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

A – RT-PCR with total RNA from camel's blood.

<p>B – RT-PCR with total RNA from camel's heart muscle. 1 – primers CamORF1/2 to <i>hspA1A/B</i> genes and <i>grp78</i>, 2 – primers PT-1A and RT-2A to <i>hspA1A/B</i> genes, and 3 – primers RT-1L and RT-2L to <i>hspA1L</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027205#pone-0027205-t003" target="_blank">Table 3</a>). RT – negative control of RT-PCR, samples without reverse transcriptase.</p

The structure of <i>Hsp70</i> translation start and surrounding sequences in various organisms.

<p>Nucleotides disturbing optimal context for translation initiation are marked by bold shrift. The position of ATG is underlined. si – upstream (silenced) ATG. Kozak cons. – consensus sequence optimal for translation initiation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027205#pone.0027205-Kozak1" target="_blank">[29]</a>.</p

Comparison of 5′-regulatory regions of <i>hspA1</i> genes from camel and other mammals.

<p>(<b>A</b>) Identification of conserved motifs in the region between <i>hsp1L</i> and <i>hsp1A</i> (designated LA) and upstream of <i>hspA1B</i> (designated B) from <i>Camelus dromedarius</i> (C), <i>Bos taurus</i> (B), <i>Sus scrofa</i> (S), <i>Equus caballus</i> (E), <i>Homo sapiens</i> (H), <i>Mus musculus</i> (M), <i>Pteropus vampyrus</i> (P), <i>Tursiops truncatus</i> (T), and <i>Canis familiaris</i> (D). Transcription start sites are indicated by arrows, and ATG codons – by triangles. Intron sequences of <i>hsp1L</i> genes (located 16 bp upstream from the ATG codon, as indicated by a vertical dotted line) were removed to reduce sequence heterogeneity. Motifs are numbered in the order of identification by MEME, and numbers at the bottom indicate approximate base pairs in the alignment. (<b>B</b>) Matches between selected motifs from panel A and binding sites of known transcription factors in the TRANSFAC database identified by TOMTOM. Shown are the logos with the corresponding p- and q-values for each TF. The remaining motifs do not yield any matches to binding sites of known TFs.</p

General organization of the <i>hsp70</i> cluster in camel and human.

<p>A – restriction maps of two overlapping recombinant phages, C3 and N10, used in the analysis (H – <i>Hind</i>III, X – <i>Xho</i>I, R – <i>Eco</i>RI, B – <i>Bam</i>HI). B – general structure of the <i>C. dromedarius HspA1</i> cluster. C – general structure of <i>H. sapiens HSPA1</i> cluster provided for comparison. The length of the intergenic region between <i>hspA1A</i> and <i>hspA1B</i> genes is given in bps.</p

Identity of camel <i>hspA1</i> genes with orthologues from other organisms.

ψ<p><i>Grp78</i> taken from different mammalian species exhibits 100% identity at amino acid level.</p

List of primers used in the experiments.

<p>List of primers used in the experiments.</p

Southern blot of camel genomic DNA with PCR-probe to <i>hspA1A/B</i> genes.

<p>1 – <i>Bam</i>HI, 2 – <i>Xba</i>I, 3 – <i>Xba</i>I/<i>Bam</i>HI, 4 – <i>Eco</i>RI. M – fragment length markers.</p