Organisms and cDNA library

The Bristol N2 strain of C. elegans was used throughout this study according to the methods compiled by Lewis and Fleming (1995). Synchronized populations of the nematode were obtained as described (Maebuchi et al., 1999). C. elegans λ ZAPII cDNA library, made with poly(A)-rich RNA from the whole animal, was a generous gift from Dr. Alan Coulson (The Sanger Center, Cambridge, UK). Saccharomyces cerevisiae strain MB328 (MATa, his3, leu2, ura3, pex1) was isolated by the method of Erdmann et al. (1989) and identified as a pex1 mutant by genetic complementation analysis.

Cloning of the pex-1 cDNA

The primers S1 (sense, 5′-TCCCCCGGGGGAAGACGTCGGTGGAATGTTTG) and A1 (antisense, 5′-GCTCTAGAGGCTAACGTCACTTTCTGACCTATC) amplified an 808-bp sequence when the cDNA library was used as a template for polymerase chain reaction (PCR); the sequence corresponded to the open reading frame (ORF) c11h1.6. The product was cloned into the plasmid pUC119 (Takara) generating pEMB835 (Fig. 1A). The sequences extending to the 5′ and 3′ termini of the mRNA were obtained by RACE (rapid amplification of cDNA ends) (Frohman et al., 1988). Products amplified by two successive rounds of 5′ RACE were cloned into the plasmid pBluescript SK(−) (Stratagene) providing the plasmids pEMB838 and pEMB842. Primers used were as follows: S2 (5′-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT), S3 (5′-GACTCGAGTCGACATC G), A2 (5′-CGGGATCCGCCAACGAATCCAGCTCATCAAAG), A3 (5′-AACTGCAGCACTG GCTCCAATATATTTCGAC), A4 (5′-CGGAATTCCTCCAACGGATTCGAAAAGTTG), A5 (5′-AACTGCAGGTCTCGAATATTTCACAACGG), A6 (5′-CGGGATCCCTCGGCTTCTTCA GTTCTTTTCCG), and A7 (5′-AACTGCAGCGGAACGACTTTCAAACGAATTGC). The 3′ untranslated region of the pex-1 cDNA was obtained by 3′ RACE with primers S4 (5′-GGGGAGCTCTGGGTAAAATCGAAGATGGACAAG), S1, and (dT)₁₇, and cloned into pBluescript SK(−) generating the plasmid pEMB855. The probe P1 was prepared by PCR with the primers S1 and A4.

Fig. 1

Schematic structures of the composite cDNAs encoding PEX-1 (A) and PEX-6 (B). Thin lines indicate the cDNA clones (A: pEMB835, pEMB838, pEMB842, and pEMB855; B: pEMB732 and pEMB807) and thick lines indicate probes (A: P1; B: P2 and P3). The open box represents the ORF, in which bars show the first and second AAA cassettes (1 and 2), and the open rectangles denote the poly(A) sequence. Arrowheads mark positions of sequences corresponding to PCR primers. The nucleotides on the mRNA-identical strand are numbered; position +1 is A of the start codon of the ORF. The nucleotide sequences of pex-1 and pex-6 are available at DDBJ/EMBL/GenBank under the accession numbers  AB054992 and  AB010968, respectively.

Cloning of the pex-6 cDNA

A cDNA that carried the ORF f39g3.7 was obtained by screening the λZAPII cDNA library as described (Bun-ya et al., 1997) and cloned into pBluescript SK(−); the resulting plasmid was pEMB732 (Fig. 1B). The probe P2 used for screening was prepared by PCR with primers S5 (5′-GAAGAGTGCGAATGTGTTC) and A8 (5′-CCGATGAGTTTCAGAAG C). The 5′ region of pex-6 cDNA was amplified by 5′ RACE with primers S2, S3, A8, A9 (5′-CTCGGATCCGTGGTAATTGTGAAGGAGG), and A10 (5′-CTCGGATCCAGAATCCCT AGAGACCAACATG). The RACE product was identified with the probe P3 that was amplified with the primers S6 (5′-AATTGATTTATTCTACTTTGAGG) and A10, and the longest product was cloned into pBluescript SK(−) to generate the plasmid pEMB807.

Other methods

Cells of mixed-stage animals were disrupted by grinding in liquid nitrogen and the poly(A)-rich RNA was isolated using a μMACS mRNA isolation kit (Miltenyi Biotec). The pex-1 and pex-6 mRNA were detected by northern blot analysis with the antisense RNA probes, which were labeled with a digoxigenin-labeled nucleic acid detection kit (Boehringer Mannheim). The templates used for in vitro synthesis of the sense and antisense RNA probes were derivatives of pEMB842 and pEMB732; the derivatives carried the cDNA fragments of pex-1 (nucleotides +1 to +450) and pex-6 (nucleotides −70 to +305). The preparation of CeIF (C. elegans homolog of initiation factor A4) mRNA and the procedure of whole-mount in situ hybridization were performed as described (Maebuchi et al., 1999). TBLASTN searches of the C. elegans ACEDB database were run on the server at The Sanger Center ( http://www.sanger.ac.uk). The CLUSTAL W program (Thompson et al., 1994) was used for sequence alignments.

Composite cDNA encoding PEX-1 and PEX-6

We searched the C. elegans genome database for orthologs of SF2 proteins and found two open reading frames, c11h1.6 and f39g3.7, which encode polypeptides with the highest similarity to Pex1p/PEX1 and Pex6p/PEX6, respectively. Gene-specific probes were prepared by PCR amplification of a C. elegans cDNA library with appropriate primers: S1 and A1 for pex-1 (Fig. 1A) and S5 and A8 for pex-6 (Fig. 1B). Since c11h1.6 encodes only the region of the highly conserved AAA cassette, we applied the RACE procedure directly to isolate the 5′ and 3′ portions of the pex-1 cDNA. Three cDNA fragments cloned in pEMB842, pEMB838, and pEMB855 corresponded to the 5′ distal, central, and 3′ distal regions of the mRNA, respectively, and constituted a cDNA of 3391 nucleotides (nt) (Fig. 1A). The main body of pex-6 cDNA was obtained by plaque hybridization because the region encoded by f39g3.7 extended beyond the AAA cassette sequences. This clone, pEMB732, together with clone pEMB807 obtained by 5′ RACE constituted the cDNA of 2303 nt (Fig. 1B). The lengths of the pex-1 and pex-6 cDNA coincided well with those of mRNA detected by northern blot analysis of the poly(A)-rich RNA from a mixed-phase culture; 3.4 kb for pex-1 and 2.3 kb for pex-6 (Fig. 2). Therefore, we concluded that the composite cDNA corresponded to the respective full-length mRNA. No spliced leader (SL) sequence was detected at the 5′ end of either cDNA.

Fig. 2

Northern blot analysis. The poly(A)-rich RNA (about 5 μg) prepared from mixed-phase cultures was separated by electrophoresis in the presence of 2.2 M formaldehyde, transferred onto nylon membranes and probed with pex-1 (A) or pex-6 (B) digoxigenin-labeled antisense RNA. The sizes of markers are shown on the left and those of the transcripts detected are shown on the right.

Analysis of the cDNA sequences, which have been deposited in DDBJ/EMBL/GenBank under the accession numbers  AB054992 (pex-1) and  AB010968 (pex-6), revealed that the ORF of pex-6 was identical to f39g3.7 but that of pex-1 was not identical to any ORF(s) predicted by the GENEFINDER program. The pex-I cDNA (and its ORF) was composed of 24 exons, encoded by a genomic region containing three ORFs, c11h1.4, c11h1.5, and c11h1.6 (Fig. 3). The region predicting c11h1.4, which consisted of 14 exons, encoded the first 15 exons of the pex-1 ORF, ten of which were shared with c11h1.4. The spacer region between c11h1.5 and c11h1.6 encoded the 16th and 17th exons of pex-1. Exons 18 through 24 were shared with c11h1.6, although the 18th exon was extended to the 5′ side by 10 nucleotides from the start codon of c11h1.6. There was no significant deviation from the splice-site consensus sequences with two exceptions of 5′ splice sites having AG/GC (instead of AG/GU) in the 6th and 15th introns, which were not recognized by the program. The ORF c11h1.5 that has a different orientation from the others was encompassed in the 15th intron. The pex-1 and pex-6 genes encoded proteins of 996 and 720 amino acid residues, respectively.

Fig. 3

Genomic organization of the pex-1 gene. The closed rectangles above the line, which indicates part of the genomic sequence cloned in the cosmid c11h1, are exons for three ORFs predicted by the GENEFINDER program. The names and orientation of the ORFs are as indicated. The rectangles below the line denote exons determined by the pex-1 cDNA sequence; closed rectangles indicate the translated region and open rectangles indicate the untranslated regions. The asterisks mark exons not correctly predicted. The numbers indicate the first and last exons as well as the 15th through 18th exons for pex-1.

Identification of PEX-1 and PEX-6

To identify the tentatively termed pex-1 and pex-6 genes as genuine peroxin genes, we compared their amino acid sequences with those of biologically established SF2 members of the AAA protein family because no pex mutants were available in C. elegans and the expression of PEX-1 in a pex1 mutant (strain MB328) of the yeast S. cerevisiae did not complement the mutation (data not shown). There were five pairs of SF2 sequences, which were confirmed to be peroxins by their mutant phenotype. Their sources and the number of amino acid residues in parentheses of Pex1p/PEX1 and Pex6p/PEX6 were: S. cerevisiae (1043, 1030) (Erdmann et al., 1991; Voorn-Brouwer et al., 1993), Pichia pastoris (1157, 1165) (Heyman et al., 1994; Spong and Subramani, 1993), Yarrowia lipolytica (1023, 1025) (Titorenko et al., 2000; Nuttley et al., 1994), Hansenula polymorpha (1074, 1135) (Kiel et al., 1999), and Homo sapiens (1283, 980) (Reuber et al., 1997; Ports-teffen et al., 1997; Yahraus et al., 1996). The sequence similarities, especially in the region outside the AAA cassettes, were moderate and C. elegans PEX-6 was smaller than the putative orthologs. We therefore compared the sequences of three motifs in the two AAA cassettes; Walker motifs A and B for nucleotide binding and SRH with unknown function (Fig. 4). The first AAA cassette was unique for SF2 proteins and the second located closest to the C-terminus was characteristic for all AAA proteins. PEX-1 and PEX-6 of C. elegance showed a high degree of similarity to respective five cognate proteins not only in the motif sequences but also in distances between the motifs. In addition to a considerable sequence similarity in the respective N-terminal region (data not shown), three crucial residues (Fig. 4, marked by asterisks) identified the C. elegans PEX-1 and PEX-6 as genuine orthologs of Pex1p/PEX1 and Pex6p/PEX6, respectively.

Fig. 4

Motifs in the first and second AAA cassettes. PEX-1 and PEX-6 (the DDBJ/EMBL/GenBank accession numbers are  AB054992 and  AB010968, respectively) of C. elegans (Cel) were aligned with the Pex1p/PEX1 and Pex6p/PEX6 orthologs, respectively and thereafter the AAA cassette regions were taken from the final alignments. The abbreviations of their sources and the accession numbers are: Ppa, P. pastoris ( Z36987 and  Z22556); Yli, Y. lipolytica ( AF208231 and  L23858); Sce, S. cerevisiae ( M58676 and  L20789); Hpo, H. polymorpha ( AF129873 and  AF129874); Hsa, H. sapiens ( AF030356 and  U56602). The numbers on the left represent the position of the first amino acid residue and those in parentheses indicate the residue number present between the motifs. Similar residues present in four or more sequences are shaded gray, and then four or more identical residues among them are blackened. The asterisks denote residues that distinguish the Pex1p/PEX1 and Pex6p/PEX6 sequences. The consensus sequences for Walker motifs A and B as well as SRH are shown above the alignments; h and x represent hydrophobic residues and any amino acid residue, respectively.

Spatio-temporal pattern of gene expression

The mRNAs of both pex-1 and pex-6 were detectable throughout the life cycle of C. elegans (Fig. 5). The extent of expression relative to that of CeIF, which is a homolog of eucaryotic initiation factor 4A and was used as the control for equal sample loading (Krause, 1995), was higher at the larval stage L3 and lower at the young adult stage (YA) than the other stages: embryo (E), larvae (L1, L2 and L4), or egg-laying adult (EA). The developmental patterns of gene expression for pex-1 and pex-6 were apparently parallel to each other, though the results were semiquantitative because of their extremely low level of expression. The tissue distributions of the pex-1 and pex-6 mRNA were examined by whole-mount in situ hybridization using antisense RNA probes (Fig. 6, A and B). Specimen used was L4 larva because in younger larval stages the somatic cell division is not completed and mature cuticle at adult stages tends to reduce the accessibility of RNA probes. Both of these mRNAs were detected mainly in intestinal cells.

Fig. 5

Amounts of pex-1 and pex-6 mRNA at each developmental stage. Poly(A)-rich RNA (5 μg of each) from embryos (E), larvae (L1, L2, L3, and L4), young adults (YA) and egg-laying adults (EA) were assessed by northern blot analysis as in Fig. 2. The pex-1, pex-6, and CeIF mRNAs were detected with the appropriate probes.

Fig. 6

Expression of the pex-1 and pex-6 mRNA in situ. Fixed L4 larvae were probed with the digoxigenin-labeled antisense RNA of pex-1 (A) or pex-6 (B) or with the sense RNA of pex-1 (C) or pex-6 (D) and incubated with alkaline phosphatase-labeled sheep anti-(digoxigenin) Fab fragment. Alkaline phosphatase catalyzed the formation of a dark blue precipitate in cells containing the pex-1 or pex-6 mRNA.