Advertisement

Molecular Genetics and Genomics

, Volume 289, Issue 6, pp 1209–1216 | Cite as

B chromosomes showing active ribosomal RNA genes contribute insignificant amounts of rRNA in the grasshopper Eyprepocnemis plorans

  • Mercedes Ruiz-Estévez
  • Liesbeth Badisco
  • Jozef Vanden Broeck
  • Francisco Perfectti
  • María Dolores López-León
  • Josefa Cabrero
  • Juan Pedro M. Camacho
Original Paper

Abstract

The genetic inertness of supernumerary (B) chromosomes has recently been called into question after finding several cases of gene activity on them. The grasshopper Eyprepocnemis plorans harbors B chromosomes containing large amounts of ribosomal DNA (rDNA) units, some of which are eventually active, but the amount of rRNA transcripts contributed by B chromosomes, compared to those of the standard (A) chromosomes, is unknown. Here, we address this question by means of quantitative PCR (qPCR) for two different ITS2 amplicons, one coming from rDNA units located in both A and B chromosomes (ITS2A+B) and the other being specific to B chromosomes (ITS2B). We analyzed six body parts in nine males showing rDNA expression in their B chromosomes in the testis. Amplification of the ITS2B amplicon was successful in RNA extracted from all six body parts analyzed, but showed relative quantification (RQ) values four orders of magnitude lower than those obtained for the ITSA+B amplicon. RQ values differed significantly between body parts for the two amplicons, with testis, accessory gland and wing muscle showing threefold higher values than head, gastric cecum and hind leg. We conclude that the level of B-specific rDNA expression is extremely low even in individuals where B chromosome rDNA is not completely silenced. Bearing in mind that B chromosomes carry the largest rDNA cluster in the E. plorans genome, we also infer that the relative contribution of B chromosome rRNA genes to ribosome biogenesis is insignificant, at least in the body parts analyzed.

Keywords

B chromosome Gene expression qPCR rDNA rRNA genes 

Notes

Acknowledgments

We thank an anonymous reviewer for helpful suggestions that substantially improved the manuscript, and Karl Meunier for language revision. This study was supported by a grant from the Spanish Ministerio de Ciencia e Innovación (CGL2009-11917) and Plan Andaluz de Investigación (CVI-6649), and was partially performed by FEDER funds. M Ruíz-Estévez was supported by a FPU fellowship from the Spanish Ministerio de Ciencia e Innovación. The authors also gratefully acknowledge the KU Leuven Research Foundation (GOA/11/02) and the Research Foundation of Flanders (Belgium) for financial support.

Integrity of research

All experiments comply with the current Spanish and Belgian laws. All institutional and national guidelines for the care and use of laboratory animals were followed. The authors declare that they have no conflict of interest.

Supplementary material

438_2014_880_MOESM1_ESM.doc (1005 kb)
Supplementary material 1 (DOC 1005 kb)

References

  1. Banaei-Moghaddam AM, Meier K, Karimi-Ashtiyani R, Houben A (2013) Formation and expression of pseudogenes on the B chromosome of rye. Plant Cell 25:2536–2544PubMedCentralPubMedCrossRefGoogle Scholar
  2. Cabrero J, Alché JD, Camacho JPM (1987) Effects of B chromosomes of the grasshopper Eyprepocnemis plorans on nucleolar organizer regions activity. Activation of a latent NOR on a B chromosome fused to an autosome. Genome 29:116–121CrossRefGoogle Scholar
  3. Cabrero J, López-León MD, Bakkali M, Camacho JPM (1999) Common origin of B chromosomes variants in the grasshopper Eyprepocnemis plorans. Heredity 83:435–439PubMedCrossRefGoogle Scholar
  4. Camacho JPM (2005) B chromosomes. In: Gregory TR (ed) The evolution of the genome. Elsevier, San Diego, pp 223–286CrossRefGoogle Scholar
  5. Camacho JPM, Shaw MW, López-León MD, Pardo MC, Cabrero J (1997) Population dynamics of a selfish B chromosome neutralized by the standard genome in the grasshopper Eyprepocnemis plorans. Am Nat 149:1030–1050PubMedCrossRefGoogle Scholar
  6. Camacho JPM, Cabrero J, López-León MD, Bakkali M, Perfectti F (2003) The B chromosomes of the grasshopper Eyprepocnemis plorans and the intragenomic conflict. Genetica 117:77–84PubMedCrossRefGoogle Scholar
  7. Carchilan M, Kumke K, Mikolajewski S, Houben A (2009) Rye B chromosomes are weakly transcribed and might alter the transcriptional activity of A chromosome sequences. Chromosoma 118:607–616PubMedCrossRefGoogle Scholar
  8. Chapuis MP, Tohidi-Esfahani D, Dodgson T, Blondin L, Ponton F, Cullen D, Simpson SJ, Sword GA (2011) Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust. BMC Mol Biol 12:7PubMedCentralPubMedCrossRefGoogle Scholar
  9. Gunderson JH, Sogin ML, Wollett G, Hollingdale M, de la Cruz VF, Waters AP, McCutchan TF (1987) Structurally distinct, stage-specific ribosomes occur in Plasmodium. Science 238:933–937PubMedCrossRefGoogle Scholar
  10. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98Google Scholar
  11. Hannan KM, Hannan RD, Rothblum LI (1998) Transcription by RNA polymerase I. Front Biosci 3:376–398Google Scholar
  12. Heitz E (1931) Die Ursache der gesetzmässigen Zahl, Lage, From und Grösse pflanzlicher Nukleolen. Planta 12:775–844CrossRefGoogle Scholar
  13. Hothorn T, Bretz F, Westfall P (2008) Simultaneous inference in general parametric models. Biom J 50:346–363PubMedCrossRefGoogle Scholar
  14. Leach CR, Houben A, Bruce F, Pistrick K, Demidov D, Timmis JN (2005) Molecular evidence for transcription of genes on a B chromosome in Crepis capillaris. Genetics 171:269–278PubMedCentralPubMedCrossRefGoogle Scholar
  15. Long EO, David IB (1980) Repeated genes in eukaryotes. Annu Rev Biochem 49:727–764PubMedCrossRefGoogle Scholar
  16. López-León MD, Neves N, Schwarzacher T, Heslop-Harrison JS, Hewitt GM, Camacho JPM (1994) Possible origin of B chromosome deduced from its DNA composition using double FISH technique. Chromosom Res 2:87–92CrossRefGoogle Scholar
  17. McClintock DJL (1934) The relation of a particular chromosomal element to the development of the nucleoli in Zea mays. Z Zellforsch Mikrosk Anat 21:294–328CrossRefGoogle Scholar
  18. Montiel EE, Cabrero J, Camacho JPM, López-León MD (2012) Gypsy, RTE and Mariner transposable elements populate Eyprepocnemis plorans genome. Genetica 140:365–374 PubMedCrossRefGoogle Scholar
  19. Montiel EE, Cabrero J, Ruiz-Estévez M, Burke WD, Eickbush TH, Camacho JPM, López-León MD (2014) Preferential occupancy of R2 retroelements on the B chromosomes of the grasshopper Eyprepocnemis plorans. PLoS ONE 9(3):e91820Google Scholar
  20. Mosgoeller W (2004) Nucleolar ultrastructure in vertebrates. In: Olson MOJ (ed) The Nucleolus. Kluwer, New York, pp 10–20Google Scholar
  21. Pinheiro J, Bates D, DebRoy S, Sarkar D, The R Development Core Team (2013) nlme: linear and nonlinear mixed effects models. R package version 3.1-113Google Scholar
  22. R Development Core Team (2008) R: a language and environment for statistical computing. In: R Foundation for Statistical Computing. Vienna, Austria. ISBN 3-900051-07-0. http://www.R-project.org
  23. Reeder RH (1999) Regulation of RNA polymerase I transcription in yeast and vertebrates. Prog Nucl Acid Res Mol Biol 62:293–327CrossRefGoogle Scholar
  24. Rufas JS, Iturra P, de Souza W, Esponda P (1982) Simple silver staining procedure for the localization of nucleolus and nucleolar organizer under light and electron microscopy. Arch Biol 93:267–274Google Scholar
  25. Ruiz-Estévez M, López-León MD, Cabrero J, Camacho JPM (2012) B-chromosome ribosomal DNA is functional in the grasshopper Eyprepocnemis plorans. PLoS One 7(5):e36600. doi: 10.1371/journal.pone.0036600 PubMedCentralPubMedCrossRefGoogle Scholar
  26. Ruiz-Estévez M, López-León MD, Cabrero J, Camacho JPM (2013) Ribosomal DNA is active in different B chromosome variants of the grasshopper Eyprepocnemis plorans. Genetica 141:337–345PubMedCrossRefGoogle Scholar
  27. Sollner-Webb B, Tower J (1986) Transcription of cloned eukaryotic ribosomal RNA genes. Annu Rev Genet 55:801–830Google Scholar
  28. Teruel M (2009) Origen, expresión y efectos fenotípicos de un parásito genómico. Universidad de Granada, PhDGoogle Scholar
  29. Teruel M, Cabrero J, Perfectti F, Camacho JPM (2007) Nucleolus size variation during meiosis and NOR activity of a B chromosome in the grasshopper Eyprepocnemis plorans. Chromosom Res 15:755–765CrossRefGoogle Scholar
  30. Teruel M, Cabrero J, Perfectti F, Camacho JPM (2009) Quantitative analysis of NOR expression in a B chromosome of the grasshopper Eyprepocnemis plorans. Chromosoma 118:291–300PubMedCrossRefGoogle Scholar
  31. Teruel M, Ruíz-Ruano F, Marchal JA, Sánchez-Baca A, Cabrero J, Camacho JPM, Perfectti F (2014) Disparate molecular evolution of two types of repetitive DNA in the genome of the grasshopper Eyprepocnemis plorans. Heredity 112:531–542PubMedCrossRefGoogle Scholar
  32. Trifonov VA, Dementyeva PV, Larkin DM, O’Brien PCM, Perelman PL, Yang F, Ferguson-Smith MA, Graphodatsky AS (2013) Transcription of a protein-coding gene on B chromosomes of the Siberian roe deer (Capreolus pygargus). BMC Biol 11:90PubMedCentralPubMedCrossRefGoogle Scholar
  33. Van Hiel MB, Van Wielendaele P, Temmerman L, Van Soest S, Vuerinckx K, Huybrechts R, Broeck JV, Simonet G (2009) Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different development conditions. BMC Mol Biol 10:56PubMedCentralPubMedCrossRefGoogle Scholar
  34. van Vugt JFA, Salverda M, de Jong H, Stouthamer R (2003) The paternal sex ratio chromosome in the parasitic wasp Trichogramma kaykai condenses the paternal chromosomes into a dense chromatin mass. Genome 46:580–587PubMedCrossRefGoogle Scholar
  35. Van Wormhoudt A, Gaume B, Le Bras Y, Roussel V, Huchette S (2011) Two different and functional nuclear rDNA genes in the abalone Haliotis tuberculata: tissue differential expression. Genetica 139:1217–1227PubMedCrossRefGoogle Scholar
  36. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3. RESEARCH0034.1-0034.11Google Scholar
  37. Zhou Q, Zhu H, Huang Q, Zhao L, Zhang G, Roy SW, Vicoso B, Xuan Z, Ruan J, Zhang Y, Zhao R, Ye C, Zhang X, Wang J, Wang W, Bachtrog D (2012) Deciphering neo-sex and B chromosome evolution by the draft genome of Drosophila albomicans. BMC Genom 13:109CrossRefGoogle Scholar
  38. Zurita S, Cabrero J, López-León MD, Camacho JPM (1998) Polymorphism regeneration for a neutralized selfish B chromosome. Evolution 52:274–277CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Mercedes Ruiz-Estévez
    • 1
  • Liesbeth Badisco
    • 2
  • Jozef Vanden Broeck
    • 2
  • Francisco Perfectti
    • 1
  • María Dolores López-León
    • 1
  • Josefa Cabrero
    • 1
  • Juan Pedro M. Camacho
    • 1
  1. 1.Departamento de Genética, Facultad de CienciasUniversidad de GranadaGranadaSpain
  2. 2.Animal Physiology and Neurobiology, Zoological InstituteK.U. LeuvenLouvainBelgium

Personalised recommendations