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© 1997 Oxford University Press 1087-1094

Footnote

Compilation and analysis of intein sequences

Compilation and analysis of intein sequences Francine B. Perler* , Gary J. Olsen 1 and Eric Adam

New England Biolabs Inc., 32 Tozer Road, Beverly , MA 01915, USA and 1 Department of Microbiology, University of Illinois, B301 C&LSL, 601 South Goodwin Avenue, Urbana , IL 61801, USA

Received November 20, 1996; Revised and Accepted January 24, 1997

ABSTRACT

We have compiled a list of all the inteins (protein splicing elements) whose sequences have been published or were available from on-line sequence databases as of September 18, 1996. Analysis of the 36 available intein sequences refines the previously described intein motifs and reveals the presence of another intein motif, Block H. Furthermore, analysis of the new inteins reshapes our view of the conserved splice junction residues, since three inteins lack the intein penultimate His seen in prior examples. Comparison of intein sequences suggests that, in general, (i) inteins present in the same location within extein homologs from different organisms are very closely related to each other in paired sequence comparison or phylogenetic analysis and we suggest that they should be considered intein alleles; (ii) multiple inteins present in the same gene are no more similar to each other than to inteins present in different genes; (iii) phylogenetic analysis indicates that inteins are so divergent that trees with statistically significant branches cannot be generated except for intein alleles.

INTRODUCTION

Protein splicing is defined as removal of an in ternal pro tein segment ( intein ) from a precursor protein and ligation of the ex ternal pro tein segments ( exteins ) to form a native peptide bond ( 1 ). Extein ligation differentiates protein splicing from other forms of self-proteolysis, such as cleavage of glycosylasparaginase ( 2 ) or the hedgehog protein ( 3 ). Protein splicing elements were first described in 1990 as in-frame insertions in the Saccharomyces cerevisiae VMA gene that were unrelated to the sequence of homologous ATPases ( 4 , 5 ). Moreover, the mature VMA protein had an electrophoretic mobility that was similar to the homolog lacking the intein and not to the predicted size of the VMA gene. A second protein with the predicted size of the intein was also detected. Most inteins contain the dodecapeptide motifs characteristic of homing endonucleases (which were first discovered in mobile self-splicing introns) and several inteins have demonstrated endonuclease activity ( 6 - 10 ). Intein genes that encode active homing endonucleases are potential mobile genetic elements ( 6 , 11 , 12 ).

Although several inteins were identified experimentally (inteins 1-3, 6, 7, 11, 12, 14, 15 and 18 in Table 1 ) ( 4 , 5 , 10 , 13 - 16 ; Cole,S., personal communication; Liu,P.X.-Q., personal communication), most of the recently described inteins were predicted from DNA sequences ( 9 , 15 , 17 - 20 ). This latter class of inteins is termed theoretical in Table 1 , since spliced products have not been experimentally observed. A combination of four criteria have been used to identify protein splicing elements in newly sequenced genes ( 9 , 15 , 17 , 18 ): (i) an in-frame insertion in a gene that has a previously sequenced homolog lacking the insertion; (ii) the presence of intein Blocks C and E (Table 2 ), which are also found in homing endonucleases, where they are called dodecapeptide motifs, DOD motifs, P1 and P2 motifs and LAGLI-DADG motifs ( 8 , 9 ); (iii) the presence of several other conserved intein motifs (Table 2 ; 9 ); (iv) the presence of four conserved splice junction residues (Ser, Thr or Cys at the intein N-terminus, the dipeptide His-Asn at the intein C-terminus and Ser, Thr or Cys following the downstream splice site) ( 1 , 9 , 21 - 24 ). The last three criteria help differentiate true inteins from in-frame inserts that result from interspecies sequence variability or other types of insertion sequences. As discussed below, these criteria have been refined as more inteins have been discovered.

ANALYTICAL METHODS

Alleles with >41% identity to the prototype intein first identified in that location, as determined using the default parameters of the BESTFIT pairwise comparison program ( 25 ), were not included in the multiple sequence analysis, since they would bias the search for conserved motifs and the calculation of their significance. This percent identity was chosen because it is just above the highest identity among the poorly related intein alleles (see below). The Mka gyrA, Mfl gyrA, Mgo gyrA, Mxe gyrA, Psp pol-1, Psp pol-3 and Mja pol-2 inteins were not included while building the alignment nor were they included in the block calculations.

Conserved motifs were detected and evaluated with the MACAW 2.0.5 program ( 26 ). MACAW does not allow gaps in the aligned sequence blocks. Briefly, the Gibbs sampling method ( 27 ) was used for identifying the sequence blocks and the block boundaries were readjusted to maximize the motif score (minimizing the p value) using the BLOSUM62 comparison matrix ( 28 ). The MACAW program calculates the chance probability for the appearance of an alignment score by a statistical formula using an extreme value distribution model of alignment scores ( p value) ( 26 ). All the final block calculations resulted in p values <10 -20 (i.e. the calculation limit of the MACAW 2.0.5 program on the Power Macintosh 8100/80) when the whole length of the 29 most diverse intein sequences (as defined above) was taken as the search sequence space.

The least squares distance phylogenetic tree was inferred using programs in version 3.5 of the PHYLIP package ( 29 ). The number of amino acid replacements per sequence position separating each pair of sequences was estimated using the PAM option of the PROTDIST program. The sampling variance of the distance values was estimated from 100 bootstrap resamplings of the sequence data using the SEQBOOT and PROTDIST programs. The phylogenetic tree that best fits (by a least squares criterion) these sequence-to-sequence distances was found with the FITCH program, using the subreplicates option to weight each pairwise distance by one over its estimated variance ( 30 ). Global rearrangements and multiple taxon addition orders were used to find an optimal tree. Because of possible errors in Block E of the Psp pol-3 intein sequence, three positions were replaced by unidentified residues (Xs) in the phylogenetic analysis, yielding FLEGXXXGDG.

THE CATALOG

The information summarized in Table 1 comprises all intein sequences that to our knowledge have been published or were available from public databases [NCBI sequence libraries or The Institute for Genomic Research (TIGR) Web page, http://www.tigr.org] as of September 18, 1996. Inteins whose sequences were not available have not been included in this list. Updates to this catalog can be obtained via Email from perler@neb.com and new inteins can be registered at this same address. The registry will also be accessible in the near future on the New England Biolabs Web site (http://www.neb.com). The REBASE database (http://www.neb.com/rebase) also collects information about inteins, with emphasis on endonuclease activity ( 31 ).


Table 1. Inteins 1-4 are from eukarya, inteins 5-13 are from eubacteria and inteins 14-36 are from archaea. The Ceu clpP intein has also been referred to as IS2 (20). *The exact intein junction was deduced from conserved intein features and not extein similarity. Endonuclease activity has been demonstrated; however, there are no published activity assays for the other inteins. Allele lists the prototype intein at this same position in a homologous extein gene. N-term and C-term list the residues present at the respective ends of each intein, including the first extein residue following the C-terminal splice junction. Size indicates the number of amino acids in each intein. Loc lists the extein amino acid preceding the intein. The Loc of the Mxe gyrA intein was inferred from the other gyrA alleles, since the complete Mxe gyrA gene has not been sequenced (GenBank accession no. U67876). [sect]Cole,S., personal communication. [infinity]Liu,P.X.-Q., personal communication. Other abbreviations: Theor, theoretically derived; Exp, experimentally determined; (/), splice junction; Acc No., accession no.; Ref, reference; pol, DNA polymerase; hyp, hypothetical protein; IF-2, translation initiation factor, FUN12/bIF-2 family; PEP synthase, phosphoenolpyruvate synthase; RNR or anaerobic rNTP reductase, anaerobic ribonucleoside triphosphate reductase; Rpol, RNA polymerase subunit; GF-6P transaminase, glutamine-fructose 6-phosphate transaminase; Replication factor C, replication factor C 37 kDa subunit; C.tropicalis , Candida tropicalis ; C.eugametos , Chlamydomonas eugametos ; P.purpurea , Porphyra purpurea ; Ssp or Synechocystis , Synechocystis spp.; Psp , Pyrococcus spp.; M.tuberculosis , Mycobacterium tuberculosis ; M.kansasii , Mycobacterium kansasii ; M.flavescens , Mycobacterium flavescens ; M.gordonae , Mycobacterium gordonae ; M.xenopi , Mycobacterium xenopi .

According to intein nomenclature conventions ( 1 ), the intein names listed in Table 1 include organism and extein gene designations as well as a numerical suffix when more than one intein is present in the same extein gene in the same organism (as in the case of the Tli and Mja pol inteins, Mja RNR inteins and Mja RFC inteins). DNA polymerase inteins from various Pyrococcus isolates ( Psp pol inteins 1-3) were numbered in order of entry into the intein registry and are not present in the same organism (Table 1 ). The Mle recA intein and the Mtu recA intein are located at different positions in recA (after G205 or K251 respectively). There are also many examples of inteins present in the same location in homologous extein genes from different organisms (dnaB, VMA, pol and gyrA). If endonuclease activity has been demonstrated, the intein is also given an endonuclease designation following the restriction enzyme nomenclature convention with the addition of the prefix PI-. To date, four inteins have demonstrated endonuclease activity: PI- Sce I ( Sce VMA intein), PI- Tli I ( Tli pol intein-2), PI- Tli II ( Tli pol intein-1) and PI- Psp I ( Psp pol intein-1) ( 7 , 10 , 32 ; Perler,F.B., unpublished data).

Except for the Sce VMA intein, the Tli pol-2 intein and the Psp pol-1 intein, for which N-terminal amino acid sequences have been determined ( 10 , 24 , 33 ), the size and splice junction residues listed in Table 1 have been deduced using the criteria listed above for theoretical inteins ( 4 , 5 , 9 , 10 , 13 - 20 , 34 ). Exact intein boundaries are usually obvious after comparison with inteinless homologs, especially since many inteins are present in conserved motifs in extein genes, such as DNA polymerases and gyrases ( 15 , 35 ). The TIGR Web site alignments were used to determine M.jannaschii intein boundaries, except for the Mja hyp-1 intein, where the Bacillus subtilis YqkH protein (GenBank accession no. D84432) provided a better extein match than the B.subtilis YqxK protein ( 36 ). Extein sequences flanking each M.jannaschii intein were not always similar to the sequence of the inteinless homolog. In these cases, the intein boundaries were deduced by comparison with conserved sequences in Blocks A and G (see below and Table 2 ) and are marked with an asterisk in Table 1 . However, because of the high degree of conservation of the intein junctions and other residues in Blocks A and G, the presence of an asterisk does not imply reduced confidence in junction assignment.

Table 2 . Conserved motifs found in inteins


Eight conserved intein motifs were identified by multiple sequence analysis (MACAW) of the 29 inteins listed in capital letters, as described in the text. Intein sequences in lower case are highly similar alleles that were not included in the multiple sequence analysis. These motifs are similar to the previously defined intein blocks ( 9 ) with the addition of Block H. Sce HO, the yeast mating type endonuclease, has been included in the table because of its similarity to inteins. The position in the protein of the last amino acid in each block is listed to the right of the block. The consensus line represents conserved residues or amino acid groups present in at least 15 of the 29 inteins included in the multiple sequence analysis. The four absolutely conserved residues are marked with an asterisk under the consensus line residue. Dashes indicate no match to that block. [infinity]The deposited DNA sequence yields a non-consensus Psp pol-3 intein Block E sequence of flegyssama. However, if a frameshift resulting from insertion of a T at nt 3846 were made, the DNA sequence would then yield the conserved motif listed in this table, while three frameshifts in this region could give a sequence nearly identical to that of the other intein alleles. Intein names and abbreviations are as in Table 1. Definition of symbols in the consensus line: capital letters indicate conserved amino acids (standard single letter code); p, polar residue (S, T or C; purple); h, hydrophobic residue (G, A, V, L, I or M; green); a, acidic residue (D or E; red); b, basic residue (H, K or R; blue); r, aromatic residue (F, Y or W; orange).

Inteins have been found in all three domains of life (Table 1 ): (i) inteins 1-2 are in eucaryal nuclear genes ( Sce VMA and Ctr VMA) and inteins 3-4 are in eucaryal chloroplast genes ( Ceu clpP and Ppu dnaB); (ii) inteins 5-13 are from eubacteria ( Mycobacterium and Synechocystis spp.); (iii) inteins 14-36 are from thermophilic Archaea ( Thermococcus litoralis , Pyrococcus isolates and Methanococcus jannaschii ). Inteins are found in the same types of organisms and chromosomal locations as mobile introns ( 37 ). The large number of inteins reported in Mycobacterium leprae and M.jannaschii are due, in part, to genome sequencing projects. However, only one intein has been found in the genomes of Synechocystis spp. ( 38 ) and S.cerevisiae and no inteins have been detected in Haemophilus influenzae Rd ( 39 ), Mycoplasma genitalium ( 40 ) and other viral or phage genome sequences present in GenBank as of September 18, 1996. Whether the 18 inteins in 14 different M.jannaschii genes ( 17 ) reflect an abundance of inteins in this particular species or in Archaea in general awaits a complete analysis of more small genomes. For now we note that extensive sequencing of archaeal RNA polymerase genes ( 41 - 45 ) and DNA polymerase genes ( 35 ) suggests that these inteins are not widely distributed in Archaea.

Although many inteins are located in enzymes that interact with nucleic acids, several inteins are located in metabolic enzymes, such as phosphoenolpyruvate synthase, anaerobic ribonucleoside triphosphate reductase, UDP-glucose dehydrogenase, ClpP protease/chaperone, vacuolar ATPase proton pump (VMA) and glutamine-fructose 6-phosphate transaminase (Table 1 ).

The inteins listed in Table 1 range in size from 335 to 548 amino acids, except for the Ppu dnaB intein (150 amino acids) and the Mxe gyrA intein (198 amino acids). The central domain present in other inteins is missing in the Mxe gyrA (GenBank accession no. U67876) and Ppu dnaB ( 18 ) inteins (Table 2 ). These small inteins may have lost those residues required for endonuclease activity and may thus represent minimal inteins. Alternatively, they may represent an intein remnant that is no longer capable of splicing.

We suggest that inteins present in the same position in an extein homolog from different organisms should be designated intein alleles . Psp pol intein-1 and Tli pol intein-1 alleles have the same endonuclease specificities (Perler,F.B., unpublished data). Pairwise amino acid sequence comparisons indicate that the 11 inteins present in identical locations in DNA polymerase or gyrA genes are more similar to their alleles than to any other intein (at least ~60% identity, except for the Mja pol-2 intein, which is only 40.4% identical to the Tli pol-1 intein). Identity among non-allelic inteins is quite low, generally ranging from 15 to 30%. The VMA inteins are 36.6% identical and branch together in phylogenetic trees (Fig. 1 ). The only intein alleles that fail to phylogenetically group together are the dnaB alleles (23% identical), possibly because 46 out of 95 residues used in this analysis are absent in the Ppu dnaB mini-intein. However, it is difficult to determine whether very dissimilar intein alleles arose from different ancestors or by divergence.


Figure 1 . Unrooted phylogenetic tree based on the conserved intein motifs. The 95 columns of aligned residues in Table 2 were subjected to phylogenetic analysis using a least squares distance method (see Analytical Methods). Branch lengths shown are proportional to the estimated number of amino acid replacements per sequence position; the scale bar corresponds to an average of one replacement per position. Except for the grouping of alleles and the grouping of Mja Hyp-2 with Mja RFC-3 and Mja TFIIB with Mja RNR-2, all branches appear in <50% of the bootstrap replicates. Abbreviations as in Table 1.

Conserved residues and the protein splicing mechanism

Protein splicing is so rapid that the precursor protein is rarely observed. The intein plus the first downstream extein residue contain sufficient information for splicing in foreign proteins ( 13 , 24 , 33 ). However, the exteins may affect splicing rates or efficiency. Using a chimeric intein construct, in vitro splicing of a purified precursor was demonstrated ( 33 ) and the chemical mechanism of protein splicing was determined ( 21 , 33 , 46 - 49 ). Protein splicing requires four nucleophilic attacks mediated by three of the four conserved splice junction residues: (i) a Ser, Thr or Cys at the intein N-terminus; (ii) an Asn at the intein C-terminus; (iii) a Ser, Thr or Cys at the downstream extein N-terminus. The intein penultimate His assists in the C-terminal cleavage reaction.

Although Ser, Thr and Cys are chemically similar, it was initially speculated that splicing of thermostable inteins could not involve Cys because of high growth temperatures ( 24 ). It is now clear that inteins from thermophiles can utilize Cys, since all archaeal inteins listed in Table 1 are from thermophiles. However, with the still small sample size currently available, Thr has yet to be observed at an intein N-terminus and Ser has yet to be observed at the N-terminus of an intein from a mesophile (Table 1 ).

The requirement of a conserved His at the C-terminal splice junction must now be modified in light of the Ceu clpP, Mja PEP Syn and Mja Rpol A' inteins that have Gly or Phe at this position (Table 1 ). However, splicing of these inteins has yet to be demonstrated in their native organisms, although splicing of the Ceu clpP intein in Escherichia coli requires changing the intein penultimate Gly to His (Liu,P.X.-Q., personal communication). Although Phe and Gly residues are unlikely to fulfil the role of assisting in C-terminal cleavage, since they cannot assist in acid/base catalysis, there is no a priori chemical requirement for this residue to be adjacent to the Asn in the primary amino acid sequence; the residue performing this function merely has to be near the Asn in three-dimensional space.

Conserved intein motifs

Twenty six new intein sequences have been determined since Pietrokovski first defined the seven conserved intein motifs termed Blocks A-G ( 9 ). The majority of the inteins included in the present analysis are found in archaea. Whether or not this biases the motifs will have to await the discovery of new eubacterial and eucaryal inteins. Seven highly similar allelic inteins were not included in the initial motif analysis using MACAW, but are listed in lower case in Table 2 . The intein blocks depicted in Table 2 yielded the maximum score obtainable using the MACAW program. However, all of these motifs could be expanded (except for limitations due to adjacent motifs or the sequence boundaries) and still yield highly significant scores. The size of some of the previously described motifs ( 9 ) has been modified in our analysis. For example, Block A has been reduced to 13 amino acids, although there is a less conserved, but still highly significant, block extending to residue 23.

Most positions in the intein motifs contain functionally or structurally similar amino acids, rather than a single predominant residue. In fact, only one His in Block B, two Gly in Block C (excluding inteins lacking this block) and one Asn in Block G are present in all inteins (marked by an asterisk under the consensus residue in Table 2 ). The consensus line in Table 2 lists amino acid groups (acidic, basic, aromatic, hydrophobic and polar) and conserved residues that are present in at least 15 of the 29 inteins used in the MACAW analysis. Note that many of these conserved residues can participate or assist in nucleophilic catalysis and the conserved Pro and Gly residues can affect secondary structure, being potential helix breaking residues. All blocks contain several hydrophobic residues.

Block A begins at the N-terminus of the intein and contains the chemically essential Ser or Cys residue. The sequence following the autocleavage site in hedgehog proteins fits the Block A consensus ( 50 ). Block B contains a polar residue (most often Thr) three amino acids prior to the only His conserved in all inteins. A similar motif is present in serine proteases and hedgehog proteins ( 51 ). The mechanism of cleavage in hedgehog proteins and at the intein N-terminus is similar ( 3 , 46 , 48 , 52 ). Thus, it is reasonable to suspect, and has been previously suggested ( 9 ), that the His in Block B may be involved in N-terminal splice junction cleavage. Block D is characterized by a conserved basic amino acid (most often Lys) and a Pro residue.

A 19 amino acid motif, called Block H, was found between blocks E and F. It overlaps with a previously identified, but unpublished, motif reported in the PRINTS database (http://www.biochem.ucl.ac.uk/bsm/dbbrowser/PRINTS/PRINTS.html) ( 53 ). Block H is characterized by one or more Ser or Thr residues in positions 1-3, a central hydrophobic region containing several Leu and a Gly at position 18 followed by a hydrophobic residue. Block F contains an aromatic residue on both sides of several acidic and hydrophobic residues. If gaps were introduced into Block F, the presence of the extended consensus sequence, rVYDLpVa(1-3 residues)(H or E)NFh (see Table 2 legend for abbreviations) would be clearer. Block G is characterized by the three conserved C-terminal splice junction residues preceded by four hydrophobic residues and contains the first extein residue following the intein.

Blocks C and E are the dodecapeptide motifs that are required for endonuclease activity ( 8 , 54 , 55 ). Note that eight inteins have not maintained both blocks or the conserved acidic residues in these blocks which have been implicated in endonuclease activity ( 8 , 54 , 55 ), suggesting that these inteins may no longer be active endonucleases. A different Mle recA intein Block E sequence was assigned by the MACAW program in the present analysis. The previously published sequence of Block E was VLAIWYMDDG ( 9 , 23 ). Although this new motif assignment does not maintain the ~100 residue distance between Blocks C and E present in other inteins, it provides an equally good match to consensus dodecapeptide motifs ( 8 ). The absence of Block D could account for the reduced distance between blocks C and E in this intein.

The S.cerevisiae HO endonuclease contains all of the intein motifs except the conserved splice junction residues (Table 2 ; 9 ). HO endonuclease, which is essential for mating type switching, is also a member of the dodecapeptide endonuclease family. Despite the presence of these conserved motifs and after addition of the conserved splice junction residues from the Psp pol-1 intein or the Sce VMA intein, HO does not splice at the protein level when placed in-frame between the E.coli maltose binding protein and a fragment of Dirofilaria immitis paramyosin (Platko,J. and Perler,F.B., unpublished data) .

Phylogeny of intein sequences

Pairwise comparison of most inteins indicated a low degree of sequence similarity. Multiple sequence analysis identified motifs composed of groups of conserved residues, but not highly conserved specific amino acids. These factors made it difficult to determine the relationships among inteins present in the same or related organisms, in different domains of life or in different extein homologs. Therefore, the phylogenetic relationships of the 36 inteins were determined using programs in the PHYLIP package ( 29 ). This analysis revealed that, except for the intein alleles, there is no clustering of inteins on the basis of phylogenetic domain, organism classification, genus, species or location in the extein gene (Fig. 1 ). It further suggests that the 18 M.jannaschii inteins did not arise from recent intein duplications. Among non-allelic inteins, the only branches which appear in >50% of the bootstrap replicates are those associating Mja Hyp-2 with Mja RFC-3 (which was seen 83% of the time) and Mja TFIIB with Mja RNR-2 (54%). However, the observed relationships are not chaotic. Except for the dnaB inteins, all sets of allelic inteins grouped together in 99-100% of the 100 bootstrap samples.

Allelic inteins are more closely related than non-allelic inteins. Is this due to recent intein mobility events or to the acquisition of an intein by a common ancestor? Since intein alleles are not present in all closely related isolates or organisms, there must be a mechanism for intein gain or loss. For example, inteins are absent in DNA polymerases from 11 of 17 Archaea analyzed, with only six alleles of Tli pol-1, one allele of Tli pol-2 and two alleles of Psp pol-2 ( 17 , 35 ). Depending on the Mycobacterium species, not all isolates contain the recA or gyrA inteins ( 14 , 15 ) and of six Archaea examined, only M.jannaschii contains an RNA polymerase intein ( 17 , 41 - 45 ).

Gain of inteins is supported by several lines of evidence. Intein mobility has been demonstrated in yeast ( 6 ). Intein gene mobility is initiated when an inteinless allele enters the cell via sexual reproduction, conjugation, transduction, phage infection, plasmid transfer, etc. The inteinless allele is then cleaved by the intein endonuclease (homing endonucleases do not cut their own genomic DNA when the intein is present) ( 6 - 8 , 12 , 32 ). This endonuclease activity, combined with extein homology, substantially increases the rate of gene conversion by the double-strand break repair recombination pathway ( 6 , 11 , 12 , 38 , 56 , 57 ). As predicted, allelic inteins Tli pol-1 and Psp pol-1 are isoschizomers with the same endonuclease specificity (Perler,F.B., unpublished data). A second line of evidence for lateral transmission of inteins is the observation that codon usage in the gyrA inteins is different from extein codon usage, suggesting that the inteins have been recently acquired from a different species ( 15 ). Finally, the DNA polymerases from GB-D and GI-J Thermococcales isolates (98% identical over the 96 amino acid GI-J fragment sequenced) are more similar than the GB-D and T.litoralis DNA polymerases (78% identical), although there is no intein in the GI-J DNA polymerase while there are allelic inteins in the GB-D and T.litoralis DNA polymerases (60% identity between inteins) ( 35 ).

If intein alleles are ancient and can be lost with time, the mechanism for intein loss has to be very specific to avoid inactivating mutations in the extein gene. Recombination could lead to intein loss if the intein was no longer an active homing endonuclease, however, if the intein was an active homing endonuclease, lateral transmission should predominate. Recombination in haploid organisms such as Mycobacterium spp., M.jannaschii and Thermococcales can only occur if merodiploids are occasionally formed. Yet the presence of inteins in haploid individuals is very variable. Barring an unknown efficient mechanism for intein loss other than by rare recombination events, the prevalence of intein loss would require selection against inteins.

Taken together, these data suggest that the presence of intein alleles is most often due to lateral transmission rather than the early acquisition of an intein by a common ancestor. On the other hand, there is no phylogenetic evidence that non-allelic inteins have spread by lateral transmission, although it is possible that they arose by an illegitimate lateral transmission event within the same genome followed by significant divergence.

Identifying inteins

How one identifies new inteins depends on whether you are analyzing the sequence of a specific gene or searching databases for new inteins. A large in-frame insertion in a sequenced gene that is absent in other sequenced homologs suggests that this gene may contain an intein. The sequence should then be examined for the presence of the conserved intein junction residues and the intein blocks, including the dodecapeptide motifs. Not all inteins will have a His as the penultimate residue. However, since most inteins end in His-Asn, the His-Asn-(Ser, Thr, Cys) C-terminal intein motif is still a valid tool for identifying intein boundaries. Not all intein blocks need be present (Table 2 ). Since several amino acids are found within each position in a block, the putative intein sequence should be checked for the presence of a member of the amino acid group present at that position (Table 2 ).

In examining databases, inteins can be identified by searching with the conserved intein blocks ( 9 , 18 ) or complete intein amino acid sequences. Once a match has been found, the entire sequence should be re-analyzed for the presence of other conserved intein motifs and database searches should be performed to find matches to the putative extein sequences. The presence of the conserved splice junction residues and the conserved blocks are not sufficient to label a sequence an intein in the absence of comparison with an inteinless extein homolog, although the presence of all the blocks would be highly indicative of the presence of an intein in an extein gene that does not have a sequenced homolog. In the absence of experimentally demonstrating protein splicing, it should be emphasized that the combined use of these criteria, rather than the use of any single criterion, yields the most significant results.

ACKNOWLEDGEMENTS

We thank Shmuel Pietrokovski, Stewart Cole, Paul X.-Q.Liu, Amalio Telenti and Mike Reith for helpful discussions and providing us with their unpublished results and Sanjay Kumar, Bill Jack, Chris Noren, Maurice Southworth and Ming Xu for helpful discussions. We thank Shmuel Pietrokovski for sharing information concerning new inteins. We thank Donald G.Comb for support and encouragement.

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