Full text of DOI:10.1007/BF03187217

NOTES
ing inteins. Then look for the homologous proteins that
maybe contain intein by searching NR database with pro-
gram TBLASTN for the homology of these proteins (the
homologous protein without intein lacks a segment of
peptide in the position of insertion of intein) and further
identify inteins based on the four criteria described in ref.
[1]. Then multiple sequence alignment was performed
using CLUSTAL[10] to find out the sequence characteristic
of these inteins. Then, search NR database for these
charateristic sequences with program TBLASTN; search
for the homology of the sequence with the low value of E
(expect value). Repeat the above steps until no inteins
found any more.
Since the lengths of different inteins vary widely, the
parameters of multiple-sequence alignment were set as
follows: gap opening (penalty): 10; gap extention (pen-
alty): 0.05; delay divergent sequences: 20%; protein
weight matrix: gonnet series. Protein gap parameters were
set as default. First, we aligned all sequences for several
times (without reseting all gaps ) and put the low-scoring
sequences into profile 2 and realigned the sequences in
profile 1 for several times, then aligned profile 2 to profile
1. The poorly aligned columns were selected and re-
aligned. The above steps were repeated until the quality
curve line stops improving.
Analysis of the characteristic
sequence of intein and revi-
sion of its motifs
XIE Jun, HUANG Jingfei, SHI Xiufan & LIU Ciquan
Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming
650223, China
Correspondence should be addressed to Liu Ciquan (e-mail: Xiesangjun
@263.net)
Abstract
Since the first intein (Sce VMA) was found in
Saccharomydes cerevisiae ATPases gene in 1990, more and
more inteins were identified. It is necessary to analyze the
new inteins to understand the sequence charateristics of
inteins. By searching protein and nucleic acid database sys-
tematically, 101 inteins were found, of which 69 inteins con-
tain homing endonuclease motifs. We only analyze the 69
inteins since most inteins are the classic inteins with homing
endonuclease motifs. We found that the distribution of these
inteins is particular among species and protein. By multiple
sequence alignment, some new sequence characteristics were
found and the motifs described previously were revised.
Keywords: intein distribution, location of intein, sequence charater-
istics.
2
Result and conclusion
Inteins are defined as internal protein segments and
removed from the proteins precursors by protein splicing
with the external protein segments (exteins) ligated to
form a native peptide bond[1]. Most inteins contain LAG-
LI-DADG motifs that define a family of homing endonu-
cleases[2]. The activity that the endonucleases provide is a
DNA site-specific endonuclease capable of recognizing
and cleaving the inteinless or intronless allele genes and
initiating intein or intron gene transposition. By this
method inteins spread in the population.
Since the first intein was found in Saccharomydes
cerevisiae ATPases gene[3,4], more and more inteins were
found. However, most inteins were predicted from DNA
sequences. By searching NR database (integrating the
databases of GenBank[5], DDBJ[6], PDB[7] and EMBL[8])
with the program of TBLASTN (comparing a protein
query sequence against a nucleotide sequence database
dynamically translated in all reading frames) in the server
of BLAST for inteins, 69 inteins with homing endonucle-
ase motifs were found. By analyzing them, we found that
the distribution and location of inteins show features, and
moreover, some new sequence charateristics were found
and motifs described previously were revised. So this
work may be useful for understanding the characteristics
of intein’s sequences, structure and function.
(ν) Distribution and sequence characteristic of both
terminals of inteins. By the methods described above,
101 inteins were found and 69 of them contained
LAGLI-DADG motifs (due to limited space, these inteins
are not listed here, they are available on request, xiesang-
jun@263.net). Since most inteins contain the motifs of
homing endonuclease which is necessary for transposition
of inteins, only the 69 inteins were chosen for further
analysis. We used protein sequences to search for the nu-
cleic acid sequences, and the kinds of the exteins protein
encoded by which were got from homologous proteins.
For the nomenclature of intein refer to ref. [1] and alleles
refer to intein database (http://www.neb.com).
The 69 inteins were distributed in archaea, eubac-
terin and eucaryote (both in nucleolus or organelle),
showing the universality of distribution of inteins. How-
ever, homology comparison found that in eucaryote, the
inteins were distributed only in unicellular organism and
virus of eucaryote, and the inteins in multicellular organ-
ism were not reported. Of the 69 inteins, 18 are in
methanococcus jannaschii, 9 in Pyrococcus horikoshii
OT3 (archaea), 10 in Pyrococcus abyssi. Of course, it is
partly due to the case that the complete genome sequences
of these species are known to date. However, there is only
one intein in Synechocystis spp. and S. Cerevisiae, whose
complete genome sequences are also known. Moreover, in
the other 28 species and the 7 chromosomes with com-
plete genome sequence known (http://igweb.integtated
1
Method
Firstly, search SWISSPROT[9] database with the
keyword “intein” and find 30 protein sequences contain-
758
Chinese Science Bulletin Vol. 46 No. 9 May 2001

genomics.com/GOLD/completegenomes.html), no intein
was found. The NR database, up to the date of May 26,
2000 which we searched, contains 6351829 nucleic acids
sequences (eucaryote: 6263389; eubacterin: 82884; ar-
chaea: 4556), the intein sequences with LAGLI-DADG
were only found in 24 species, and moreover, they are not
equably distributed among the 24 species. This suggested,
to a certain extent, that the distribution of inteins is dif-
ferent among species.
conservation is just lower than the His and Asn residues of
Block G. We think that the residue should belong to Block
A.
Block G is located at C-terminus of inteins and
characterized by the terminal residues of Asn and the pe-
nultimate residue His. They are important in the third step
of self-splicing of intein: Asn functions as nucleophilic
attacks. The penultimate His assists in this step by pro-
ü
viding proton[11,14 17]. The ultimate residue of Block G is
More than 50% of the 69 inteins are located in en-
zymes or protein factors related to DNA replication, repair,
transcription or translation, although some inteins are dis-
tributed in metabolic enzymes, such as Glutamine-fruct-
ose-6-phosphate transaminase, Vacuolar ATPase subunit
A, which suggests that the distribution of inteins varies
among different proteins. ꢀ
the first residue of downstream extein. It is necessary for
the intein’s self-splicing: in the second step of nucleo-
philic attacks, this residue provides hydroxy or sulfhydryl
group and links upstream extein by acyl rearrange-
ü
ment[11 16]. Table 1 shows that the residue is also conser-
vative and only Thr, Ser, Cys have been reported in this
position up to date. Most of the two middle residues are
Gln and Gly. On each side of the two residues are three
hydrophobic residues. The first residue of Block G is His
residue. There are only 8 residues[1] in Block G described
previously. Compared with other blocks, the 5 residues are
also conservative in the 69 inteins.
Compared with intein database, Block I is new and
located between Blocks A and B. Its obvious character is
the fifth residue, of which 82% are Asp and Glu. Both of
the two residues are acidic, and the first and second nu-
cleotide acids of the two residues codons are G and A,
respectively. They are near to the reaction point in three-
dimensional structure. But their functions in the self-
splicing reaction are still unknown. In the middle of Block
I, there are two conservative residues (the second residue
Lys and the third residue Ile), on their sides are several
hydrophobic residues.
ꢀ
Of the 69 inteins, 50 are found in archaea, 15 in
eubacterin, 4 in eucaryote or their viruses. It shows obvi-
ously that most inteins prefer to be located in archaea,
fewer inteins are in eubacterin, which is contrary to the
distribution of introns. However, the spread approaches of
intein and intron are similar. They both transpose by
homing endonuclease cutting the inteinless alleles, and
what is more, their homing endonucleases are similar in
structure, function and action[11]
.
There is no obvious sequence characteristics for both
terminal sequences of the position of intein insertion ex-
cept that the first residue of the back terminus is always
Cys, Thr or Ser, which is consistent with the theory that
the information of self-splicing of intein included in
inteins themselves and the first residues of the down-
stream exteins.
(ξ) Intein motifs. Multiple sequence alignment of
the 69 inteins showed that the motifs described previously
need revision. Compared with intein motifs listed in
http://www.neb.com, there is the following changes in the
motifs of table 1: Block A is defined by adding two amino
acids at the terminal. The number of residues of Block G
increases to 13 residues from 8 residues previously de-
scribed. Block I is a new one.
For the features of other blocks refer to ref. [1]. In
addition, according to the result of our search and analysis,
Block B of Pfu RIR1-2 should be EEKVEYETVHGRVK
(while the previous is EEKVEYETVHGKVL), Block H
of PsP KOD Pol-2 should be TKSELLANQLVLLLNS
VGV (the previous is NEKRALANQLVLLLNSVGV).
For the above analysis suggests that there are no
absolutely conserved residues except a Gly in Block A,
His in block B, His, Asn, Ser in Block G. However, most
positions in intein motifs contain functionally or
structurally similar amino acids which can participate or
assist in nucleophilic catalysis.
In conclusion, this note includes the following three
aspects: (ν) 69 inteins were got by searching NR data-
base; (ξ) the characteristics of distribution and the loca-
tion of inteins were shown by analyzing our results; (ο)
by multiple sequence aligning of the 69 inteins, motifs I
were found, which were not included in intein database
and the motifs described previously were revised.
Block A is located at N-terminus of inteins and
characterized by the first residues Cys, Thr or Ser, which
ü
are essential for the first step of splicing[11 16]. The termi-
nal or subterminal residue Gly is located in the loop of
two Eꢀstrands in the three-dimensional structure. There is
no report about the function of this residue. Since the
transpose of the upstream extein from N-terminus to
C-terminus requires some flexibility in the three-dimen-
sional structure of intein N-terminus, we predict that the
Gly provides the flexibility. If Block A is defined by only
13 residues, the conservation residue Gly in the 14th of
many intein’s Block A will be ignored. Considering that
90% of inteins contain the residue in this position whose
Acknowledgements This work was supported by the National Natural
Science Foundation of China (Grant Nos. 39770418 and 39570395).
Chinese Science Bulletin Vol. 46 No. 9 May 2001
759

NOTES
Table 1 Conserved motifs found in inteins
1Rꢁꢀ1DPHDꢂ
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a) For the intein names refer to ref. [1]; b) the position in the protein of the last amino acid in each block is listed on the right of the block; c)
definition of symbols in the consensus line˖capital letters indicate conserved amino acids (standard single letter code); p, polar residue (S,T or C);
h, hydrophobic residue (G, A, V, L, I or M); a, acidic residue (D or E); b, basic residue (H, K or R); r, aromatic residue (F,Y or W).
760
Chinese Science Bulletin Vol. 46 No. 9 May 2001

10. Thompson, J. D., Gibson, T. J., Plewniak, F. et al., The ClustalX
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(Received August 18, 2000)
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