The Non-Random Pattern of Insertion of IS2 into the hemB Gene of Escherichia coli

The hemB gene of Escherichia coli has been identified as a hot spot for the insertion of the transposable element IS2. The insertional specificity of IS2 is still unclear. This study reports on the attempt to sequence a statistically significant number of insertions in hemB, in order to determine whether there might be a basis for future studies to determine a molecular basis of IS2 insertional specificity. The results indicate that IS2 inserts in a non-random manner into a 240 bp segment at the 5′ end of the gene (region I). Twenty-one of 24 insertions occurred in region I. Three insertions have been identified in the two middle 250 bp segments of the 975 bp gene, and none in the 3′ terminal segment. A seventeen bp sequence showing 88.2% identity with a segment of IS2, 221 bp from the 3′ terminus has been identified in region I. Four instances of repeated insertion between the same pair of nucleotides have been observed at four different sites.     

Factors determining the frequency of plasmid cointegrate formation mediated by insertion sequence IS3 from Escherichia coli.

Summary Transposition events mediated by plasmid-borne copies of the insertion sequence IS3 of Escherichia coli are difficult to detect because of a low frequency of cointegrate formation. We found that cointegration activity could be strongly enhanced by using plasmid constructions in which a second IS3 element, disabled by a large deletion, was placed adjacent to an intact IS3 copy. Attempts to construct plasmids containing two adjacent intact IS3 copies were unsuccessful, probably because of instability. Transpositional hyperactivity of tandemly duplicated IS sequences was previously described for spontaneous duplications of IS21 and IS30 and may well be a more general phenomenon. The frequency of cointegration events was also strongly increased in an E. coli strain deficient in Dam methylation, suggesting that IS3, like some other Dam site-containing IS elements, is regulated by the Dam methylation system. Insertion sites were strongly clustered within the target lambda repressor gene; however no sequence specificity determinants could be identified. All insertions analyzed carried the IS element in the same orientation; target sequence duplications were mostly 3 bp, but in some cases 4 by long. To obtain information about the roles of the open reading frames (ORFs) in IS3, we constructed plasmid-borne mutant elements in which potentially functional reading frames were inactivated by site-directed mutations; the mutants were introduced into partial tandem constructions and tested in cointegration assays. Mutations inactivating the putative initiation codons of ORF I and 11 in the intact element reduced insertion activity to less than 4% of the wild type, whereas the introduction of a termination codon into ORF IV had no effect on cointegration frequency. We conclude that translation of ORFs I and II is essential for cointegration activity and that the mutagenized ATG codons most probably serve as the normal initiation codons in the wild-type element. In contrast, ORF IV could either be non-functional or its gene product could be supplied in trans from chromosomal elements.     

A cloned DNA fragment from bacteriophage P1 enhances IS2 insertion

Summary A 1.75 kb DNA segment of the bacteriophage P1 genome is known to serve as a preferred target for IS2 insertions. The presence of this fragment in a plasmid expressing the galK gene dramatically increases the proportion of IS2 insertions among spontaneous galK ^- mutants. Subfragments from two different parts of the 1.75 kb segment independently stimulate IS2 insertion, while another subfragment does not. In the plasmids studied IS2 elements not only insert into the cloned P1 fragment but also into parts of the galK gene with similar probability and mostly in one orientation. Many insertion sites are unique but several specific sites within the preferred target are repeatedly used for IS2 integration. The experimental data are compatible with a proposed cooperative mechanism, according to which more than one attracting sequence on the same plasmid might significantly enhance the probability of a particular target region to attract IS2.     

Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA.

Summary The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots). Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites. We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences. Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 by away from this hotspot. Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam ^– mutation did not affect Tn5 insertion relative to an I end sequence in target DNA. These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.     

Integration of IS3 into IS2 generates a short sequence duplication.

Summary The Gal⁺ allele IS2-43 is known to segregate Gal^- clones. Among 11 Gal^- segregants, one was shown to be due to the integration of IS3 into IS2-43. Precise excision of the integrated IS3 element occured at a rate of 5x10^-9/cell/generation. DNA sequence analysis revealed that the termini of the IS3 element have the relation of imperfect inverted repeats and it is now flanked by a 3bp or 4bp duplication, a size which has not been seen before with other elements.     

Absence of insertions among spontaneous mutants of Salmonella typhimurium

Summary While insertion sequences (IS) in Escherichia coli transpose frequently to generate spontaneous insertion mutants, such mutations are rare in Salmonella typhimurium: the only documented insertion mutation is a hisD mutation caused by the Salmonella-specific IS element IS200. To obtain more examples of IS200 insertion mutations and to seek additional types of IS elements in Salmonella, we selected and characterized 422 independent, spontaneous His^– mutants and some 2100 additional mutants that are not necessarily independent. None of the mutants showed the absolute polar effect characteristic of insertion mutations or the reversion properties characteristic of insertions (low spontaneous reversion frequency and no reversion induction by chemical mutagens). A few mutants, showing a high spontaneous reversion frequency, were screened physically. No insertion mutations were found. Thus insertion mutations appear to be rare in S. typhimurium, in strong contrast to E. coli and despite the possession in Salmonella of at least one type of insertion element (IS200). These results suggest that in Salmonella transposition of the endogenous elements has been controlled. The transposition ability of the elements may have been reduced or favored target sites removed from the host genome.     

Isolation and characterization of a temperature-sensitive amber suppressor mutant of Escherichia coli K12

Summary By mutagenizing an E. coli strain carrying an amber suppressor supD ^- (or su _I ⁺), we isolated a mutant whose amber suppressor activity was now temperature-sensitive. The mutant suppressor gene was named sup-126, which was found to be cotransduced with the his gene by phage P1vir at the frequency of ca. 20%. At 30° C it suppresses many amber mutations of E. coli, phage T4, and phage . At 42° C, however, it can suppress none of over 30 amber mutations tested so far. The sup-126 mutation is unambiguous and stable enough to be useful for making production of an amber protein temperature-sensitive.     

Inactivation of the Serratia marcescens gene for the lipoprotein in Escherichia coli by insertion sequences, IS1 and IS5; sequence analysis of junction points

Summary A pBR322-derived plasmid pKEN221 carrying a Serratia marcescens lpp gene overproduces the outer membrane lipoprotein in an Escherichia coli lpp ^–cell. However, when this strain was continuously cultured in a rich medium for about thirty generations, many Lpp^– mutants were accumulated. Out of six mutants analyzed, three were found to carry insertion mutation in the lpp gene in pKEN221. From restriction enzyme mapping and hybridization analysis of the mutant plasmid DNA, it was found that two mutants were caused by insertion sequence IS1 and one by IS5. Nucleotide sequence analysis of these mutant DNAs revealed that both IS1 and IS5 insertions occured in the A-T rich 5 untranslated region of the lpp gene. While the IS1 insertion resulted in a direct duplication of a nine-base-pair sequence in the original pKEN221 DNA at the junction with IS1, the IS5 insertion resulted in a direct duplication of a four-base-pair sequence. IS5 was found to contain inverted-repeat sequences of twelve nucleotides at its exact ends. This is the first example of the nucleotide sequence analysis of an IS5 insertion mutation. By Southern blot hybridization, the E. coli chromosomal DNA was found to contain about ten copies of IS5.     

Electron microscopy of polar insertions in the lac operon of Escherichia coli

Summary Several strongly polar mutations in the omega region of the z gene of the lac operon result from insertions consisting of only two specific sequences of DNA, one about 870 and the other 1170 nucleotide pairs long (based on single-strand measurements). No sequence homology was detected between the shorter (IS1) and longer (IS3) insertions. The IS1 insertion was shown to possess a specific attachment site, but it can be inserted with either orientation at several sites in the z gene. Four insertion sites in the omega region of gene z were identified and the position of the lac5 substitution and the SR2 deletion in the plac DNA were determined by heteroduplex mapping.     

A selection cartridge for rapid detection and analysis of spontaneous mutations including insertions of transposable elements in Enterobacteriaceae

Summary We present a method that allows positive selection and rapid analysis of mutations in Enterobacteriaceae. Mutations are detected in a 2630 bp selection cartridge inserted in two different bacterial mutlicopy plasmid vectors. Spontaneous mutations in Escherichia coli, Enterobacter cloacae and Citrobacter freundii include insertions, deletions and point mutations. The small size of the target sequence facilitates rapid analysis of DNA rearrangements by cleavage with restriction enzymes and of any type of mutation by DNA sequence analysis. While in E. coli insertions of the mobile elements IS1, IS2 and IS5 were readily found, insertions of putative new transposable elements were detected in Enterobacter cloacae. The selection cartridge can thus serve as a tool for studying the spectrum of insertion mutations in Enterobacteriaceae and probably other Gramnegative bacteria, and the dependency of this spectrum on physiological and environmental factors and the host's genetic background can be investigated.     

Amber mutants of Salmonella-phage P22 in genes engaged in the establishment of lysogeny

Summary Phage mutants were isolated with amber mutations in genes necessary for establishment of lysogeny. These mutants form turbid plaques on su ⁺ strain 527R1 and clear plaques of different types on LT2. According to complementation tests, fourteen mutants fall in the c ₂ gene, four in the c ₃ gene but no amber mutants were found belonging to the c ₁ gene. Pulse labelling experiments to follow DNA synthesis after phage infection were done with the mutants classified by complementation tests. Furthermore the labelling experiments demonstrated that the nonleaky c ₃ amber mutants displayed the same DNA synthesis pattern as c ₁ missense mutants. Since these c ₃ amber mutants complement missense c ₁ mutants it is concluded that the c ₃ and c ₁ genes must act together for the first transient repression of DNA synthesis, i.e., seven minutes after infection. It is suggested that clear plaque forming c ₁ amber mutants cannot be isolated because of polarity leading to defectivity of lysogenic as well as of lytic functions.The majority of the experiments presented are a part of the dissertation of H. D. Dopatka at the University of Göttingen.     

Insertions of transposable elements in the promoter proximal region of the gene cluster for Escherichia coli H+-ATPase: 8 base pair repeat generated by insertion of IS1

Summary A plasmid pKY159 (Yamaguchi and Yamaguchi 1983) carrying a promoter proximal portion of the gene cluster of the proton-translocating ATPase (H⁺-ATPase) of Escherichia coli causes growth inhibition of wild-type cells. Insertion of a transposable element in this plasmid released this inhibitory effect. In analyzing this inhibitory effect, we determined the insertion points at the nucleotidesequence level of transposable elements on 30 independent derivatives of pKY159. Insertions of IS1, IS5, and were found between the promoter and the gene for a possible component of 14,000 daltons of the H⁺-ATPase. Of 31 insertions, 26 were of IS1 and were located at the same site, indicating that this site is a hotspot for IS1 insertion and that IS1 insertion is much more frequent than that of IS5 of in this region. Four different sites for IS1 insertion were found; in two of these an 8 base pair (bp) duplicate of the target sequence (AAAAACGT and AAACGTTG) was generated, while in the other two a 9 bp duplicate was found. In all cases in this study the nucleotide sequence of IS1 was the same as that of IS1-K. In the two cases with an 8 bp duplicate in different sites, a common 6 bp sequence (AAACGT) was found. These results suggested that generation of the 8 bp duplicate is related to the common sequence rather than a mutation in IS1 suggested by Iida et al. (1981) and also suggested that the essential length of the duplicate is 8 bp or less than 8 bp. A 6 bp sequence (GTGATG) homologous to the end portion of IS1 was found at the hotspot, but not at other sites, suggesting that this homology contributed to the high frequency of IS1 insertion. The direction of IS1 insertion at the hotspot was the same in 25 of 26 instances, suggesting that the direction of IS1 insertion is determined by the structure of the target and/or its nearby sequence.Abbreviations bp base pairs - 14 K protein a possible component of the H⁺-ATPase with molecular weight of 14,000 (see Kanazawa and Futai 1982 for details) - EDTA ethylenediaminetetraacetate     

DNA sequence of a class I pseudogene from theTla region of the murine MHC: Recombination at a B2 Alu repetitive sequence

Summary We have determined the DNA sequence of a BALB/cTla region class I gene from the major histocompatibility complex (MHC) that had been identified previously as encoding a murine antigen by DNA-mediated gene transfer. Analysis of the DNA sequence shows, however, that this gene, the T1^c gene from theTla ^c genotype, could not encode a TL antigen or any other functional class I molecule due to the presence of numerous stop codons and frameshift mutations in the coding regions. This result suggests that the earlier transformation data may have been incorrect or perhaps that the clone containing the T1^c gene contains sequences that induced expression of a serologically reactiveTla gene in the genome of the recipient L cell. The T1^c gene is structurally related to the previously sequenced T13^c gene that encodes a serologically defined TL antigen. The 3 half of the T1^c gene including exons 4, 5, 6, and the 3 untranslated region has about 85% nucleotide similarity (including introns) with the corresponding parts of the T13^c gene; however, the 5 half of the T1^c gene has little homology with the T13^c gene. There is a sharp line of demarcation between the homologous and nonhomologous regions, and this border occurs precisely at a B2 Alu repeat sequence present in the T13^c gene. This suggests that a recombination event took place here and that an Alu repeat sequence that is known to have characteristics of transposable elements played some role in a recombination or gene conversion event.     

Recombination in the lambda repressor gene: evidence that very short patch (VSP) mismatch correction restores a specific sequence

Summary The mutation am6 in the cI gene of bacteriophage is identified as a CT transition in a 5CC _T ^A GG sequence. In four-factor crosses of am6 with nearby mutations in cI, the frequencies of cI⁺ recombinants are much higher than expected from the physical distances. A very short patch (VSP) mismatch repair system is presumed to recognize am6/am ⁺ mispairs in the heteroduplexes that accompany recombination between the outside markers. Mutation am6 is corrected to am ⁺; correction of am ⁺ to am6 was not detected. Clear-plaque mutation 1-1 in cI is a TC transition in a 5CTTGG sequence, resulting in the sequence 5CC _T ^A GG. When 1-1 was crossed with nearby mutations in gene cI, there were no excess cI⁺ recombinants, which would result from repair of CCTGG (1-1) to CTTGG (cI⁺). However, in crosses of cI⁺ phages with mutation 1-1, there was an excess of cI^- recombinants, indicating that cI⁺ was repaired to 1-1. Preferential repair does not require adenine or cytosine methylation: when repairing a mismatch, the VSP repair system apparently identifies specific mispaired bases by sequence alone.     

Nature of deletions formed in response to IS2 in a revertant of the gal3 insertion of E. coli.

Summary The gal3 mutation of E. coli, which arose by the insertion of IS2 in the OP region of the gal operon, reverts spontaneously by excision of the IS2 to produce inducible revertants or by mutational alterations of IS2 to produce constitutive revertants. However, gal3() strains bearing chlD-pgl deletions produce constitutive revertants alone. We proposed that deletions formed in the presence of IS2 terminate specifically at its right end, so that revertants arising by excision of IS2 fuse the gal genes to other promoters. Therefore, the revertants are exclusively constitutive.The above hypothesis was tested by electron microscopy of IS2-specific deletions. Spontaneous chlD-pgl deletions were isolated from gal ^c331 (a revertant of gal3 which retains IS2) and transferred to gal genomes. Electron microscopy of DNA heteroduplexes from these phages confirmed that all of the deletions examined have one end-point fixed at the right end of IS2, whereas their other end-points are variable. In each case, the complete IS2 element was apparently retained. This specificity was also detectable in a revertant (gal ^c200) which retains only the right 1/5 portion of the IS2. The frequencies of these deletions were generally increased in constitutive revertants of gal3. Since a galO ^cmutant did not show a similar increase, it seems that this effect depends upon a base sequence provided by IS2. Moreover, the presence of prophage contributes to the specificity and, in some instances, the frequency of IS2-specific deletions.A mechanism for the formation of the IS2-specific deletions has been proposed. A base sequence located at, or near, the right end of IS2 is recognized and nicked by a specific endonuclease. The nick is enlarged by unidirectional, exonucleolytic degradation to produce deletions extending outwards from the insertion. In constitutive revertants, the nicking site may be exposed to endonucleolytic attack more frequently.     

Frameshift and double-amber mutations in the bacteriophage T4 uvsX gene: analysis of mutant UvsX proteins from infected cells

Summary The bacteriophage T4 uvsX gene encodes a 43 kDa, single-stranded DNA-dependent ATPase, double-stranded DNA-binding protein involved in DNA recombination, repair and mutagenesis. Mutants of uvsX have a DNA-arrest phenotype and reduced burst size. Western blot immunoassay of UvsX peptides made by a number of amber mutants revealed amber peptides ranging from 25–32 kDa. Wild-type UvsX protein was also detected in lysates of cells infected with uvsX amber mutants, suggesting that their mutations are suppressed by translational ambiguity. We investigated the effects of mutations near the 5 end of uvsX. A frameshift mutation was engineered at codon 33. Western immunoblots for UvsX protein demonstrated that the frameshift mutant expresses no detectable wild-type UvsX; instead, a 37 kDa reactive peptide was detected. In order to determine if this peptide represents truncated UvsX protein, the mutation was regenerated in the cloned uvsX gene and expressed in transformed Escherichia coli. Endopeptidase digestion of the 37 kDa protein from the cloned gene generated peptide fragments indistinguishable from those obtained from wild-type UvsX. A double-amber mutant of uvsX was also generated by oligonucleotide site-directed mutagenesis. No UvsX protein was detected in lysates of cells infected with the uvsX-am64am67 double mutant. Plaque size and sensitivity to UV inactivation for both the double-amber and the frame-shift mutants were indistinguishable from those of other uvsX mutants. Mutations in uvsY had no demonstrable effect on efficiency of plating or UV sensitivity of uvsX mutants. Thus, null mutants of uvsX are viable.     

Regulation of the expression of iso 2-cytochrome c gene in S. cerevisiae: cloning of the positive regulatory gene CYP1 and identification of the region of its target sequence on the structural gene CYP3

Summary CYP1 is a trans acting regulatory locus modulating both iso 1- and iso 2-cytochrome c synthesis. Genetical analysis of various mutated alleles has allowed us to identify the gene product as a positive regulatory element.The region of the target sequence of the CYP1 product on the iso 2-cytochrome c structural gene was located by molecular and genetic analysis of two cis acting mutations located at the CYP3 locus: CYP3-36 and CYP3-4, which have been shown to arise from the integration of TY1 elements near the promoter site. Determination of the amount of iso 2-cytochrome c synthesized by strains bearing various genetic constructions, in which the cis acting mutations were associated with different alleles of the CYP1 trans acting locus, showed that TY1 inserted into CYP3-36 extinguishes the activation function due to a mutated overproducer allele CYP1-18, while CYP3-4 amplifies this function. This result identifies at least a part of the target sequence of the CYP1 product within the region separating the two TY1 insertions.To clone the CYP1 gene, we took advantage of the iso 2-cytochrome c overproducer phenotype of the mutated allele CYP1-18, which confers a Lactate⁺ phenotype on an iso 1-cytochrome c-deficient strain. Such a phenotype allowed the isolation of a recombinant plasmid YEpJFM1 carrying the mutated allele, able to complement on lactate medium a lactate ^- recipient strain. The identity of the YEpJFM1 sequence with the chromosomal gene was confirmed by homologous recombination at the CYP1 locus.     

Functional characterization of the prokaryotic mobile genetic element IS26

Summary IS26L and IS26R are the 820 bp long elements found as direct repeats at both ends of the kanamycin resistance transposon Tn2680. They can mediate cointegration in E. coli K12 which contains no IS26 in its chromosome. Cointegration occurs in rec ⁺ or recA ^- strains with similar frequency. Upon cointegration mediated by either IS26R or IS26L, the element is duplicated and integrated into one of many different sites. Both IS26L and IS26R carry 14 bp perfect terminal inverted repeats and generate 8 bp direct repeats at their target sequences. Deletion formation mediated by IS26R was also observed. These functional and structural features of IS26 are characteristic of a prokaryotic mobile genetic element.