INTERNATIONAL JOURNAL OF VETERINARY AND ANIMAL MEDICINE (ISSN:2517-7362)

The HSPB1, HSPB2, CRYAB (HSPB5), HSPB6 and HSPB8 loci from the family of small heat shock protein genes – HSPB, chosen for their possible functions in prion diseases, were localized on cattle, sheep and goat chromosomes. The experiments were performed by FISH technique with bovine BAC clones CH240-362H14, CH240-134C10, CH240-422N21 and CH240-279N2, as probes. The genes were assigned, respectively, to the following cattle/ goat 25q22, 15q14-21, 18q24, 17q24-25 and sheep 24q22, 15q14-21, 14q24, 17q24-25 genome regions. The BAC clone (CH240-134C10) overlapping sequences of the HSPB2 and CRYAB genes, located at the close proximity in the Bovidae genomes, was mapped to the homologous chromosome region BTA/OAR/CHI15q14-q21. Physical localization of HSPB genes on chromosomes of cattle, sheep and goats confirmed the data of their genome sequence assemblies as well as assigned the attachment of these loci to the linkage and syntenic groups of genes in the bovid species. Precise molecular and cytogenetic identification of genes controlling resistance/susceptibility to neurodegenerative disorders is essential for the expectation of the genetic selection effects, aimed to prevent of prion diseases in breeding populations. The studies performed may help to unequivocally elucidate the role of the HSPB genes in the development of neurodegenerative diseases in breeding animals.

Domestic bovids chromosomes; Fluorescence in situ hybridization; Small heat shock protein genes; Seurodegenerative disorders

The authors declare that they have no competing interests.

Small heat shock proteins (HSPB) are intracellular molecular chaperones which bind misfolded proteins and prevent them from aggregating. The ten polypeptides belonging to the human HSPB family (HSPB1 to HSPB10) have molecular weights between 12 and 43 kDa, distinguishing them in size from large heat shock proteins. A common feature of most these proteins is the presence of a conserved sequence of about 80 – 100 residues, which is generally referred to as the α-crystallin domain, located in the C-terminal region and folded into a β-sandwich conformation [1]. Although HSPB share both common structural and functional characteristics, they differ in tissue distribution and expression. Among them, HSPB1, CRYAB (alternative name HSPB5), HSPB6 and HSPB8 are ubiquitously expressed, and are constitutively present in the brain at low levels, whereas HSPB2 is expressed in muscle and heart and have also some expression in the brain in response to cellular stress [2]. Naturally occurring mutations of genes encoding HSPB proteins, mainly in the conserved regions, have functional pathological consequences involving myopathies and neurodegenerative disorders, including transmissible spongiform encephalopathies (TSEs) [3]. Majority of the latest studies demonstrate and notably emphasize protective functions of endogenous expressed HSPB in infectious protein aggregation diseases in humans, rodents and domestic bovids (among them prion diseases: Creutzfeldt-Jakob disease - CDJ, bovine spongiform encephalopathy - BSE and scrapie) [2-5]. Recently, some loci modulating resistance/susceptibility to prion diseases (other than PRNP – prion protein locus) have been reported in cattle and sheep. Several quantitative trait loci (QTL) influencing BSE and scrapie incubation period have been identified on Bos taurus chromosomes (BTA5, BTA6, BTA10, BTA13, BTA17, BTA19, BTA20, BTAX) and Ovis aries (OAR6, OAR18) autosomes (6-9). On the basis of the newest experiments in different human and bovid transmissible spongiform encephalopathy models, some of the heat shock protein loci (from the families HSP90, HSP70 and HSPB, each named according to its mass) were chosen as a putative positional and functional candidate genes contributing polygenic response to prion diseases [10-11]. Chromosomal assignment of the HSP genes is a good tool to identify additional, new QTL associated with resistance/susceptibility for TSEs in domestic bovids [12]. Although assembled genome sequences are available for several Bovidae species, however due to existence of errors in gene location (e.g., in the Btau_4.0 assembly), it is important to verify these assemblies using the FISH mapping method [13].

The aim of the presented study was chromosomal assignment of the HSPB1, HSPB2, CRYAB (HSPB5), HSPB6 and HSPB8 small heat shock protein genes, chosen for their possible functions in prion diseases in cattle, sheep and goats.

Cattle, sheep and goat lymphocytes were cultured and treated with BrdU (10 µg/ml) and H33258 (20 µg/ml) (Sigma) 6 h before harvesting to obtain, counterstained by DAPI, late-replicating banded chromosome preparations for FISH detection (according to the protocol reported by Iannuzzi and Di Berardino) [14]. The bovine BAC clones overlapping HSPB1, HSPB2, CRYAB (HSPB5), HSPB6 and HSPB8 genes, as indicated in Table 1, were screened by database searching (http://www.ncbi.nlm.nih.gov/clone; http://www.chori. org/bacpac/bovine240.htm) and obtained from CHORI-240 Bovine BAC Library (BACPAC Resources) (http://bacpac.chori.org/libraries. php). It was not possible to select separate BAC clones for the HspB2 and CRYAB (HSPB5) genes, due to their adjacent location (http:// www.ncbi.nlm.nih.gov/gene/508671;http://www.ncbi.nlm.nih. gov/gene/281719) in the bovine genome, thus the clone containing sequences of both the genes (CH240-134C10) was used. Each selected clone was verified to contain the studied gene with PCR amplification using specific primers, which are displayed in Table 1. The BAC DNA, extracted according to the alkaline lysis miniprep protocol (Qiagen), was labelled with biotin-16-dUTP by standard nick translation kit (Roche) and applied as the probes in the FISH experiments on bovid chromosomes. Labelled probes with an excess of bovine competitor DNA were denatured for 5 min at 75°C, preannealed for 15 min at 37°C, and applied onto chromosome preparations, denatured previously in formamide for 1 min at 70°C. Hybridizations were carried out overnight (up to three days in cross-species experiments) at 37°C. After detection step with the use of FITCavidin (fluorescein isothiocyanate-avidin) (Vector Laboratories) and anti-avidin antibodies (Sigma), slides were counterstained with DAPI (4,6-diamidino-2-phenylindole) solution (0.24 µg/ml) in Antifade (Vector Laboratories) to obtain DAPI-banded chromosomes (with patterns corresponding to the Q bands). Slides were analyzed under fluorescence microscope (Nikon) equipped with computer-assisted image analysis system (Cyto Vision). Chromosome identification followed the standard cattle, sheep and goat karyotypes and ideograms, according to the international chromosome nomenclature for domestic bovids ISCANDB 2000 [15].

The experiments allowed for the successful assignment of all BAC clones containing five small heat shock protein genes - HSPB1, HSPB2, CRYAB, HSPB6 and HSPB8 to the cattle (BTA), sheep (OAR) and goat (CHI) chromosomes by FISH, as shown in Figure 1 and 2, and displayed in Table 2. The BAC clone (CH240-134C10), overlapping sequences of the HSPB2 and CRYAB (HSPB5) genes located at the close proximity (http://www.ncbi.nlm.nih.gov/gene/) in bovid and other mammalian genomes, was mapped to the identical cattle, sheep and goat chromosome regions BTA/OAR/CHI15q14-q21.

In this study we present localizations of five HSPB genes (HSPB1, HSPB2, CRYAB (HSPB5), HSPB6, HSPB8) in cattle/goat 25q22, 15q14- 21, 18q24, 17q24-25 and sheep 24q22, 15q14-21, 14q24, 17q24-25 genome regions, which are reported in Table 2. The HSPB2 and CRYAB (HSPB5) loci (located at the distance of 1.035 kb in cattle, 0.482 kb in sheep and 1.472 kb in goat genome) were mapped to the homologous bovid chromosomes and chromosome bands (15q14-21), extending the cytogenetic maps of the 15 autosome in three studied species. Similarly in humans, as indicated in Table 2, these two neighboring genes (composed of the two and three exons, respectively) are localized in the same HSA11q22.3-23.1 genome region. What’s interesting, these two genes are arranged in a head-to-head manner with an inter-genic sequence of less than 1 kb, raising a possibility of shared regulatory elements for their expression [16].

In general, as displayed in the mentioned Table 2, all the physical positions presented in this study were in agreement with their corresponding human locations (HGNC) (http://www.genenames. org), based on the comparative painting, radiation hybrid or marker mapping data between bovids and humans [17-21]. It is worth to note also, that all these five HSPB loci map in homologous chromosomes and chromosome bands of the three studied species, as expected given the high degree of autosome homologies among bovids which are very close to each other from the evolutionary point of view [22]. Furthermore, the experiments reported in this paper confirmed in part results of our previous provisional comparative mapping of the HSPB genes in domestic bovids [23-24] and definitely proved that FISH-based mapping is still useful to validate the data on physical gene location, construct precise genome maps as well as improve domestic bovids genomes assemblies [13]. This study adds further information to the previous cytogenetic maps of the domestic bovids (BOVMAP http://dga.jouy.inra.fr/cgi-bin/lgbc/main.pl?BASE=) [21- 25] and precisely assigns for the first time, five loci of small heat shock protein genes involved in prion diseases on chromosomes of cattle, sheep and goat, which are the major domestic bovid species of great economic importance.

eat economic importance. At present, some of the heat shock proteins genes have been analyzed as possible candidate genes (other than the PRNP gene) involved in spongiform encephalopathy resistance/susceptibility in the domestic bovids, probably by modulating the incubation period of prion disease. The four members of the heat shock protein gene families HSP90 (the inducible form HSP90AA1), HSP70 (HSPA13, the constitutive form HSP72/HSPA1A, the inducible HSP73/HSPA8) and HSPB (HSPB1, HSPB5) have been chosen for their known roles as chaperones and apoptosis modulators, as well as their possible protective effect against the stress related infectious protein aggregation and neuronal degeneration in prion diseases [2,10,26,27, 30-33]. Structural and functional analysis of the HSP90AA1 gene and distribution of polymorphisms among sheep with different responses to scrapie revealed variability in the HSP90AA1 5’ flanking region, associated with scrapie incubation period [28-29]. The subsequent studies displayed negative correlation between prion protein deposition and expression of HSP90, HSP73 and HSPB1 (HSP27) genes, suggesting that high levels of HSP gene or protein expression are associated to the prevention or degradation of prion protein aggregates and the presence of reactive astrocytosis in natural scrapie [10]. The above mentioned experiments confirmed previous findings, which had been proved that HSPB1 gene expression level contribute to the development of the prion protein deposits and morphological lesions such as spongiosis or gliosis of classical scrapie in sheep [5]. The similar studies showed increased HSPB1 expression as a stress response of the central nervous system in a mouse model of BSE [4], however research on the other small heat shock protein genes has been limited to prions and tauropathies in humans. The HSPB5 expression has been shown to be dramatically elevated in both glia and neurons from Creutzfeldt-Jakob disease brains, whereas HSPB2, HSPB6 and HSPB8 in Alzheimer’s disease, but it is currently unknown why there were HSPB elevated levels in these diseases [1-3]. There have been no published studies determining that loss or gain of HSPB would have the effects on tauropthies. However, given the similarities in protein aggregation between prions and tauropthies, it is likely that these HSPB genes and proteins are playing similar roles in both disease states.

The reported FISH-mapping results may broaden the knowledge on genome organization and cytogenetic maps of domestic bovids, enabling to specify precisely HSPB loci related to prion disease development in cattle, sheep and goats.

The presented chromosomal localization of HSPB genes may be a basis for identifying new QTLs associated with response for TSE in Bovidae species.

Physical localization of five HSPB genes in the cattle, sheep and goats genomes assigned their attachment to the linkage and syntenic groups of genes, which is essential for the expectation of the genetic selection effects, aimed to prevent of neurodegenerative disorders in breeding populations.

The study was financed by the Ministry of Agriculture and Rural Development, Poland, Project No: 03-17-23-09. 

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