Functional organization of hsp70 cluster in camel (Camelus dromedarius) and other mammals

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 6 (2011): e27205, doi:10.1371/journal.pone.0027205.Heat shock protein 70 (Hsp70) is a molecular chaperone providing tolerance to heat and other challenges at the cellular and organismal levels. We sequenced a genomic cluster containing three hsp70 family genes linked with major histocompatibility complex (MHC) class III region from an extremely heat tolerant animal, camel (Camelus dromedarius). Two hsp70 family genes comprising the cluster contain heat shock elements (HSEs), while the third gene lacks HSEs and should not be induced by heat shock. Comparison of the camel hsp70 cluster with the corresponding regions from several mammalian species revealed similar organization of genes forming the cluster. Specifically, the two heat inducible hsp70 genes are arranged in tandem, while the third constitutively expressed hsp70 family member is present in inverted orientation. Comparison of regulatory regions of hsp70 genes from camel and other mammals demonstrates that transcription factor matches with highest significance are located in the highly conserved 250-bp upstream region and correspond to HSEs followed by NF-Y and Sp1 binding sites. The high degree of sequence conservation leaves little room for putative camel-specific regulatory elements. Surprisingly, RT-PCR and 5′/3′-RACE analysis demonstrated that all three hsp70 genes are expressed in camel's muscle and blood cells not only after heat shock, but under normal physiological conditions as well, and may account for tolerance of camel cells to extreme environmental conditions. A high degree of evolutionary conservation observed for the hsp70 cluster always linked with MHC locus in mammals suggests an important role of such organization for coordinated functioning of these vital genes.This work was supported by the Russian Foundation for Basic Research, project 09-04-00643 and 09-04-00660, project from ‘‘Genofond dynamics’’ program, and Grant of the Program of Molecular and Cellular Biology RAN to Dr. Evgen’ev; and by the Ministry of Education and Science of Russian Federation (State contract 14.740.11.0757 and Russia President Grant to young scientists MK-1418.2010.4. The research was supported by State Contract N16.552.11.7034 of Ministry of Education and Science

Expression of Drosophila virilis Retroelements and Role of Small RNAs in Their Intrastrain Transposition

Transposition of two retroelements (Ulysses and Penelope) mobilized in the course of hybrid dysgenesis in Drosophila virilis has been investigated by in situ hybridization on polytene chromosomes in two D. virilis strains of different cytotypes routinely used to get dysgenic progeny. The analysis has been repeatedly performed over the last two decades, and has revealed transpositions of Penelope in one of the strains, while, in the other strain, the LTR-containing element Ulysses was found to be transpositionally active. The gypsy retroelement, which has been previously shown to be transpositionally inactive in D. virilis strains, was also included in the analysis. Whole mount is situ hybridization with the ovaries revealed different subcellular distribution of the transposable elements transcripts in the strains studied. Ulysses transpositions occur only in the strain where antisense piRNAs homologous to this TE are virtually absent and the ping-pong amplification loop apparently does not take place. On the other hand small RNAs homologous to Penelope found in the other strain, belong predominantly to the siRNA category (21nt), and consist of sense and antisense species observed in approximately equal proportion. The number of Penelope copies in the latter strain has significantly increased during the last decades, probably because Penelope-derived siRNAs are not maternally inherited, while the low level of Penelope-piRNAs, which are faithfully transmitted from mother to the embryo, is not sufficient to silence this element completely. Therefore, we speculate that intrastrain transposition of the three retroelements studied is controlled predominantly at the post-transcriptional level

Copy number of <i>Penelope</i>, <i>Ulysses</i> and <i>gypsyDv</i> in polytene chromosomes of <i>D. virilis</i> strains 9 and 160.

<p>Copy number was determined by <i>in situ</i> hybridization analysis within the last two decades (1991–2008). When performing <i>in situ</i> hybridization analysis in 2008, we excluded a few <i>Ulysses</i> sites that were polymorphic in 1991 (did not contain <i>Ulysses</i> in 100% of larvae). Asterisks indicate site 49F where all three TEs were found.</p

The pattern of piRNAs distribution along transposons in testes.

<p>Distribution of <i>Ulysses</i>-piRNAs in testes of strain 9 (A) and strain 160 (C). The distribution of piRNAs homologous to <i>gypsyDv</i> in testes of strain 9 (B) and strain 160 (D). Sense small RNAs are indicated in red, antisense – in blue.</p

Maternal deposition and distribution levels of <i>Penelope</i>-derived small RNAs.

<p>siRNAs at (A, B) and piRNAs at (C, D) in strain 160 and its 0–2 h embryos. Sense small RNAs are indicated in red, antisense–in blue.</p

Transcription levels of selected <i>D. virilis</i> TEs.

<p>(A) semiquantitative RT-PCR data for ovaries and carcasses; (B) Quantitative RT-PCR analysis of TE transcription levels in ovaries. Since RT-PCR failed to reveal any transcription of <i>Penelope</i> and <i>Helena</i> in strain 9, we do not include the results of comparative analysis of these TEs by qRT-PCR in the panel; (C) Northern blot detection of <i>Ulysses</i> and <i>gypsyDv</i> sense transcripts in strains 9 and 160. Poly-A RNAs isolated from strain 9, strain 160 and <i>D. melanogaster</i> yw^67c23 strain ovaries were used. The size of marker RNA is given in nt at the right. The filter was rehybridized with a fragment of constitutively expressed <i>D. melanogaster rp49</i> to monitor the level of loaded RNA.</p

Organization and evolution of <it>hsp70 </it>clusters strikingly differ in two species of Stratiomyidae (Diptera) inhabiting thermally contrasting environments

<p>Abstract</p> <p>Background</p> <p>Previously, we described the heat shock response in dipteran species belonging to the family Stratiomyidae that develop in thermally and chemically contrasting habitats including highly aggressive ones. Although all species studied exhibit high constitutive levels of Hsp70 accompanied by exceptionally high thermotolerance, we also detected characteristic interspecies differences in heat shock protein (Hsp) expression and survival after severe heat shock. Here, we analyzed genomic libraries from two Stratiomyidae species from thermally and chemically contrasting habitats and determined the structure and organization of their <it>hsp70 </it>clusters.</p> <p>Results</p> <p>Although the genomes of both species contain similar numbers of <it>hsp70 </it>genes, the spatial distribution of <it>hsp70 </it>copies differs characteristically. In a population of the eurytopic species <it>Stratiomys singularior</it>, which exists in thermally variable and chemically aggressive (hypersaline) conditions, the <it>hsp70 </it>copies form a tight cluster with approximately equal intergenic distances. In contrast, in a population of the stenotopic <it>Oxycera pardalina </it>that dwells in a stable cold spring, we did not find <it>hsp70 </it>copies in tandem orientation. In this species, the distance between individual <it>hsp70 </it>copies in the genome is very large, if they are linked at all. In <it>O. pardalina </it>we detected the <it>hsp68 </it>gene located next to a <it>hsp70 </it>copy in tandem orientation. Although the <it>hsp70 </it>coding sequences of <it>S. singularior </it>are highly homogenized via conversion, the structure and general arrangement of the <it>hsp70 </it>clusters are highly polymorphic, including gross aberrations, various deletions in intergenic regions, and insertion of incomplete <it>Mariner </it>transposons in close vicinity to the 3'-UTRs.</p> <p>Conclusions</p> <p>The <it>hsp70 </it>gene families in <it>S. singularior </it>and <it>O. pardalina </it>evolved quite differently from one another. We demonstrated clear evidence of homogenizing gene conversion in the <it>S. singularior hsp70 </it>genes, which form tight clusters in this species. In the case of the other species, <it>O. pardalina</it>, we found no clear trace of concerted evolution for the dispersed <it>hsp70 </it>genes. Furthermore, in the latter species we detected <it>hsp70 </it>pseudogenes, representing a hallmark of the birth-and-death process.</p

Remarkable Site Specificity of Local Transposition Into the Hsp70 Promoter of Drosophila melanogaster

Heat-shock genes have numerous features that ought to predispose them to insertional mutagenesis via transposition. To elucidate the evolvability of heat-shock genes via transposition, we have exploited a local transposition technique and Drosophila melanogaster strains with EPgy2 insertions near the Hsp70 gene cluster at 87A7 to produce numerous novel EPgy2 insertions into these Hsp70 genes. More than 50% of 45 independent insertions were made into two adjacent nucleotides in the proximal promoter at positions −96 and −97, and no insertions were into a coding or 3′-flanking sequence. All inserted transposons were in inverse orientation to the starting transposon. The frequent insertion into nucleotides −96 and −97 is consistent with the DNase hypersensitivity, absence of nucleosomes, flanking GAGA-factor-binding sites, and nucleotide sequence of this region. These experimental insertions recapitulated many of the phenotypes of natural transposition into Hsp70: reduced mRNA expression, less Hsp70 protein, and decreased inducible thermotolerance. The results suggest that the distinctive features of heat-shock promoters, which underlie the massive and rapid expression of heat-shock genes upon heat shock, also are a source of evolutionary variation on which natural selection can act

Small RNA-based silencing strategies for transposons in the process of invading Drosophila species

Colonization of a host by an active transposon can increase mutation rates or cause sterility, a phenotype termed hybrid dysgenesis. As an example, intercrosses of certain Drosophila virilis strains can produce dysgenic progeny. The Penelope element is present only in a subset of laboratory strains and has been implicated as a causative agent of the dysgenic phenotype. We have also introduced Penelope into Drosophila melanogaster, which are otherwise naive to the element. We have taken advantage of these natural and experimentally induced colonization processes to probe the evolution of small RNA pathways in response to transposon challenge. In both species, Penelope was predominantly targeted by endo-small-interfering RNAs (siRNAs) rather than by piwi-interacting RNAs (piRNAs). Although we do observe correlations between Penelope transcription and dysgenesis, we could not correlate differences in maternally deposited Penelope piRNAs with the sterility of progeny. Instead, we found that strains that produced dysgenic progeny differed in their production of piRNAs from clusters in subtelomeric regions, possibly indicating that changes in the overall piRNA repertoire underlie dysgenesis. Considered together, our data reveal unexpected plasticity in small RNA pathways in germ cells, both in the character of their responses to invading transposons and in the piRNA clusters that define their ability to respond to mobile elements