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.     

Transposition of IS 117, the 2.5 kb Streptomyces coelicolor A3(2) 'minicircle': roles of open reading frames and origin of tandem insertions

IS 117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of 0RF1 of IS 117, presumed to encode a transposase, abolished transposition. Deletion or mutation of 0RF2 and 0RF3, which overlap each other on opposite strands of IS 117, caused a c. 20-fold reduction in integration frequency of the circular form of IS 117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS 117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. 0RF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild-type IS 117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS 117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS 117 derivatives yielded tandem chromosomal insertions, which arose by co-transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration.     

An Acetobacter xylinum insertion sequence element associated with inactivation of cellulose production.

An insertion sequence (IS) element, IS1031, caused insertions associated with spontaneous cellulose deficient (Cel-) mutants of Acetobacter xylinum ATCC 23769. The element was discovered during hybridization analysis of DNAs from Cel- mutants of A. xylinum ATCC 23769 with pAXC145, an indigenous plasmid from a Cel- mutant of A. xylinum NRCC 17005. An IS element, IS1031B, apparently identical to IS1031, was identified on pAXC145. IS1031 is about 950 bp. DNA sequencing showed that the two elements had identical termini with inverted repeats of 24 bp containing two mismatches and that they generated 3-bp target sequence duplications. The A. xylinum ATCC 23769 wild type carries seven copies of IS1031. Southern hybridization showed that 8 of 17 independently isolated spontaneous Cel- mutants of ATCC 23769 contained insertions of an element homologous to IS1031. Most insertions were in unique sites, indicating low insertion specificity. Significantly, two insertions were 0.5 kb upstream of a recently identified cellulose synthase gene. Attempts to isolate spontaneous cellulose-producing revertants of these two Cel- insertion mutants by selection in static cultures were unsuccessful. Instead, pseudorevertants that made waxlike films in the liquid-air interface were obtained. The two pseudorevertants carried new insertions of an IS1031-like element in nonidentical sites of the genome without excision of the previous insertions. Taken together, these results suggest that indigenous IS elements contribute to genetic instability in A. xylinum. The elements might also be useful as genetic tools in this organism and related species.     

Spontaneous transposition in the bacteriophage lambda cro gene residing on a plasmid.

A new mutagenesis assay system based on the phage lambda cro repressor gene residing on a plasmid was developed. The assay detects mutations in cro that decrease the binding of the repressor to the OR operator in an OR PR-lacZ fusion present in a lambda prophage. Mutations arose spontaneously during growth of E. coli cells harboring cro plasmids at a frequency of 3-6 x 10(-6). Analysis of some 200 cro mutants from several 'wild-type' strains revealed a substantial fraction of 25-70% insertion events caused by transposition of IS elements. Most of the insertions were caused by IS1, but IS5 insertions were observed too. In strains harboring Tn10, IS10 was responsible for most insertions. Restriction nuclease digestion analysis revealed a preference for insertion of IS10 into the C-terminal half of cro, despite the absence of sequences which are known hot spots for Tn10 insertions. The frequency of IS1 insertions into cro decreased 25-60-fold and that of IS10 insertions decreased 200-fold in cells carrying the recA56 mutation, suggesting that RecA is involved in transposition of these elements. During the logarithmic phase of growth, the mutation frequency was constant for at least 22 generations; however, upon continuous incubation at the stationary phase, the mutation frequency gradually increased, yielding a 3-fold increase in the frequency of insertion and a 4-5-fold increase in point mutation. Genomic Southern analysis of chromosomal IS elements in cells which underwent a transposition from the chromosome into the cro plasmid revealed that the number and distribution of IS1 and IS5 were usually unaltered compared to cells which did not undergo a transposition event. In contrast, essentially each IS10 transposition was accompanied by multiple events which led to changes in the number and distribution of chromosomal IS10 elements.     

Mechanism of Is1 Transposition in E. COLI: Choice between Simple Insertion and Cointegration

Insertion element IS1 and IS1-based transposon Tn9 generate cointegrates (containing vector and target DNAs joined by duplicate copies of IS1 or Tn9) and simple insertions (containing IS1 or Tn9 detached from vector sequences). Based on studies of transposon Tn5 we had proposed a conservative (non-replicative) model for simple insertion. Others had proposed that all transposition is replicative, occurring in a rolling circle structure, and that the way DNA strands are joined when replication terminates determines whether a simple insertion or a cointegrate is formed.--We selected for the transposition of amp and cam resistance markers from pBR322::Tn9 plasmids to an F factor in recA-E. coli and identified products containing three and four copies of IS1, corresponding to true cointegrates (from monomeric plasmids), and simple insertions (from dimeric plasmids). The simple insertions with four copies of IS1 outnumbered those with three by a ratio of about 3:1, whereas true cointegrates containing three copies of IS1 were more numerous than those with four.--A straightforward rolling circle model had predicted that the simple insertions containing three copies of IS1 should be more frequent than those with four. Because we obtained the opposite result we propose that simple insertions only arise when the element fails to replicate or if replication starts but then terminates prematurely. The two classes of products, simple insertions and cointegrates, reflect alternative conservative and replicative fates, respectively, of an early intermediate in transposition.     

ISMapper: identifying transposase insertion sites in bacterial genomes from short read sequence data

Background

Insertion sequences (IS) are small transposable elements, commonly found in bacterial genomes. Identifying the location of IS in bacterial genomes can be useful for a variety of purposes including epidemiological tracking and predicting antibiotic resistance. However IS are commonly present in multiple copies in a single genome, which complicates genome assembly and the identification of IS insertion sites. Here we present ISMapper, a mapping-based tool for identification of the site and orientation of IS insertions in bacterial genomes, directly from paired-end short read data.

Results

ISMapper was validated using three types of short read data: (i) simulated reads from a variety of species, (ii) Illumina reads from 5 isolates for which finished genome sequences were available for comparison, and (iii) Illumina reads from 7 Acinetobacter baumannii isolates for which predicted IS locations were tested using PCR. A total of 20 genomes, including 13 species and 32 distinct IS, were used for validation. ISMapper correctly identified 97 % of known IS insertions in the analysis of simulated reads, and 98 % in real Illumina reads. Subsampling of real Illumina reads to lower depths indicated ISMapper was able to correctly detect insertions for average genome-wide read depths >20x, although read depths >50x were required to obtain confident calls that were highly-supported by evidence from reads. All ISAba1 insertions identified by ISMapper in the A. baumannii genomes were confirmed by PCR. In each A. baumannii genome, ISMapper successfully identified an IS insertion upstream of the ampC beta-lactamase that could explain phenotypic resistance to third-generation cephalosporins. The utility of ISMapper was further demonstrated by profiling genome-wide IS6110 insertions in 138 publicly available Mycobacterium tuberculosis genomes, revealing lineage-specific insertions and multiple insertion hotspots.

Conclusions

ISMapper provides a rapid and robust method for identifying IS insertion sites directly from short read data, with a high degree of accuracy demonstrated across a wide range of bacteria.     

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.     

Generation of isogenic K54 capsule-deficient Escherichia coli strains through TnphoA-mediated gene disruption

Transposon muta genesis, using IS50^L::phoA(Tn-phoA), was performed in a K54/O4/H5 blood isolate of Escherichia coli (CP9), to generate a library of random mutants. Five hundred and twenty-six independent CP9 TnphoA mutants were isolated with active gene fusions to alkaline phosphatase. From this mutant library, eight capsule-deficient strains were detected and were found to have a single copy of TnphoA. Sixteen additional capsule deficient mutants with TnphoA inserts were subsequently obtained that did not possess active PhoA fusions. In conjunction with the initial eight capsule-deficient isolates we have defined genes on three different XbaI fragments as being involved in capsule production. Generalized transduction with the bacteriophage T4 established that these insertions were responsible for the loss of capsule and that they are linked. These capsule-deficient strains can be used to assess the pathogenic role of the K54 capsular polysaccharide.     

Suppression of polarity of insertion mutations within the gal operon of E. coli.

Summary Phenotypic revertants of galOP::IS1 and galOP::IS2 mutations have been isolated after mutagenesis with nitrosoguanidine, they are probably caused by mutations in gene suA. The polarity suppressor mutations described in this study and a known mutation in gene suA isolated by D. Morse (Morse and Guertin, 1972) suppress polarity caused by IS1 more effectively than that caused by IS2 or IS4. Furthermore, suppressibility is influenced by the site and orientation of IS integration.The synthesis of the three enzymes in galOP::IS suA double mutants is constitutive and the ratio of the three enzymes is altered in comparison to the wild type. The reasons for constitutive synthesis of the galactose enzymes and for the altered ratio of enzyme synthesis are discussed.     

The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth

Sequence determination of the chloroplast clpP gene from two distantly related Chlamydomonas species (C. reinhardtii and C. eugametos) revealed the presence of translated large insertion sequences (IS1 and IS2) that divide the clpP gene into two or three sequence domains (SDs) and are not found in homologous genes in other organisms. These insertion sequences do not resemble RNA introns, and are not spliced out at the mRNA level. Instead, each insertion sequence forms a continuous open reading frame with its upstream and downstream sequence domains. IS1 specifies a potential polypeptide sequence of 286 and 318 amino acid residues in C. reinhardtii and C. eugametos, respectively. IS2 encodes a 456 amino acid polypeptide and is present only in C. eugametos. The two Chlamydomonas IS1 sequences show substantial similarity; however, there is no significant sequence similarity either between IS1 and IS2 or between these insertion sequences and any other known protein coding sequences. The C. reinhardtii clpP gene was further shown to be essential for cell growth, as demonstrated through targeted gene disruption by particle gun-mediated chloroplast transformation. Only heteroplasmic transformants could be obtained, even under mixotrophic growth conditions. The heteroplasmic transformants were stable only under selection pressure for the disrupted clpP, rapidly segregated into wild-type cells when the selection pressure was removed, and grew significantly more slowly than wildtype cells under phototrophic conditions.     

Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion

Escherichia coli was used as a model to study initial adhesion and early biofilm development to abiotic surface. Tn10 insertion mutants of Escherichia coli K-12 W3110 were selected for altered abilities to adhere to a polystyrene surface. Seven insertion mutants that showed a decrease in adhesion harbored insertions in genes involved in lipopolysaccharide (LPS) core biosynthesis. Two insertions were located in the rfaG gene, two in the rfaP gene, and three in the galU gene. These adhesion mutants were found to exhibit a deep-rough phenotype and to be reduced, at different levels, in type 1 fimbriae production and motility. The loss of adhesion exhibited by these mutants was associated with either the affected type 1 fimbriae production and/or the dysfunctional motility. Apart from the pleiotropic effect of the mutations affecting LPS on type 1 fimbriae and flagella biosynthesis, no evidence for an involvement of the LPS itself in adhesion to polystyrene surface could be observed. Received: 1 December 1998 / Accepted: 3 April 1999     

Repeated sequences including RS 1100 from Pseudomonas cepacia AC1100 function as IS elements

Summary Several lines of evidence were obtained that the previously identified, repeated sequence RS 1100 of Pseudomonas cepacia strain AC1100 undergoes transposition events. DNA sequences flanking the chlorohydroxy hydroquinone (CHQ) degradative genes of this organism were examined from sources, including several independently isolated cosmid clones from an AC1100 genomic library and genomic DNAs of two independently maintained wild-type AC1100 isolates. Hybridization and restriction endonuclease mapping studies revealed these sequences to be similar except for their numbers and distributions of RS1100 copies. A recombinant plasmid containing the immediate chq gene region and excluding any copies of RS1100 was conjugated into AC1100 mutant RHA5 which was shown to have undergone a deletion of its corresponding DNA. Hybridization and restriction mapping analyses of several reisolated plasmids revealed the presence of RS1100 sequences at different positions within either the vector or insert portions. One such plasmid contained tandem copies of RS1100 with an intervening DNA sequence also of AC1100 origin. Similar experiments involving introduction of the promoter probe plasmid pKT240 into wild-type AC1100 cells resulted in the acquisition of high-concentration streptomycin resistance by a number of recipients. The reisolated plasmids in most cases also conferred streptomycin resistance to Escherichia coli transformants and in each case were found to contain insertions close to the upstream portion of the aphC structural gene. These insertions alternatively contained RS1100 sequences or a newly identified 3400 by repeated sequence from AC1100. Based on these results, RS1100 has been redesignated as insertion sequence IS931 and the 3400 bp repeated sequence has been designated as IS932.[/ab]Abbreviations aphc aminoglycoside phosphotransferase gene - BSM basal salts medium - chq chlorohydroxy hydroquinone degradative gene(s) - dCTP deoxycytidine triphosphate - IS insertion sequence - Tft 2,4,5-T degradative phenotype     

Characterization of Insertions of IS476 and Two Newly Identified Insertion Sequences, IS1478 and IS1479, in Xanthomonas campestris pv. campestris

Thirty-two plasmid insertion mutants were independently isolated from two strains of Xanthomonas campestris pv. campestris in Taiwan. Of the 32 mutants, 14 (44%), 8 (25%), and 4 (12%) mutants resulted from separate insertions of an IS3 family member, IS476, and two new insertion sequences (IS), IS1478 and IS1479. While IS1478 does not have significant sequence homology with any IS elements in the EMBL/GenBank/DDBJ database, IS1479 demonstrated 73% sequence homology with IS1051 in X. campestris pv. dieffenbachiae, 62% homology with IS52 in Pseudomonas syringae pv. glycinea, and 60% homology with IS5 in Escherichia coli. Based on the predicted transposase sequences as well as the terminal nucleotide sequences, IS1478 by itself constitutes a new subfamily of the widespread IS5 family, whereas IS1479, along with IS1051, IS52, and IS5, belongs to the IS5 subfamily of the IS5 family. All but one of the IS476 insertions had duplications of 4 bp at the target sites without sequence preference and were randomly distributed. An IS476 insertion carried a duplication of 952 bp at the target site. A model for generating these long direct repeats is proposed. Insertions of IS1478 and IS1479, on the other hand, were not random, and IS1478 and IS1479 each showed conservation of PyPuNTTA and PyTAPu sequences (Py is a pyrimidine, Pu is a purine, and N is any nucleotide) for duplications at the target sites. The results of Southern blot hybridization analysis indicated that multiple copies of IS476, IS1478, and IS1479 are present in the genomes of all seven X. campestris pv. campestris strains tested and several X. campestris pathovars.     

Somatic mobility of the maize element Ac and its utility for gene tagging in aspen

We have investigated the somatic activity of the maize Activator (Ac) element in aspen with the objective of developing an efficient transposon-based system for gene isolation in a model tree species. The analysis of the new insertion sites revealed the exact reconstitution of the Ac, however, aberrant transposition events were also found. Characterization of the genomic sequences flanking the Ac insertions showed that about one third (22/75) of the sequences were significantly similar to sequences represented in public databases and might correspond to genes. The frequency of Ac landing into coding regions was about two-fold higher when compared to the frequency of T-DNA hitting the predicted genes (5/32) in the aspen genome. Thus, Ac is demonstrated to be a potentially useful heterologous transposon tag in a tree species. This is the first report on transposon-based gene tagging in a tree species describing the excision and reinsertion of transposable element into new genomic positions. We also suggest a heterologous transposon tagging strategy that can be used in aspen somatic cells to obtain dominant gain-of-function mutants and recessive loss-of-function mutants overcoming the regeneration time barrier of a long-lived tree species.     

A family of IS1031 elements in the genome of Acetobacter xylinum: nucleotide sequences and strain distribution

An insertion sequence (here called IS 1031A) from Acetobacter xylinum ATCC 23769 has recently been isolated. This study describes the complete nucleotide sequence of IS 1031A as well as the sequences of two novel iso-IS 1031 elements, IS1031C and IS1031D, from A. xylinum ATCC 23769. The three ISs are all exactly 930 bp long, have imperfect terminal inverted repeats of 24 bp for IS1031A and 21 bp for IS1031C and IS1031D, are flanked by three base pair direct repeats, and contain an open reading frame encoding a putative basic protein of 278 amino acids. Because of nucleotide substitutions, IS1031C and IS1031D differ from IS 1031A by 12.9% while IS1031C differs from IS1031D by only 0.6%. Hybridization analyses of total DNA from nine A. xylinum strains showed that all strains contained IS 1031-like elements varying in copy number from three to at least 16. None of three Acetobacter aceti strains examined contained IS1031-like elements. Taken together, the results suggest that A. xylinum contains a family of IS 1031 elements with considerably diversified nucleotide sequences.     

Insertion sequence-caused large-scale rearrangements in the genome of Escherichia coli

A majority of large-scale bacterial genome rearrangements involve mobile genetic elements such as insertion sequence (IS) elements. Here we report novel insertions and excisions of IS elements and recombination between homologous IS elements identified in a large collection of Escherichia coli mutation accumulation lines by analysis of whole genome shotgun sequencing data. Based on 857 identified events (758 IS insertions, 98 recombinations and 1 excision), we estimate that the rate of IS insertion is 3.5 × 10⁻⁴ insertions per genome per generation and the rate of IS homologous recombination is 4.5 × 10⁻⁵ recombinations per genome per generation. These events are mostly contributed by the IS elements IS1, IS2, IS5 and IS186. Spatial analysis of new insertions suggest that transposition is biased to proximal insertions, and the length spectrum of IS-caused deletions is largely explained by local hopping. For any of the ISs studied there is no region of the circular genome that is favored or disfavored for new insertions but there are notable hotspots for deletions. Some elements have preferences for non-coding sequence or for the beginning and end of coding regions, largely explained by target site motifs. Interestingly, transposition and deletion rates remain constant across the wild-type and 12 mutant E. coli lines, each deficient in a distinct DNA repair pathway. Finally, we characterized the target sites of four IS families, confirming previous results and characterizing a highly specific pattern at IS186 target-sites, 5′-GGGG(N6/N7)CCCC-3′. We also detected 48 long deletions not involving IS elements.     

A new dispensable genetic locus of the terminus region involved in control of cell division in Escherichia coli

Summary Temperature-sensitive mutants defective in cell division were isolated after localised mutagenesis of the terminus region of the Escherichia coli chromosome. The defective gene in one of these mutants, dicA, was mapped at 34.9 min by linkage with manA and with three physically characterized Tn10 insertions. Temperature-sensitivity conferred by mutation dicA1 in a recA backround was suppressed by the presence of hybrid plasmids carrying the wild-type gene. In addition, the mutation was suppressed either by tranposon inactivation of a nearby gene, dicB, or by deletion of the entire dicA-dicB interval. These results define the dicA-dicB locus as a new dispensable genetic cluster involved in the control of cell division.     

Early and multiple Ac transpositions in rice suitable for efficient insertional mutagenesis

A GFP excision assay was developed to monitor the excision of Ac introduced into rice by Agrobacterium-mediated transformation. The presence of a strong double enhancer element of the CaMV 35S promoter adjacent to the Ac promoter induced very early excision, directly after transformation into the plant cell, exemplified by the absence of Ac in the T-DNA loci. Excision fingerprint analysis and characterization of transposition events from related regenerants revealed an inverse correlation between the number of excision events and transposed Ac copies, with single early excisions after transformation generating Ac amplification. New transpositions were generated at a frequency of 15–50% in different lines, yielding genotypes bearing multiple insertions, many of which were inherited in the progeny. The sequence of DNA flanking Ac in three representative lines provided a database of insertion tagged sites suitable for the identification of mutants of sequenced genes that can be examined for phenotypes in a reverse genetics strategy to elucidate gene function. Remarkably, two-thirds of Ac tagged sites showing homology to sequences in public databases were in predicted genes. A clear preference of transposon insertions in genes that are either predicted by protein coding capacity or by similarity to ESTs suggests that the efficiency of recovering knockout mutants of genes could be about three times higher than random. Linked Ac transposition, suitable for targeted tagging, was documented by segregation analysis of a crippled Ac element and by recovery of a set of six insertions in a contiguous sequence of 70 kb from chromosome 6 of rice.     

Linker insertion mutagenesis based on IS21 transposition: isolation of an AMP-insensitive variant of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa

The bacterial insertion sequence IS21 when repeated in tandem efficiently promotes non-replicative cointegrate formation in Escherichia coli. An IS21-IS21 junction region which had been engineered to contain unique SalI and BglII sites close to the IS21 termini was not affected in the ability to form cointegrates with target plasmids. Based on this finding, a novel procedure of random linker insertion mutagenesis was devised. Suicide plasmids containing the engineered junction region (pME5 and pME6) formed cointegrates with target plasmids in an E.coli host strain expressing the IS21 transposition proteins in trans. Cointegrates were resolved in vitro by restriction with SalI or BglII and ligation; thus, insertions of four or 11 codons, respectively, were created in the target DNA, practically at random. The cloned Pseudomonas aeruginosa arcB gene encoding catabolic ornithine carbamoyltransferase was used as a target. Of 20 different four-codon insertions in arcB, 11 inactivated the enzyme. Among the remaining nine insertion mutants which retained enzyme activity, three enzyme variants had reduced affinity for the substrate ornithine and one had lost recognition of the allosteric activator AMP. The linker insertions obtained illustrate the usefulness of the method in the analysis of structure-function relationships of proteins.