Functional analysis of a highly conserved abundant larval transcript-2 (alt-2) intron 2 repeat region of lymphatic filarial parasites

Infect Genet Evol. 2014 Jun:24:177-82. doi: 10.1016/j.meegid.2014.03.017. Epub 2014 Mar 26.

Abstract

The filarial-specific protein abundant larval transcript-2 (ALT-2) is expressed exclusively in the infective larval stage (L3) and is a crucial protein for establishing immunopathogenesis in human hosts. The alt-2 gene has a conserved minisatellite repeat (29 or 27bp) in intron 2 (IR2) whose significance within lymphatic filarial species is unknown. Here, we report the role of IR2 in the regulation of alt-2 gene expression using an in vitro model. Using electrophoretic mobility shift assays, we identified the presence of a putative nuclear protein binding region within IR2. Subsequent transient expression experiments in eukaryotic cell lines demonstrated that the IR2 downregulated the expression of a downstream luciferase reporter gene, which was further validated with RT-PCR. We therefore identify IR2 as a suppressor element that regulates L3 stage-specific expression of alt-2.

Keywords: Evolution; Gene regulation; Intron DNA repeat; Silencer; Transcription.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Helminth / biosynthesis
  • Antigens, Helminth / genetics*
  • Antigens, Helminth / immunology
  • Brugia malayi / genetics*
  • Brugia malayi / immunology
  • CHO Cells
  • Cell Line
  • Cricetulus
  • DNA, Helminth / genetics
  • Electrophoretic Mobility Shift Assay
  • Elephantiasis, Filarial / parasitology*
  • Gene Expression Regulation
  • Genes, Reporter / genetics
  • Helminth Proteins / genetics
  • Hep G2 Cells
  • Humans
  • Immune Evasion
  • Introns / genetics*
  • Larva / genetics
  • Luciferases / genetics
  • Minisatellite Repeats / genetics
  • Nuclear Proteins / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / immunology
  • Sf9 Cells
  • Silencer Elements, Transcriptional / genetics
  • Spodoptera
  • Wuchereria bancrofti / genetics*
  • Wuchereria bancrofti / immunology

Substances

  • ALT-2 protein, Brugia malayi
  • Antigens, Helminth
  • DNA, Helminth
  • Helminth Proteins
  • Nuclear Proteins
  • Recombinant Proteins
  • Luciferases