!"#$%&'& ()& Genomic organization and comparative chromosome mapping of U1 snRNA gene in cichlid fish, with emphasis in Oreochromis niloticus* D.C. Cabral-de-Mello1,*, G.T. Valente2, R.T. Nakajima2 and C. Martins2 1UNESP – Univ Estadual Paulista, Instituto de Biociências/IB, Departamento de Biologia, Rio Claro, São Paulo, Brazil 2UNESP – Univ Estadual Paulista, Instituto de Biociências/IB, Departamento de Morfologia, Botucatu, São Paulo, Brazil Short running title: Genomic organization and mapping of U1 snRNA in cichlid fish * Corresponding author: UNESP - Univ Estadual Paulista, Instituto de Biociências/IB, Departamento de Biologia, CEP 18618-000 Botucatu, SP, Brazil Phone/Fax: 55 14 38116264. e-mail: [email protected] *Cabral-de-Mello DC, Valente GT, Nakajima RT, Martins C (2012) Genomic organization and comparative chromosome mapping of the U1 snRNA gene in cichlid fish, with an emphasis in Oreochromis niloticus. Chromosome Research 20(2): 279-292. !"#$%&'& (*& Abstract To address the knowledge of genomic and chromosomal organization, and evolutionary patterns of U1 snRNA gene in cichlid fish this gene was cytogenetically mapped and comparatively analyzed in 19 species belonging to several clades of the group. Moreover, the genomic organization of U1 snRNA was analyzed using as reference the Oreochromis niloticus genome. The results indicated a high conservation of one chromosomal cluster of U1 snRNA in the African and Asian species with some level of variation mostly in the South American species. The genomic analysis of U1 revealed a distinct scenario of that observed under the cytogenetic mapping. It was observed just an enrichment of U1 gene in the linkage group (LG) 14, that do not correspond to the same chromosome that harbors the U1 cluster identified under the cytogenetic mapping. Moreover it was revealed the presence of several distinct transposable elements in the U1 gene flanking regions that could be involved in the spreading of this sequence, being the origin of new snRNA large clusters hampered. The results reinforce the utility of integrative analysis, as the use of cytogenetic and bioinformatics methods, to address the genomic and chromosomal evolutionary patterns of multigene families among vertebrates. Moreover, the U1 gene represents a useful new chromosomal marker for purposes of cytogenetic studies. Key-words: chromosomal evolution, cytogenetic, FISH, genome, multigene family !"#$%&'& (+& Introduction In eukaryotes protein-coding mRNA introns are excised and the exons spliced together in the primary transcript by the spliceosome machinery. The spliceosome consists of large RNA- protein complex containing of up to 200 proteins and five types of metabolically stable non- coding RNAs known as U small nuclear RNAs (snRNAs), U1, U2, U4, U5 and U6 (Bringmann and Lührmann 1986; Nilsen 2003; Valadkhan 2005). The U snRNA genes were isolated from a variety of eukaryotes and they were reported to be in multiple copies dispersed thought the genome, however some cases of tandemly repeats were also found (see for example, Wise and Weiner 1980; Marzluff et al. 1983; Mattaj and Zeller 1983; Watanabe- Nagasu et al. 1983; Van Arsdell and Weiner 1984). Recently Marz et al. (2008) investigated in detail the evolutionary history of snRNAs gene families using data of completely or partially sequenced metazoan genomes, revealing that they behave like mobile elements exhibiting very little syntenic conservation. At chromosomal point to view, the mapping of snRNA genes was performed in few species. For example, the U1 snRNA gene/pseudogene was cytogenetically mapped in humans (Lund et al. 1983; Lindgren et al. 1985) and mouse (Lund and Nesbitt 1988), and in fish the U2 snRNA gene was mapped in four Batrachoididae and two Moronidae representatives (Merlo et al. 2010; Úbeda-Manzanaro et al. 2010). Moreover, in two crustaceans (Asellus aquaticus and Proasellus coxalis) and in another fish (Solea senegalensis) a repetition of 5S rRNA gene linked to snRNAs sequences were also mapped (Pelliccia et al. 2001; Barzotti et al. 2003; Manchado et al. 2006). The family Cichlidae includes more than 3.000 species (Helfaman et al. 1997, Salzburger and Meyer, 2004), being one of the most species-rich families of vertebrates (Nelson, 2006), distributed on Africa, Latin America and Madagascar, with few species in South India and the Middle East (Genner et al. 2007). The cichlid representatives found in the !"#$%&'& (,& lakes of Africa have served as model systems for the study of evolution (Kornfield and Smitth 2000, Kocher 2004, Genner et al 2007) and some species have received increasing scientific attention due their great importance to aquaculture (Pullin 1991). Due its great scientific importance some cichlid genomes have been sequenced, i.e. Oreochromis niloticus, Pundamilia nyererei, Haplochromis burtoni, Neolaprologus brichardi and Metraclima zebra (Cichlid genome consortium), permitting the integration of nucleotide sequence information and chromosomal data in the understanding of karyotypic and genomic diversification in the group. To advance in the knowledge about chromosome evolution and genomic organization of multigene families in cichlids and to contribute in the construction of a cytogenetic map in this group, here it was performed the mapping of U1 snRNA gene by fluorescence in situ hybridization (FISH) in 19 cichlid species. Moreover, analysis of the genomic organization of U1 snRNA sequence was addressed using as reference the Nile tilapia, O. niloticus. The results obtained are discussed on the focus of the chromosomal conservation of one cluster of U1 snRNA genes in diverse cichlid lineages and a spread pattern of genomic organization of this gene in O. niloticus. In this way, this paper reinforces the importance of integration between cytogenetic and genomic analysis in the elucidation of genome evolution. Material and Methods Animals, DNA extraction and chromosome obtaining The 19 analyzed species of African, South American and Asian cichlids were obtained from commercial source, from wild stocks (mainly from the Lake Malawi, East Africa) maintained at the Tropical Aquaculture Facility of University of Maryland, USA and directly from Brazilian hydrographic systems (Table 1). Metaphase chromosome spreads were obtained from kidney cells according to the protocols of Bertollo et al. (1978). Genomic DNA !"#$%&'& (-& extraction was performed according standard phenol-chloroform procedures (Sambrook and Russel, 2001) using liver previously stored in 100% ethanol. Analysis of U snRNAs genes and their flanking regions in Oreochromis niloticus genome The genes of U1 snRNAs (NR_004430), U2 (NR_002716) and U5 (NR_002756) of human were used as queries in blast (blastn) search against O. niloticus genome (Tilapia_broad_v1 genome), allocated in BouillaBase database (www.buillabase.org) (at 20- 05-2011 for U1 and 10-07-2011 for both U2 and U5), to reveal the presence of these genes in the Nile tilapia genome. The matches with significant similarities were retrieved using the cut off E-values ! 5e-13 for U1, ! 4e-11 for U2 and ! 3e-10 for U5. The putative U1 sequences of O. niloticus were retrieved with their relative scaffolds and the U1 copies were used as the queries in blast (megablast) search against NCBI nucleotide collection (http://www.ncbi.nlm.nih.gov/nucleotide/). Moreover these sequences were assembled using an assembly tool of Geneious 4.8.5 software and the human U1 gene (NR_004430) as reference. New queries for each U1 annotated entries were built for a second step of analysis involving 1,000 bp upstream and 1,000 bp downstream flanking regions (FRs) of each U1 gene copy. When upstream and downstream FRs analyzed included the U1 gene, it was named FRU; the analyzes of upstream and downstream FRs excluding the U1 gene was named FR. However U1 annotated entries of each FR were excluded in attempt to decrease the redundancy of results; the Ns were excluded in FRs as well. Each FRs were used as queries in blast (megablast and discontinuous_megablast) searches against NCBI nucleotide collection and each FRUs were used as queries in the searches against Repbase database (Jurka et al. 2005) at Genetic Information Research Institute (Giri) (http://www.girinst.org/repbase/) using the CENSOR software (Kohany et al. !"#$%&'& (.& 2006). The analysis in Giri became able to find repeated sequences such as transposable elements (TEs) in the flanking FRs and FRUs regions. To further the comparison among the FRUs regions, all sequences were submitted to assembling using the assembling tool of Geneious 4.8.5. Moreover it was performed a search for open reading frames (ORFs), using a tool of Geneious 4.8.5 searching by a minimum size of 15 aminoacid (aa) residues, in attempt to check the potential protein-coding regions and possible capacity of transpositions of the TEs found by analysis in Repbase database (Jurka et al. 2005). The search for potential protein- coding of the TEs was performed in all scaffolds, except in scaffold 255 due its absence of TEs in the FRUs reveled in the previous analysis in Repbase. After, the aminoacid (aa) residues were submitted to searches against Repbase database (Jurka et al. 2005), as already described. When necessary some sequences were re-submitted to blast searches at NCBI nucleotide collection and all annotation procedures and alignment editing were performed using this Geneious 4.8.5. Sequences isolation, probes obtaining and labeling Partial sequence of U1 snRNA gene was obtained by Polymerase