topical↗