Identi®cation of two processed pseudogenes of the ... - Springer Link

Ó Springer-Verlag 1998

Mol Gen Genet (1998) 258: 117±122

ORIGINAL PAPER

J. M. HernaÂndez á B. Blat á C. Iruela á F. Vila J. HernaÂndez-Yago

Identi®cation of two processed pseudogenes of the human Tom20 gene

Received: 12 September 1997 / Accepted: 29 November 1997

Abstract The open reading frame (ORF) of the human Tom20 gene (hTom20) was ampli®ed by PCR from a HeLa cDNA library using primers based on the sequence of HUMRSC145 and cloned into a pET15b vector. Ampli®cation of human genomic DNA using these primers yielded a DNA fragment of the same size as that of the ORF of hTom20 cDNA. Sequencing of this fragment revealed that: (1) it has the same number of base pairs as the ORF of hTom20 cDNA (438 bp); and (2) the two sequences di€er by 14 single base pair substitutions (97% similarity) causing eight changes in the amino acid sequence and two premature stop codons. Further ampli®cation of human genomic DNA adaptor-ligated libraries using primers based on HUMRSC145 revealed three di€erent sequence-related genomic regions; one corresponding to the fragment referred above, another corresponding to the hTom20 gene, and a third fragment of which the sequence di€ers from the ORF of hTom20 cDNA by only 22 base pair substitutions and a deletion of 4 bp. We conclude that, in addition to the hTom20 gene, there are two genomic DNA sequences (W1Tom20 and W2Tom20) that are processed pseudogenes of hTom20. Aspects concerning their evolutionary origin are discussed. Key words Mitochondria á Import receptor á Human Tom20 á Nucleotide sequence á Processed pseudogenes

Introduction The human homolog of the Saccharomyces cerevisiae/ Neurospora crassa mitochondrial protein import recep-

Communicated by K. Illmensee J. M. HernaÂndez á B. Blat á C. Iruela á F. Vila á J. HernaÂndez-Yago (&) Instituto de Investigaciones CitoloÂgicas, FundacioÂn Valenciana de Investigaciones BiomeÂdicas, Amadeo de Saboya 4, E-46010 Valencia, Spain

tor, Mas20p/MOM19 (Goping et al. 1995; Seki et al. 1995; Hanson et al. 1996), termed Tom20 in the new nomenclature (Pfanner et al. 1996), has recently been identi®ed. This receptor plays a crucial role in the biogenesis of mitochondria as a subunit of the translocase of the outer mitochondrial membrane that recognizes directly most cytosolic mitochondrial protein precursors (Schneider et al. 1991; Ramage et al. 1993; Harkness et al. 1994; Moczko et al. 1994; reviewed by Lill and Neupert 1996). The human Tom20 gene (hTom20) has been localized at chromosome 1 (Nomura et al. 1994), more precisely at 1q42, and the existence of at least one sequence-related pseudogene of hTom20 has been mentioned (Seki et al. 1995). In this paper we report the identi®cation of two human genomic DNA sequences we found in the course of studies to characterize the hTom20 gene. Their features strongly suggest they are processed pseudogenes which arose from two di€erent mRNAs of the hTom20 gene.

Materials and methods Isolation, cloning, and ampli®cation of the open reading frame of hTom20 cDNA The open reading frame (ORF) of hTom20 cDNA was ampli®ed by PCR from a HeLa cell cDNA library, using primers 1 and 2 based on the sequence of HUMRSC145 submitted to GenBank (N. Nomura, N. Miyajima, Y. Kawarabayasi, and S. Tabata, DDBT accession number D13641, 1993). The sense primer 1 was 5¢GCGGATCCCATATGGTGGGTCGGAACAGCGCCA-3¢, with a NdeI site (boldface type), and the antisense primer 2 was 5¢GCGGATCCTCATTCCACATCATCTTCAG-3¢ with a BamHI site (boldface type). The reaction mixture contained 5 ll of 10 ´ BIOTAQ bu€er from Ecogen [200 mM Tris-HCl, pH 8.55, 160 mM (NH4)2SO4, 25 mM MgCl2, 1.5 mg/ml BSA], 200 lM dNTPs, 10 pmol of each primer, 2.5 U Taq polymerase (BIOTAQ, Ecogen), 3 mM MgCl2, and 100 ng of HeLa cDNA library. The PCR reaction was carried out as follows: 95° C for 1 min; 35 cycles of 94° C for 30 s and 72° C for 90 s; and ®nally 72° C for 5 min. The PCR product was cloned into NdeI±BamHI-linearized vector pET15b (Novagen), resulting in phTom20. Recombinant phTom20 DNA (1 ng) was ampli®ed by PCR using primers 1 and 2 as described above.

118 Electrophoresis of samples was performed in 0.8% agarose gels and visualized on a UV screen after ethidium bromide staining. Synthesis of hTom20 in vitro In vitro transcription of phTom20 with T7 RNA polymerase and translation in a rabbit reticulocyte lysate system were carried out as previously described (GonzaÂlez-Bosch et al. 1991). Published procedures were used to analyze labeled translation products by SDS-PAGE (12% w/v gel) (Laemmli 1970) and ¯uorography (Bonner and Laskey 1974). Genomic DNA isolation and ampli®cation Genomic DNA was isolated from 5 ml of human blood according to Moreno et al. (1989). Ampli®cation of W1Tom20 and W2Tom20 was performed by PCR and the reaction mixture contained 5 ll of 10 ´ bu€er (BIOTAQ, composition described above), 200 lM dNTPs, 20 pmol of each primer, 2.5 U Taq polymerase (BIOTAQ, Ecogen), 2 mM MgCl2, and 500 ng of human genomic DNA. Primers used were primers 1 and 2 for W1Tom20 and the sense primer 3 (5¢-ACCCACCAACATTCCCAGCCATTGG-3¢) and the antisense primer 4 (5¢-TTGCATTTGTCAGATGGTCTACGCCC-3¢) for W2Tom20. Primers 3 and 4 are based on the 5¢-¯anking sequence of W2Tom20 and HUMRSC145, respectively. The PCR reaction was carried out as follows: 94° C for 3 min; 40 cycles of 94° C for 30 s, 63° C for 45 s, and 72° C for 150 s; and ®nally 72° C for 10 min. Figure 1 shows the relative location of the primers that have been used for PCR ampli®cation and/or sequencing in di€erent steps of this work. PCR products were analyzed by electrophoresis in 2% agarose gels and visualized on a UV screen after ethidium bromide staining.

moterFinder DNA library. The program used for primary PCR was: 7 cycles of 94° C for 2 s and 72° C for 3 min; 37 cycles of 94° C for 2 s and 67° C for 3 min; and ®nally 67° C for 4 min. The same conditions were used for nested PCR but using 22 instead of 37 cycles. PCR products obtained from these walkings were directly cloned into vector pT-Adv (Clontech) using the A overhang left by the Taq and Pwo DNA polymerases in PCR reactions. The cloning method was followed as described by the supplier. Recombinant DNAs were further analyzed by restriction enzyme digestion (using EcoRI) and sequencing. Sequences presented in this paper result from the analysis of at least three independent clones. DNA sequencing DNA sequencing was carried out using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer) on double-stranded DNA according to the manufacturer's instructions with a Perkin-Elmer thermal cycler and ABI 373A DNA sequencer and ABI sequence analysis system. Recombinant DNAs obtained from walkings, as well as PCR products, were sequenced with the same primers used for their isolation.

Results and discussion Figure 2A shows that the PCR product of the ORF of hTom20 cDNA from a HeLa cDNA library (lane 2), the

Isolation and cloning of PCR products from genomic DNA libraries Genomic DNA fragments, adaptor-ligated from human libraries of the Human PromoterFinder DNA Walking kit (Clontech), were ampli®ed using gene-speci®c primers described below. These libraries allowed us to identify, isolate, and clone sequences upstream and downstream in genomic DNA from a known sequence. Three di€erent walkings were prepared: (1) a walking upstream of the ORF of hTom20 cDNA, using the antisense primers 5 (5¢TCAGGTAACTTGGAAAGCCCAGCTCTCTCC-3¢) and 6 (5¢CGTTCTCGAAGCCTGTTCTTGAAGTTGG-3¢) and the adaptor primers AP1 and AP2, provided in the kit; (2) a walking downstream of the ORF of hTom20 cDNA, using the sense primers 7 (5¢-TGGGTACTGCATCTACTTCGACCGC-3¢) and 8 (5¢-AGCTGGGCTTTCCAAGTTACCTGACC-3¢) and the adaptor primers AP1 and AP2; and (3) a walking downstream of the ORF of hTom20 cDNA, using the sense primers 8 (see above) and 9 (5¢-ACTCTTCCACCACCAGTGTTCCAGATGC-3¢) and the adaptor primers AP1 and AP2. Primers 5±9 are based on the sequence of HUMRSC145. The reaction mixture contained 5 ll of bu€er 1 (Boehringer Mannheim), 200 lM dNTPs, 10 pmol of each primer, 2.5 U Taq and Pwo DNA polymerases (polymerase mix from Boehringer Mannheim, containing 300 ng of TaqStart antibody from Clontech), and 1 ll of the corresponding human ProFig. 1 Scheme of sequences shown in Fig. 4, with the relative location of the primers that were used for PCR and/or sequencing

Fig. 2 A Isolation and cloning of the open reading frame (ORF) of human (h)Tom20 cDNA. Electrophoresis in a 0.8% agarose gel of EcoRI-digested phage SSP1 DNA (molecular mass reference marker) (lane 1); product of PCR ampli®cation from a HeLa cDNA library using primers 1 and 2 (lane 2); BamHI-linearized phTom20 (lane 3); NdeI±BamHI-digested phTom20 showing two bands corresponding to the vector and the insert (ORF of hTom20 cDNA) (lane 4); PCR product of phTom20 using primers 1 and 2 (lane 5). B Synthesis in vitro of hTom20. SDS-PAGE and further ¯uorography of the radiolabeled polypeptide (hTom20). Lane 1 Rainbow [14C]-methylated protein molecular weight markers (Amersham), lane 2 without exogenous mRNA, lane 3 plus transcript from phTom20

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insert of the recombinant plasmid phTom20 (lane 4, lower band), and the product of PCR ampli®cation of phTom20 (lane 5) have a similar size. Sequencing of both PCR products revealed the previously reported sequence of 438 bp of the ORF of hTom20 cDNA (Goping et al. 1995; Seki et al. 1995; Hanson et al. 1996) (Fig. 4, labeled C). Figure 2B shows that the 35S-labeled transcription±translation product in reticulocyte lysate corresponds to a polypeptide of about 16 kDa, as expected for hTom20. In the course of experiments carried out to characterize the hTom20 gene, we found that ampli®cation by PCR of human genomic DNA isolated from human blood, using primers 1 and 2, yielded a prominent band of the same size as the ORF of hTom20 cDNA (Fig. 3 lane 3). The absence of bands in control samples (Fig. 3, lane 2) precluded a possible contamination with hTom20 cDNA as an alternative explanation for this result. Sequencing of the PCR product using primers 1±4 yielded a sequence, hereafter called Y1Tom20 (Fig. 4, labeled Y1), which reveals that: (1) it has the same length (438 bp) as the ORF of the hTom20 cDNA; (2) both sequences are 97% identical; and (3) di€erences are due to single base pair substitutions. Identical results were obtained with human genomic DNA isolated from blood of two other unrelated people. We decided, therefore, to further investigate the signi®cance of this genomic sequence. With the exception of some genes encoding the ®ve histones, the a and b interferons, and several mammalian virus proteins, all known vertebrate protein coding genes contain introns. As a matter of fact, although the hTom20 gene has not yet been characterized, the sequence of the homologous gene, Mom19, in N. crassa (Schneider et al. 1991) has been identi®ed, revealing the presence of three introns. The absence of introns in the homologous gene, Mas20, in S. cerevisiae (Ramage et al. 1993) should be interpreted taking into account reported observations showing that introns are rare in the protein coding genes of S. cerevisiae, although they occur more frequently in some other yeasts (Singer and Berg 1991).

Fig. 3 Ampli®cation of human genomic DNA by PCR using primers 1 and 2. Electrophoresis in a 2% agarose gel of a 50-bp DNA ladder (molecular mass reference marker) (lane 1); control without DNA (lane 2); ampli®ed human genomic DNA, using primers 1 and 2 (lane 3)

Therefore, it was unexpected that ampli®cation of human genomic DNA with primers 1 and 2 yielded a unique band of the same size as the ORF of the hTom20 cDNA (Fig. 3, lane 3). Sequencing of this product led us to realize that this sequence has characteristics of processed pseudogenes, which appear more like DNA copies of mRNA than like genes. Pseudogenes are DNA sequences found to be both related to functional genes and defective. Most of them lack introns found in their functional counterparts and constitute a subclass termed processed pseudogenes that appear to be generated by reverse transcription of mRNA and transposition. According to the generally accepted explanation, these pseudogenes are inserted randomly into the genome (for reviews, see Vanin 1985; Weiner et al. 1986). Thus, any functional constraints to preserve sequence integrity are lost. Many of these pseudogenes have a polyA sequence immediately 3¢ to the point at which the homology between the pseudogene and its functional counterpart ceases and many of them are ¯anked by direct repeats of 7±17 bp. In addition, they often have multiple genetic lesions in the coding region, including deletions, insertions, and single base pair substitutions, which frequently result in premature stop codons. The human genomic DNA sequence, Y1Tom20, ®ts with the concept of a processed pseudogene because it has a high similarity with the corresponding cDNA, it has no intervening sequences, and it shows several changes. In addition, this would agree with a comment of Seki et al. (1995) who noticed, in the course of mapping studies to determine the chromosomal location of the hTom20 gene, the presence of at least one sequence-related pseudogene of hTom20, although no information was provided relative to this sequence(s). The substitutions reported in Y1Tom20 would cause eight changes in the amino acid sequence and two premature stop codons at positions 278 and 314 (Fig. 4), and therefore the hypothetical expression of this sequence would result in a truncated polypeptide of 28 amino acids compared to the functional hTom20 of 145 residues. To further characterize the Y1Tom20 sequence as well as its ¯anking regions, ampli®cation by PCR of human genomic DNA libraries contained in the Human PromoterFinder DNA Walking kit (Clontech) was performed as described in Materials and methods. These PCR products were directly cloned into vector pT-Adv using the T/A cloning method (Clark 1988; Mead et al. 1991) and positive clones were selected. Sequencing of recombinant DNAs revealed the existence of three related genomic sequences: (1) one corresponding most probably to a portion of the real genomic hTom20 gene, crossing an intron±exon junction (data not shown); (2) another sequence con®rming the pseudogene described above (Y1Tom20); and (3) another that was a portion of a second processed pseudogene that we call Y2Tom20. To complete the sequence of Y2Tom20, a PCR ampli®cation of human genomic DNA was carried out

120

using primers 3 and 4, as described in Materials and methods that yielded a product of the expected size (Fig. 5), which was sequenced. Comparison of the hTom20 cDNA with Y2Tom20 (Fig. 4) reveals that both sequences di€er in two deletions of 4 and 8 bp, ®ve insertions of 7, 4, 2, 2, and 1 bp, and 51 single base pair substitutions, which result in a 95% identity. It should be noted that two point mutations are located at the initiation codon and, therefore, that this sequence could not be translated. The similarity region of sequences Y1Tom20 and Y2Tom20 extended from 11 bp upstream of the 5¢ end of the cDNA to the 3¢ end of Y2Tom20. Comparison of Y1Tom20 and Y2Tom20 with the cDNA sequence (Fig. 4) shows that the changes are in good agreement with the pattern of mutations found in other processed pseudogenes. In particular, the base substitutions C®T (14 substitutions for Y1Tom20 and 11 for Y2Tom20) and G®A (9 substitutions for Y1Tom20 and 11 for Y2Tom20) are the most and the second most common, respectively (Gojobori et al. 1982). The dinucleotide CpG has long been known to have an unusually high frequency of mutation (Kricker et al. 1992) and, since a majority of cytosines in CpG dinucleotides are methylated, they result, upon deamination, in thymidines that are not perceived as a mutation by the repair machinery (Lindahl 1974). When comparing the similarity region of the cDNA and Y1Tom20, we found that 13 of the 26 CpG dinucleotides present in the cDNA are changed in Y1Tom20 to 8 TpG and 5 CpA. In the similarity region of the cDNA and Y2Tom20, 7 of the 25 CpG dinucleotides present in the cDNA changed to 3 CpA, 2 TpG, 1 TpT, and 1 CpC. Indeed, 25% and 16% of all point mutations in Y1Tom20 and Y2Tom20, respectively, involve a CpG dinucleotide, whereas if point mutations occurred at random, the expected value would be 6.25% (Ozer et al. 1993). Due to the lack of functional constraints on these processed pseudogenes, their rate of spontaneous mutation can be considered approximately equal to the spontaneous mutation rate of other better characterized mammalian pseudogenes. If we use the estimated value of 4.7 ´ 10)9 per nucleotide per year calculated for mammalian globin pseudogenes (Li et al. 1987; Sudo b Fig. 4 Comparison of hTom20 cDNA, Y1Tom20, and Y2Tom20 sequences. Alignment of the nucleotide sequences of hTom20 pseudogenes (labeled Y1 and Y2) and the corresponding functional cDNA (labeled C) is shown. The 5¢-¯anking sequences of Y1Tom20 and Y2Tom20 are reported in lower case letters. The nucleotide sequence corresponding to the ORF of hTom20 cDNA is in bold face type. Bases dissimilar to the hTom20 cDNA are depicted below the cDNA sequence. Deletions in pseudogene sequences, relative to the cDNA sequence, are indicated by dashes. Insertions in the pseudogene sequences, relative to the cDNA sequence , are indicated by asterisks. The start (ATG) and stop (TGA) codons, relative to the cDNA sequence, are doubly underlined. In-frame stop codons in pseudogene sequences that were created as a result of point mutations are underlined. Polyadenylation signals are in bold face type and underlined. The polyA tract is written in italic type

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introns, and contain multiple genetic lesions that would preclude their transcription and translation into functional polypeptides. These results strongly suggest that Y1Tom20 and Y2Tom20 are processed pseudogenes of the hTom20 gene and probably arose from di€erent mRNAs of hTom20. The presence of these two processed pseudogenes in the human genome should be kept in mind to avoid misinterpretations in potential genetic diagnosis, upon characterization of the complete hTom20 gene, of pathologies linked to mitochondrial disorders hinting at defects in the mitochondrial protein import machinery. Fig. 5 Ampli®cation of human genomic DNA by PCR. Electrophoresis in a 2% agarose gel of EcoRI-digested phage SSPI DNA (molecular mass reference marker) (lane 1); control without DNA (lane 2); ampli®ed human genomic DNA (sample 1), using primers 3 and 4 (lane 3)

et al, 1990), and assume that the sequence of the functional Tom20 gene has remained static since their divergence and that all the 54 and 61 nucleotide di€erences out of 1017 and 1086 possible sites were amassed by the Y1Tom20 and Y2Tom20, then these pseudogenes originated 10 million [(51/1086)/4.7 ´ 10)9] and 10.7 million [(51/1017)/4.7 ´ 10)9] years ago, respectively. A search for transcription initiation signals in the sequences Y1Tom20 and Y2Tom20, upstream of the 5¢ end of the cDNA, was unsuccessful. In contrast, a search for transcription termination signals downstream of the 3¢ end of the ORF revealed a polyadenylation signal, ATTAAA, at position 990 of the cDNA sequence followed by the sequence GGTGTGCT at position 1048, which is known to be a second signal required to trigger the reaction leading to cleavage and polyadenylation of transcripts that frequently occurs within or near a CA dinucleotide located between both signals (Singer and Berg 1991). These sequences are also present in both pseudogenes and, interestingly, similarity of Y2Tom20 with the cDNA sequence, but not of Y1Tom20, ceases just one base before the dinucleotide CA at position 1019 showing a polyA tract that is characteristic of mRNAs and also of processed pseudogenes 3¢ ends (Fig. 4). Although nothing is known about the existence of di€erent mRNAs arising from the hTom20 gene, these observations support the idea that pseudogenes Y1Tom20 and Y2Tom20 could have arisen from mRNAs of hTom20 with di€erent polyadenylation sites. This agrees with other reports showing that functional genes can give rise to di€erent mRNAs by using di€erent polyadenylation sites, and that processed pseudogenes corresponding to each of the di€erent mRNA species have been identi®ed (Chen et al. 1982; Gwo-Shu Lee et al. 1983; Masters et al. 1983; Scarpulla 1984; Shimada et al. 1984). In conclusion, we have identi®ed two sequences of human genomic DNA (Y1Tom20 and Y2Tom20) that are essentially identical to the cDNA of hTom20, lack

Acknowledgements We are most grateful to Dr. A. Alconada (EMBL, Heidelberg, Germany) for his help and discussions and Dr. F. Thompson for carefully reading the manuscript. We also thank Eloisa Barber, Sara HernaÂndez, and Amparo MartõÂ nez for their help in various phases of this work. This work was supported by the Fondo de Investigaciones Sanitarias de la Seguridad Social (93/0635, 96/2080).

References Bonner WM, Laskey RA (1974) A ®lm detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46:83±88 Chen MJ, Shimada T, Moulton AD, Harrison M, Nienhuis AW (1982) Intronless human dihydrofolate reductase genes are derived from processed RNA molecules. Proc Natl Acad Sci USA 79:7435±7439 Clark JM (1988) Novel non-template nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res 16:9677±9686 Gojobori T, Li W-H, Graur D (1982) Patterns of nucleotide substitution in pseudogenes and functional genes. J Mol Evol 18:360±369 GonzaÂlez-Bosch C, Marcote MJ, HernaÂndez-Yago J (1991) Role of polyamines in the transport in vitro of the precursor ornithine transcarbamylase. Biochem J 279:815±820 Goping IS, Millar DG, Shore GC (1995) Identi®cation of the human mitochondrial protein import receptor, huMas 20p. Complementation of Dmas20 in yeast. FEBS Lett 373:45±50 Gwo-Shu Lee M, Lewis SA, Wilde CD, Cowan NJ (1983) Evolutionary history of a multigene family: an expressed human btubulin gene and three processed pseudogenes. Cell 33:477±487 Hanson B, Nuttall S, Hoogenraad N (1996) A receptor for the import of proteins into human mitochondria. Eur J Biochem 235:750±753 Harkness TA, Nargang FE, Klei I van der, Neupert W, Lill R (1994) A crucial role of the mitochondrial protein import receptor MOM19 for the biogenesis of mitochondria. J Cell Biol 124:637±648 Kricker MC, Drake JW, Radman M (1992) Duplication-targeted DNA methylation and mutagenesis in the evolution of eukaryotic chromosomes. Proc Natl Acad Sci USA 89:1075±1079 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680±685 Li W-H, Tanimura M, Sharp PM (1987) An evaluation of the molecular clock hypothesis using mammalian DNA sequences. J Mol Evol 25:330±342 Lill R, Neupert W (1996) Mechanisms of protein import across the mitochondrial outer membrane. Trends Cell Biol 6:56±61 Lindahl T (1974) An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues. Proc Natl Acad Sci USA 71:3649±3653 Masters JN, Yang JK, Cellini A, Attardi G (1983) A human dihydrofolate reductase pseudogene and its relationship to

122 multiple forms of speci®c messenger RNA. J Mol Biol 167:23± 36 Mead DA, Pey NK, Hernstadt C, Marcil RA, Smith LM (1991) A universal method for the direct cloning of PCR ampli®ed nucleic acid. Biotechnology 9:657±663 Moczko M, Ehmann B, GaÈrtner F, HoÈnlinger A, SchaÈfer E, Pfanner N (1994) Deletion of the receptor MOM19 strongly impairs import of cleavable preproteins into Saccharomyces cerevisiae mitochondria. J Biol Chem 269:9045±9051 Moreno RF, Brooth FR, Hung IH, Tilzer LL (1989) Ecient DNA isolation within a single gel barrier tube. Nucleic Acids Res 17:8393 Nomura N, Miyajima N, Sazuka T, Tanaka A, Kawarabayasi Y, Sato S, Nagase T, Seki N, Ishikawa K, Tabata S (1994) Prediction of the coding sequence of unidenti®ed human genes. I. The coding sequence of 40 new genes (KIAA0001± KIAA0040) deduced by analysis of randomly sampled cDNA clones from human immature myeloid cell line KG-1. DNA Res 1:27±35 Ozer J, Chalkley R, Sealy L (1993) Isolation of the CCAAT transcription factor subunit EFIA cDNA and a potentially functional EFIA processed pseudogene from Bos taurus: insights into the evolution of the EFIA/dbpB/YB-1 gene family. Gene 124:223±230 Pfanner N, Douglas MG, Endo T, Hoogenraad NJ, Ensen RE, Meijer M, Neupert W, Schatz G, Schmitz UK, Shore GC (1996) Uniform nomenclature for the protein transport machinery of the mitochondrial membrane. Trends Biochem Sci 21:51±52 Ramage L, Junne T, Hahne K, Lithgow T, Schatz G (1993) Functional cooperation of mitochondrial protein import receptors in yeast. EMBO J 12:4115±4123

Scarpulla RC (1984) Processed pseudogenes for rat cytochrome c are preferentially derived from one of three alternate mRNAs. Mol Cell Biol 4:2279±2288 Schneider H, SoÈllner T, Dietmeier K, Eckerskorn C, Lottspeich F, TruÈlzsch B, Neupert W, Pfanner N (1991) Targeting of the master receptor MOM19 to mitochondria. Science 254:1659± 1662 Seki N, Moczko M, Nagase T, Zufall N, Ehmann B, Dietmeier K, SchaÈfer E, Nomura N, Pfanner N (1995) A human homolog of the mitochondrial protein import receptor Mom19 can assemble with the yeast mitochondrial receptor complex. FEBS Lett 375:307±310 Shimada T, Chen MJ, Nienhuis AW (1984) A human dihydrofolate reductase intronless pseudogene with an Alu repetitive sequence: multiple RNA insertion at a single chromosomal site. Gene 31:1±8 Singer M, Berg P (1991) Genes and genomes. University Science Book, Mill Valley, California Sudo K, Masato N, Luedeman MM, Deaven LL, Li SS-L (1990) Human lactate dehydrogenase-B processed pseudogene: nucleotide sequence analysis and assignment to the X-chromosome. Biochem Biophys Res Commun 171:67±74 Vanin EF (1985) Processed pseudogenes: characteristics and evolution. Annu Rev Genet 19:253±272 Weiner AM, Deininger PL, Efstratiadis A (1986) Nonviral retroposons: genes, pseudogenes, and transposable elements generated by the reverse ¯ow of genetic information. Annu Rev Biochem 55:631±661