Copyright 0 1995 by the Genetics Society of America Complete Sequence and Gene Organization of the Mitochondrial Genome of the Land Snail Albinuria cornlea Evi Hatzoglou, George C. Rodakis and Rena Lecanidou Department of Biochemistry, Cell and Molecular Biology, and Genetics, University of Athens, Panepistimiopolis, Athens 157 01, Greece Manuscript receivedJanuary 31, 1995 Accepted for publication May 15, 1995 ABSTRACT The complete sequence (14,130 bp) of the mitochondrial DNA (mtDNA) of the land snail Ahinaria coerulea was determined. It contains 13 protein, two rRNA and 22 tRNA genes. Twenty-four of these genes are encoded by one and 13 genes by the other strand. The gene arrangement shares almost no similarities with that of two other molluscs for which the complete gene content and arrangement are known, the bivalve Mytilus edulis and the chiton Kathanna tunicata; the protein and rRNA gene order is similar to that of another terrestrial gastropod, Cepaeu nemoralis. Unusual features include the following: (1) the absence of lengthy noncoding regions (there are only 141 intergenic nucleotides interspersed at different gene borders, the longest intergenic sequence being 42 nucleotides), (2) the presence of several overlapping genes (mostlytRNAs), (3) the presence of tRNA-like structures and other stem and loop structures within genes. An RNA editing system acting on tRNAs must necessarily be invoked for posttranscriptional extension of the overlapping tRNAs. Due to these features, and also because of the small size of its genes (e.g.,it contains the smallest rRNA genes among the known coelomates), it is one of the most compact mitochondrial genomes known to date. N the last few years, there has been an accelerated aswell as Cqbuea nemoralis, whose partial gene order I accumulation of sequence data on animal mitochon- (excluding tRNA genes) has been published (TERREIIT drial genomes. Most of the complete mitochondrial et al. 1994). DNA (mtDNA) sequences that have been published The mtDNA gene content of coelomate metazoans concerndeuterostomes [vertebrates and echino- is constant: it consists of 13 protein genes, two genes derms: human (ANDERSON et al. 1981), mouse (BIBB for the small and large ribosomal RNA subunits, and et al. 1981), rat (GADALETAet al. 1989), cow (ANDER- 22tRNA genes, some of which are transcribed from SON et al. 1982), fin whale (ARNASON et al. 1991), blue one andsome from the othermtDNA strand. An excep whale (ARNASON and GULLBERG1993), harbor seal, tion is observed in molluscs. Mytilusis missing a protein Phoca vitulina (ARNASON andJOHNsSON 1992),Ameri- gene (ATPuse8), while it contains an extra tRNA gene can opossum, Didelphis virgzniana UANKEet al. 1994), and has all of its genes transcribed from one strand chicken (DESJARDINSand MORAIS 1990),Xenopus (HOFFMANNet al. 1992); Katharina, on the other hand, (ROE et al. 1985), carp (CHANGet al. 1994), sea ur- contains the standard setof 37 mitochondrial genes, as chins Paracentrotus lividus (CANTATOREet al. 1989) well as two extra tRNA genes, that may or may not be and Strongylocentrotus purpuratus (JACOBS et al. 1988), functional (BOOREand BROWN1994b). sea star, Asterina pectinijiera (AsAKAWA et al. 1991)], Besides coding regions, there are also noncoding while the available sequences for protostome coelo- sequences in animal mtDNA and, more specifically, a mates are limited to fourarthropods [Drosophila major noncoding segment,which in deuterostomes (D- yalzuba (CLARYand WOLSTENHOLME1985a), bee (CRO- loop) contains a combination of sequence elements ZIER and CROZIER1993), mosquito, Anopheles quadrima- that are related with control of both replication and culatus (MITCHELLet al. 1993), Artemia (PEREZet al. transcription (JACOBS et al. 1988; CLAYTON 1991, 1992; 1994, EMBLdata bank accession number X69067)l and WOLSTENHOLME 1992; SHADEL and CLAY~ON1993). one mollusc [the chiton Katharina tunicata (BOOREand The length of this region is extremely variable [121 BROWN1994b)l. Within the phylum of molluscs, con- nucleotides (nt), sea urchin (JACOBSet al. 1988); over siderable information is also availablefor Mytilus edulis, 20 kb, pine weevil(BOYCE et al. 1989)l. The length whose partial sequence but complete mtDNA gene con- variation is usually due to the presence of short (e.g., tent and organization is known (HOFFMANet al. 1992), 10 bp) (GHMZZANIet al. 1993) or longer [e.g., 260 bp (BROUGHTONand DOWLINC1994) or 1.2 kb (GJETVAJ Cmespondingauthw:Rena Lecanidou, Department of Biochemistry, et al. 1992)] repeated sequences. Size variation in non- Cell and Molecular Biology, and Genetics, University of Athens, Panepistimiopolis,Athens 157 01, Greece. coding sequencesinevitably results in total length varia- E-mail: [email protected] tion, which can be observed even at thelevel of individ- Genetics 140 1353-1366 (August, 1995) 1354 E. 1354 Hatzoglou, G. C. Rodakis and R. Lecanidou uals (reviewed by MORITZ et al. 1987). Extremes in size variation have been documented in molluscs:seven species of scallops have sizes ranging from 16.2 to 42 kb (SNYDERet al. 1987; LAROCHEet al. 1990; GJETVAJet al. 1992). The mtDNA gene organization is considered to be relatively constant within eachmetazoan phylum, where the observed variations mainly concern tRNA gene rearrangements (WOLSTENHOLME1992) and nu- cleotide substitutions (CROZIER et al. 1989; PALUMBI and BENZIE 1991; AVISE et al. 1992; MARTIN et al. 1992). However, molluscs are the largest exception to this rule, since large variations in gene organiza- tion (e.g., between Mytilus and Katharina) (BOORE and BROWN 1994b) and sequence [Albinaria tunita vs. Mytilus (LECANIDOUet al. 1994) and Katharina us. Mytilus (BOORE and BROWN1994b)l have been found. We have recently pointed out thefirst indications for this great diversity of molluscan mtDNA, as Albinaria sequences are more similar to the corresponding se- quences of Drosophila than of Mytilus, and have sug- FIGURE1.-Gene map of the A. coeruleu mtDNA molecule. gested that this may be attributed to either a polyphy- The outer circle represents the sense strand for genes tran- scribed clockwise and the inner circle for genes that are tran- letic origin or to a high and differentiated evolutionaIy scribed counterclockwise. tRNA genes are depicted by the rate of molluscan mtDNA (DOURISet al. 1995; LECANI- one-letter amino acid code. Positive numbers at gene bound- DOU et al. 1994). Boom and BROWN(1994b), on the aries indicate noncoding nucleotides; negativenumbers indi- basis of the observation that structural features of Ka- cate overlappingnucleotides of adjacent genes. Abbreviations tharina resemble more those of Drosophila than of used: ATPase68, ATP synthase subunits 6 and 8; COI-IIZ, cyte Mytilus, have proposed a fast mtDNA molecular clock chrome c oxidase subunits I, 11, and 111; Cytb, cytochrome b apoenzyme; NDl-6and ND4L, NADH dehydrogenase subunits for Mytilus. 1-6 and 4L; s-rRNA and l-rRNA, small and large subunits of In this paper, we present the complete mtDNA se- ribosomal RNA. quence of the land snail A. coerulea. It is the smallest mtDNA among coelomate metazoans (14,130 bp), does KS (Stratagene). Further subcloning was performed after not contain any noncoding sequence longer than 42 deletions of clones and subclones using the enzyme Exo- nt, andits gene organization seems to have almost noth- nuclease I11 (HENIKOFF1987). Clones and derived sub- clones were amplified after transformation withCaC12/ ing in common with that of other known metazoans, RbC1, using as hosts Escherichia coli JM83. DNA was ex- with the exception of the major genes of another terres- tracted by mini plasmid preparations with alkaline lysis trial gastropod, C. nemoralis. (SAMBROOKet al. 1989). Both mtDNA strands werecompletely sequenced. Se- quence determination was performed from the ends of clones MATERIALSAND METHODS and subclones by the dideoxynucleotide chain termination method (SANGERet al. 1977) using Sequenase V 2.0 (US Bie The mtDNA of A. coemleu was cloned in three consecu- chemical) and universal Ml3/pUC primers.Samples were tive HindIII segments: 6.55 kb (13A3, bases 1561-8123), resolved in 4 or 5% polyacrylamide, 7.5 M urea gels, and 5.4 kb (13A12, bases8124-13278), and 2.4 kb (13F6, bases autoradiography was performed using Kodak X-Omat film. 13279-1560) (Figure 2) (DOURISet al. 1995). The follow- Sequences wereidentified by comparison with data from the ing evidence indicates that the entire mitochondrial ge- EMBL data bank or from cited publications.The tRNA genes nome is represented in these three Hind111 clones: (1) were identified by their potential to form the characteristic sequencing of an EcoRI clone (15A6, bases 282-2950) (Fig- for mt-tRNA stem and loop structures. Sequence analysis was ure 2) showed that it overlaps clones 13F6 and 13A3, which performed using computer programs developed by PUSTELL therefore must be adjacent; (2) clones 13A12 and 13F6 and KAFATOS (1984, 1986). Other computer programs used must also be adjacent, because a sequenced clone from are cited in the text. a closely related Albinaria species (A28 from A. turrita) Northern hybridization was used to identify the location (LECANIDOUet al. 1994) overlaps them, and (3) theHindIII of the tRNALysgene. RNA was extracted from the feet (mus- site between clones 13A3 and 13A12 (base 8124) (Figure cle) of 12 snails, treated with Proteinase K, extracted with 2) lies in a conserved region of the ND4 gene (bases 8113 phenol/chloroformand precipitated with ethanol ac- to 8137 correspond