Resumen de Evolution of the hsp70 gene family at the nucleotide, genome organization and gene expression levels in drosophila subobscura
ABSTRACT Numerous studies have confirmed the adaptive value of the rich chromosomal inversion polymorphism in the drosophilid D. subobscura. However, until recently very little was known about the molecular basis behind its maintenance in natural populations. In search of candidate loci, a previous heat shock experiment quantified Hsp70 protein levels in homokaryotypic strains for the OST, O3+4+8 and O3+4 arrangements. Unexpectedly, individuals of the warm climate-associated O3+4 arrangement showed increased levels in absence of thermal stress that did not boost after the heat shock. Unfortunately, by the time this experiment was performed there was very little data available on the molecular organization of the Hsp70IR locus in D. subobscura. The previously mentioned results led to the present thesis work, whose objectives are to locate the Hsp70IR locus in the karyotype and to know the genomic organization, molecular characteristics and gene expression patterns in several representative chromosomal arrangements that comprise the genomic region where the hsp70 gene family is located: O3+4+16+2, O3+4+8, O3+4 and OST. Using the sequence of a clone from an OST line genomic library and contigs from the unassembled genome of D. subobscura, we designed a probe from the hsp70 coding region that enabled us to determine the location of the locus by in situ (ISH) hybridization. Concomitantly, we completed the sequencing of a 9-10 kb region in the Hsp70IR locus in 12 lines isogenic for the aforementioned arrangements and in D. madeirensis and D. guanche to shed light on the evolution of this locus in the last 1.8 - 2.8 million years (myr). ISH results showed a single hybridization site in the 94A band in the distal segment (SI) of the O chromosome coincident in the 4 studied karyotypes: O3+4+16+2, O3+4+8, O3+4 and OST. The sequences corresponding to the 12 isogenic lines and to D. madeirensis and D. guanche indicated that in these three species of the subobscura cluster, the Hsp70IR locus consists of two 2.5 - 3.0 kb long paralogous copies in divergent orientation separated by a 0.5 - 1.4 kb nonduplicated central spacer region. The two copies show a high degree of conservation between the different gene arrangements and species analyzed, while the central spacer region is highly polymorphic. Among the most relevant aspects of polymorphism analyses, we highlight the high degree of conservation in the coding regions (CDSs) and the cis-regulatory elements (CREs) in the proximal promoter of all the analyzed hsp70 genes, which might indicate that these are functional in all studied lines, and that their regulation might be similar. Curiously, at the sequence level, the paralogous 5'-UTR and CDS regions tend to be significantly more similar within the same arrangement and, in some cases, within the same line, probably as a result of ectopic gene conversion. Lastly, we carried out the quantification of basal hsp70 mRNA and protein levels in adult males and females of six lines isogenic for the cold climate-associated OST and six for the warm climate-associated O3+4 arrangements. Basal mRNA quantification results indicate that the two arrangements exhibit similar levels, yet significant differences are observed between males and females of the warm O3+4 arrangement. Regarding the quantification of basal Hsp70 protein levels, these suggest that there are no differences between sexes nor between the two arrangements, but instead we observe a significant interaction between sex and arrangement. Overall, the results for both, mRNA and protein data, indicate that hsp70 expression might be influenced by sex.
