1. RESULTS AND DISCUSSION
Has the structure of the Sry gene caused XY sexual reversed females in Akodon montensis?
Lopes, Silvia R1; Timmers, LFSM2; Norberto de Souza, Osmar2; Yonenaga-Yassuda, Yatiyo3; Fagundes, Valéria1.
Foto: Paulo Gonçalves
1Laboratório de Genética Animal, UFES, Vitória, ES; 2Laboratório de Bioinformática, Modelagem e Simulação de Biossistemas, PUCRS, Porto Alegre, RS; 3Laboratório de Citogenética de Vertebrados, USP, São Paulo, SP
INTRODUCTION
XY sexual reversed females have been reported in nine species of the South American rodent Akodon (Cricetidae, Akodontini) and mutation in the Sry gene was mentioned to be responsible for sexual reversion for three of them. We investigated the DNA and the protein structure of Sry gene of 19 individuals of Akodon montensis from three localities in Brazil: five XY females and four males from Iguape, São Paulo (IGU), five males from Dourados, Mato Grosso do Sul (DOU), and five males from Maquiné, Rio Grande do Sul (MAQ).
RESULTS AND DISCUSSION
MATERIALS AND METHODS
The 734 bp PCR-fragment included the complete Sry (533 bp) and flanking regions. Interpopulational genetic divergences varied from 2% (IGUxMAQ) to 5% (MAQxDOU) while intrapopulational divergences ranged from 1% (IGU and MAQ) to 5% (DOU).
Genetic divergence was lower between males and XY females (1%) than among males (3,8%) or XY females (2,4%). We observed that Sry sequences of male and XY female from IGU are more similar to each other than to other populations.
The number of amino acid residues (AA) of the eight target sequences ranged from 155 to 180 AA, while the human SRY protein contains 204 AA. HMG-box region of Sry gene presented 137 AA and was responsible for AA variation in the protein structure. C-G transversions at sites 48 and 52 were responsible for distinguish MAQ from the other two populations, which suggest a population-specific marker variation in the protein. All models revealed a conservative terciary structure formed by three α1-helices, (Ala3-Glu18), α 2 (Asn24-Lys36) and α 3 (Glu40-Lys60) (Fig. 1).
Strikingly, the 3D-structure of the putative Sry protein showed no difference among males and XY females (Fig.2). Thus, we are prone to suggest that, if the Sry protein is responsible for sexual reversed females in A. montensis, pre- or post-transcriptional mechanisms of gene regulation are more significant than the SRY gene structure it self.
DNA was extracted from frozen liver sample and the Sry gene was amplified and sequenced. Pairwise and multiple sequence alignments were carried out with the CLUSTAL W1.6 program. Sequence homology was analyzed using BLAST 2.2 and genetic divergence using MEGA v.4. Amino acid sequences and molecular weight of the predicted proteins were estimated using the Translate program in ExPASY at the Swiss Institute of Bioinformatics platform. As many sequences presents the same aminoacids sequence we used 8 sequences to construct models of Sry sequences of A. montensis. Approach of molecular modeling by homology implemented in MODELLER 9v7 was adopted to design the models, using the the domain structure human HMG-Box as template for the experiments. The models were subjected to an assessment of energy, which is implemented in the program MODELLER9v7. Besides the evaluation of energy were generated Ramachandran plots to analyze the folding and quality stereo-chemistry, through the program PROCHECK. Analysis of protein-DNA interaction was performed with the program LigPlot in order to obtain clues about the mode of association of macromolecules and on the influence of mutations in the sequences present in the interaction with DNA. The figures were generated with the program PyMOL .
Figure 1. Multiple alignment performed with Clustal W, of the eight sequences modeled and analyzed. Note the region conserved in most sequences and will position 46 to position 180. Sry human protein (PDB code: 1J46) was used as a template to model the eight proteins Rodent. Only the region HMG-Box has the 3D structure in PDB. This region is located between positions 56 and 140 of this multiple alignment. In the figure is delimitated helices- α 1 (Ala3-Glu18), α 2 (Asn24-Lys36) and α 3 (Glu40-Lys60). Marked with orange the sites 48 and 52 responsible for exclusively distinguish MAQ from the other two populations.
This work was supported by:
Figure 2. Tertiary structure of the HMG-box region of the eight sequences associated with the modeled DNA. (A) CIT160 male; (B) CIT200 XY female, (C) CIT204 male, (D) CTA1559 male, (E) CTA1560 male, (F) CTA1562 male,(G) CTA1563 male, (H) LGA411 male, (I) CIT160 male structure without DNA association, (J) CIT200 XY female structure without DNA association and (K) superposition of I and J. The α-helices are colored in dark blue, loops are colored in dark green, both with the type represented Cartoon, and double-stranded DNA is represented by the molecular surface colored by atom type. The images were generated with the program PyMOL.