GLOBAL JOURNAL OF BIOCHEMISTRY
Detection, PCR-amplification and characterization of a 4 intron-containing
non-symbiotic hemoglobin gene from the moss Physcomitrella patens
Fernando Martínez-Ocampo, Consuelo Vázquez-Limón, Raúl Arredondo-Peter*
Laboratorio de Biofísica y Biología Molecular, Departamento de Bioquímica y Biología Molecular,
Facultad de Ciencias, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001,
Col. Chamilpa, 62210 Cuernavaca, Morelos, México
*
Author for Correspondence: Raúl Arredondo-Peter, email: [email protected]
Received 14 Mar 2012; Accepted 12 Apr 2012; Available Online 13 Apr 2012
Abstract
It has been well established that land plant non-symbiotic hemoglobin (nshb), symbiotic hemoglobin (shb) and leghemoglobin (lb)
genes are interrupted by 3 introns (IVS-I, IVS-II and IVS-III). Here, we report the detection, PCR-amplification and DNA sequencing of a 4
intron-containing nshb (nshb2) gene from the Physcomitrella patens DNA. Sequence analysis revealed that nshb2 is interrupted by introns IVSI, IVS-II and IVS-III, and by an extra intron (pre-IVS-I) interrupting coding sequences upstream to IVS-I. Phylogenetic analysis showed that P.
patens nsHb2 clusters with bryophyte nsHbs, however nsHb2 is divergent from P. patens nsHb1. Modeling of the tertiary structure of P. patens
nsHb2 showed that this protein could fold into the myoglobin-fold. The existence of intron pre-IVS-I in P. patens nshb2 but not in other known
land plant nshbs, shbs and lbs suggests that this intron inserted after gene duplication that originated nshb2. If this is correct intron insertion
possibly occurred in land plant nshbs other than P. patens nshb2. This will be verified as novel nshbs become available from newly sequenced
land plant genomes.
Keywords: Gene structure; Hemoglobin; Intron; Non-symbiotic; Physcomitrella patens; Plants
Abbreviations: Hb, hemoglobin; IVS, intervening sequence (intron); Lb, leghemoglobin; Mb, myoglobin; nsHb, non-symbiotic hemoglobin;
ORF, open reading frame; sHb, symbiotic hemoglobin; tHb, truncated hemoglobin
1. Introduction
Globins (Hbs) are a large family of proteins that have
been detected in organisms from the three kingdoms of life
[1]. In land plants three types of Hbs exist: symbiotic Hbs
(sHbs), non-symbiotic Hbs (nsHbs) and truncated Hbs (tHbs)
[2-4]. Symbiotic Hbs (or leghemoglobins (Lbs) when isolated
from legume species) are only localized in nodules of N2fixing land plants [5-9]. In contrast, nsHbs and tHbs are
widely distributed in land plants and are localized in tissues
from symbiotic and non-symbiotic plant organs [10-16].
Phylogenetic analysis of land plant Hbs suggests that these
proteins vertically evolved and diversified with land plants,
that the nsHb, sHb and Lb and tHb lineages evolved from
different ancestors previously to the emergence of
Archaeplastida (i.e. algae and land plants), and that sHbs and
Lbs originated from a nsHb ancestor [4, 17, 18].
During the last decades the availability of sequenced
genomes permitted the identification and characterization of
hb genes from several land plants, ranging from primitive
bryophytes to evolved monocots and dicots. Known land plant
nshb, shb and lb genes are interrupted by 3 introns (IVS-I,
IVS-II and IVS-III) mostly conserved at positions B12.2,
E15.0 and G7.0 (i.e. intron position within the myoglobin
(Mb)-fold and phase). Thus, the apparent gene structure for
land plant nshbs, shbs and lbs is 4 exons/3 introns. In contrast,
intron position and number is variable in known land plant thb
genes [18]. During the search for nshb gene copies in the
genome of the moss Physcomitrella patens at the COSSMOS
database (see subsection 2.1.) we detected a nshb (nshb2) that
is interrupted by 4 introns. This observation suggested that
gene structure for nshb2 is 5 exons/4 introns. Here, we report
the detection, PCR-amplification and characterization of 4
intron-containing nshb2 from the P. patens DNA. Results
from this work revealed that nshbs with gene structure other
than 4 exons/3 introns exist in land plants.
2. Experimental
2.1. Database search and mapping of the nshb2 gene in the
P. patens genome
Physcomitrella patens nshb (nshb1) gene and protein
sequences (GenBank accession number EF028055 and
ABK20873, respectively) were used as probes to search for
nshb gene copies in the P. patens genome at the COSMOSS
database (http://www.cosmoss.org/, last accessed on
September 2011) using the Blast tool [19]. Putative nsHb
sequences were subjected to a FUGE analysis (http://wwwcryst.bioc.cam.ac.uk, last accessed on September 2011) to
determine the most similar globin structure and presence of
proximal His at the Mb-fold position F8. Putative globins had
to satisfy the following criteria: length higher than 100 amino
acids, a FUGE Z score higher than 6 (which corresponds to
99% specificity [20]) with globin structures, and the presence
of proximal His at position F8. Scaffolds containing copies of
the nshb gene were used for mapping nshbs. This included the
detection of open reading frames (ORFs) 10-16 and 5-9 Kb
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Archaeplastida nsHbs and Lbs, respectively,
were performed using the ClustalX program [23].
Archaeplastida nsHb and Lb sequences were
obtained
from
the
GenBank
database
(http://www.ncbi.nlm.nih.gov, last accessed on
September 2011) using the accession numbers
reported by Garrocho-Villegas et al. [4],
Fernández et al. [24] and Vinogradov et al. [18].
Sequence alignment was manually verified using
the procedure described by Kapp et al. [25] based
on the Mb-fold [26]. Phenogram was obtained
from aligned sequences using the MEGA
program (version 5.05) [27] and the Neighbor
Joining method [28] with 1000000 bootstrap
replicates.
2.4. Molecular modeling and analysis of the
predicted P. patens nsHb2 tertiary structure
The tertiary structure of P. patens nsHb2
was modeled using the automated mode of the IFigure 1. Mapping of the nshb1 (A) and 2 (B) genes in P. patens scaffolds _71 and
TASSER
server
_660, respectively. Numbers in parenthesis indicate the scaffold total length.
(http://zhanglab.ccmb.med.umich.edu/IArrows indicate open reading frames (ORFs), and ORF length, transcription
TASSER/, last accessed on November 2011) [29direction, and localization in the +/- strand.
31] and the crystal structure of rice Hb1 [32]
(PDB ID 1D8U) as a template. Model reliability
up- and downstream to nshbs, respectively, and ORF length,
was
evaluated
using
the
Verify3D
program
transcription direction and localization in the +/- strand.
(http://nihserver.mbi.ucla.edu/Verify_3D/, last accessed on
November 2011) [33]. Model was edited using the VMD
2.2. Plant growth, isolation of total DNA, PCR-amplification
program [34] and Adobe Photoshop® software. Distance of
and DNA sequencing
amino acid residues at the heme prosthetic group were
Physcomitrella patens var. Gransden was grown
calculated using the distance tool of the SwissPDBViewer
essentially as described by Garrocho-Villegas et al. [21].
program as described by Gopalasubramaniam et al. [35].
Briefly, protonemas were cultured in Knop medium in a plant
growth chamber (Biotronette Plant Growth Chamber, Lab3. Results and Discussion
Line) at 20oC with 16/8 h light/dark periods for 1 to 2 months,
plants were harvested, immediately frozen in liquid N2 and
3.1. Detection and mapping of nshb2 in the P. patens
stored at -70oC until used. Total DNA was isolated from 100
genome
mg
of
P.
patens
protonemas
using
the
The search for copies of nshb genes at the
cetyltrimethylammonium bromide (CTAB) method [22].
COSMOSS database using P. patens nshb1 sequences
Primers were designed for PCR-amplification using sequences
(GenBank accession number EF028055 and ABK20873) as
at the start and stop codons from the P. patens nshb2 gene
probe showed that a number of scaffolds containing hb-like
detected at the COSMOSS database (see subsection 2.1.).
sequences exist into the P. patens genome. After sequence
Primer sequences were: (sense) primer P. patens Hb2fwd: 5'evaluation only nshb sequences from scaffold_71 and
ATGGCATCATCGATTGGCG-3’, and (antisense) primer P.
scaffold_660 satisfied the criteria to be considered authentic
patens Hb2rvs: 5'-TCAAGTATGAGATTTAGCTGCTGC-3’.
globins (see subsection 2.1.). Scaffold_71 contains the P.
Total P. patens DNA (1.6 ng) was used as template for PCRpatens nshb1 sequence used as probe, and scaffold_660
amplification. PCR components and concentrations were 14
contains a copy (nshb2) of nshb1. Mapping of nshb1 and 2
pmol of each sense and antisense primer, 0.2 mM of each
revealed that these genes are located at the – and + strands
dNTP (Invitrogen), and 1 U of Taq DNA polymerase
from scaffolds _71 and _660, respectively, that genes coding
(Invitrogen) in 1X PCR buffer containing 2 mM MgCl2 in a
for a PAP2_haloperoxidase and hypothetical proteins are
final volume of 25 l. PCR-amplification was carried out for
flanking to nshb1, and that no ORFs exist 16 and 5 Kb up35 cycles at 58oC for annealing using a thermalcycler
and downstream to nshb2, respectively (Figure 1). Translation
(Minicycler, MJ Research). PCR products were detected in a
of nshb1 and 2 into predicted nsHb1 and 2 polypeptides and
1.2% agarose gel after staining with ethidium bromide,
sequence comparison between nshb1 and 2 genes revealed that
isolated from the gel using the GeneClean kit (Q-BIOgene)
nshb1 and 2 are interrupted by 3 and 4 introns, respectively.
and sequenced by PCR in both orientations using the sense
Gene nshb1 is interrupted by IVS-I, IVS-II and IVS-III
and antisense primers at the Institute of Biotechnology of the
previously recognized in known land plant nshbs, shbs and
National Autonomous University of México.
lbs. Gene nshb2 is interrupted by IVS-I, IVS-II and IVS-III
identically to those in nshb1, and by an extra intron
2.3. Sequence alignment and phenetic analysis
interrupting coding sequences upstream to IVS-I (see
Pairwise and multiple sequence alignment of P.
subsection 3.2.). Because sequences deposited in genome
patens nsHb1 and nsHb2, and P. patens nsHb2 and selected
databases occasionally result from sequencing and assembly
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GLOBAL JOURNAL OF BIOCHEMISTRY
ag/gg, tg/gt and ag/ac, and ag/gt and ag/gt, respectively) from
P.. patens nshb2 and other land plant nshb
nshbs,
s, such as P. patens
nshb1 (GenBank accession number EF028055), rice hb1 [36]
and Parasponia hb [37],
[37], are highly
highly conserved.
3.3. Sequence alignment and phenetic analysis of P. patens
nsHb2 and selected Archaeplastida hemoglobins
Sequence alignment showed that nsHb1 and 2 are 63
and 78% identical and similar, respectively, and that these
proteins contain the highly conserved distal (H84) and
proximal (H119) histidine. Also, pairwise sequence alignment
showed that intron pre-IVS-I
pre
I from nsHb2 is located at position
preA11.1, and that introns IVS-I,
IVS I, IVS
IVS-II
II and IVS-III
IVS III are
located in nsHb1 and 2 at positions B12.2
B12.2,, E15.0 and G7.0,
respectively (Figure
(
4). Phylogenetic analysis of selected
Archaeplastida nsHbs and Lbs showed that P. patens nsHb2
clusters with bryophyte nsHbs, however nsHb2 is divergent to
P.. patens nsHb1 and Ceratodon purpureus nsHb (Figure
(
5).
The above observations indicate that P.. patens nsHb1 and 2
are conserved and evolved from a common ancestor.
However, the existence of intron pre-IVSpre
-I in nshb2 but not in
nshb1 and other known land plant nshb
nshbss suggests that pre
preIVS I inserted into nshb2 after gene duplication that originated
IVS-I
nshb2 (see section 4).
Figure 2. PCR-amplification
amplification of P. patens nshb2 (arrow).
Samples were subjected to electrophoresis in a 1.2% agarose gel
and stained with ethidium bromide. Line 1, molecular size
markers (1 kb plus, Invitrogen); line 2, a 5 ll aliquot from the
total PCR reaction. Molecular size markers are shown in bp.
artifacts we verified the existence of nshb2 in the P. patens
DNA by PCR-amplification
PCR amplification and DNA sequencing.
3.2. PCRPCR-amplification,
amplification, DNA sequencing and analysis of
P. patens nshb2
Total DNA isolated from P. patens protonemas was
used as template for PCR-amplification
PCR amplification in combination with
primers designed from the nshb2 sequence detected in
scaffold_660 (see subsection 2.2.). Figure 2 shows that a
single fragment of 1,832 bp in size was amplified from the P.
patens total DNA. This fragment was isolated and sequenced
in both orientations. Sequence comparison showed that 1,832
bp fragment and nshb2 from scaffold_660 are identical, thus
indicating that nshb2 exists in the P. patens DNA. Sequence
analysis showed that nshb2 is interrupted by introns at
positions 61, 341, 613 and 1,519 ((Figure
Figure 3). Introns at
positions 341, 613 and 1,519 interrupt nshb2 identically to
IVS-I,
IVS I, IVS-II
IVS II and IVS-III
IVS II from known land plant nshbs,
nshb
including to P. patens nshb1.. Intron at position 61 interrupts
nshb2 at coding sequences located upstream to IVS
IVS-I,
I, thus we
named this intron as pre-IVS
pre IVS-I.
I. Sequences flanking the
exon/intron boundaries to prepre-IVS-II are tg/gt and ag/ag. With
the exception of the 5´-end
5´ end of exon/intron boundary in IVS-II
IVS
(tg/gt), which is identical to the 55-end
end of exon/intron boundary
in pre-IVSpre
-I,
I, the exon/intron boundaries among pre
pre-IVS-II and
IVS-I,
IVS I, IVS-II
IVS II and IVS-III
IVS III are different ((Figure 3). However,
exon/intron boundaries in IVSIVS-I, IVS-II
II and IVS-III
IVS III (ga/gt and
Global J. Biochem. 2012,
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3.4. Molecular modeling and analysis of the predicted P.
patens nsHb2 tertiary structure
The crystal structure of few land plant nsHbs has
been reported [17, 32, 38, 39],
39], but no crystallographic analysis
has been performed
performed on bryophyte nsHbs. However, the
predicted structure of C. purpureus and P
P. patens nsHbs was
reported by Garrocho-Villegas
Garrocho Villegas et al. [21] and is deposited in
the Caspur database
database (http://mi.caspur.it/PMDB/
(http://mi.caspur.it/PMDB/,
http://mi.caspur.it/PMDB/
ID:
PM0074985), respectively. Elucidating the tertiary structure of
nsHbs is important to understand the evolution and clarify the
function of these proteins in land plants. We modeled the
tertiary structure of predicted P. patens nsHb2 using the
automated mode of the I-TASSER
I TASSER server and the crystal
structure of rice Hb1 [32] (PDB ID 1D8U) as a template.
Evaluation using the Verify 3D program indicated that model
for P. patens nsHb2 is reliable, as values along the sequence
were higher than 0. Figure 6 shows that the predicted structure
of P. patens nsHb1 and 2 are highly similar, and that P. patens
nsHb2 could fold into the Mb-fold.
Mb fold. Minor differences between
the predicted structure of P. patens nsHb1 and 2 were detected
at the conformation of pre-helix
pre helix A and CD
CD- and GH-loop.
GH loop.
However, a major difference was that length of helix H is
longer in predicted P. patens nsHb2 than nsHb1. The
examination of amino acids that are essential for binding of
ligands to the Fe-heme
Fe heme showed that position of proximal and
distal His are similar among P. patens nsHb1
Hb1 and 2 and
hexacoordinate land plant nsHbs, such as rice Hb1 [32] and
nd
maize Hb [40] (not shown). This observation suggests that Fe
Feheme in P. patens nsHb1 and 2 is hexacoordinate.
4. Conclusions
Based on available sequences, for many years it was
known that land plant nshbs, shbs
shb and lbss are interrupted by 3
introns. As result from these observations it was postulated
that the ancestor to land plant nshbss was interrupted by 3
introns and that the 4 exons/3 introns gene structure
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1
1
............................................................
ATGGCATCATCGATTGGCGCCGCACCAGTTTCCGTTGCGCCAGAAACTGCACATGGTGCT
AMAAAAASAASAAIAAGAAAAAAAAPAAVAASAAVAAAAAPAAEAATAAAAAHAAGAAA
60
20
61
............................................................
Ggtaagcacggcgccgcatttgctcagctccatatttcgccgtcaccattacccccagtt
120
121
............................................................
ttcttaggaagttctgcttgggaatagaagcgcggctgagtatcttagaagctgttttgg
180
181
............................................................
tgtcccaatctcgattgaagaacctttctgtaactaatcatttgtaaccgtgaattcgtt
240
241
21
............................................................
gcagAGAAGGTTTACTCCAAGGAGAGCGTGGCCCTGGTGAAGCAGTCTTGGGAGCTTCTA
AAAAEAAKAAVAAYAASAAKAAEAASAAVAAAAALAAVAAKAAQAASAAWAAEAALAAL
300
39
301
40
............................................................
AAAGTGGACGCACAAGCAAACGCCGTCGCTTTCTTCAAAGAgtaaatacatctcacattc
AKAAVAADAAAAAQAAAAANAAAAAVAAAAAFAAFAAKAAE
360
53
361
............................................................
cgatattgtcgtgactggctgtagctcgagttttagtggatctgcctgtcgcagatgtga
420
421
............................................................
tgggcagtggtgctcgtgttactgccaatagagtcgtttcctgtctttcgtgaggtgcta
480
481
54
............................................................
agttggtggcatcatcagGGTATTCGAGATTGCACCAGCAGCCAAGGGGTTCTTCTCCTT
AAAAAAAAAAAAAAAAAAAAVAAFAAEAAIAAAAAPAAAAAAAAKAAGAAFAAFAASAAF
540
67
541
68
............................................................
CATGCAGGACACCAGCATCCCCTTCGAGGATAACCCCAAAGTGAAATTCCATGCTCTTCA
AAMAAQAADAATAASAAIAAPAAFAAEAADAANAAPAAKAAVAAKAAFAAHAAAAALAAQ
600
87
601
88
............................................................
GGTCTTCAAGTTGgtaagcactgagaatcctaaattccaactccctgagttttgagaaac
AAVAAFAAKAAL
660
91
661
............................................................
gtctgcacagctattgttatccgttacctagatgcaggtatatgttttgagattataact
720
721
............................................................
aagaacatcctgtcagggtggccgtaacaacattcttccttcatcacgcgcatgcccagt
780
781
............................................................
tttaggtacagtgatggaccacgtttgggccctgtgctctttgccttgcctgtgattaaa
840
841
............................................................
caagcaattctttagagcgatcgtgtaaaggaaatagttacagtcattctcaggggatct
900
901
............................................................
gacagatggtagagctgtgttcgaccagtacatccggctgcatgacgagtgtctggcatg
960
961
............................................................
ctcatagagcttggttgtccttttaattttcttcgttgaagacttgaatctaaaacttat
1020
1021
............................................................
tttcgtcctaatcattcagtttaattgctacgtcattcaagggtttacaatctagttgtg
1080
Figure 3. Nucleotide and predicted protein sequence of P. patens nshb2 amplified by PCR (Figure 2). Coding (exon) and non-coding
(intron) sequences are shown in upper and lowercase letters, respectively. Sequences flanking the exon/intron boundaries are underlined.
Distal (H84) and proximal (H119) His residues are shown with black background. The P. patens nshb2 gene sequence was deposited in the
GenBank database under the accession number JQ692084.
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1081
............................................................
attagatggaacctatatcttgataacatggatcaactgagtaatttccggcagataagc
1140
1141
............................................................
atcattttgaaacttacggaaaccattcattacgttattccacttcagtcattcgttata
1200
1201
............................................................
tccctcgtgatctttgcccgtcctcatcaacgtgatgtttcgcccaacctcggttatcag
1260
1261
............................................................
ttcttggctaaattactttgtgaaaacttaagaatgaagtcccaagaaccgacttgaata
1320
1321
............................................................
ccggaaatcggcatgcttctaaagtagtaagccagacttgaactttaaatttagggctcc
1380
1381
92
............................................................
ttacacttcttaatggattcagACAGGGGACGCGGCGGCGCAACTCGGAGAGAAAGGCGC
AAAAAAAAAAAAAAAAAAAAAAATAAGAADAAAAAAAAAAAQAALAAGAAEAAKAAGAAA
1440
104
1441
105
............................................................
ATACGAGCTTCTCCAGTCTCGTCTTCACAGTCTGGCCGCCAAGCATCTCAGTAAGGGAGT
AAYAAEAALAALAAQAASAARAALAAHAASAALAAAAAAAAKAAHAALAASAAKAAGAAV
1500
124
1501
125
............................................................
TCAAGATGCTCATTTCGAGgtaagatccctgcctcatgccattaacttgaccccggtcat
AAQAADAAAAAHAAFAAE
1560
130
1561
............................................................
ctgcacatgcttccagcgatgctcattcccaattccgacaggcgtagacgaacatttcta
1620
1621
131
............................................................
atacgtgaaatgtaaatggaaaattaacccatgatcggttataccagGTGGTAAAAGAGG
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAVAAVAAKAAE
1680
134
1681
135
............................................................
CTTTGCTGCGGACGATCGCGGCAGGCTTGCCAGACCTCTGGTCCCCCGCATTGAAACAGG
AAALAALAARAATAAIAAAAAAAAGAALAAPAADAALAAWAASAAPAAAAALAAKAAQ
1740
154
1741
155
............................................................
CATGGGCCGATGCCTACGACGCACTTGCCACCACTCTCAAGAATGAAATGCACGCTCAAG
AAAWAAAAADAAAAAYAADAAAAALAAAAATAATAALAAKAANAAEAAMAAHAAAAAQ
1800
174
1801
175
..............................
CCGCCGCGGCAGCAGCTAAATCTCATACTTGA
AAAAAAAAAAAAAAAAAAKAASAAHAATAA*
1832
184
Figure 3. (Continued)
conserved in land plant nshbs, shbs and lbs [4, 21, 41]. These
postulates were confirmed over the last years as novel nshbs,
shbs and lbs became available from newly sequenced land
plant genomes. However, our search in land plant genomes
revealed the existence of a nshb gene (nshb2) interrupted by 4
introns in P. patens (see subsections 3.1. and 3.2.). This
observation indicated that gene structure other than 4 exons/3
introns may exist in land plants.
Introns interrupting P. patens nshb2 correspond to
canonical IVS-I, IVS-II and IVS-III from known land plant
nshbs, shbs and lbs plus pre-IVS-I. Thus, an intriguing
question is: what was the origin of intron pre-IVS-I in P.
patens nshb2? One possibility is that intron pre-IVS-I already
existed in an ancestral algal nshb and conserved into first land
plant nshbs. This implicates that nshbs from a number of
primeval land plants, such as liverworts and mosses other than
P. patens, are also interrupted by intron pre-IVS-I, and that a
nshb copy only interrupted by 3 introns (i.e. introns IVS-I,
IVS-II and IVS-III) originated from an ancestral 4 introncontaining nshb after (pre-IVS-I) intron loss. The resulting 3
intron-containing nshb was conserved and the ancestral 4
intron-containing nshb was lost during the subsequent
evolution of land plants. Other possibility is that nshb2
inserted into the P. patens genome by horizontal gene transfer
from a 4 intron-containing hb donor. This implicates that hb
donor was interrupted by introns pre-IVS-I, IVS-I, IVS-II and
IVS-III. However, a candidate for a 4 intron-containing hb
donor is not known. We consider the above possibilities
unlikely and favor the following most parsimonious
hypothesis: a 3 intron (IVS-I, IVS-II and IVS-III)-containing
nshb1 duplicated into the P. patens genome originating nshb2,
which was subsequently interrupted by insertion of intron preIVS-I thus resulting in 4 intron (pre-IVS-I, IVS-I, IVS-II
and IVS-III)-containing P. patens nshb2. If this hypothesis
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Figure 4. Sequence alignment of P. patens nsHb1 and 2. Conserved amino acid residues, and distal and proximal His residues are shown
with asterisks and black background, respectively. Intron (IVS) position within the Mb-fold
Mb fold is indicated in parenthesis an
and
d with gray
background. Helix regions were calculated from the structure of rice Hb1 [32].
[32]
Figure 5. Phenetic relationships of P. patens nsHb1 and 2 (arrow) and selected Archaeplastida nsHbs and Lbs. Phenogram was constructed
from Hb sequences reported by Garrocho
Garrocho-Villegas
Villegas et al. [4],, Fernández et al. [24] and Vinogradov et al. [18]..
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8.
9.
10.
11.
Figure 6. Overlay of predicted P. patens nsHb1 (red) and 2 (blue)
tertiary structures. Helices (including prehelix A) are indicated
with letters A to H. The predicted structure of P. patens nsHb1
and 2 is deposited in the Protein Model Database (http://mi.
caspur.it/PMDB/) under the ID number PM0074985 and
PM0077988, respectively.
is correct, it is possible that intron insertion similar to that
postulated for P. patens nshb2 has occurred in other land plant
nshbs. This hypothesis will be verified as novel nshbs become
available from newly sequenced land plant genomes.
12.
13.
14.
Acknowledgements
Work in the authors´ laboratory has been financed by
SEP-PROMEP (grant number UAEMor-PTC-01-01/PTC23)
and Consejo Nacional de Ciencia y Tecnología (CoNaCyT
grant numbers 25229N and 42873Q), México. C. V.-L. is a
postdoctoral fellow supported by CoNaCyT.
15.
16.
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Cite this article as:
Fernando Martínez-Ocampo et al.: Detection, PCR-amplification and characterization of a 4 intron-containing non-symbiotic
hemoglobin gene from the moss Physcomitrella patens. Global J. Biochem. 2012, 3: 12
Global J. Biochem. 2012, 3: 12
8
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© 2012 Simplex Academic Publishers. All rights reserved.