AUTHORS: I.A.Shahmuradov & V.V.Solovyev
LAST UPDATE: 15 Novembef 2013
VERSION: 5.2013
ACCESS: http://softberry.com
ACTION: Search for statistically nonrandom motifs of various (up to 30) known human/animal and plant transcription Regulatory Elements (REs) which is available simultaneously and, at least, in ONE copy in P% or more of analyzing DNA sequences (parameter "P" is given by USER (default = 50%).
SEARCH CONDITION:
(1) Expected Mean Numbers of any regulatory motif found must be less than a given number (default: 0.05).
(2) regulatory motif(s) are available simultaneously and, at least, in ONE copy in P% or more of analyzing DNA sequences.
As NsiteH and NsiteM programs, Nsite is based on the previously proposed real site and/or their IUPAC consensus based probabilistic approach of searching for putative REs in nucleotide sequences and statistically estimating motifs found (Shahmuradov et al., Genetika (Russ.), 1986, 22, 357-367; Solovyev,V.V. and Kolchanov,N.A. In: "Computer analysis of genetic macromolecules. Structure, Function and Evolution", World Scientific, 1993,16-20).
The main features of the approach are the follows:
(i) RE may consist of a single box (a continuous DNA segment) or two boxes,
spaced by some DNA sequence, where only length, but not nucleotide content, of this spacer is important
for functioning of such a composite site.
(ii) A real RE or its IUPAC consensus contains both variable positions
(where the presence of a certain group of nucleotides is permissible), and strictly conserved positions
(where a strong identity between real site/consensus and predicted motif is required).
(iii) The nonequivalence of these positions should be taken into
account, i.e., complete homology at conserved positions is needed, and a violation of homology
in the variable positions should be permissible.
(iv) The homology between the RE and motif on query DNA sequence may a
chance happening, therefore, estimation of its statistical significance is of major importance.
A conclusion on the functional significance of the revealed homology can be reached, only if the
homology is significantly nonrandom, i.e., the homology is not a chance event.
(v) Characteristics such as nucleotide frequencies should not be used
when describing the consensus because of its small size. Instead, one should use estimates based on
the number of nucleotides of various types in the consensus.
(vi) Although all available databases on REs usually annotate a fixed
distance between two boxes of composite elements, some variability of the spacer length, seems, to
have a place. Therefore, a search algorithm for composite REs should allow some limited flexibility
in the sense of spacer length, relaying on both the known experimental data and theoretical assumption.
One of the important components of this approach is preparation of RE data set. Nsite uses the following 3 sets of human/animal and plant REs.
[1]: Set of human/animal REs prepared by merging and verification (excluding longer than 50 bp REs; elimination of redundancies) RE/TF information from ooTFD (Ghosh,D. Nucleic Acids Res, 2000, 28, 308- 310). It includes 8030 real/consensus REs.
[2]: Set of human/animal REs prepared by merging and verification RE/TF information from RegsiteAN DB developed Softberry Inc. It includes 1500 real/consensus REs.
[3]: Set of plant REs prepared by merging and verification RE/TF information from RegsitePL DB developed by Softberry Inc. The current version of this set includes 2359 real/consensus REs, but it is regularly updated.
Moreover, user can perform a search for motifs of REs from his own dataset in a format described below.
Input query sequences of length 100000 nucleotides or less must be given in FASTA format.
1. USER's set must include only sequences of real REs and/or their consensuses.
2. Every real RE/consensus is described in 3 lines:
LINE 1: Name/description of RE/consensus
LINE 2: Sequence of of RE/consensus
LINE 3: <par1> <par2> <par3> <par4>
3. Sequence (LINE2) may include both standard nucleotides (A/a, T/t, G/g,C/c)
and their any combinations in according to IUPAC abbreviations:R - A or G,
Y - T or C, K - G or T, M - A or C, S - G or C, W - A or T, B - G or T or C,
D - A or G or T, H - A or C or T, V - A or G or C, N - A or G or C or T.
In the case of composite REs, 2 boxes are seperated by "-".
Length of RE/consensus sequence must not exceed 80 symbols, including "-" in the case of composite elements.
Capital letters indicate Conservative nucleotides (positions) in which mismatch is not allowd.
4. In LINE 3:
<par1> - a maximal number of mismatches for the 1st box
<par2> - a maximal number of mismatches for the 2nd box (for composite REs)
If RE contains a single box, then <par2> = 0;
If any mismatch is not allowed, then <par1> = <par2> = 0 )
<par3> - minimal distance between boxes of composite RE
<par4> - maximal distance between boxes of composite RE (for a single-box REs <par3> = <par4> = 0 )
All <par1>, <par2>, <par3> and <par3> are given as INTEGER in 4i5 format.
RE 1
agTGGcgAggcg
2 0 0 0
RE2
caggccTGc-CCAGctgg
1 1 8 10
RE 3
RRTGTGGWWW
0 0 0 0
Every OUTPUT file begins with description of the Program's allocation, Search Parameters, as well as used abbreviations (in the case of using Data sets created by us). The next 2 includes name and length of the first query sequence. At last, name of REs, statistical estimation and sequences of motifs found are given.
For example:
Program NsiteM | Version 5.2013
Search for motifs of Regulatory Elements (REs) available in a given (by USER) portion of
2356 QUERY Sequences | SET of REs: RegsitePDB: 2359 Plant Transcription REs [Last update: 05.02.2012; Softberry Inc
____________________________________________________________
Search PARAMETRS:
Expected Mean Number : 0.0500000
Statistical Siginicance Level : 0.9500000
Level of homology between known RE and motif: 90%
Variation of Distance between RE Blocks : 20%
NOTE: RE - Regulatory Element/Consensus | AC - Accession No of RE in a given DB
OS - Organism/Species | BF - Binding Factor or One of them
Mism. - Mismatches | Mean. Exp. Number - Mean Expected Number | Up.Conf.Int. - Upper Confidence Interval
============================================================
Graphic View of RE motifs found in 80% or more of analyzed sequences
MOTIFS
12345678
_______________
SEQs
1| .++..+.+
2| ++.+++++
3| ++.+++++
4| .++..+.+
5| ++++++++
6| ++++++++
7| ++++++++
8| +++++.++
9| +++++.++
10| ++++++++
Motifs of RE No 1 | NAME: 175. AC: RSP00175//OS: arabidopsis (Arabidopsis thaliana) /GENE: synthetic oli | SEQ: cacgtggc
Found in the following 8 ( 80.0%) out of 10 analyzed sequences:
2 3 5 6 7 8 9 10
Motifs of RE No 2 | NAME: 204. AC: RSP00204//OS: arabidopsis (Arabidopsis thaliana) /GENE: AtEm6/RE: ABR | SEQ: gacACGTggc
Found in the following 10 ( 100.0%) out of 10 analyzed sequences:
1 2 3 4 5 6 7 8 9 10
Motifs of RE No 3 | NAME: 524. AC: RSP00524//OS: carrot (Daucus carota) /GENE: Dc3/RE: E4-core /BF: DPBF | SEQ: ACACgtG
Found in the following 8 ( 80.0%) out of 10 analyzed sequences:
1 4 5 6 7 8 9 10
Motifs of RE No 4 | NAME: 723. AC: RSP00723//OS: tobacco (Nicotiana plumbaginifolia) /GENE: rbcS 8B/RE: | SEQ: cacgtggc
Found in the following 8 ( 80.0%) out of 10 analyzed sequences:
2 3 5 6 7 8 9 10
Motifs of RE No 5 | NAME: 1041. AC: RSP01034//OS: arabidopsis (Arabidopsis thaliana) /GENE: RD29B/RE: ABR | SEQ: ACGTGgC
Found in the following 8 ( 80.0%) out of 10 analyzed sequences:
2 3 5 6 7 8 9 10
Motifs of RE No 6 | NAME: 1159. AC: RSP01151//OS: maize (Zea mays) /GENE: Em/RE: Em1a /BF: EmBP-1 (+VP1) | SEQ: GacACGTggc
Found in the following 8 ( 80.0%) out of 10 analyzed sequences:
1 2 3 4 5 6 7 10
Motifs of RE No 7 | NAME: 1602. AC: RSP01570//OS: rice (Oryza sativa, indica) /GENE: Synthetic oligonucle | SEQ: cACGTGGC
Found in the following 8 ( 80.0%) out of 10 analyzed sequences:
2 3 5 6 7 8 9 10
Motifs of RE No 8 | NAME: 1850. AC: RSP01816//OS: tobacco (Nicotiana tabacum) /GENE: PNZIP/RE: G-box /BF: | SEQ: gcCACGTGtc
Found in the following 10 ( 100.0%) out of 10 analyzed sequences:
1 2 3 4 5 6 7 8 9 10
============================================================
LIST of sequences presented in the graphic view of results
1. EXAMPLE Group (10 genes) | Arabidopsis, Chr 1: GENE: F21B7.21 [At1g03600] |898875..899654 |SUPPORT | 1 exon(s)
|PROD: photosystem II protein family |5"-UTR: 40 | -500: +1 | +1 ... CDS start
2. EXAMPLE Group (10 genes) | Arabidopsis, Chr 1: GENE: F4H5.23 [At1g06680] |2047878..2049417 |SUPPORT | 4 exon(s)
|PROD: photosystem II oxygen-evolving complex 23 (OEC23) |5"-UTR: 61 | -500: +1 |
3. EXAMPLE Group (10 genes) | Arabidopsis, Chr 1: GENE: F9I5.11 [At1g52230] |19520367..19521199 |SUPPORT | 3 exon(s)
|PROD: photosystem I subunit VI precursor |5"-UTR: 66 | -500: +1 | +1 ... CDS st
4. EXAMPLE Group (10 genes) | Arabidopsis, Chr 2: GENE: T6B20.8 [At2g30570] |complement(13025520..13027271) |SUPPORT | 2 exon(s)
|PROD: photosystem II reaction center 6.1KD protein |5"-UTR: 141 | -50
5. EXAMPLE Group (10 genes) | Arabidopsis, Chr 3: GENE: MSL1.18 [At3g16140] |complement(5468517..5469482) |SUPPORT | 3 exon(s)
|PROD: photosystem I subunit VI precursor |5"-UTR: 61 | -500: +1 | +1
6. EXAMPLE Group (10 genes) | Arabidopsis, Chr 3: GENE: MSA6.6 [At3g21055] |complement(7376643..7377192) |SUPPORT | 1 exon(s)
|PROD: photosystem II 5 kD protein precursor |5"-UTR: 114 | -500: +1 | +
7. EXAMPLE Group (10 genes) | Arabidopsis, Chr 3: GENE: F18B3.100 [At3g50820] |complement(18901857..18903407) |SUPPORT | 3 exon(s)
|PROD: photosystem II oxygen-evolving complex 33 (OEC33) |5"-UTR: 11
8. EXAMPLE Group (10 genes) | Arabidopsis, Chr 4: GENE: T5F17.110 [At4g28660] |14149922..14151103 |SUPPORT | 2 exon(s)
|PROD: photosystem II protein W - like |5"-UTR: 92 | -500: +1 | +1 ... CDS sta
9. EXAMPLE Group (10 genes) | Arabidopsis, Chr 4: GENE: F16A16.140 [At4g28750] |complement(14202779..14203961) |SUPPORT | 3 exon(s)
|PROD: photosystem I subunit PSI-E - like protein |5"-UTR: 67 | -5
10. EXAMPLE Group (10 genes) | Arabidopsis, Chr 5: GENE: K1F13.25 [At5g66570] |26585884..26587504 |SUPPORT | 3 exon(s)
|PROD: photosystem II oxygen-evolving complex 33 (OEC33) |5"-UTR: 86 | -500: +1
============================================================
Motifs of REs found in 80% or more of analyzed sequences
............................................................
1. QUERY: > EXAMPLE Group (10 genes) | Arabidopsis, Chr 1: GENE: F21B7.21 [At1g03600] |898875..899654 |SUPPORT | 1 exon(s)
|PROD: photosystem II protein family |5"-UTR: 40 | -500: +1 | +1 ... CDS start
Length of Query Sequence: 500 bp | Nucleotide Frequencies: A - 0.37 G - 0.15 T - 0.27 C - 0.21
............................................................
RE: 204. AC: RSP00204//OS: arabidopsis (Arabidopsis thaliana) /GENE: AtEm6/RE: ABRE/6.2 /BF: ABI5
Found in 10 (100.00 %) SEQs (out of 0; 0.00% SEQs with motifs of this Group)
Motifs on "+" Strand: Mean Exp. Number 0.00232 Found 1
393 GACACGTGtC 402 (Mism.= 1)
Motifs on "-" Strand: Mean Exp. Number 0.00223 Found 1
402 GACACGTGtC 393 (Mism.= 1)
............................................................
RE: 524. AC: RSP00524//OS: carrot (Daucus carota) /GENE: Dc3/RE: E4-core /BF: DPBF-1; DPBF-2;
Found in 8 ( 80.00 %) SEQs (out of 0; 0.00% SEQs with motifs of this Group)
Motifs on "+" Strand: Mean Exp. Number 0.01810 Found 1
394 ACACGTG 400 (Mism.= 0)
Motifs on "-" Strand: Mean Exp. Number 0.01313 Found 2
401 ACACGTG 395 (Mism.= 0)
155 ACACGTG 149 (Mism.= 0)
............................................................
RE: 1159. AC: RSP01151//OS: maize (Zea mays) /GENE: Em/RE: Em1a /BF: EmBP-1 (+VP1)
Found in 8 ( 80.00 %) SEQs (out of 0; 0.00% SEQs with motifs of this Group)
Motifs on "+" Strand: Mean Exp. Number 0.00185 Found 1
393 GACACGTGtC 402 (Mism.= 1)
Motifs on "-" Strand: Mean Exp. Number 0.00190 Found 1
402 GACACGTGTC 393 (Mism.= 1)
............................................................
RE: 1850. AC: RSP01816//OS: tobacco (Nicotiana tabacum) /GENE: PNZIP/RE: G-box /BF: NtbZIP
Found in 10 (100.00 %) SEQs (out of 0; 0.00% SEQs with motifs of this Group)
Motifs on "+" Strand: Mean Exp. Number 0.00143 Found 1
393 GaCACGTGTC 402 (Mism.= 1)
Motifs on "-" Strand: Mean Exp. Number 0.00152 Found 1
402 GaCACGTGTC 393 (Mism.= 1)
............................................................
Totally 9 motifs of 4 different REs have been found
------------------------------------------------------------
2. QUERY: > EXAMPLE Group (10 genes) | Arabidopsis, Chr 1: GENE: F4H5.23 [At1g06680] |2047878..2049417 |SUPPORT | 4 exon(s)
|PROD: photosystem II oxygen-evolving complex 23 (OEC23) |5"-UTR: 61 | -500: +1 |
Length of Query Sequence: 500 bp | Nucleotide Frequencies: A - 0.37 G - 0.17 T - 0.26 C - 0.19
............................................................
RE: 175. AC: RSP00175//OS: arabidopsis (Arabidopsis thaliana) /GENE: synthetic oligonucleotides/RE: ABRE /BF: ABFs
Found in 8 ( 80.00 %) SEQs (out of 0; 0.00% SEQs with motifs of this Group)
Motifs on "+" Strand: Mean Exp. Number 0.00175 Found 1
301 CACGTGGC 308 (Mism.= 0)
Motifs on "-" Strand: Mean Exp. Number 0.00175 Found 1
306 CACGTGGC 299 (Mism.= 0)
............................................................
RE: 204. AC: RSP00204//OS: arabidopsis (Arabidopsis thaliana) /GENE: AtEm6/RE: ABRE/6.2 /BF: ABI5
Found in 10 (100.00 %) SEQs (out of 0; 0.00% SEQs with motifs of this Group)
Motifs on "+" Strand: Mean Exp. Number 0.00288 Found 1
299 GcCACGTGGC 308 (Mism.= 1)
Motifs on "-" Strand: Mean Exp. Number 0.00226 Found 1
308 GcCACGTGGC 299 (Mism.= 1)
............................................................
RE: 723. AC: RSP00723//OS: tobacco (Nicotiana plumbaginifolia) /GENE: rbcS 8B/RE: G-box /BF: HY5
Found in 8 ( 80.00 %) SEQs (out of 0; 0.00% SEQs with motifs of this Group)
Motifs on "+" Strand: Mean Exp. Number 0.00175 Found 1
301 CACGTGGC 308 (Mism.= 0)
Motifs on "-" Strand: Mean Exp. Number 0.00175 Found 1
306 CACGTGGC 299 (Mism.= 0)
............................................................
RE: 1041. AC: RSP01034//OS: arabidopsis (Arabidopsis thaliana) /GENE: RD29B/RE: ABRE 1/2 /BF: ABI3; ABI5; AREB1
Found in 8 ( 80.00 %) SEQs (out of 0; 0.00% SEQs with motifs of this Group)
Motifs on "+" Strand: Mean Exp. Number 0.00921 Found 1
302 ACGTGGC 308 (Mism.= 0)
Motifs on "-" Strand: Mean Exp. Number 0.01005 Found 1
305 ACGTGGC 299 (Mism.= 0)
............................................................
RE: 1159. AC: RSP01151//OS: maize (Zea mays) /GENE: Em/RE: Em1a /BF: EmBP-1 (+VP1)
Found in 8 ( 80.00 %) SEQs (out of 0; 0.00% SEQs with motifs of this Group)
Motifs on "+" Strand: Mean Exp. Number 0.00234 Found 1
299 GCCACGTGGC 308 (Mism.= 1)
Motifs on "-" Strand: Mean Exp. Number 0.00189 Found 1
308 GCCACGTGGC 299 (Mism.= 1)
............................................................
RE: 1602. AC: RSP01570//OS: rice (Oryza sativa, indica) /GENE: Synthetic oligonucleotides/RE: Em1a /BF: OSBZ
Found in 8 ( 80.00 %) SEQs (out of 0; 0.00% SEQs with motifs of this Group)
Motifs on "+" Strand: Mean Exp. Number 0.00175 Found 1
301 CACGTGGC 308 (Mism.= 0)
Motifs on "-" Strand: Mean Exp. Number 0.00175 Found 1
306 CACGTGGC 299 (Mism.= 0)
............................................................
RE: 1850. AC: RSP01816//OS: tobacco (Nicotiana tabacum) /GENE: PNZIP/RE: G-box /BF: NtbZIP
Found in 10 (100.00 %) SEQs (out of 0; 0.00% SEQs with motifs of this Group)
Motifs on "+" Strand: Mean Exp. Number 0.00148 Found 1
299 GCCACGTGgC 308 (Mism.= 1)
Motifs on "-" Strand: Mean Exp. Number 0.00186 Found 1
308 GCCACGTGgC 299 (Mism.= 1)
............................................................
Totally 14 motifs of 7 different REs have been found
------------------------------------------------------------