Steinar Johansen

The genome sequence of Atlantic cod reveals a unique immune system

Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates, but we show that Atlantic cod has lost the genes for MHC II, CD4 and invariant chain (Ii) that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions. We find a highly expanded number of MHC I genes and a unique composition of its Toll-like receptor (TLR) families. This indicates how the Atlantic cod immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHC II. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.

A new nomenclature of group I introns in ribosomal DNA.

The current nomenclature system of group I introns (see Cech, 1988;Michel & Westhof, 1990) has become insufficient to distinguish and categorize the complex collection of more than 900 group I introns in ribosomal DNA (rDNA) of nuclear, mitochondrial, chloroplast, and eubacterial genomes (http://www+rna+icmb+utexas+edu/; GenBank; our unpubl+ results) in a rational way+ The majority of these group I introns (;750) are found in nuclear rDNA of fungi and protists, but the distribution appears highly scattered since most species analyzed lack introns+ Many of the rDNA introns are optional among strains of a particular species or between closely related species, and some have been shown in experimental settings to be true mobile genetic elements (see Belfort & Roberts, 1997)+ All group I rDNA introns are found at a limited number of insertion sites (;75) in highly conserved regions of the small subunit (SSU) and large subunit (LSU) rRNA genes, and some of these sites (;10) are shared by introns from the nuclei, mitochondria, or chloroplasts+ There are numerous examples of multiple group I introns in a single rRNA gene, and as many as eight nuclear introns have been noted in the SSU rDNA of the lichen ascomycete Lecanora dispersa (accession number L37734) and in the LSU rDNA of the myxomycete Fuligo septica (our unpubl+ results)+ Finally, group I introns that occupy the same site in rDNA, but in distantly related hosts, tend to share a number of structural features as well as high levels of primary sequence similarities compared to introns at different insertion sites (e+g+, Suh et al+, 1999)+ We propose an alternative nomenclature system for the rDNA group I introns based on (1) three-letter abbreviation of host scientific name, (2) one letter abbreviation of host gene, and (3) insertion site in the rDNA according to the Escherichia coli SSU or LSU rRNA sequence numbering (accession number AB035922)+ Examples of renaming are Nja+S516 (former NjaSSU1) from Naegleria jamiesoni SSU rDNA at position 516, and Tth+L1925 (former TtLSU1) from Tetrahymena thermophila LSU rDNA at position 1925 (see Table 1, lines 1 and 4)+ Typical examples of the new rDNA group I intron nomenclature are included in Table 1 (lines 1–6)+ When appropriate, introns in different genome types could be distinguished by adding an abbreviation in front of L or S (see Table 1, lines 7–12)+An example is group I introns at position 2449 in LSU rDNA of Physarum polycephalum nuclei (Ppo+nL2449), Saccharomyces cerevisiae mitochondria (Sce+mL2449), and Chlamydomonas pallidostigmatica chloroplast (Cpa+cL2449)+Flexibility in the nomenclature becomes necessary in a few exceptional cases+Distantly related introns present at the same insertion site in different strains of the same species are named numerically, for example the two very different group I introns at position 956 in SSU rDNA in Didymium iridis isolates Pan2 and CR8 are named Dir+S956-1 and Dir+S956-2, respectively (Table 1, lines 13 and 14)+ Finally, the three-letter abbreviation of host scientific names may sometimes be insufficient+ An example is introns at position 1516 in SSU rDNA of different Lecanora species+ The introns in L. albescenc, L. allophana, L. concolor, and L. contractula should be named Lalb+S1516, Lall+S1516, Lconc+S1516, and Lcont+S1516, respectively (Table 1, lines 15–18)+

Large-scale sequence analyses of Atlantic cod.

The Atlantic cod (Gadus morhua) is a key species in the North Atlantic ecosystem and commercial fisheries, with increasing aquacultural production in several countries. A Norwegian effort to sequence the complete 0.9Gbp genome by the 454 pyrosequencing technology has been initiated and is in progress. Here we review recent progress in large-scale sequence analyses of the nuclear genome, the mitochondrial genome and genome-wide microRNA identification in the Atlantic cod. The nuclear genome will be de novo sequenced with 25 times oversampling. A total of 120 mitochondrial genomes, sampled from several locations in the North Atlantic, are being completely sequenced by Sanger technology in a high-throughput pipeline. These sequences will be included in a new database for maternal marker reference of Atlantic cod diversity. High-throughput 454 sequencing, as well as Evolutionary Image Array (EvoArray) informatics, is used to investigate the complete set of expressed microRNAs and corresponding mRNA targets in various developmental stages and tissues. Information about microRNA profiles will be essential in the understanding of transcriptome complexity and regulation. Finally, developments and perspectives of Atlantic cod aquaculture are discussed in the light of next-generation high-throughput sequence technologies.

Group I introns: Moving in new directions.

Group I introns are genetic elements interrupting functional genes. They are removed from precursors at the RNA level and most catalyze their own splicing. The catalytic part of these constitutes one of the major classes of catalytic RNAs, the group I ribozymes. However, group I introns have a lot more to offer than their own elimination by splicing. Intron RNA can circularize in at least three different ways and introns are mobile both at the DNA and RNA level. Some group I introns have a very complex organization incorporating functional genes and other sequence elements and have established deep relationships with their host genomes. Finally, group I introns can develop into new ribozymes with new biological functions

Variable numbers of simple tandem repeats make birds of the order Ciconiiformes heteroplasmic in their mitochondrial genomes

We have analyzed a variable domain of the mitochondrial DNA control region of 18 avian species. intra-individual length variation was identified and characterized in 15 species. The occurrence of heteroplasmy among species is phylogenetically consistent with a current classification of birds. Polymerase chain reaction amplifications, direct sequencing, and Southern analysis of mitochondrial DNA showed that the heteroplasmy is due to variable numbers of direct repeats in a tandem organization, located in the control region close to the tRNAPhe gene. The tandem repeats consist of short sequence motifs that vary in size from 4 to 32 base pairs between species. Sequence complexity of the repeat motifs was low, with almost exclusively Ts and Gs in the heavy-strand. Extensive variation in the copy number of the repeats was seen both intra-specifically and within individuals. This is the first report of mitochondrial heteroplasmy characterized at the sequence level in birds.

In vivo mobility of a group I twintron in nuclear ribosomal DNA of the myxomycete Didymium iridis

DiSSU1 is an optional group I twintron present in the nuclear extrachromosomal ribosomal DNA of the myxomycete Didymium iridis. DiSSU1 appears to be complex both in structure and function. At the RNA level it has a twin‐ribozyme organization composed of two group I ribozymes with different functions, separated by an open reading frame. Here, we show that DiSSU1 is mobile when haploid intron‐containing and intron‐less amoebae are mated. The mobility process is fast, being completed in 5–10 nuclear cycles after mating in the developing zygote and plasmodia. Analyses of progeny from genetic crosses confirm intron mobility. DiSSU1 is the first example of a mobile group I twintron. The intron‐encoded protein was expressed in Escherichia coli and found to be an endonuclease, I‐Dir I, that cleaves an intron‐less ribosomal DNA allele at the intron‐insertion site, and is probably involved in intron homing. The endonuclease I‐Dir I seems to be a rare example of a protein that is expressed from a ribozyme‐processed RNA polymerase I transcript in vivo.

In vivo expression of the nucleolar group I intron‐encoded I‐DirI homing endonuclease involves the removal of a spliceosomal intron

The Didymium iridis DiSSU1 intron is located in the nuclear SSU rDNA and has an unusual twin‐ribozyme organization. One of the ribozymes (DiGIR2) catalyses intron excision and exon ligation. The other ribozyme (DiGIR1), which along with the endonuclease‐encoding I‐DirI open reading frame (ORF) is inserted in DiGIR2, carries out hydrolysis at internal processing sites (IPS1 and IPS2) located at its 3′ end. Examination of the in vivo expression of DiSSU1 shows that after excision, DiSSU1 is matured further into the I‐DirI mRNA by internal DiGIR1‐catalysed cleavage upstream of the ORF 5′ end, as well as truncation and polyadenylation downstream of the ORF 3′ end. A spliceosomal intron, the first to be reported within a group I intron and the rDNA, is removed before the I‐DirI mRNA associates with the polysomes. Taken together, our results imply that DiSSU1 uses a unique combination of intron‐supplied ribozyme activity and adaptation to the general RNA polymerase II pathway of mRNA expression to allow a protein to be produced from the RNA polymerase I‐transcribed rDNA.

Sex-biased miRNA expression in Atlantic halibut (Hippoglossus hippoglossus) brain and gonads

The role of miRNA in fish sexual development is not elucidated yet. We profiled miRNAs in gonads and brains of Atlantic halibut using SOLiD sequencing technology. We found tissue- and sexually dimorphic expression of several miRNAs, including miR-29a, miR-34, miR-143, miR-145, miR-202-3p, miR-451, and miR-2188. miR-9 and miR-202 were abundant in brain and gonads, respectively. In the next step, we selected some miRNAs showing differential expression patterns between sexes and performed RT-qPCR on 3 age groups: juveniles, 3-year-, and 5-year-olds. In brains, miR-451 was significantly down-regulated in juveniles compared to adults. let-7a, miR-143, and miR-202-3p were up-regulated in gonads of mature males compared to immature females at the same age. We investigated the effect of suppressing aromatase cytochrome P450 enzyme on miRNA expression at the onset of sex differentiation through masculinization with Fadrozole or 17-α-methyltestosterone. We found significant differences in miRNA expression between masculinized individuals and untreated controls. miR-202-3p was significantly down-regulated in female juveniles compared to male juveniles. The expression levels of let-7a and miR-451 were restored after termination of the masculinization treatment. Our data give a first insight into miRNA involvement in sexual development in teleosts.

Two group I ribozymes with different functions in a nuclear rDNA intron.

DiSSU1, a mobile intron in the nuclear rRNA gene of Didymium iridis, was previously reported to contain two independent catalytic RNA elements. We have found that both catalytic elements, renamed GIR1 and GIR2, are group I ribozymes, but with differing functionality. GIR2 carries out the several reactions associated with self‐splicing. GIR1 carries out a hydrolysis reaction at an internal processing site (IPS‐1). These conclusions are based on the catalytic properties of RNAs transcribed in vitro. Mutation of the P7 pairing segment of GIR2 abrogated self‐splicing, while mutation of P7 in GIR1 abrogated hydrolysis at the IPS‐1. Much of the P2 stem and all of the associated loop could be deleted without effect on self‐splicing. These results are accounted for by a secondary structure model, in which a long P2 pairing segment brings the 5′ splice site to the GIR2 catalytic core. GIR1 is the smallest natural group I ribozyme yet reported and is the first example of a group I ribozyme whose presumptive biological function is hydrolysis. We hypothesize that GIR1‐mediated cleavage of the excised intron RNA functions in the generation and expression of the mRNA for the intron‐encoded endonuclease I‐DirI.

Structure and evolution of myxomycete nuclear group I introns: a model for horizontal transfer by intron homing

SummaryWe have examined five nuclear group I introns, located at three different positions in the large subunit ribosomal RNA (LSU rRNA) gene of the two myxomycete species, Didymium iridis and Physarum polycephalum. Structural models of intron RNAs, including secondary and tertiary interactions, are proposed. This analysis revealed that the Physarum intron 2 contains an unusual core region that lacks the P8 segment, as well as several of the base-triples known to be conserved among group I introns. Structural and evolutionary comparisons suggest that the corresponding introns 1 and 2 were present in a common ancestor of Didymium and Physarum, and that the five introns in LSU rRNA genes of these myxomycetes were acquired in three different events. Evolutionary relationships, inferred from the sequence analysis of several different nuclear group I introns and the ribosomal RNA genes of the intron-harbouring organisms, strongly support horizontal transfer of introns in the course of evolution. We propose a model that may explain how myxomycetes in natural environments obtained their nuclear group I introns.