Open Access

The ABC transporter gene family of Caenorhabditis elegans has implications for the evolutionary dynamics of multidrug resistance in eukaryotes

  • Jonathan A Sheps1,
  • Steven Ralph1, 3,
  • Zhongying Zhao2,
  • David L Baillie2 and
  • Victor Ling1Email author
Genome Biology20045:R15

DOI: 10.1186/gb-2004-5-3-r15

Received: 13 October 2003

Accepted: 13 January 2004

Published: 11 February 2004

Abstract

Background

Many drugs of natural origin are hydrophobic and can pass through cell membranes. Hydrophobic molecules must be susceptible to active efflux systems if they are to be maintained at lower concentrations in cells than in their environment. Multi-drug resistance (MDR), often mediated by intrinsic membrane proteins that couple energy to drug efflux, provides this function. All eukaryotic genomes encode several gene families capable of encoding MDR functions, among which the ABC transporters are the largest. The number of candidate MDR genes means that study of the drug-resistance properties of an organism cannot be effectively carried out without taking a genomic perspective.

Results

We have annotated sequences for all 60 ABC transporters from the Caenorhabditis elegans genome, and performed a phylogenetic analysis of these along with the 49 human, 30 yeast, and 57 fly ABC transporters currently available in GenBank. Classification according to a unified nomenclature is presented. Comparison between genomes reveals much gene duplication and loss, and surprisingly little orthology among analogous genes. Proteins capable of conferring MDR are found in several distinct subfamilies and are likely to have arisen independently multiple times.

Conclusions

ABC transporter evolution fits a pattern expected from a process termed 'dynamic-coherence'. This is an unusual result for such a highly conserved gene family as this one, present in all domains of cellular life. Mechanistically, this may result from the broad substrate specificity of some ABC proteins, which both reduces selection against gene loss, and leads to the facile sorting of functions among paralogs following gene duplication.

Background

ATP-binding cassette (ABC) transporters are one of the largest families of transport proteins constituting the single largest gene family, comprising about 5% of the genome, in Escherichia coli [1]. ABC transporters are grouped into several structural classes, or subfamilies, on the basis of amino acid sequence and domain organization [2] (Figure 1). The presence of a strongly conserved ATP-binding motif defines membership in the family and the basic functional organization of an ABC transporter in the membrane is the same from bacteria to humans, and in all subclasses [35]. A complex of at least two ATP-binding domains, coupled to two blocks of membrane-spanning helices, appears to be the minimum requirement for a functional transporter. Often these domains are found in tandem within a single molecule, but in many cases are distributed across separate proteins that must then assemble in the membrane. ABC transporters are collectively able to accommodate an unusually large array of different substrates. This diversity of function is manifest at the family level, but also in individual members of the family, for example those associated with multidrug resistance (MDR).
https://static-content.springer.com/image/art%3A10.1186%2Fgb-2004-5-3-r15/MediaObjects/13059_2003_Article_819_Fig1_HTML.jpg
Figure 1

Structural diversity of ABC transporters. Illustration of the various domain organizations found among members of the ABC transporter family in C. elegans. TM indicates a transmembrane domain typically containing six predicted membrane-spanning helices. ABC indicates an ATP-binding cassette domain. The color codes for each structure are used throughout the figures to show the lack of concordance between structural categories and families defined on the basis of sequence similarity.

Decottignies and Goffeau [6] catalogued the entire ABC transporter family of the yeast Saccharomyces cerevisiae and in so doing delineated six of the major subgroups of eukaryotic ABC transporters. Allikmets et al. [7] catalogued all the then known 33 human ABC transporters, including those known only from partial expressed sequence tag (EST) sequences, and divided these into seven subfamilies. This scheme has been adopted, with a revised nomenclature, by the Human Genome Organisation (HUGO) [8] in order to provide a unified nomenclature for both human and mouse ABC transporters. Of these seven subfamilies, one, ABCA, has no exact equivalent in the yeast genome [9, 10]. Genes considered to be part of subfamily ABCA have been identified in the slime mold Dictyostelium discoideum, as well as in malaria parasites [11] and Caenorhabditis elegans (this paper). With the completion of the human and Drosophila melanogaster genomes, a joint summary of the ABC transporter complements of both genomes was published [12]. This identified a new subfamily, ABCH, which appears to be the most divergent yet. One, previously unclassified yeast ABC gene, YDR061w [13], appears to be a structurally aberrant member of subfamily H.

The phenotypes of five ABC transporter knockouts have been reported in C. elegans. Four of these involve genes expected, by homology to mammalian genes, to be involved in drug resistance: three P-glycoproteins (Pgp-1, Pgp-3 and Pgp-4) (subfamily B) and one multi-drug resistance protein (MRP) [14, 15] (subfamily C). These ABC transporter mutants are associated with sensitivity to environmental insult [16]. Pgp-3 mutant strains of C. elegans are more sensitive to the drugs chloroquine and colchicine. Pgp-1 and mrp-1 strains are hypersensitive to toxic pigments produced by some bacteria [17]. All the nematode P-glycoproteins examined so far seem to be highly expressed in intestinal cells [18], and in the excretory cell, which functions somewhat like a kidney in C. elegans. The mrp-1, pgp-1 and pgp-3 mutant strains have been reported to be hypersensitive to the heavy metals cadmium and arsenite [15]. The fifth reported knockout is of the product of the ced-7 gene [19]. Mutant alleles of ced-7 cause a defect in engulfment of the cell corpses left behind by apoptosis. ced-7 is a member of the ABCA subfamily, and has a similar phenotype to the abca1 gene in humans. ABCA1 protein is required for engulfment of apoptotic cells by macrophages and is thought to regulate membrane fluidity through an increase in phosphatidylserine exposure on the outer leaflet of the cell membrane [20].

The term orthology is used to describe genes separated from one another by speciation events while paralogy describes those separated by gene duplication events [21]. Of particular interest, from the point of view of functional annotation, are the cases where a pair of genes, one from each of a pair of organisms, are found. In these cases it is reasonable to presume that the orthologous genes may share a conserved function retained from the same single gene present in the common ancestor of the two organisms. However, where a single gene (or set of duplicated genes) in one genome is most closely related to a set of duplicated (paralogous) genes in another genome this is sometimes termed co-orthology [22], and then no particular orthologous pair can be unambiguously specified. In the case of co-orthologs the argument for retention of analogous functions between members of the sets of descendant genes is much weaker. Comparison of two complete genomes, those of C. elegans and S. cerevisiae [23], demonstrated a high fraction of ortholog pairs in gene families involved in core biological functions. Specifically, Chervitz et al. [23] found, when pairing conserved yeast genes with their most similar worm homologs (subject to a BLAST score cut-off of < 10-100), 57% of these highly conserved gene pairs involved orthologous, rather than paralogous, pairs of genes. In this category of core functions they included trafficking, and, as possibly the largest family of trafficking genes in animal genomes, ABC transporters should be expected to share in this high level of one-to-one correspondence between genomes. We expected therefore that this would allow us to assign predicted functions to newly discovered C. elegans ABC proteins on the basis of their already-characterized mammalian orthologs. Following a comprehensive phylogenetic analysis of ABC transporters from four eukaryote genomes, we found that the frequency of orthologous pairs among ABC transporters was substantially lower than we expected. Particular domain organizations and substrate specificities seem to have evolved independently several times in multiple lineages. This is expected to complicate the functional analysis of ABC transporter function in newly characterized genomes.

Results and discussion

Here we present a classification of all ABC transporters encoded in the C. elegans genome, based on a phylogenetic analysis which includes the 49 currently known human ABC proteins for which there are reliable, public, sequence data. We took the approach of analyzing primarily the conserved ATP-binding cassettes from each protein, regardless of the structural class from which the domain is drawn. This allows evaluation of the evolutionary history of each protein in the family, without biases that might result from gene-fusion events resulting in convergent acquisition of similar domain structures by distantly related proteins. In addition, we re-evaluated the relationships of transporters within statistically reliable clusters whose members are closely related enough that structural variations do not lead to errors in alignment. We did this to capture additional phylogenetic information, which may be apparent in the less conservative transmembrane domains, at a level of analysis where it is less likely to be misleading.

An example of our first-pass approach is given in Figure 2, which shows an analysis of isolated ATP-binding cassette domains from the human ABC transporters only. In particular, we find that all seven subfamilies recognized by Allikmets et al. [7] are recovered with significant bootstrap support. Their finding, that subfamily B is more closely related to the carboxy-terminal component of subfamily C than the two halves of ABCC molecules are with one another, is supported by our results.
https://static-content.springer.com/image/art%3A10.1186%2Fgb-2004-5-3-r15/MediaObjects/13059_2003_Article_819_Fig2_HTML.jpg
Figure 2

Tree of human ATP-binding cassette domains. The evolution of the ABCB subfamily from within the ABCC subfamily, and the structural diversity of subfamily B is shown here. Each cluster of ABC domains within each subfamily, except for subfamily B, is collapsed to form a single, representative, branch; n-term: amino-terminal ABC; c-term: carboxy-terminal ABC. The phylogeny of ATP-binding cassettes from human ABC transporters was produced according the following procedure. Predicted amino-acid sequences were aligned using ClustalX [54]. Aligned sequences were used to generate matrices of mean distances among proteins, and these matrices were used to generate a phylogenetic tree according to the neighbor-joining algorithm [55], refined using the SPR branch-swapping technique under the minimum evolution criterion, implemented by PAUP*4.0b10 [56]. Bootstrapping [57] was used to determine the relative support for the various branches of the tree (1,000 replicates), and nodes with less than 50% support were collapsed to form polytomies. The structures of the proteins in which the domains are embedded are indicated according to the color scheme in Figure 1. It should be noted that branch lengths in the figures are not to scale and do not represent distances between protein sequences. The original alignment files are available as Additional data files 18.

A collection of transporters

We found a total of 60 confirmed ABC transporters in the annotated protein set derived from the C. elegans genome sequence. This represents approximately 0.3% of the total number of genes (approximately 19,000) in the worm genome. Only 8 of the 60 predicted genes lack any corresponding mRNA (Table 1), and only one (F56F4.6) is structurally aberrant in a way that would suggest it is likely to be a pseudogene.
Table 1

Characterization of the 60 C. elegans ABC proteins

Subfamily

ORF name/CGC name

Chromosome

GenBank accession number

Size (amino acids)

Predicted topology

cDNA if known

RNAi phenotype

A

Abt

C24F3.5/ Abt-1

IV

CAA18775

1,429

(6TM-ABC)2

 

None

  

C48B4.4/ Ced-7

III

NP_499115

1,704

(8TM-ABC)2

Complete

None

  

F12B6.1/ Abt-2

I

AAB54153

1,547

(6TM-ABC)2

Partial

None

  

F55G11.9/ Abt-3

IV

CAB05222

1,431

(8/4TM-ABC)2

 

None

  

F56F4.6

I

AAB54203

260

ABC

 

None

  

Y39D8C.1/ Abt-4

V

AAC69223

1,802

(6/8TM-ABC)2

Partial

None

  

Y53C10A.9/ Abt-5

I

CAA22142

1,564

(6TM-ABC)2

Partial

None

B

Pgp (full molecules)

C05A9.1/ Pgp-5

X

CAA94202

1,283

(6TM-ABC)2

Partial

None

  

C34G6.4/ Pgp-2

I

AAB52482

1,265

(6TM-ABC)2

Partial

None

  

C47A10.1/ Pgp-9

V

CAB03973

1,294

(6TM-ABC)2

Partial

None

  

C54D1.1/ Pgp-10

X

AAC48149

1,283

(4TM-ABC)2

Partial

None

  

DH11.3/ Pgp-11

II

CAA88940

1,270

(6TM-ABC)2

Partial

None

  

F22E10.1/ Pgp-12

X

CAA91799

1,318

(6TM-ABC)2

Partial

None

  

F22E10.2/ Pgp-13

X

CAA91800

1,291

(6TM-ABC)2

 

None

  

F22E10.3/ Pgp-14

X

CAA91801

1,327

(6TM-ABC)2

Partial

None

  

F22E10.4/ Pgp-15

X

CAA91802

1,270

(6TM-ABC)2

 

None

  

F42E11.1/ Pgp-4

X

CAA91463

1,266

(6TM-ABC)2

Partial

None

  

K08E7.9/ Pgp-1

IV

CAB01232

1,321

(6TM-ABC)2

Partial

None

  

T21E8.1/ Pgp-6

X

CAA94220

1,225

(6TM-ABC)2

Partial

None

  

T21E8.2/ Pgp-7

X

CAA94219

1,269

(6TM-ABC)2

 

None

  

T21E8.3/ Pgp-8

X

CAA94203

1,243

(6TM-ABC)2

Partial

None

  

ZK455.7/ Pgp-3

X

CAA91467

1,268

(6TM-ABC)2

Partial

None

 

Haf (half molecules)

C30H6.6/ Haf-1

IV

CAB02812

586

4TM-ABC

Partial

None

  

F43E2.4/ Haf-2

II

AAC71121

761

8TM-ABC

Partial

None

  

F57A10.3/ Haf-3

V

CAB09418

733

6TM-ABC

Partial

None

  

W04C9.1/ Haf-4

I

AAC68724

787

8TM-ABC

Complete

Weak embryonic lethality, slow growth

  

W09D6.6/ Haf-5

III

CAB04947

801

8TM-ABC

Complete

None

  

Y48G8AL.11/ Haf-6

I

AAK29911

565

4TM-NBF

Partial

 
  

Y50E8A.16/ Haf-7

V

CAB60586

807

6TM-ABC

Partial

 
  

Y57G11C.1/ Haf-8

IV

CAB16503

633

4TM-ABC

 

None

  

ZK484.2/ Haf-9

I

AAK39394

815

8TM-ABC

Complete

None

C

Mrp/Cft

C18C4.2/ Cft-1

V

AAK52175

1247

(5/6TM-NBF)2

Partial

None

  

E03G2.2/ Mrp-3

X

CAA92148

1,398

(6TM-ABC)2

Partial

None

  

F14F4.3/ Mrp-5

X

CAB54225

1,427

(6TM-ABC)2

Partial

Slow growth, Clear

  

F20B6.3/ Mrp-6

X

AAA82317

1,396

(6TM-ABC)2

Partial

Egg laying defect

  

F21G4.2/ Mrp-4

X

CAB02667

1,573

(10/6TM-ABC)2

Partial

None

  

F57C12.4/ Mrp-2

X

AAB07022

1,525

(10/6TM-ABC)2

Complete

None

  

F57C12.5/ Mrp-1

X

AAD31550

1,528

(12/6TM-ABC)2

Complete

None

  

Y43F8C.12/ Mrp-7

V

CAA21622

1,119

(12/2TM-ABC)2

Partial

 
  

Y75B8A.26/ Mrp-8

X

CAA22110

1,144

(4/6TM-ABC)2

Partial

 

D

 

C44B7.8

II

AAA68339

665

4TM-ABC

Partial

 
  

C44B7.9

II

AAA68340

661

4TM-ABC

Partial

None

  

C54G10.3

V

CAA99810

660

6TM-ABC

Complete

None

  

T02D1.5

IV

CAB0590

734

6TM-ABC

Partial

None

  

T10H9.5

V

AAC19238

598

6TM-ABC

Complete

None

E

 

Y39E4B.1

III

CAB54424

610

ABC-ABC

Partial

Embryonic lethality

F

 

F18E2.2

V

CAA99835

622

ABC-ABC

Partial

None

  

F42A10.1

III

AAA19072

712

ABC-ABC

Partial

None

  

T27E9.7/ GCN20-2

III

CAB04880

622

ABC-ABC

Complete

None

G

 

C05D10.3

III

AAA20989

598

ABC-4TM

Partial

None

  

C10C6.5

IV

CAB05682

610

ABC-6TM

Partial

None

  

C16C10.12

III

CAA86750

610

ABC-4TM

Partial

None

  

F02E11.1

II

AAB66050

658

ABC-4TM

Partial

None

  

F19B6.4

IV

CAA93461

695

ABC-6TM

Partial

None

  

T26A5.1

III

AAC77504

608

ABC-4TM

Partial

None

  

Y42G9A.6

III

AAF60554

684

ABC-6TM

Partial

None

  

Y47D3A.11

III

CAB57891

547

ABC-6TM

Partial

None

  

Y49E10.9

III

CAB11549

454

ABC-4TM

 

None

H

 

C56E6.1

II

AAA81093

1,667

ABC-12TM

 

Larval arrest

  

C56E6.5

II

AAA81094

595

ABC-6TM

Partial

None

Subfamily names given are according to the HUGO nomenclature (A-H) [33] as well as the CGC (Caenorhabditis Genetics Centre [61]) gene names for each subfamily. TM, transmembrane domain, where the number preceding it is the predicted number of membrane spanning helices or the number in the amino-terminal/carboxy-terminal TM domains, respectively. ABC, ATP-binding cassette. The existence of known cDNAs, whether complete or partial, is listed according to information in WormBase release WS112 [62]. RNAi phenotypes of genes on chromosome I are given according to [63], those on chromosomes II, IV, V and X are from [64], those of genes on chromosome III are from [65].

Thirty ABC transporters are described in the yeast genome, or approximately 0.5% of its approximately 6,000 proteins [13]. At present 49 human ABC transporters have been identified and, at least partially, cloned. They are included here (Figures 3,4,5,6,7 and Table 2) to illustrate their relationships with nematode proteins, which might then shed light on their biological roles. Inclusion of human as well as D. melanogaster ABC transporters in our tree allows us to explicitly classify C. elegans ABC transporters according to the current eight-subfamily taxonomic scheme for ABC transporters [12].
https://static-content.springer.com/image/art%3A10.1186%2Fgb-2004-5-3-r15/MediaObjects/13059_2003_Article_819_Fig3_HTML.jpg
Figure 3

Phylogenetic tree of ABCA proteins in three eukaryote genomes. A phylogeny derived and displayed according to the procedure outlined in the legend to Figure 2, except that complete protein sequences were used, not just those of the ATP-binding cassettes. The genome of origin for each protein is indicated by prefixes before each gene name, according the following scheme: Ce, C. elegans; Dm, D. melanogaster; Hs, H. sapiens; Sc, S. cerevisiae.

https://static-content.springer.com/image/art%3A10.1186%2Fgb-2004-5-3-r15/MediaObjects/13059_2003_Article_819_Fig4_HTML.jpg
Figure 4

Phylogenetic tree of ABCB proteins in four eukaryote genomes. A phylogeny derived and displayed according to the procedure outlined in the legend to Figure 3. Shown here is the division between the half transporters, which are most of the ABCB genes in mammals, and the full-transporters (called P-glycoproteins (P-gps)) that have evolved from them. Four lineages of P-gps (exemplified by genes F22E10.1-4, T21E8.1-3, C47A10.1 and C54D1.1) have been lost in both flies and mammals, and of the two remaining P-gp lineages, one has been lost in each of the fly and human lines of descent. Subsequent duplications within the single remaining P-gp lineage in both flies and mammals have not been sufficient to keep pace with continuing P-gp duplications in the worm genome.

https://static-content.springer.com/image/art%3A10.1186%2Fgb-2004-5-3-r15/MediaObjects/13059_2003_Article_819_Fig5_HTML.jpg
Figure 5

Phylogenetic tree of ABCC proteins in four eukaryote genomes. A phylogeny derived and displayed according to the procedure outlined in the legend of Figure 3.

https://static-content.springer.com/image/art%3A10.1186%2Fgb-2004-5-3-r15/MediaObjects/13059_2003_Article_819_Fig6_HTML.jpg
Figure 6

Phylogenetic trees of ABCD, ABCE, and ABCF proteins in four eukaryote genomes. Phylogenies derived and displayed according to the procedure outlined in the legend of Figure 3.

https://static-content.springer.com/image/art%3A10.1186%2Fgb-2004-5-3-r15/MediaObjects/13059_2003_Article_819_Fig7_HTML.jpg
Figure 7

Phylogenetic trees of ABCG and ABCH proteins in four eukaryote genomes. Phylogenies derived and displayed according to the procedure outlined in the legend of Figure 3.

Table 2

Alphabetic list, by taxon, of protein sequences used in this study

S. cerevisiae

Accession number

D. melanogaster

Accession number

C. elegans

Accession number

H. sapiens

Accession number

ADP1

NP_009937

171D11.2

AAF45509

C05A9.1

CAA94202

ABCA1

NP_005493

ATM1

NP_014030

Atet

AAF51027

C05D10.3

AAA20989

ABCA2

NP_001597

BPT1

NP_013086

Brown

AAF47020

C10C6.5

CAB05682

ABCA3

CAA65825

CAF16

NP_116625

CG10226

AAF50670

C16C10.12

CAA86750

ABCA5

NP_061142

GCN20

NP_116664

CG10441

AAF53737

C18C4.2

AAK52175

ABCA6

NP_525023

MDL1

NP_013289

CG10505

AAF46706

C24F3.5

CAA18775

ABCA7

AF328787

MDL2

NP_015053

CG11069

AAF56361

C30H6.6

CAB02812

ABCA8

AB020629

PDR10

NP_014973

CG11147

AAF52284

C34G6.4

AAB52482

ABCA9

NP_525022

PDR11

NP_012252

CG11460

AAF55727

C44B7.8

AAA68339

ABCA10

XP_085647

PDR12

NP_015267

CG11897

AAF56869

C44B7.9

AAA68340

ABCA12

NP_056472

PDR15

NP_010694

CG11898

AAF56870

C47A10.1

CAB03973

ABCA13

NP_689914

PDR5

NP_014796

CG12703

AAF49018

C48B4.4

CAA82384

ABCB5

AAO73470

PXA1

NP_015178

CG14709

AAF54656

C54D1.1

AAC48149

ABCB7

AB005289

PXA2

NP_012733

CG1494

AAF50838

C54G10.3

CAA99810

ABCB9

AC002486

SNQ2

NP_010294

CG1703

AAF48069

C56E6.1

AAA81093

ABCC10

NP_258261

Ste6

NP_012713

CG1718

AAF50837

C56E6.5

AAA81092

ABCC11

NP_149163

YBT1

NP_013052

CG17338

AAF53736

DH11.3

CAA88940

ABCC12

NM_033226

YCF1

NP_010419

CG17646

AAF51341

E03G2.2

CAA92148

ABCC13

NP_742021

YDR061w

NP_010346

CG1801

AAF50836

F02E11.1

AAB66050

ABCF1

AAH34488

YDR091C

NP_010376

CG1819

AAF50847

F12B6.1

AAB54153

ABCF2

NP_005683

yEF3

NP_013350

CG1824

AAF48177

F14F4.3

CAB54225

ABCF3

NP_060828

yEFB

P53978

CG18633

AAF56360

F18E2.2

CAA99835

ABCG5

AF320293

YER036C

NP_010953

CG2316

AAF59367

F19B6.4

CAA93461

ABCG8

AF320294

YHL035C

NP_011828

CG3164

AAF51548

F20B6.3

AAA82317

ABCR (A4)

AF001945

YKR103W

NP_013030

CG3327

AAF51122

F21G4.2

CAB02667

ALDP (D1)

CAA79922

YNR070w

NP_014468

CG4225

AAF55241

F22E10.1

CAA91799

ALDR (D2)

NP_005155

YOL075C

NP_014567

CG4562

AAF55707

F22E10.2

CAA91800

BCRP (G2)

XP_032425

YOR011w

NP_878167

CG4794

AAF55726

F22E10.3

CAA91801

BSEP (B11)

AF091582

YOR1

NP_011797

CG4822

AAF51552

F22E10.4

CAA91802

CFTR (C7)

AAC13657

YPL226W

S65245

CG5651

AAF50342

F42A10.1

AAA19072

MABC1 (B8)

AF047690

  

CG5789

AAF56312

F42E11.1

CAA91463

MABC2 (B10)

XP_001871

  

CG5853

AAF52835

F43E2.4

AAC71121

MDR1 (B1)

4505769

  

CG5944

AAF49305

F55G11.9

CAB05222

MDR3 (B4)

AAA36207

  

CG6052

AAF49312

F56F4.6

AAB54203

MRP1 (C1)

AAB46616

  

CG6162

AAF56584

F57A10.3

CAB09418

MRP2 (C2)

CAA65259

  

CG6214

AAF53223

F57C12.4

AAB07022

MRP3 (C3)

AB010887

  

CG7346

AAF50035

F57C12.5

AAD31550

MRP4 (C4)

NP_005836

  

CG7491

AAF53328

K08E7.9

CAB01232

MRP5 (C5)

AAB71758

  

CG7627

AAF52648

T02D1.5

CAB05909

MRP6 (C6)

AF076622

  

CG7806

AAF52639

T10H9.5

AAC19238

MTABC3 (B6)

NP_005680

  

CG7955

AAF47525

T21E8.1

CAA94220

PMP69 (D4)

AF009746

  

CG8473

AAF48511

T21E8.2

CAA94219

PMP70 (D3)

CAA41416

  

CG8799

AAF58947

T21E8.3

CAA94203

RNAse LI (E1)

CAA53972

  

CG8908

AAF57490

T26A5.1

AAC77504

SUR1 (C8)

AAB02278

  

CG9270

AAF53950

T27E9.7

CAB04880

SUR2 (C9)

AF061323

  

CG9281

AAF48493

W04C9.1

AAC68724

TAP1 (B2)

CAA40741

  

CG9330

AAF49142

W09D6.6

CAB04947

TAP2 (B3)

AAA59841

  

CG9663

AAF51130

Y39D8C.1

AAC69223

WHITE 1 (G1)

AAC51098

  

CG9664

AAF51131

Y39E4B.1

CAB54424

WHITE 2 (G4)

NP_071452

  

CG9892

AAF51223

Y42G9A.6

NP_498332

  
  

CG9990

AAF56807

Y43F8C.12

CAA21622

  
  

Mdr49

AAF58437

Y47D3A.11

CAB57891

  
  

Mdr50

AAF58271

Y48G8AL.11

AAK29911

  
  

Mdr65

AAF50669

Y49E10.9

CAB11549

  
  

Scarlet

AAF49455

Y50E8A.16

CAB60586

  
  

Sur

AAF52866

Y53C10A.9

CAA22142

  
  

White

AAF45826

Y57G11C.1

CAB16503

  
    

Y75B8A.26

CAA22110

  
    

ZK455.7

CAA91467

  
    

ZK484.2

AAK39394

  

Typing ABCs to subfamily

We define membership of a particular gene in an ABC transporter subfamily primarily on the basis of the position of its ATP-binding domains in our first phylogenetic tree (not shown). Genes that fell unambiguously within a clade containing genes already assigned to given subfamily, were included in that subfamily. Where we could not assign a gene to a particular clade with a significant bootstrap value, the assignment was made on the basis of which subfamily's members scored highest when that gene was used as query in a BLAST search. The subfamilies are sometimes named according to the well-characterized mammalian genes that typify each of them, for example, P-gp (P-glycorprotein), MRP, White gene homologs, RNAse L inhibitor, GCN20 homologs, ABC1 and ALDP [7]. These correspond to the HUGO-defined subfamilies B, C, G, E, F, A and D, respectively. Re-analysis of the full-length sequences confirmed the placement all C. elegans genes within the preexisting subfamilies, with substantial bootstrap support (Figures 3,4,5,6,7).

Instances of orthology

In the set of worm and human ABC transporters, only 8 of 49 possible pairs (16%) of sister genes contained a single human protein and a nematode homolog (Table 3). Similarly, 10% of ABC transporters were found in orthologous pairs when the comparison is made between yeast and worm genomes. A more comprehensive comparison of worm and yeast genomes [23] came to the overall conclusion that 57% of genes in highly conserved gene families were found in orthologous pairs, and the study suggested that such gene families provide a conserved core proteome which forms the basis of eukaryote biochemistry. ABC transporters are conserved in all eukaryotic and prokaryotic genomes, so it is interesting to note that they are found in orthologous pairs much less frequently than most gene families that are roughly as well conserved. Clearly, ABC transporter evolution has not been typical of strongly conserved gene families, and while we might have inferred that ABC-transporter-mediated metabolism differs radically among eukaryotes, this seems improbable, given the broadly comparable set of substrates associated with ABC transporters in all eukaryotes where they have been studied.
Table 3

Frequency of orthologous pairs among ABC transporters

 

Sc

Ce

Dm

Hs

Sc

 

10%

17%

10%

Ce

3

 

14%

16%

Dm

5

8

 

22%

Hs

5

8

11

 

Numbers below the diagonal represent the number of orthologous pairs of ABC transporters, according to our phylogeny, found in pairwise comparisons between each of the four genomes in this study. Percentages above the diagonal are calculated from the corresponding number given for that pair, divided by the smaller of the two counts of ABC transporters in that pair of genomes. Ce, C. elegans; Dm, D. melanogaster; Hs, H. sapiens; Sc, S. cerevisiae.

Within the P-gp-related ABCB subfamily, the only one-to-one pairings found between C. elegans and human genes are those of W09D6.6 (Haf-5) and MTABC3 (B6), and Y48G8AL.11 (Haf-6) and MABC1 (B8). These are both half-transporters localized to the mitochondria. MTABC3 (B6) is involved in iron homeostasis [24] and its rat ortholog, PRP, is overexpressed during hepatocarcinogenesis [25]. Two other mitochondrial ABC transporters in humans, MABC2 (B10) and ABCB7, have orthologs in flies and/or yeast, but not nematodes.

Among ABCC molecules, whose range of functions broadly overlaps with P-gps, only C18C4.2 (Cft-1) and CFTR (C7) are indicated as orthologs in our analysis. However, the bootstrap value on this pairing is very low (51%, see Figure 5), so we cannot attach much confidence to this observation. It may simply be that C18C4.2 (Cft-1) is a highly divergent member of subfamily C, and does not bear much functional similarity to CFTR (C7). Although not forming simple pairs with any nematode gene, human MRP5 (C5), a transporter of nucleotide analogs [26, 27], and ABCC11 and ABCC12 appear to be co-orthologous to worm F14F4.3 (Mrp-5), which may provide some hint as to the function of the latter.

All four of the C. elegans members of subfamilies E and F (Figure 6) form strongly supported and unambiguous pairs with their homologs in D. melanogaster, Homo sapiens, and yeast. This unusually strong conservation, compared to the other subfamilies of ABC genes, argues for involvement in something indispensable, at least on an evolutionary timescale. The three genes in subfamily F, which lack transmembrane domains, are generally regarded as forming ribosome associated proteins involved in regulation of mRNA translation, rather than transporters. The RNase L inhibitor (E1), also known as the oligoadenylate-binding protein (OABP), is thought to be involved in the regulation of the interferon-induced antiviral response [28] that bears some similarities to the mechanism thought to underlie the now common molecular biology technique of double-stranded RNA-directed interference (RNAi). It also seems to have a role in muscle differentiation [29] in mammals. The critical role of the RNase L inhibitor is underlined by its conservation even in a highly reduced genome. In the rather minimal genome of the endosymbiotic Guillardia theta nucleomorph (302 genes) the RNase L inhibitor is the only ABC protein found [30]. The yeast ortholog of the RNase L inhibitor protein, YDR091c, is essential for growth, as is YER036c, the yeast ortholog of T27E9.7/ABCF2 [31]. On the other hand GCN20, the yeast version of F42A10.1/ABCF3, is not essential, although mutants do have specific defects in translation.

Processes of gene duplication and loss

While the conservation of simple orthologous gene pairs is a rare observation in our study, the numbers of genes in most ABC transporter subfamilies are about the same, despite numerous instances of gene duplication and loss. For example, within ABCB the number of half-transporters in each genome is almost identical. Furthermore, most mammalian half-transporters in subfamily B are found in clusters of functionally related, or at least co-localized, genes (the TAP (B2 and B3) genes, and the four mitochondrial ABCB genes, MABCs1 and 2 (B8 and B10), MTABC3 (B6) and ABCB7 [32]), paired with similarly sized groups of C. elegans genes. Likewise the number of genes in subfamilies A, C and D is much the same between genomes. However, it does appear that C. elegans, relative to humans, has undergone a massive expansion in the P-gp (full or pseudo-dimer configuration) subclass of subfamily B, and subfamily G, the 'White-like' genes. The likelihood that ABC transporter lineages have been lost repeatedly in evolution is evident from the phylogeny. The single group of P-gps in mammals contains only four members, while C. elegans has 15 P-gps, of which only three are closely related to their mammalian homologs. A literal reading of the tree (Figure 4) would suggest the presence of five additional P-gp lineages in the common ancestor of nematodes, flies and mammals that have been lost, independently, in both mammals and flies. These losses, and the species-specific expansion of the remaining lineages of genes, underlines the peculiarly dynamic composition of this group of multifunctional transport proteins.

Conclusions

The completion of the C. elegans and D. melanogaster genome projects [33, 34] make it possible to analyze entire gene families in metazoans. The advantage of performing a combined analysis of all known ABC proteins from two organisms is that it allows unambiguous identification of orthologous pairs of genes, as well as allowing the pattern of evolution by a process of gene duplication, lineage sorting, and functional convergence to be explicitly modeled.

Saurin et al. [35] surveyed the ABC transporters, considering both eukaryotic and prokaryotic systems, and found that there is a fundamental phylogenetic division among ABC transporters involved in import versus export processes. The importer class of ABCs is found only in prokaryotes, whereas exporters are found in all domains of life [35]. However, that survey, while covering all classes of ABC transporter, was not comprehensive with respect to any of the organisms surveyed. Most recently, Schriml and Dean [10] compared the human ABC family to that of the mouse Mus musculus, and found almost perfect identity between the two genomes. We have integrated previous information with the complete inventory of ABC transporters from the genome of the nematode worm C. elegans. We find that most of the ABC transporters in the worm can be classified into the existing human transporter taxonomy. We find 60 ABC transporters in the worm genome, representing an overall doubling in size of the ABC transporter family relative to yeast, whose genome contains one third as many protein-coding genes. No ABC genes were found that could be classified among the bacterial import proteins.

At least three subfamilies of ABC transporter contain members capable of a conferring an MDR phenotype, and transporters from at least two different subfamilies cause MDR in human tumors [36]. A multi-drug transporter is a single protein capable of specifically recognizing several structurally distinct classes of compounds, and which catalyzes their efflux from the cell or sequestration in a subcellular compartment. Proteins of the P-glycoprotein (P-gp) group (ABCB) transport hydrophobic compounds and function in transport of lipids and bile from the liver as well as generally defending the body from toxic natural products in the diet [37]. P-gps are also a component of the blood-brain barrier and function in tolerance of drugs normally minimally toxic to mammals, such as ivermectin [38]. Multi-drug resistance mediated by MRP group (ABCC) proteins depends on a slightly different mechanism. MRPs seem to function by co-transporting toxic compounds with glutathione, or as glutathione conjugates [36]. An MDR phenotype is also associated with some members of the ABCG group of transporters, in both yeast [39] and humans [40]. The MDR phenotype appears to have evolved not just once, but at least three times in the history of ABC transporters. Given the distribution of MDR-causing and non-MDR genes among mammalian P-gps; it seems reasonable to infer that MDR genes may well have arisen more than once among the P-gps themselves. It has been observed [41, 42] that the entire ABC transporter family is characterized by a highly adaptable common mechanism for coupling substrate binding to ATP hydrolysis and extrusion. It has been pointed out that, because P-gp recognizes substrate directly within the cytoplasmic leaflet of the plasma membrane [43], it does so at a much higher effective substrate concentration than would be the case if it recognized aqueous substrate. As a result, P-gp drug-binding sites can operate at relatively low affinity, and this, in turn, facilitates recognition of multiple substrates. This flexibility may be the key to explaining the range of tasks performed by ABC transporters, but also their apparently anomalous evolutionary history.

The mammalian P-gps include proteins capable of producing an MDR phenotype (MDR1 (B1)), as well as members with, apparently, specificity restricted to single physiological substrates such as phosphatidylcholine (MDR3 (B4)). As none of these have simple, orthologous, relationships with any of the C. elegans P-gps, no detailed predictions of function in nematode P-gps can be drawn on the basis of phylogeny alone. C. elegans P-gps do differ from one another in their ability to cause resistance to various environmental toxins [16], with no apparent correlation between phenotype and genetic distance from their mammalian homologs. Both human abca1 and nematode ced-7 mutants present similar apoptotic phenotypes, despite their rather distant relationship (Figure 3). ABCA1 mutations also cause defects in high-density lipoprotein cholesterol transport, and it is still an open question as to whether the analogous function of these two homologs in apoptosis accurately predicts a sharing of other functions. Similar limitations on the extent to which function may be predicted from sequence alone are likely to obtain in those subfamilies whose members are noted for variability and multiplicity of function, that is, subfamilies A, B, C and G.

Schriml and Dean [10] speculated that the distinct clustering of amino- and carboxy-terminal halves of ABCA proteins suggests that full ABC transporters have generally evolved from half-transporters. The pattern of structural change within the closely related subfamilies ABCD, ABCC and ABCB does suggest that the half-transporter configuration was the ancestral one for at least these three subfamilies (Figure 2). It also reveals instances where half-transporters have evolved from duplicated genes, as in the origination of ABCB from a fragment of an ABCC gene, and that, in turn, some ABCB genes have duplicated again, in giving rise to the P-gp genes.

A comprehensive comparison of worm and yeast genomes [23] noted that while most of the nematode genome did not closely resemble that of yeast, there was a strongly conserved 20% of the nematode genome that had a high degree of homology to a corresponding 40% of the yeast genome. Within this highly conserved subset of genes, there was a very frequent finding of orthology between members of the two genomes. As many as 57% of the most closely related gene pairs contained exactly one worm and one yeast gene. The obvious inference is that one corresponding gene was present in the common ancestor of the two species. Their overall picture of genome evolution is one in which a conserved cadre of proteins performs core biological functions required by all eukaryotes. These would remain essentially invariant throughout eukaryotes, and one expects analogous functions to be carried out by orthologous genes across large evolutionary distances. These gene families are presumably protected over the long run by their essential and irreplaceable roles in basic biochemical functions required by all organisms. However, as Chervitz et al. [23] point out, only a minority of gene families fit this mode, with most genes belonging to poorly conserved or taxonomically restricted families.

We expected that the frequency of simple orthologous gene pairs typical of highly conserved gene families shared by both yeast and worm would hold true for our comparison between nematode and human versions of such a highly conserved gene family as ABC transporters. However, this generality clearly does not apply to ABC transporters, despite their strong conservation across all domains of life. It seems reasonable to suppose that the rather loose relationship between substrate specificity and amino acid sequence that characterizes ABC transporters allows for much more potential exchange and sorting of biological functions among homologous genes than is typical. In turn, this pervasive pre-adaptation for functional overlap enables organisms to survive the occasional loss of substantial numbers of ABC transporters and to rapidly re-evolve lost functionality by co-opting homologous genes.

The evolutionary dynamic we propose here is reminiscent of an explanation put forward by Huynen et al. [44] to explain a pattern observed in a comparative analysis of 11 microbial genomes. They found that the frequency distribution of gene-family sizes within each completely sequenced genome tended to follow a power-law distribution across a 30-fold range of genome sizes. Their model is one in which genes are duplicated or deleted randomly in time, but the gene families are coherent with respect to the probability of duplication or deletion in each time unit in the simulation. In other words, the probability of duplicating or deleting a gene may change over time, but every member of a gene family always has the same probability of duplication or deletion as every other member of the family. So, whereas a given family can be either favored for expansion or targeted for deletion in a given time period, all members of the family are equally favored or disfavored by selection at the same time. Huynen et al. argued that this property of 'dynamic coherence' in a gene family could arise if all gene-family members have more or less the same function, so that they are all favored or disfavored by selection at the same time, depending on how much that function is needed.

Under a power-law distribution, gene families would tend to be subject to fluctuations of a size on the same order as the gene-family size itself [44]. We should then expect that typical gene families will have undergone very substantial episodes of expansion and near-extinction, and in Huynen et al.'s model all gene families do become extinct within a finite time. It is evident that ABC transporters are highly atypical for a strongly conserved gene family, in that the family as a whole is highly conserved across genomes despite being subject to the same large fluctuations in size, which would tend to eventually eliminate gene families whose members are not individually indispensable. It should be noted that the ABC family does not seem uniformly subject to one or the other mode of evolution. Subfamilies E and F, which are not involved with transport, but rather have roles in translation and gene regulation, fit the 'strongly conserved' [23] model very well, retaining simple orthologous relationships over long spans of time. Only the transporter subfamilies themselves, because of their highly adaptable substrate-recognition capability, are subject to large fluctuations in size. We propose that finding large sets of paralogous genes, and infrequently conserved orthologs, in a gene family reflects ongoing cycles of gene loss and reacquisition of analogous functions in distantly related, newly expanded, lineages. Furthermore, we suggest that this is in fact the expected outcome of dynamic coherence, a mode shared, perhaps, by most of the less-conservative gene families, as well as the ABC genes.

We expect that future functional studies, to determine the extent of parallel and convergent evolution among ABC transporters, will eventually allow us to discern the fundamental roles of ABC transporters that ensure their long-term survival as a group. Also of interest will be whether the functional suites of genes fulfilling these roles are bounded in any way that resembles the phylogenetic subdivisions into which we presently categorize these proteins.

Materials and methods

Identification of ABC transporter genes

A computer file, WormPep16 [45], containing 16,332 protein sequences predicted from the completed C. elegans genome was searched using the FASTA program [46]. Our initial query sequences were those of known C. elegans ABC proteins (for example, Pgp-1, the D. melanogaster white gene homolog T26A5.1, and so on). Matching protein sequences returned by FASTA were checked by BLAST [47], using either the NCBI [48] or Baylor College of Medicine (BCM) servers [49]. Only those with highly significant matches to annotated ABC proteins in the sequence database were retained. The most poorly matched, verified ABC protein from each FASTA run was used as the query sequence for an additional FASTA search, and this process was repeated until no new ABC proteins were found. At a later stage in the analysis, representative members of different ABC transporter subfamilies were used as query sequences to search the updated WormPep81 file using a BLAST server at the Sanger Centre [45]. Searches were conducted using multiple queries until all proteins already included in our dataset were found. No additional ABC proteins were identified, though some sequences were found to have been included in our dataset twice under different names. These redundant sequences were eliminated. FASTA searches were run on a SUN Microsystems UltraSPARC 5 computer. All other computer operations were carried out on an Apple Power Macintosh G3. Yeast and human ABC transporter sequences were obtained from NCBI and are described in the literature [10, 13].

Identification of ABC protein features

BLAST + Beauty searches on the BCM server identified the location of the conserved Walker A and ABC signature motifs (Prosite motifs [50] PS00017 and PS00211, respectively) associated with the ATP-binding cassette(s) of each protein. The number and positions of transmembrane domains in each ABC protein were predicted by using TopPred II v1.3 [51] and then vetting the program's results by eye to exclude spurious transmembrane segments. Chromosomal locations of each ABC protein in the C. elegans genome were looked up in the C. elegans database AceDB [52].

Phylogenetic analyses

Using the information derived from each protein sequence (as above) we extracted only the sequence of each predicted ATP-binding cytoplasmic domain. These domains were assembled into a single file using the SeqApp1.9 multiple sequence editor [53], and aligned using ClustalX [54]. In those cases where two ATP-binding cassettes (ABCs) are present in a single protein with no intervening transmembrane domains (Subfamilies E and F, see Figure 1), the entire sequence was divided into two at an arbitrary point halfway between the two predicted ABC domains. As a result, 'two-domain' proteins are represented twice in our initial analysis. Once this approach had been used to assign genes to particular well-supported subgroups, we realigned the sequences and reanalyzed the relationships within each group using full-length amino acid sequence data.

Aligned sequences were used to generate matrices of mean distances between proteins, and these matrices were used to generate phylogenetic trees according to the neighbour-joining algorithm [55], refined using the SPR branch-swapping technique under the minimum evolution criterion, implemented by PAUP*4.0b10 [56]. Bootstrapping (1,000 replicates) was done according to the method of Felsenstein [57], using the same parameters described above. Phylogenetic trees were visualized and manipulated using TreeView 1.6.2 [58] and MacClade 3.0.4 [59].

Additional data files

The following additional data are included with the online version of this article: the protein sequence alignments for the ABCA subfamily (Additional data file 1), the ABCB subfamily (Additional data file 2), the ABCC subfamily (Additional data file 3), the ABCD subfamily (Additional data file 4), the ABCE and ABCF subfamilies (Additional data file 5), the ABCG subfamily (Additional data file 6), the ABCH subfamily (Additional data file 7), and the protein sequences from the nucleotide-binding folds only (Additional data file 8). In addition to the four genomes discussed in this paper, mouse (M. musculus) ABC transporter genes are included in some of these alignments. All eight files are in Nexus format, which is a plain-text format designed for use with the programs PAUP [56] and MacClade [59]. A Nexus Data Editor for Windows is also available [60].

Declarations

Acknowledgements

We thank Fang Zhang, whose insight and curiosity were essential, on more than one occasion, to the initiation and completion of this work. We are grateful to Yuji Kohara for the elucidation of C. elegans cDNAs. The helpful comments of anonymous reviewers made a substantial contribution to the final draft.

Authors’ Affiliations

(1)
British Columbia Cancer Research Centre, BC Cancer Agency
(2)
Department of Molecular Biology and Biochemistry, Simon Fraser University
(3)
Genome BC and the Departments of Botany and Forest Sciences, University of British Columbia

References

  1. Blattner FR, Plunkett G, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, et al.: The complete genome sequence ofEscherichia coliK-12.Science 1997, 277:1453–1474.View ArticleGoogle Scholar
  2. Croop JM: Evolutionary relationships among ABC transporters.Methods Enzymol 1998, 292:101–116.View ArticleGoogle Scholar
  3. Higgins CF: ABC transporters: from microorganisms to man.Annu Rev Cell Biol 1992, 8:67–113.View ArticleGoogle Scholar
  4. Childs S, Ling V: The MDR superfamily of genes and its biological implications.In Important Advances in Oncology(Edited by: DeVita VT, Hellman S, Rosenberg SA). Philadelphia: J.B. Lippincott 1994, 21–36.Google Scholar
  5. Linton KJ, Higgins CF: TheEscherichia coliATP-binding cassette (ABC) proteins.Mol Microbiol 1998, 28:5–13.View ArticleGoogle Scholar
  6. Decottignies A, Goffeau A: Complete inventory of the yeast ABC proteins.Nat Genet 1997, 15:137–145.View ArticleGoogle Scholar
  7. Allikmets R, Gerrard B, Hutchinson A, Dean M: Characterization of the human ABC superfamily: isolation and mapping of 21 new genes using the expressed sequence tags database.Hum Mol Genet 1996, 5:1649–1655.View ArticleGoogle Scholar
  8. Nomenclature for Human ABC-Transporter Genes[http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html]
  9. Klein I, Sarkadi B, Varadi A: An inventory of the human ABC proteins.Biochim Biophys Acta 1999, 1461:237–62.View ArticleGoogle Scholar
  10. Schriml LM, Dean M: Identification of 18 mouse ABC genes and characterization of the ABC superfamily inMus musculus.Genomics 2000, 64:24–31.View ArticleGoogle Scholar
  11. Broccardo C, Luciani M, Chimini G: The ABCA subclass of mammalian transporters.Biochim Biophys Acta 1999, 1461:395–404.View ArticleGoogle Scholar
  12. Dean M, Rzhetsky A, Allikmets R: The human ATP-binding cassette (ABC) transporter superfamily.Genome Res 2001, 11:1156–1166.View ArticleGoogle Scholar
  13. Bauer BE, Wolfger H, Kuchler K: Inventory and function of yeast ABC proteins: about sex, stress, pleiotropic drug and heavy metal resistance.Biochim Biophys Acta 1999, 1461:217–236.View ArticleGoogle Scholar
  14. Lincke CR, The I, Vangroen M, Borst P: The P-glycoprotein gene family ofCaenorhabditis elegans-cloning and characterization of genomic and complementary DNA sequences.J Mol Biol 1992, 228:701–711.View ArticleGoogle Scholar
  15. Broeks A, Gerrard B, Allikmets R, Dean M, Plasterk RHA: Homologues of the human multidrug resistance genes MRP and MDR contribute to heavy metal resistance in the soil nematodeCaenorhabditis elegans.EMBO J 1996, 15:6132–6143.Google Scholar
  16. Broeks A, Janssen HWRM, Calafat J, Plasterk RHA: A P-glycoprotein protectsCaenorhabditis elegansagainst natural toxins.EMBO J 1995, 14:1858–1866.Google Scholar
  17. Mahajan-Miklos S, Tan M-W, Rahme LG, Ausubel FM: Molecular mechanisms of bacterial virulence elucidated using aPsudomonas auruginosa-Caenorhabditis eleganspathogenesis model.Cell 1999, 96:47–56.View ArticleGoogle Scholar
  18. Lincke CR, Broeks A, The I, Plasterk RHA, Borst P: The expression of two P-glycoprotein (pgp) genes in transgenicCaenorhabditis elegansis confined to intestinal cells.EMBO J 1993, 12:1615–1620.Google Scholar
  19. Wu Y, Horvitz HR: TheC. eleganscell corpse engulfment geneced-7encodes a protein similar to ABC transporters.Cell 1998, 93:951–960.View ArticleGoogle Scholar
  20. Hamon Y, Broccardo C, Chambenoit O, Luciani M-F, Toti F, Chaslin S, Freyssinet J-M, Devaux PF, McNeish J, Marguet D, Chimini G: ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine.Nat Cell Biol 2000, 2:399–406.View ArticleGoogle Scholar
  21. Fitch W: Distinguishing homologous from analogous proteins.Syst Zool 1970, 19:99–113.View ArticleGoogle Scholar
  22. Taylor JS, Van de Peer Y, Braasch I, Meyer A: Comparative genomics provides evidence for an ancient genome duplication event in fish.Philos Trans R Soc London B Biol Sci 2001, 356:1661–1679.View ArticleGoogle Scholar
  23. Chervitz SA, Aravind L, Sherlock G, Ball CA, Koonin EV, Dwight SS, Harris MA, Dolinski K, Mohr S, Smith T, et al.: Comparison of the complete protein sets of worm and yeast; orthology and divergence.Science 1998, 282:2022–2028.View ArticleGoogle Scholar
  24. Mitsuhashi N, Miki T, Senbongi H, Yokoi N, Yano H, Miyazaki M, Nakajima N, Iwanaga T, Yokoyama Y, Shibata T, Seino T: MTABC3, a novel mitochondrial ATP-binding cassette protein involved in iron homeostasis.J Biol Chem 2000, 275:17536–17540.View ArticleGoogle Scholar
  25. Furuya KN, Bradley G, Sun D, Schuetz EG, Schuetz JD: Identification of a new P-glycoprotein-like ATP-binding cassette transporter gene that is overexpressed during hepatocarcinogenesis.Cancer Res 1997, 57:3708–3716.Google Scholar
  26. Wijnholds J, Mol CA, van Deemter L, de Haas M, Scheffer GL, Baas F, Beijnen JH, Scheper RJ, Hatse S, De Clercq E, et al.: Multidrug-resistance protein 5 is a multispecific organic anion transporter able to transport nucleotide analogs.Proc Natl Acad Sci USA 2000, 97:7476–7481.View ArticleGoogle Scholar
  27. Jedlitschky G, Burchell B, Keppler D: The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides.J Biol Chem 2000, 275:30069–30074.View ArticleGoogle Scholar
  28. Le Roy F, Laskowska A, Silhol M, Salehzada T, Bisbal C: Characterization of RNABP, an RNA binding protein that associates with RNase L.J Interferon Cytokine Res 2000, 20:635–644.View ArticleGoogle Scholar
  29. Bisbal C, Silhol M, Laubenthal H, Kaluza T, Carnac G, Milligan L, Le Roy F, Salehzada T: The 2'-5' oligoadenylate/RNase L/RNase L inhibitor pathway regulates both MyoD mRNA stability and muscle cell differentiation.Mol Cell Biol 2000, 20:4959–4969.View ArticleGoogle Scholar
  30. Douglas S, Zauner S, Fraunholz M, Beaton M, Penny S, Deng LT, Wu X, Reith M, Cavalier-Smith T, Maier UG: The highly reduced genome of an enslaved algal nucleus.Nature 2001, 410:1091–1096.View ArticleGoogle Scholar
  31. SaccharomycesGenome Database[http://genome-www.stanford.edu/Saccharomyces]
  32. Allikmets R, Raskind WH, Hutchinson A, Schueck ND, Dean M, Koeller DM: Mutation of a putative mitochondrial iron transporter gene (ABC7) in X-linked sideroblastic anemia and ataxia (XLSA/A).Hum Mol Genet 1999, 8:743–749.View ArticleGoogle Scholar
  33. Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, Amanatides PG, Scherer SE, Li PW, Hoskins RA, Galle RF, et al.: The genome sequence ofDrosophila melanogaster.Science 2000, 287:2185–2195.View ArticleGoogle Scholar
  34. The C. elegans Sequencing Consortium: Genome sequence of the nematodeC. elegans: a platform for investigating biology.Science 1998, 282:2012–2018.View ArticleGoogle Scholar
  35. Saurin W, Hofnung M, Dassa E: Getting in or out: early segregation between importers and exporters in the evolution of ATP-binding cassette (ABC) transporters.J Mol Evol 1999, 48:22–41.View ArticleGoogle Scholar
  36. Cole SPC, Deeley R: Multidrug resistance mediated by the ATP-binding cassette transporter protein MRP.BioEssays 1998, 20:931–940.View ArticleGoogle Scholar
  37. van Helvoort A, Smith AJ, Sprong H, Fritzsche I, Schinkel AH, Borst P, van Meer G: MDR1 P-glycoprotein is a lipid translocase of broad specificity, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine.Cell 1996, 87:507–517.View ArticleGoogle Scholar
  38. Schinkel AH: The physiological function of drug-transporting P-glycoproteins.Semin Cancer Biol 1997, 8:161–170.View ArticleGoogle Scholar
  39. Balzi E, Wang M, Leterme S, Van Dyck L, Goffeau A: PDR5, a novel yeast multidrug resistance conferring transporter controlled by the transcription regulator PDR1.J Biol Chem 1994, 269:2206–2214.Google Scholar
  40. Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, Rishi AK, Ross DD: A multidrug resistance transporter from human MCF-7 breast cancer cells.Proc Natl Acad Sci USA 1998, 95:15665–15670.View ArticleGoogle Scholar
  41. Loo TW, Clarke DM: Functional consequences of phenylalanine mutations in the predicted transmembrane domain of P-glycoprotein.J Biol Chem 1993, 268:19965–19972.Google Scholar
  42. Zhang F, Sheps JA, Ling V: Complementation of transport-deficient mutants ofEscherichia coliα-hemolysin by second-site mutation in the transporter hemolysin B.J Biol Chem 1993, 268:19889–19895.Google Scholar
  43. Shapiro AB, Ling V: Transport of LDS-751 from the cytoplasmic leaflet of the plasma membrane by the rhodamine-123-selective site of P-glycoprotein.Eur J Biochem 1998, 254:181–188.View ArticleGoogle Scholar
  44. Huynen MA, van Nimwegen E: The frequency distribution of gene family sizes in complete genomes.Mol Biol Evol 1998, 15:583–589.View ArticleGoogle Scholar
  45. The Sanger Institute:Caenorhabditisgenome sequencing projects[http://www.sanger.ac.uk/Projects/C_elegans]
  46. Pearson WR: Rapid and sensitive sequence comparison with FASTP and FASTA.Methods Enzymol 1990, 183:63–98.View ArticleGoogle Scholar
  47. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool.J Mol Biol 1990, 215:403–410.View ArticleGoogle Scholar
  48. NCBI BLAST home page[http://www.ncbi.nlm.nih.gov/BLAST]
  49. BCM search launcher[http://searchlauncher.bcm.tmc.edu]
  50. Hofmann K, Bucher P, Flaquet L, Bairoch A: The PROSITE database, its status in 1999.Nucleic Acids Res 1999, 27:215–219.View ArticleGoogle Scholar
  51. Claros MG, von Heijne G: TopPred II: an improved software for membrane protein structure predictions.Comput Appl Biosci 1994, 10:685–686.Google Scholar
  52. AceDB home page[http://www.acedb.org]
  53. UIBio archive: SeqApp[http://iubio.bio.indiana.edu/soft/molbio/seqapp/]
  54. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucleic Acids Res 1997, 25:4876–4882.View ArticleGoogle Scholar
  55. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees.Mol Biol Evol 1987, 4:406–425.Google Scholar
  56. Swofford DL: Paup* (Phylogenetic analysis using parsimony and other methods). Sunderland, MA: Sinauer 1997.Google Scholar
  57. Felsenstein J: Confidence limits on phylogenies: an approach using the bootstrap.Evolution 1985, 39:783–791.View ArticleGoogle Scholar
  58. Page RDM: TreeView: an application to display phylogenetic trees on personal computers.Comput Appl Biosci 1996, 12:357–358.Google Scholar
  59. Maddison WP, Maddison DR: MacClade: analysis of phylogeny and character evolution.3.0 Edition Sunderland, MA: Sinauer 1992.Google Scholar
  60. NEXUS data editor for Windows[http://taxonomy.zoology.gla.ac.uk/rod/NDE/nde.html]
  61. Genetic nomenclature forCaenorhabditis elegans[http://biosci.umn.edu/CGC/Nomenclature/nomenguid.htm]
  62. WormBase home page[http://www.wormbase.org]
  63. Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrmann M, Ahringer J: Functional genomic analysis ofC. eleganschromosome I by systemic RNA interference.Nature 2000, 408:325–330.View ArticleGoogle Scholar
  64. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, et al.: Systematic functional analysis of theCaenorhabditis elegansgenome using RNAi.Nature 2003, 421:231–237.View ArticleGoogle Scholar
  65. Gönczy P, Echeverri C, Oegema K, Coulson A, Jones SJ, Copley RR, Duperon J, Oegema J, Brehm M, Cassin E, et al.: Functional genomic analysis of cell division inC. elegansusing RNAi of genes on chromosome III.Nature 2000, 408:331–336.View ArticleGoogle Scholar

Copyright

© Sheps et al. 2004

This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.