Views
5 years ago

Evolution of a RNA Polymerase Gene Family in Silene - Systematic ...

Evolution of a RNA Polymerase Gene Family in Silene - Systematic ...

2004 POPP AND

2004 POPP AND OXELMAN—EVOLUTION OF A RNA POLYMERASE GENE FAMILY IN SILENE 915 genes (RPA2, RPB2, and RPC2), encoding the second largest subunits of these holoenzymes, are single-copy and are located on chromosomes 1, 4, and 5, respectively, whereas the fourth (RPD2)ispresent in two, presumably recently diverged paralogues located on chromosome 3 (The Arabidopsis Genome, 2000). The phylogeny of the tribe Sileneae (Caryophyllaceae) has recently been investigated using nrDNA (the ITS regions) and cpDNA (the rps16 intron) data (Oxelman and Lidén, 1995; Desfeux and Lejeune, 1996; Oxelman et al., 1997, 2001). We choose 29 species from Sileneae to study the evolution and the phylogenetic utility of the RNAP introns and to compare the results with the analyses of the ITS and rps16 data. In this paper we aim to (1) test the phylogenetic hypothesis based on ITS and rps16 data in Sileneae (Oxelman et al., 2001); (2) provide future studies of Sileneae with backbone information from several, presumably unlinked regions, thus facilitating inferences of gene duplications and allopolyploidizations; and (3) investigate the topological congruence among trees inferred from the data sets. To accomplish this we develop a general method for rapid design of primers targeting all members of a gene family; in this case, a region coding for the second largest subunits of the RNA polymerase family. We aim at a complete sampling, i.e., all orthologous and paralogous sequences, of intron regions between two highly conserved amino acid motifs (GDK and GEMERD; Fig. 1) in the genes encoding the second largest subunits (RPA2, RPB2, RPC2, and RPD2, respectively) in the RNAP family. MATERIAL AND METHODS Plant Materials Plant materials used in this study are presented with voucher data and GenBank/EMBL accession numbers in Table 1. Total genomic DNA was extracted as described in Oxelman et al. (1997), or in a few cases using either DNeasy Plant Mini Kit (QiaGen) or Plant DNA Isolation Kit (Boehringer Mannheim) according to the manufacturer’s manual. PCR and Sequencing Typically, 0.625 U Taq polymerase from Advanced Biotechnologies were used in 25 µL PCR reactions, with 1.5 to 2.5 mM Mg 2+ , 200 µMofeach dNTP, 0.5 to 1.0 µM of each primer, 0.01% bovine serum albumin (Boehringer Mannheim), and 0.5 to 1.0 µL total genomic DNA of unknown concentration. The rps16 and ITS regions were amplified using PCR cycling with an initial 5 min denaturation at 95 ◦ C, followed by 35 cycles of 30 s denaturation at 95 ◦ C, 1 min annealing at 56 to 58 ◦ C, 2 min extension at 72 ◦ C, and ended with 10 min final extension at 72 ◦ C. Primers rpsF/rpsR2R (rps16; Oxelman et al., 1997) and P17/26S- 82R (ITS; Popp and Oxelman, 2001) were used for PCR, and rpsF2a/rpsR3R (rps16; Popp et al., in press) and P16b/ITS4R (ITS; Popp et al., in press, and White et al., 1990, respectively) where used for sequencing. To obtain the first RNAP intron sequences we used a low-stringency nested PCR approach (Fig. 2). The first PCR was performed with all combinations of the four RNAP-specific primers kindly provided by B. D. Hall, Washington University, Seattle, USA (Table 2) to amplify the region between the highly conserved amino acid motifs GDK and GEMERD of RPA2, RPB2, RPC2, and RPD2 simultaneously (Fig. 1). After an initial 5 min denaturation at 95 ◦ C, the cycling started with 30 s at 95 ◦ C. Annealing began with3sat50 ◦ C followed by a 0.3 ◦ C increase/s up to 72 ◦ C. This was followed by 72 ◦ C for 2 to 5 min (+1 s/cycle). The cycling was repeated 34 times before 15 min extension at 72 ◦ C and subsequent soak at 4 ◦ C. The result was a heterogeneous pool of PCR products, presumably including all the sought-after RNAP introns. The second PCR (Fig. 2) used the same PCR program, degenerated, though subunit specific primers, provided by B. D. Hall (Table 2), and the pooled PCR products from the previous four reactions as template. In this PCR round, the subunit introns were separated. The resulting FIGURE 1. Structure of second largest subunits of the RNAP gene family in Arabidopsis thaliana. Accession numbers RPA2 to RPD2b: AC008030, AL035527, AB012240, AB020749, and AP000377, respectively. Boxes represents exons and lines represents introns. Lengths are proportional to scale bar. Arrows indicate the highly conserved amino acid regions GDK and GEMERD, and also approximate primer sites for RNAP10F, RNAP10FF, RNAP11R, and RNAP11bR. Note that the two paralogous RPD2 sequences in A. thaliana are not orthologous to the two paralogues in Sileneae. Downloaded from http://sysbio.oxfordjournals.org/ by guest on April 7, 2013

916 TABLE 1. Plant material and GenBank accession numbers. Taxon/chromosome number Vouchera,b rps16c ITSc RPA2c RPB2c RPC2c RPD2a/bc Agrostemma githago, 2n= 24 BO ITS-AGR 30616 (GB) 1 ,MP1049 (UPS) 2 A—541 B—951 C—279–802 D—063–642 D—154–562 D—1382 Atocion armeria, 2n= 24 BO ITS-ARM30611(GB) A—59 B—80 n.a. D—065 n.a. D—139–40 Atocion lerchenfeldiana, 2n= 24 AS 24188 (C) E—061 E—057 C—281 D—066 n.a. D—150–53 Atocion rupestre, 2n= 24 BO 2198 (GB) A—60 B—74 C—282 n.a. n.a. n.a. Eudianthe coeli-rosa, 2n=24 BO 2285 (GB) A—56 B—81 C—283 D—067, 91 n.a. D—125 Eudianthe laeta, 2n=24 BO 1876 (GB) A—55 B—82 C—284 n.a. n.a. D—116–17 Lychnis abyssinica, 2n=? GF 8418 (UPS) 1 ,OH5530 (UPS) 2 A—611 B—901 C—2852 D—0892 n.a. D—135–362 Lychnis chalcedonica, 2n=24 BO 2277 (GB) A—64 B—94 C—286 D—068 n.a. D—141–42 Lychnis coronaria, 2n=24 BO 2278 (GB) 1 ,MP1050 (UPS) 2 A—651 B—911 C—2872 D—0692 D—1572 D—092, 442 Lychnis flos-cuculi, 2n=24 BO 2200 (GB) A—63 B—93 C—288 D—070-71 n.a. D—113–15 Lychnis flos-jovis, 2n=24 BOITS-FLO30610 (GB) A—66 B—92 n.a. D—072 n.a. D—122–24 Petrocoptis pyrenaica, 2n=24 BO 2276 (GB) A—67 B—75 C—289 D—073 n.a. D—093–94 Silene acaulis, 2n=24 BO 2243 (GB) 1 ,MP1046 (UPS) 2 A—891 B—601 C—2902 D—0742 n.a. D—126–272 Silene baccifera, 2n=24 BO2287 (GB) A—69 B—89 C—291 F—139 n.a. D—121, 28–29 Silene bergiana, 2n=24 H 1182 (GB) A—91 B—35 C—292 D—076 n.a. D—132–33 Silene conica, 2n=20 BO1944 (GB) 1 ,BO1898 (GB) 2 A—701 B—321 C—2932 D—0772 n.a. D—145–462 Silene fruticosa, 2n=24 OT 934 (GB) A—88 B—65 C—294 D—078 n.a. D—134, 51–52 Silene keiskei, 2n=24, 48 BO 2345 (UPS) C—913 C—909 C—295, 68–69 D—079–80 D—158 D—095–97 Silene linnaeana, 2n=24 G 143 (MV) 1 ,M1975.VI.28 (K) 2 E—0601 E—0581 C—296–972 D—0752 n.a. D—137, 43, 492 Silene ajanensis, 2n= ? Silene nivalis, 2n=24 BO 2255 (GB) A—90 B—61 C—299 D—082 n.a. D—102-04 Silene nigrescens, 2n=? KGB217 (GB) C—915 B—58 C—298 D—081 n.a. D—100–01 Silene nocturna, 2n=24 OT 654 (GB) A—92 B—41 C—300 D—083 n.a. D—120 Silene noctiflora, 2n=24 BO 2229 (GB) A—76 B—29 n.a. F—140 n.a. D—098–99 Silene parishii, 2n=48 ME 886 (WTU) C—914 C—910 C—301–02 D—084–85 n.a. D—107, 10–12, 18 Silene pentelica, 2n=24 MP1008 (UPS) 1 ,MP1009 (UPS) 2 ,C2046 (GB) 3 AJ2949661 AJ2998122 C—3031 F—1321 n.a. D—105–063 Silene rotundifolia, 2n=48 BO 2231 (GB) A—83 B—87 C—304 D—086–87 n.a. D—147–48 Silene schafta, 2n=24 BO 2264 (GB) 1 ,MP1053 (UPS) 2 A—941 B—521 C—3052 D—0882 n.a. D—130–312 Silene zawadskii, 2n=24 BO2241 (GB) A—77 B—83 C—306 F—141 n.a. D—108-09 Viscaria vulgaris, 2n=24 MP1051 (UPS) C—912 C—911 C—307 D—090 n.a. D—119 a b Superscript numbers indicate corresponding specimen if sequences are produced from more than one voucher. BO = B. Oxelman; MP = M. Popp; AS = A. Strid et al.; GF = Gilbert and Fries; OH = O. Hedberg; H = Holmdahl; OT = Oxelman and Tollsten; G = Gubanov; M = Mikhajlova [?]; ME = M. Egger; C = Christodoulakis. cA— = Z831; B— = X868; C— = AJ629; D— = AJ634; E— = AJ409; F— = AJ296. Downloaded from http://sysbio.oxfordjournals.org/ by guest on April 7, 2013

Systematic Evolution of Ligands by Exponential Enrichment: RNA ...
Analyses of RNA Polymerase II Genes from Free-Living Protists ...
RNA polymerase III transcribes human microRNAs - Gene ...
Evolution of two modes of intrinsic RNA polymerase transcript ...
RNA Polymerase Stalling at Developmental Control Genes in - MIT
Evolution of the Globin Gene Family in Deuterostomes: Lineage ...
RNA Polymerase II progression through H3K27me3-enriched gene ...
Evolution of the Globin Gene Family in ... - Current Research
Highly Heterogeneous Rates of Evolution in the SKP1 Gene Family ...
Review Evolution and systematics of the Chelicerata
a-turan.vp:CorelVentura 7.0 - Institute of Systematics and Evolution ...
Transcription of RNA templates by T7 RNA polymerase
Evolution and microsynteny of the apyrase gene family in three ...
Molecular Systematics and Evolution of Regina and the ...
Hox gene family and the evolution of chordates
Gene-specific requirement for P-TEFb activity and RNA polymerase ...
ChIP-Seq of ERa and RNA polymerase II defines genes ... - CBMEG
Recruitment of RNA polymerase III to its target promoters - Genes ...
Evidence of Independent Gene Duplications During the Evolution of ...
An archaebacterial RNA polymerase binding site ... - Mbio.ncsu.edu
Evolution of Mitochondrial Gene Orders in Echinoderms
Antiquity and evolution of the MADS-box gene family controlling ...
Transcription Termination by Bacteriophage T7 RNA Polymerase at ...
RNA Polymerase Stalling at Developmental ... - Young Lab - MIT
Phylogenetic Relationships, Evolution, and Systematic ... - BioOne
Protein Evolution of the PEBP gene family in Plants - Uppsala ...
The Evolution of Homing Endonuclease Genes and Group I Introns ...