(Received for publication, July 18, 1995; and in revised form, August 28, 1995)
From the
At the molecular level, little is known about the transport of copper across plant membranes. We have isolated an Arabidopsis thaliana cDNA by complementation of a mutant (ctr1-3) of Saccharomyces cerevisiae defective in high affinity copper uptake. This cDNA codes for a highly hydrophobic protein (COPT1) of 169 amino acid residues and with three putative transmembrane domains. Most noteworthy, the first 44 residues display significant homology to the methionine- and histidine-rich copper binding domain of three bacterial copper binding proteins, among these a copper transporting ATPase. Mutant yeast cells expressing COPT1 exhibit nearly wild type behavior with regard to growth on a nonfermentable carbon source and resistance to copper and iron starvation. Expression of COPT1 is also associated with an increased sensitivity to copper toxicity. Additionally, COPT1 shows significant homology to an open reading frame of 189 amino acid residues on yeast chromosome VIII. This gene (CTR2) may encode an additional yeast metal transporter able to mediate the uptake of copper. A mutation in CTR2 displays a higher level of resistance to toxic copper concentrations. Overexpression of CTR2 provides increased resistance to copper starvation and is also associated with an increased sensitivity to copper toxicity. The amino acid sequence of CTR2, like Arabidopsis COPT1, contains three potential transmembrane domains. Taken together, the data suggest that a plant metal transporter, which is most likely involved in the transport of copper, has been identified.
Copper is a constituent of a great number of proteins and as such an essential micronutrient for plants. Copper-containing proteins act as terminal oxidase, mono- and dioxygenases, in the elimination of superoxide radicals as well as in electron transfer reactions, most notably in the process of respiration and photosynthesis(1) .
At the molecular level, our knowledge on the copper uptake in roots of higher plants is very rudimentary(2) . For the time being, neither a copper transporter of the cytoplasmic membrane nor of the inner membranes of mitochondria or chloroplasts has been identified. A number of uptake studies is consistent with a Michaelis-Menten saturation kinetic of copper transport across plant membranes(3, 4) . However, as pointed out by Graham(5) , it is not yet clear whether this is a protein-mediated diffusion process or an active transport.
To date, the only biochemically characterized eukaryotic copper tranporter known is the CTR1 protein that is required for high affinity copper uptake in Saccharomyces cerevisiae(6, 7) . The CTR1 gene encodes a plasma membrane protein of 406 amino acid residues and three potential transmembrane helices. The amino terminus of CTR1, which is unusually rich in methionine and serine residues, contains a repetitive motif that is also present in bacterial proteins involved in the handling of copper(6) . The analysis of CTR1 mutants revealed an unexpected relation between the uptake of copper and the ability of cells to accumulate iron. Copper is strictly required for high affinity iron uptake (6, 8) by a specific ferrous iron transporter(9) . As a prerequisite to permit continuous high affinity iron transport, an oxidation step of the ferrous to the ferric iron form is necessary. This oxidation is catalyzed through the transmembrane FET3 protein, a copper-containing oxidase(8, 10) , thus most likely explaining this copper requirement. While CTR1 mutants are impaired in the uptake of both copper and iron, FET3 mutants are defective in iron uptake only. This intimate connection between the uptake of these two metals is further demonstrated by the FRE1 gene(11, 12) . FRE1 encodes a multispanning plasma membrane protein that is required for reduction of ferric iron to ferrous iron, which is the actual transport form into the cell(13) . In fact, not only is the expression of FRE1 negatively regulated by both iron and copper(14) , but the FRE1 protein functions as a copper(II) reductase as well(15) .
Yeast mutants have proven to be a valuable tool in identifying new genes from homologous and heterologous sources via functional complementation. This approach was also effective in the identification and characterization of a number of plant membrane proteins that catalyze the transport of inorganic ions or organic molecules across plant membranes such as potassium channels(16, 17) , potassium transporters(18) , ammonium transporters(19) , sucrose transporters (20) , and amino acid permeases(21, 22) . This let us question whether yeast would be a good model organism to study copper and iron transport across plant membranes at the molecular level as well. In fact, the physiology of iron uptake by yeast is similar to the uptake of iron by roots of dicots and nongraminaceous monocots(23) . These plants, like yeast, reduce ferric iron before transport via a plasma membrane-bound iron(III) reductase and appear to have a ferrous iron transporter as well(24) . This suggests that yeast could be a suitable model organism to study the transport of iron and probably of copper as well, e.g. via the functional complementation of mutants perturbed in copper and iron acquisition across plant membranes.
We have isolated a cDNA from Arabidopsis thaliana that suppresses the mutant phenotype of a high affinity copper transport-deficient strain of S. cerevisiae. Characterization of this cDNA, the derived protein (designated COPT1 for Copper Transporter 1), and a corresponding gene (named CTR2) from yeast are described.
Figure 1:
Functional complementation of the ctr1-3 mutant strain 83 of yeast by Arabidopsis COPT1. S. cerevisiae strains CM3262 and 83 (ctr1-3) untransformed or transformed with the vector
pFL61 or plasmid pKK31 (COPT1 cDNA under the control of the PGK promoter) are shown. A, cells were streaked on a
YG plate containing glycerol as a test for respiratory competence.
Incubation lasted for 7 days at 30 °C. B, strains were
grown overnight in SDC and, cell numbers were determined by
spectrophotometry at 600 nm. Each culture was adjusted to an A of 1 and then serially 10-fold diluted in
water (lanes 1-5). 20-µl aliquots of each strain and
dilution were spotted onto YNBFC plates containing 1 mM ferrozine, 160 µM BCS, 900 µM CuSO
, or 0 µM CuSO
(no
addition of CuSO
) and incubated for 14 days, respectively,
at 30 °C prior to photography.
Copper is toxic at elevated levels, which
may relate to its ability to catalyze the formation of the extremely
reactive hydroxyl radicals (38) . To further support the
conclusion that the isolated Arabidopsis cDNA promotes copper
uptake, strains 83 (pFL61) and 83 (pKK31) were examined for the effects
of copper-mediated growth inhibition. The ctr1-3 strain
harboring plasmid pKK31 was more sensitive to toxicity on a solid plate
containing 900 µM CuSO than the same strain
transformed with the vector pFL61 only (Fig. 1B),
indicating an efficient delivery of copper to the yeast cells. This
putative metal uptake system that apparently mediates a copper
transport has been designated COPT1.
Figure 2: Nucleotide and predicted amino acid sequence of the A. thaliana COPT1 cDNA. The sequence presented corresponds to the COPT1 cDNA isolated in this study (from pKK31) via the functional complementation of the high affinity copper transporter mutant ctr1-3 of yeast. The EMBL sequence data bank accession number of the COPT1 cDNA sequence is Z49859.
A comparison of the nucleotide sequence with those in the EMBL nucleotide sequence data base and the Swiss-PROT sequence data base revealed a significant homology to only two proteins, a putative protein encoded by the open reading frame of YHR185c, recently identified by sequencing of yeast chromosome VIII(30) , and the QP protein from the protozoan parasite Theileria parva(40) . The function of these two proteins is unknown(30, 40) ; however, the yeast protein was tentatively named Copper Transporter 2 (CTR2) for reasons that will be elaborated in the following section. A sequence alignment of the predicted COPT1 protein of A. thaliana with the CTR2 protein from yeast (23.4% identity, 38.3% similarity) and the QP protein of T. parva (21.9% identity, 40.3% similarity) is shown in Fig. 3A. Whereas COPT1 and CTR2 are similar in length, the QP protein is much longer with 480 amino acid residues, of which only the carboxyl-terminal 180 residues display homology to COPT1 (Fig. 3A). Another region of sequence homology is covered by the first 44 amino-terminal residues of COPT1, which are enriched in methionine (25%) and histidine (16%) residues (Fig. 3B). A similar amino acid sequence is present in the amino terminus of the P-type ATPase CopB of Enterococcus hirae (34.8% identity, 56.5% similarity) (41) and in two proteins of Pseudomonas syringae, CopB (32.6% identity, 52.2% similarity) and CopA (26.1% identity, 58.7% similarity)(42) . These bacterial proteins are all involved in the handling of copper. The COPT1 protein of A. thaliana has no detectable homology to the high affinity copper transporter CTR1 from yeast.
Figure 3: Alignment of the predicted amino acid sequence of A. thaliana COPT1 protein in (A) with S. cerevisiae CTR2 and T. parva QP proteins and in (B) with putative copper binding sites in E. hirae CopB and the P. syringae CopB and CopA proteins. Eh, E. hirae; Ps, P. syringae. Numbers indicate amino acid positions. Identical amino acids are marked by asterisks, and similar amino acids are indicated by colons above the aligned sequences. The dots mark artificial sequence gaps introduced to improve the homologies between the proteins.
Analysis of the hydropathy profile of the amino acid sequence of the COPT1 protein using the method of Kyte and Doolittle (36) demonstrates that the A. thaliana protein is highly hydrophobic (Fig. 4). Thus, COPT1 is most likely an integral membrane protein. Employing the method of Rost et al.(37) , under the assumption that a stretch of 16 amino acid residues is sufficiently long to span the lipid bilayer(43) , revealed the presence of three potential transmembrane helices in the COPT1 protein (Fig. 4). The potential transmembrane helices extend in COPT1 from residues 63 to 84, 104 to 122, and 127 to 144 (Fig. 4). The distribution of hydrophilic and hydrophobic amino acids along the polypeptide chains is also very similar when comparing the CTR1 and QP protein with COPT1 (Fig. 4), which further supports their relatedness.
Figure 4:
Hydropathy profiles of the amino acid
sequences of the COPT1 protein from A. thaliana, the CTR2
protein from S. cerevisiae, and the QP protein from T.
parva. The method of Kyte and Doolittle (36) was employed
using a window size of 11 successive residues (hydrophilic, negative
values; hydrophobic, positive values). In each case, the predicted
three membrane-spanning -helices are indicated by cross-bars (1-3) extending in COPT1 approximately from
residues 63 to 84, 104 to 122, and 127 to 144; in CTR2 from residues 81
to 101, 135 to 151, and 152 to 168; and in QP from 371 to 391, 405 to
429, and 433 to 456.
To
examine a possible role of the CTR2 gene in copper uptake, the CTR2 disruption mutant was compared with the parental wild
type strain for the ability to grow on the nonfermentable carbon source
glycerol, as a test for respiration competence, and on a minimal medium
containing the ferrous iron chelator ferrozine, a test for high
affinity ferrous iron uptake. On these media, the high affinity copper
transport mutant ctr1-3 fails to grow(6) . As
shown in Fig. 5, a mutation of CTR2 did not result in
impaired growth on glycerol or iron-starved medium. We therefore tested
the growth response of the ctr2::HIS3 single and the ctr1-3 ctr2::HIS3 double mutant on copper-starved medium
containing the copper chelator BCS and on copper-rich medium. Whereas
the ctr2::HIS3 mutant again did not show a growth impairment
on copper-starved medium, the double mutant ctr1-3 ctr2::HIS3 was slightly stronger growth inhibited in the presence of 80
µM BCS than the ctr1-3 single mutant (Fig. 5B). However, both, the ctr2::HIS3 single and the ctr1-3 ctr2::HIS3 double mutant
strain were more resistant to copper toxicity than the respective
parental strains (800 µM CuSO in Fig. 5B). Under the same conditions, a mutation only in CTR1 does not result in an increased resistance to copper when
compared to a wild type strain (Fig. 5B). This was to
be expected, since copper excess has been shown to strongly repress CTR1 gene expression(7) . The growth responses of the CTR2 disruption mutant on copper-deprived and copper-rich
media may suggest that CTR2 allows a low affinity copper uptake. This
notion is supported by the observation that overexpression of CTR2 (pKK52) does not result in the complementation of the high
affinity copper transport mutant ctr1-3 on solid medium
containing as sole carbon source glycerol or containing ferrozine to
lower the free iron concentration (data not shown). However, cells of
the ctr1-3 ctr2::HIS3 mutant strain KK4 overexpressing CTR2 from plasmid pKK52 showed a slightly enhanced growth in
presence of the copper chelator BCS (Fig. 5C).
Overexpression of CTR2 resulted in increased sensitivity to
toxicity on a plate containing 700 µM CuSO
(Fig. 5C), which is consistent with mediation of
an efficient copper transport into the cell via CTR2 when high external
copper concentrations are present.
Figure 5:
Phenotypes resulting from various CTR2 genotypes. Culture plates either contained YG medium with glycerol
or YNBFC medium. YNBFC was with 1 mM ferrozine, BCS (40 or 80
µM), CuSO (700 or 800 µM), or no
copper addition (0 µM CuSO
). The S.
cerevisiae strains included the parental strain CM3262 (CTR1
CTR2), strain KK3 (ctr2::HIS3), strain 83 (ctr1-3), and strain KK4 (ctr1-3
ctr2::HIS3). A, cells streaked on YNBFC plates containing
ferrozine to assess high affinity ferrous iron uptake. B,
strains were grown in YPD overnight, adjusted to A
of 1, and serially diluted as described in Fig. 1. C, strains either transformed with vector pFL61 or plasmid
pKK52 (CTR2 under control of PGK promoter) were grown
for 48 h in SDC. Serial dilutions were performed as described in Fig. 1. All plates were incubated for 14 days except for the YG
and YNBFC plates (no copper addition), which were incubated only for 7
days at 30 °C prior to photography.
Consistent with a possible role of CTR2 in copper uptake is the highly hydrophobic nature of this protein (Fig. 4), suggesting that CTR2 is an integral membrane protein. Inspection of the CTR2 amino acid sequence revealed the presence of three potential transmembrane helices, like for the plant homologue COPT1 (Fig. 4). The predicted transmembrane helices extend in CTR2 from residues 81 to 101, 135 to 151, and 152 to 168 (Fig. 4).
Figure 6:
Expression of COPT1 in different
organs of Arabidopsis and Southern blot analysis of COPT1. A, Arabidopsis genomic DNA (2 µg
per track) extracted from ecotypes Columbia (C) and Landsberg erecta (L) was digested with DraI (lane
1), SacI (lane 2), EcoRV (lane
3), PstI (lane 4), and HindIII (lane 5). The blot was probed with a P-labeled NotI fragment from pKK31 covering the entire COPT1 cDNA (Fig. 2). Note, the COPT1 cDNA does not
contain SacI, EcoRV, PstI, or HindIII sites, but a single DraI site in position 728
is present (Fig. 2). B, Northern blot analysis of total
RNA (10 µg per track) extracted from A. thaliana flowers (lane 1), stems (lane 2), leaves (lane 3),
and roots (lane 4). The same COPT1 probe as in A has been used. Length standard (kb) in A and B was HindIII-digested and
P-labeled
DNA.
A Southern blot of genomic DNA from the A. thaliana ecotypes Columbia and Landsberg erecta, which was digested with DraI, SacI, EcoRV, PstI, and HindIII and probed with the entire COPT1 cDNA (Fig. 2), is shown in Fig. 6A. Washing under a mild stringency revealed only one hybridizing band in each of the tracks (Fig. 6A). This result is in agreement with the assumption that no other closely related gene is cross-hybridizing under these conditions, suggesting that COPT1 is a single copy gene. Digestion of the genomic DNA with HindIII revealed the presence of a polymorphism when comparing the two ecotypes (Fig. 6A, lane 5).
Very little is known about the molecular basis of copper transport across plant membranes. Since the functional complementation of yeast mutants for the isolation of plant cDNAs from an expression library has been used successfully to identify plant transporter cDNAs (16, 17, 18, 19, 20, 21, 22) , we started to dissect the plant copper transport system by complementation of yeast mutants.
We have isolated a full-length cDNA of a putative copper transporter (COPT1) from A. thaliana by functional complementation of the high affinity copper transport mutant ctr1-3 from yeast. The cDNA encodes a polypeptide of 169 amino acid residues, which, interestingly, shows no detectable sequence homology to the copper transporter CTR1 from yeast. The distribution of hydrophilic and hydrophobic amino acid residues along the polypeptide chain suggests the presence of three potential transmembrane helices (Fig. 4); thus, COPT1 is most likely an integral membrane protein. The presence of only three potential membrane-spanning domains does not contradict a possible role of COPT1 in metal transport since also for the high affinity copper transporter from yeast, CTR1, only three transmembrane domains have been predicted (6) . In addition, there are other metal transporters known from the cytoplasmic membrane of prokaryotes that also have only three transmembrane segments, for example magnesium and mercury transport systems(44, 45) . The COPT1 protein resides presumably in the cytoplasmic membrane since the complementation of the ctr1-3 mutant on copper-limited plates (Fig. 1B) would be otherwise difficult to interpret. The ctr1-3 mutant is well characterized and has been clearly demonstrated to suffer from a copper uptake defect across the cytoplasmic membrane(6, 7) , which is obviously overcome by heterologous expression of COPT1 (Fig. 1B). However, the COPT1 protein lacks a predictable amino-terminal leader sequence and may thus utilize one of the transmembrane domains to initiate insertion into the membrane. The CTR1 protein is lacking a leader sequence as well(6) .
A copper transporter would be predicted to first interact with copper before subsequent translocation of the copper ion across the membrane. The methionine- and histidine-rich amino terminus composed of the first 44 amino acid residues of the COPT1 protein might be such a copper binding domain since it displays considerable sequence homology to similar motifs present in the bacterial copper binding proteins CopA and CopB of P. syringae(42) and CopB of E. hirae(41) (Fig. 3B). These bacterial proteins have been demonstrated experimentally to bind copper(46, 47) . The CopB-ATPase of E. hirae is a cytoplasmic membrane protein and functions as an efflux pump of copper(46) . The Cop proteins of P. syringae mediate the sequestration of copper outside the cytoplasm; CopB is an outer membrane protein, and CopA is localized in the periplasm as part of a copper resistance mechanism(47) . Since this methionine- and histidine-rich domain is the only common denominator among these bacterial copper binding proteins, which are otherwise completely unrelated in amino acid sequence, it has been proposed that this motif represents a copper binding domain(41, 42, 46, 47) . The presence of this putative copper binding domain at the amino terminus of COPT1 is consistent with a direct role in copper transport and points to its possible ability to interact directly with copper.
The
interpretation that COPT1 might be a plant metal transporter capable of
transporting copper is based on the following observations by
heterologous expression in the well characterized high affinity copper
transporter mutant ctr1-3 of yeast. (i) Expression of
COPT1 allows the yeast strain 83 (ctr1-3) to grow on the
nonfermentable carbon source glycerol (Fig. 1A). The ctr1-3 mutant is unable to grow on a nonfermentable
carbon source due to a respiratory defect, which is a consequence of
the high affinity copper uptake defect(6) . Regaining the
ability to grow on glycerol plates in presence of COPT1 is consistent
with increased copper uptake via the plant membrane protein. (ii)
Expression of COPT1 restores the growth defect of the ctr1-3 mutant on iron-starved plates (Fig. 1B). Since the
high affinity ferrous iron transport is strictly copper dependent, the
FET3 oxidase, which is a component of the high affinity ferrous iron
transporter system, contains copper in the active
site(8, 10) ; the ctr1-3 mutant suffers
from both a copper and an iron uptake defect. Regained growth on
iron-deprived plates in presence of COPT1 is again consistent with
enhanced copper transport across the cytoplasmic membrane of the ctr1-3 mutant. (iii) Expression of COPT1 allows yeast
strain 83 (ctr1-3) to grow more efficiently on
copper-starved plates (Fig. 1B). As pointed out by
Dancis et al.(7) , the growth arrest of CTR1 mutants in low copper-containing medium does not seem to be
related to iron deficiency but to a copper deficiency, since it is not
rescued by iron addition. The most plausible explanation for this
phenotype is that A. thaliana COPT1, heterologously expressed
in the ctr1-3 mutant, promotes enhanced copper uptake
and not iron uptake. (iv) Expression of COPT1 inhibits growth of the ctr1-3 mutant on copper-rich medium (Fig. 1B, 900 µM CuSO). Thus,
the COPT1 protein mediates the efficient delivery of copper to the
cells, even at high external copper concentrations, and thereby
resulting in copper toxicity. The same visible phenotype has been
described upon overexpression of the high affinity copper transporter
CTR1 from yeast on copper-rich plates(7) . The toxic nature of
copper for yeast cells at elevated concentrations is well
known(48) . We should point out that we cannot currently
exclude the possibility that COPT1 might transport, in addition to
copper, other metals.
The COPT1 protein of A. thaliana shows a low but significant homology (23.4% identity, 38.3% similarity) to the predicted protein of an open reading frame on yeast chromosome VIII (Fig. 3A). We have named this gene CTR2 and hypothesize that it encodes a metal uptake system of low affinity for copper. In fact, Dancis et al.(6) observed that the CTR1 mutant still displays a measurable copper uptake activity, although strongly reduced when compared to the wild type. However, high levels of exogenous copper (500 µM) completely suppressed the ferrous iron uptake defect in the CTR1 mutant(6) , which is indicative for the presence of a low affinity copper uptake system in the yeast cytoplasmic membrane. The conclusion that CTR2 might mediate a low affinity copper uptake into yeast cells is based on the following results. (i) Overexpression of CTR2 results in increased sensitivity to copper toxicity on copper-rich plates (Fig. 5C), which is consistent with enhanced copper uptake via CTR2. (ii) The disruption mutant ctr2::HIS3, on the other hand, displays increased resistance to copper toxicity (Fig. 5B), indicative for a strongly reduced copper uptake. (iii) The disruption mutant ctr2::HIS3 does not show a growth defect on a nonfermentable carbon source nor on an iron- or copper-starved medium (Fig. 5, A and B). However, the double mutant ctr1-3 ctr2::HIS3 is slightly stronger growth inhibited in presence of the copper chelator BCS than the single mutant ctr1-3 (Fig. 5B). Thus, CTR2 seems to make only a minor contribution to the delivery of copper into cells under conditions of a copper starvation when the high affinity copper transporter CTR1 is still functional. (iv) Overexpression of CTR2 results in a slight growth promotion in a ctr1-3 ctr2::HIS3 mutant background (Fig. 5C) but not in a complementation of the ctr1-3 mutant on medium containing a nonfermentable carbon source or the iron chelator ferrozine. This observation strongly supports the hypothesis that CTR2 may encode a metal uptake system of a low affinity for copper, transport capacity of which is not high enough to fully complement all the growth defects of the ctr1-3 mutant, especially when only a low external copper concentration is available. The CTR2 protein is predicted, like its plant homologue COPT1, to contain three potential transmembrane domains (Fig. 4). Interestingly, the CTR2 protein does not exhibit a histidine- and methionine-rich amino-terminal domain like COPT1. Assuming that CTR2 directly binds copper, a novel metal binding motif must be involved. As has been discussed already by Dancis et al.(6) , different copper binding motifs have to be expected dependent on the strength of the binding to copper, which not only can be permanent but transient, in case of a copper transporter, as well. It wouldn't be surprising if a high affinity copper transporter would comprise a completely different copper binding domain than a low affinity copper transporter. An alternative explanation could be that CTR2 actually transports other metals instead of or besides copper, which would explain the lack of a defined copper binding motif and the inability to fully complement the ctr1-3 mutant.
The Southern data (Fig. 6A) indicate that COPT1 is encoded by a single gene in the A. thaliana genome. The observed polymorphism between ecotypes Columbia and Landsberg erecta can now be exploited to locate the position of the COPT1 gene in the Arabidopsis genome(49) . It will be very interesting to see if there are already mutants known that map to this position. In this context, recent work by Murphy and Taiz (50) should be mentioned. These authors described 59 putative copper-sensitive Arabidopsis mutants. From the Northern blot, a single COPT1 messenger of 0.9 kb was detected in leaves, flowers, and stems. However, no COPT1 transcript could be detected in roots. Thus, it is at the moment questionable if COPT1 is directly involved in copper uptake from soil via the roots or rather in the distribution within the plant, and here especially in the green parts.
In conclusion, we believe that the A. thaliana COPT1 protein is a copper transporter that presumably resides in the cytoplasmic membrane. The yeast homologue, CTR2, is most likely a metal transporter as well and might well function as a low affinity copper uptake system. We are currently trying to test this hypothesis by performing transport measurements with radioactive copper by heterologous expression of the COPT1 cDNA in yeast cells. Detailed studies will be needed to clearly define the substrate specificities and the kinetic characteristics of these novel transport proteins. It could well be possible that these transporters are capable of transporting copper and iron in addition to other metals. Functional complementation in yeast was used in the work described here to identify a putative copper transporter from plants. Via this approach, we may identify further components of plant copper transport systems. The intracellular transport of copper within yeast cells has just started to be dissected, and mutants are already available(51) . The availability of plant copper transporter genes together with the possibility of constructing transgenic plants where the expression is either down- or up-regulated will provide us with novel insights into the inorganic nutrition and partitioning of copper in higher plants.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z49859[GenBank].