The Isoforms of Ral Guanine Nucleotide Dissociation Stimulator (RalGDS) in LLC-PK1 Cells

Abstract

This study investigated the isoforms of porcine-origin Ral guanine nucleotide dissociation stimulator (RalGDS) in LLC-PK1 cells using reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. Through segmented amplification, sequence assembly, and comparative genomics analysis, seven RalGDS isoforms were identified, characterized by insertions, deletions, and frameshift mutations. These genetic variations may significantly alter RalGDS's protein structure and function, potentially impacting its role in Ral GTPase-mediated signaling pathways. This work provides foundational insights into the genetic diversity of porcine RalGDS and its implications for porcine physiology and economically significant traits.

Keywords: LLC-PK1 cells; RalGDS; frameshift mutation; isoform; molecular evolution.

Figure 1

Alignment of nucleotide sequences of the variable region among ten RalGDS genes in this study. Dots represent the deletion mutation of 430 nucleotide sequences in RalGDS dP10 and 4 nt in RalGDS dP8 and dP9 compared to other RalGDS genes. Black, pink and blue represent nucleotide homology levels of 100%, 75% or higher and 50% or higher, respectively.

Figure 2

Alignment of amino acid sequences of the hypervariable region among four RalGDS genes. The N-terminal regions of dP4 and dP5, and the C-terminal domain of dP8, dP9, and dP10 demonstrate significant divergence in their amino acid sequences when compared to other members of the RALGDS family. Black, pink and blue represent amino acid homology levels of 100%, 75% or higher and 50% or higher, respectively.

Figure 3

Phylogenetic analyses with the nucleotide sequences (a) and amino acid sequences (b) of the full-length RalGDS strains. Multiple-sequence alignments were performed using the ClustalW program implemented in MEGA 7, and phylogenetic trees were constructed using the neighbor-joining method. Bootstrap values > 50% (1000 replicates) of NJ analysis are shown above the branches.