Abstract. A 3667-base pair (bp) fragment of the mito- chondrial genome of the crustacean Daphnia pulex has been sequenced and found to contain the ...
CurrGenet(1994)25:66-72
Current 9
Partial mitochondrial D N A sequence of the crustacean
Genetics
Springer-Verlag 1994
Daphniapulex
Terence J. Van Raay, Teresa J. Crease Department of Zoology, University of Guelph, Guelph, Ontario, NIG 2W1, Canada Received: 6 January 1993 / Accepted: 13 July 1993
Abstract. A 3667-base pair (bp) fragment of the mitochondrial genome of the crustacean Daphnia pulex has been sequenced and found to contain the complete genes for the small subunit ribosomal RNA, ND2, seven tRNAs and the control region. This organization is identical to that found in Drosophila yakuba m t D N A yet D. pulex m t D N A exhibits several unique features when compared to other mitochondrial sequences. The sequenced fragment is only 62.6% A + T which is much lower than that of any other arthropod m t D N A sequenced to date. D. pulex m t D N A also exhibits length conservation having shorter coding and non-coding regions. The putative control region is 689 bp in length and includes a sequence that has the potential to fold into a hairpin structure with a perfect 20-bp pair stem and a 22-base loop.
Brown 1987; Rand and Harrison 1989; Buroker et al. 1990; Hayasaka et al. 1991 ; Okimoto et al. 1991 ; Wilkinson and Chapman 1991). Thus far it appears that gene rearrangements are common among lower invertebrates and between these and the m t D N A molecules of higher invertebrates and vertebrates (Wolstenholme 1992). In this paper we present a partial sequence of the mitochondrial genome of an Arthropod in the class Crustacea. Although limited work has been done on Artemia (Batuecas et al. 1988), the present study is the first substantial undertaking in sequencing a crustacean mitochondrial genome. The genome organization thus far determined is identical to Drosophila. Consequently, the most detailed comparisons are between D. pulex and D. yakuba mitochondrial sequences (Clary and Wolstenholme 1985 b).
Key words: Crustacea - Mitochondrial D N A sequence Materials and methods Introduction The mitochondrial genomes from a wide variety of metazoans have been sequenced in whole or in part with the vertebrates (Anderson et al. 1981, 1982; Bibb et al. 1981 ; Roe et al. 1985; Desjardins and Morais 1990, 1991) and insects (Clary and Wolstenholme 1985 b, 1987; Rand and Harrison 1989; Cornuet et al. 1991 ; Crozier and Crozier 1993) being highly represented. Other work has involved echinoderms (Jacobs et al. 1988; Cantatore et al. 1989; Asakawa et al. 1991), a mussel (Hoffmann et al. 1992), and nematodes (Okimoto et al. 1992) among others (see Wolstenholme 1992). Completely-sequenced metazoan m t D N A s contain two ribosomal subunit R N A genes, 22 t R N A genes, 12 or 13 protein coding genes, and one or two non-coding regions that regulate transcription and/ or replication. The non-coding region is often the site for length polymorphism and heteroplasmy (Moritz and Correspondence to: T. J. Crease
Mitochondrial DNA was extracted from a single parthenogenetic clone of D. pulex from Amana, Iowa, following the procedures outlined in Stanton et al. (1991). Approximately 1.0 gg of D. pulex mtDNA and 0.5 gg of pBluescript II SK(-) (Stratagene) were digested with PstI (BRL), ligated, and transformed into competent Escherichia coli (XL1-Blue; Stratagene). Three PstI fragments of 9.0, 3.4 and 2.6 kb were cloned.A fourth of about 300 bp was not isolated. The largest fragment was further digested with PstI and BamHI to yield two fragments of 6.4 and 2.6 kb which were subcloned into pBluescript II SK(-). In addition, the 6.4-kb fragment was further subcloned to yield a 1-kb fragment using BamHI and SstI. The BamHI site was previously found to be in the control region (Stanton et al. 1991). Large-scale plasmid preparations (Maniatis et al. 1982) provided mtDNA for sequencing and deletion experiments. An Erase-A-Base kit (Promega) was used to generate progressive, unidirectional, deleted subclones of the original inserts. To generate deletions from the BamHI site (Fig. 1), the 2.6-kb BamHI-PstI subclone was digested with BamHI and SacI (BRL) to yield 5' overhanging (digestion) and 3' overhanging (protecting) ends, respectively. The same fragment was also deleted from the PstI site using BglII and PstI (BRL). To determine the 3' end of the control region, deletions were generated from the 6.4-kb subclone in the direction Pstl to BamHI by double digesting with KpnI and NcoI (BRL). The 1-kb BarnHI-SstI subclone was used to obtain
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Fig. 1. Sequencing strategy for the 2.6-kb BamHI-PstI fragment and the 5' end of the 6.4-kb PstI-BamHI fragment from D. pulex mtDNA. The tRNA genes are identified by their one letter code and the direction of transcription of each gene is identified by a hollow arrow head, as is the putative direction of replication within the control region. Restriction sites shown were previously mapped
by Stanton et al. (1991) and are as follows: 1 (ApaI), 2 (BamHI), 3 (Bg/II), 4 (EeoRI), 5 (EcoRI), 6 (PstI), 7 (ScaI), 8 (XbaI). The arrows represent the direction of DNA sequencing of the nested deletion subclones. The length of the arrows represents the approximate length of readable sequence obtained from each subclone
deletions in the BamHI to SstI direction by double digesting with KpnI and ClaI. The deleted subclones were sequenced by the Sanger dideoxy method using a Sequenase 2.0 kit (United States Biochemical) and ~-S35-thio dATP. Sequencing reactions were run on 6% denaturing acrylamide gels using SequaGel 6 pre-mixed acrylamide (National Diagnostics). DNA sequences were analyzed using the DNASTAR package of computer programs. D. pulex mitochondrial protein genes, tRNA genes, and the small subunit ribosomal RNA (s-rRNA) gene were first identified by searching for similarities with D. yakuba mtDNA (Clary and Wolstenholme 1985 b). The tRNA genes were identified by the high primary-sequence similarity in the anticodon loops and stems. Secondary structure was obtained by eye using the general tRNA model of Sprinzl et al. (1989). The s-rRNA gene was first identified by primary-sequence similarity to D. yakuba. The secondary structure was then determined by eye using the model of Neefs et al. (1991). Open reading frames were identified as ND2 and COI genes by comparisons of inferred amino-acid sequences with other, known, ND2 and COI sequences.
(ile, met, and trp), ND2, and COI, are all transcribed in one direction, and if D. pulex m t D N A replication is the same as D. yakuba, then these genes would be transcribed in the opposite direction of replication (Fig. 1). The putative initiation codon of the COI gene is ATTA as opposed to the ATAA found in Drosophila (Clary and Wolstenholme 1985 b). The remaining t R N A genes (val, gln, cys and tyr) and the two r R N A genes would be transcribed in the same direction as replication (Fig. 1). There are a total of 707 intergenic nucleotides with 689 bp belonging to the putative control region. Using data obtained from restriction enzyme studies, it has been estimated that D. pulex m t D N A contains approximately 15 400 bp (Stanton et al. 1991). The smaller size of this genome relative to D. yakuba (16019 bp) is due to: (1) a smaller control region (689 bp in Daphnia vs 1077 bp in Drosophila); (2) a smaller N D 2 coding region (988 bp vs 1041 bp); and (3) a shorter s - r R N A gene (753 bp vs 789 bp) (Clary and Wolstenholme 1985 b). These differences account for 78 % of the estimated total size difference between the two mitochondrial genomes. Excluding the control region, this 3667-bp sequence of D. pulex m t D N A also contains 38 fewer intergenic nucleotides than the homologous region in D. yakuba (Clary and Wolstenholme 1985 b).
Results and discussion
Genome organization The 3667-bp fragment of the D. pulex mitochondrial genome (Fig. 2) contains the following genes in the order given; 89 bp o f the 5' end of the large subunit ribosomal R N A gene, the t R N A val gene, the small subunit ribosomal R N A (s-rRNA) gene, the putative control region, the t R N A ale, t R N A gin, t R N A m~ genes, N A D H dehydrogenase subunit 2 (ND2) gene, the t R N A trp, t R N A ors, tRN A tyr genes, and the 5' terminal 668 bp of the Cytochrome c oxidase subunit I (COI) gene. The gene order and direction of transcription are identical to that found for these genes in the D. yakuba mitochondrial genome (Clary and Wolstenholme 1985 b). Three t R N A genes
Control region The putative control region of D. pulex m t D N A contains no open reading frames longer than 42 codons, and is only 67 % A +T. Although there is little sequence conservation to other putative control regions (Clary and
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