The Role of Hsp70 in Adaptation to Adverse Conditions and Its Possible Medical Application.

In the present era of global warming and dramatically increased environmental pollution posing a threat to animal life, the understanding and manipulation of organisms' resources of stress tolerance is apparently a question of survival. Heat stress and other forms of stressful factors induce a highly organized response of organisms at the cellular level where heat shock proteins (Hsps) and in particular Hsp70 family of chaperones are among the major players in the protection from the environmental challenge. The present review article summarizes the peculiarities of the Hsp70 family of proteins protective functions being a result of many millions of years of adaptive evolution. It discusses the molecular structure and specific details of hsp70 gene regulation in various organisms, living in diverse climatic zones, with a special emphasis on the protective role of Hsp70 in adverse conditions of the environment. The review discusses the molecular mechanisms underlying Hsp70-specific properties that emerged in the course of adaptation to harsh environmental conditions. This review also includes the data on the anti-inflammatory role of Hsp70 and the involvement of endogenous and recombinant Hsp70 (recHsp70) in proteostatic machinery in various pathologies including neurodegenerative ones such as Alzheimer's and Parkinson's diseases in rodent model organisms and humans in vivo and in vitro. Specifically, the role of Hsp70 as an indicator of disease type and severity and the use of recHsp70 in several pathologies are discussed. The review discusses different roles exhibited by Hsp70 in various diseases including the dual and sometimes antagonistic role of this chaperone in various forms of cancer and viral infection including the SARS-Cov-2 case. Since Hsp70 apparently plays an important role in many diseases and pathologies and has significant therapeutic potential there is a dire need to develop cheap recombinant Hsp70 production and further investigate the interaction of externally supplied and endogenous Hsp70 in chaperonotherapy.

Genes Responsible for H2S Production and Metabolism Are Involved in Learning and Memory in Drosophila melanogaster.

The gasotransmitter hydrogen sulfide (H2S) produced by the transsulfuration pathway (TSP) is an important biological mediator, involved in many physiological and pathological processes in multiple higher organisms, including humans. Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in H2S production and metabolism. Here, we investigated the role of H2S in learning and memory processes by exploring several Drosophila melanogaster strains with single and double deletions of CBS and CSE developed by the CRISPR/Cas9 technique. We monitored the learning and memory parameters of these strains using the mating rejection courtship paradigm and demonstrated that the deletion of the CBS gene, which is expressed predominantly in the central nervous system, and double deletions completely block short- and long-term memory formation in fruit flies. On the other hand, the flies with CSE deletion preserve short- and long-term memory but fail to exhibit long-term memory retention. Transcriptome profiling of the heads of the males from the strains with deletions in Gene Ontology terms revealed a strong down-regulation of many genes involved in learning and memory, reproductive behavior, cognition, and the oxidation-reduction process in all strains with CBS deletion, indicating an important role of the hydrogen sulfide production in these vital processes.

Deletions of the cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) genes, involved in the control of hydrogen sulfide biosynthesis, significantly affect lifespan and fitness components of Drosophila melanogaster.

The gasotransmitter hydrogen sulfide (H2S) is an important biological mediator, playing an essential role in many physiological and pathological processes. It is produced by transsulfuration - an evolutionarily highly conserved pathway for the metabolism of sulfur-containing amino acids methionine and cysteine. Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in cysteine metabolism and H2S production. Here we investigated the fitness components (longevity, stress resistance, viability of preimaginal stages, and reproductive function parameters) in D. melanogaster lines containing deletions of the CBS and CSE genes. Surprisingly, in most tests, CSE deletion improved, and CBS worsened the fitness. Lines with deletion of both CBS and CSE demonstrated better stress resistance and longevity than lines with single CBS deletion. At the same time, deletion of both CBS and CSE genes causes more serious disturbances of reproductive function parameters than single CBS deletion. Thus, a complex interaction of H2S-producing pathways and cellular stress response in determining the lifespan and fitness components of the whole organism was revealed.

Role of a Heat Shock Transcription Factor and the Major Heat Shock Protein Hsp70 in Memory Formation and Neuroprotection.

Heat shock proteins (Hsps) represent the most evolutionarily ancient, conserved, and universal system for protecting cells and the whole body from various types of stress. Among Hsps, the group of proteins with a molecular weight of 70 kDa (Hsp70) plays a particularly important role. These proteins are molecular chaperones that restore the native conformation of partially denatured proteins after exposure to proteotoxic forms of stress and are critical for the folding and intracellular trafficking of de novo synthesized proteins under normal conditions. Hsp70s are expressed at high levels in the central nervous system (CNS) of various animals and protect neurons from various types of stress, including heat shock, hypoxia, and toxins. Numerous molecular and behavioral studies have indicated that Hsp70s expressed in the CNS are important for memory formation. These proteins contribute to the folding and transport of synaptic proteins, modulate signaling cascades associated with synaptic activation, and participate in mechanisms of neurotransmitter release. In addition, HSF1, a transcription factor that is activated under stress conditions and mediates Hsps transcription, is also involved in the transcription of genes encoding many synaptic proteins, whose levels are increased in neurons under stress and during memory formation. Thus, stress activates the molecular mechanisms of memory formation, thereby allowing animals to better remember and later avoid potentially dangerous stimuli. Finally, Hsp70 has significant protective potential in neurodegenerative diseases. Increasing the level of endogenous Hsp70 synthesis or injecting exogenous Hsp70 reduces neurodegeneration, stimulates neurogenesis, and restores memory in animal models of ischemia and Alzheimer's disease. These findings allow us to consider recombinant Hsp70 and/or Hsp70 pharmacological inducers as potential drugs for use in the treatment of ischemic injury and neurodegenerative disorders.

Xenobiotic-induced activation of human aryl hydrocarbon receptor target genes in Drosophila is mediated by the epigenetic chromatin modifiers.

Aryl hydrocarbon receptor (AHR) is the key transcription factor that controls animal development and various adaptive processes. The AHR's target genes are involved in biodegradation of endogenous and exogenous toxins, regulation of immune response, organogenesis, and neurogenesis. Ligand binding is important for the activation of the AHR signaling pathway. Invertebrate AHR homologs are activated by endogenous ligands whereas vertebrate AHR can be activated by both endogenous and exogenous ligands (xenobiotics). Several studies using mammalian cultured cells have demonstrated that transcription of the AHR target genes can be activated by exogenous AHR ligands, but little is known about the effects of AHR in a living organism. Here, we examined the effects of human AHR and its ligands using transgenic Drosophila lines with an inducible human AhR gene. We found that exogenous AHR ligands can increase as well as decrease the transcription levels of the AHR target genes, including genes that control proliferation, motility, polarization, and programmed cell death. This suggests that AHR activation may affect the expression of gene networks that could be critical for cancer progression and metastasis. Importantly, we found that AHR target genes are also controlled by the enzymes that modify chromatin structure, in particular components of the epigenetic Polycomb Repressive complexes 1 and 2. Since exogenous AHR ligands (alternatively - xenobiotics) and small molecule inhibitors of epigenetic modifiers are often used as pharmaceutical anticancer drugs, our findings may have significant implications in designing new combinations of therapeutic treatments for oncological diseases.

Drosophila Model for the Analysis of Genesis of LIM-kinase 1-Dependent Williams-Beuren Syndrome Cognitive Phenotypes: INDELs, Transposable Elements of the Tc1/Mariner Superfamily and MicroRNAs.

Genomic disorders, the syndromes with multiple manifestations, may occur sporadically due to unequal recombination in chromosomal regions with specific architecture. Therefore, each patient may carry an individual structural variant of DNA sequence (SV) with small insertions and deletions (INDELs) sometimes less than 10 bp. The transposable elements of the Tc1/mariner superfamily are often associated with hotspots for homologous recombination involved in human genetic disorders, such as Williams Beuren Syndromes (WBS) with LIM-kinase 1-dependent cognitive defects. The Drosophila melanogaster mutant agnts3 has unusual architecture of the agnostic locus harboring LIMK1: it is a hotspot of chromosome breaks, ectopic contacts, underreplication, and recombination. Here, we present the analysis of LIMK1-containing locus sequencing data in agnts3 and three D. melanogaster wild-type strains-Canton-S, Berlin, and Oregon-R. We found multiple strain-specific SVs, namely, single base changes and small INDEls. The specific feature of agnts3 is 28 bp A/T-rich insertion in intron 1 of LIMK1 and the insertion of mobile S-element from Tc1/mariner superfamily residing ~460 bp downstream LIMK1 3'UTR. Neither of SVs leads to amino acid substitutions in agnts3 LIMK1. However, they apparently affect the nucleosome distribution, non-canonical DNA structure formation and transcriptional factors binding. Interestingly, the overall expression of miRNAs including the biomarkers for human neurological diseases, is drastically reduced in agnts3 relative to the wild-type strains. Thus, LIMK1 DNA structure per se, as well as the pronounced changes in total miRNAs profile, probably lead to LIMK1 dysregulation and complex behavioral dysfunctions observed in agnts3 making this mutant a simple plausible Drosophila model for WBS.

Interplay between recombinant Hsp70 and proteasomes: proteasome activity modulation and ubiquitin-independent cleavage of Hsp70.

The heat shock protein 70 (Hsp70, human HSPA1A) plays indispensable roles in cellular stress responses and protein quality control (PQC). In the framework of PQC, it cooperates with the ubiquitin-proteasome system (UPS) to clear damaged and dysfunctional proteins in the cell. Moreover, Hsp70 itself is rapidly degraded following the recovery from stress. It was demonstrated that its fast turnover is mediated via ubiquitination and subsequent degradation by the 26S proteasome. At the same time, the effect of Hsp70 on the functional state of proteasomes has been insufficiently investigated. Here, we characterized the direct effect of recombinant Hsp70 on the activity of 20S and 26S proteasomes and studied Hsp70 degradation by the 20S proteasome in vitro. We have shown that the activity of purified 20S proteasomes is decreased following incubation with recombinant human Hsp70. On the other hand, high concentrations of Hsp70 activated 26S proteasomes. Finally, we obtained evidence that in addition to previously reported ubiquitin-dependent degradation, Hsp70 could be cleaved independent of ubiquitination by the 20S proteasome. The results obtained reveal novel aspects of the interplay between Hsp70 and proteasomes.

The development of modified human Hsp70 (HSPA1A) and its production in the milk of transgenic mice.

The production of major human heat shock protein Hsp70 (HSPA1A) in a eukaryotic expression system is needed for testing and possible medical applications. In this study, transgenic mice were produced containing wild-type human Hsp70 allele in the vector providing expression in the milk. The results indicated that human Hsp70 was readily expressed in the transgenic animals but did not apparently preserve its intact structure and, hence, it was not possible to purify the protein using conventional isolation techniques. It was suggested that the protein underwent glycosylation in the process of expression, and this quite common modification for proteins expressed in the milk complicated its isolation. To check this possibility, we mutated all presumptive sites of glycosylation and tested the properties of the resulting modified Hsp70 expressed in E. coli. The investigation demonstrated that the modified protein exhibited all beneficial properties of the wild-type Hsp70 and was even superior to the latter for a few parameters. Based on these results, a transgenic mouse strain was obtained which expressed the modified Hsp70 in milk and which was easy to isolate using ATP columns. Therefore, the developed construct can be explored in various bioreactors for reliable manufacture of high quality, uniform, and reproducible human Hsp70 for possible medical applications including neurodegenerative diseases and cancer.

Proteomics of the 26S proteasome in Spodoptera frugiperda cells infected with the nucleopolyhedrovirus, AcMNPV.

Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 ß subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection.

The Fate of Exogenous Human HSP70 Introduced into Animal Cells by Different Means.

Over the last decade, it has become evident that in mammals, including humans, heat shock protein 70 (HSP70), apart from its intracellular localization, is found in extracellular space, where it may execute various protective functions. Furthermore, the upregulation of HSP70 family members can be beneficial in the prevention and treatment of various human neurodegenerative diseases and cancer. Here, we demonstrate that recombinant human HSP70 after intranasal administration can penetrate various brain regions of mice in its native form and subsequently undergo rapid degradation. It was also shown that labeled HSP70 added to culture medium of different human and mouse cell lines enters the cells with strikingly different kinetics, which positively correlates with the basic levels of membrane bound Toll-like receptors (TLR) that are characteristic of these cell lines. HSP70 administration does not significantly modulate the level of TLR expression at the protein or RNA level. The degradation of the introduced recombinant HSP70 after entering the cells is likely proteasome-dependent and varies significantly depending on the cells type and origin. These results should be considered when developing HSP70-based therapies.

Activity of heat shock genes' promoters in thermally contrasting animal species.

Heat shock gene promoters represent a highly conserved and universal system for the rapid induction of transcription after various stressful stimuli. We chose pairs of mammalian and insect species that significantly differ in their thermoresistance and constitutive levels of Hsp70 to compare hsp promoter strength under normal conditions and after heat shock (HS). The first pair includes the HSPA1 gene promoter of camel (Camelus dromedarius) and humans. It was demonstrated that the camel HSPA1A and HSPA1L promoters function normally in vitro in human cell cultures and exceed the strength of orthologous human promoters under basal conditions. We used the same in vitro assay for Drosophila melanogaster Schneider-2 (S2) cells to compare the activity of the hsp70 and hsp83 promoters of the second species pair represented by Diptera, i.e., Stratiomys singularior and D. melanogaster, which dramatically differ in thermoresistance and the pattern of Hsp70 accumulation. Promoter strength was also monitored in vivo in D. melanogaster strains transformed with constructs containing the S. singularior hsp70 ORF driven either by its own promoter or an orthologous promoter from the D. melanogaster hsp70Aa gene. Analysis revealed low S. singularior hsp70 promoter activity in vitro and in vivo under basal conditions and after HS in comparison with the endogenous promoter in D. melanogaster cells, which correlates with the absence of canonical GAGA elements in the promoters of the former species. Indeed, the insertion of GAGA elements into the S. singularior hsp70 regulatory region resulted in a dramatic increase in promoter activity in vitro but only modestly enhanced the promoter strength in the larvae of the transformed strains. In contrast with hsp70 promoters, hsp83 promoters from both of the studied Diptera species demonstrated high conservation and universality.

Combination of hypomorphic mutations of the Drosophila homologues of aryl hydrocarbon receptor and nucleosome assembly protein family genes disrupts morphogenesis, memory and detoxification.

Aryl hydrocarbon receptor is essential for biological responses to endogenous and exogenous toxins in mammals. Its Drosophila homolog spineless plays an important role in fly morphogenesis. We have previously shown that during morphogenesis spineless genetically interacts with CG5017 gene, which encodes a nucleosome assembly factor and may affect cognitive function of the fly. We now demonstrate synergistic interactions of spineless and CG5017 in pathways controlling oxidative stress response and long-term memory formation in Drosophila melanogaster. Oxidative stress was induced by low doses of X-ray irradiation of flies carrying hypomorphic mutation of spineless, mutation of CG5017, and their combination. To determine the sensitivity of these mutants to pharmacological modifiers of the irradiation effect, we irradiated flies growing on standard medium supplemented by radiosensitizer furazidin and radioprotector serotonin. The effects of irradiation were investigated by analyzing leg and antenna morphological structures and by using real-time PCR to measure mRNA expression levels for spineless, Cyp6g1 and Gst-theta genes. We also examined long-term memory in these mutants using conditioned courtship suppression paradigm. Our results show that the interaction of spineless and CG5017 is important for regulation of morphogenesis, long-term memory formation, and detoxification during oxidative stress. Since spineless and CG5017 are evolutionary conserved, these results must be considered when evaluating the risk of combining similar mutations in other organisms, including humans.

Medium-sized tandem repeats represent an abundant component of the Drosophila virilis genome.

BACKGROUND: Previously, we developed a simple method for carrying out a restriction enzyme analysis of eukaryotic DNA in silico, based on the known DNA sequences of the genomes. This method allows the user to calculate lengths of all DNA fragments that are formed after a whole genome is digested at the theoretical recognition sites of a given restriction enzyme. A comparison of the observed peaks in distribution diagrams with the results from DNA cleavage using several restriction enzymes performed in vitro have shown good correspondence between the theoretical and experimental data in several cases. Here, we applied this approach to the annotated genome of Drosophila virilis which is extremely rich in various repeats. RESULTS: Here we explored the combined approach to perform the restriction analysis of D. virilis DNA. This approach enabled to reveal three abundant medium-sized tandem repeats within the D. virilis genome. While the 225 bp repeats were revealed previously in intergenic non-transcribed spacers between ribosomal genes of D. virilis, two other families comprised of 154 bp and 172 bp repeats were not described. Tandem Repeats Finder search demonstrated that 154 bp and 172 bp units are organized in multiple clusters in the genome of D. virilis. Characteristically, only 154 bp repeats derived from Helitron transposon are transcribed. CONCLUSION: Using in silico digestion in combination with conventional restriction analysis and sequencing of repeated DNA fragments enabled us to isolate and characterize three highly abundant families of medium-sized repeats present in the D. virilis genome. These repeats comprise a significant portion of the genome and may have important roles in genome function and structural integrity. Therefore, we demonstrated an approach which makes possible to investigate in detail the gross arrangement and expression of medium-sized repeats basing on sequencing data even in the case of incompletely assembled and/or annotated genomes.

Evolution and dynamics of small RNA response to a retroelement invasion in Drosophila.

Although small RNAs efficiently control transposition activity of most transposons in the host genome, such an immune system is not always applicable against a new transposon's invasions. Here, we explored a possibility to introduce potentially mobile copy of the Penelope retroelement previously implicated in hybrid dysgenesis syndrome in Drosophila virilis into the genomes of two distant Drosophila species. The consequences of such introduction were monitored at different phases after experimental colonization as well as in D. virilis species, which is apparently in the process of ongoing Penelope invasion. We investigated the expression of Penelope and biogenesis of Penelope-derived small RNAs in D. virilis and D. melanogaster strains originally lacking active copies of this element after experimental Penelope invasion. These strains were transformed by constructs containing intact Penelope copies. We show that immediately after transformation, which imitates the first stage of retroelement invasion, Penelope undergoes transposition predominantly in somatic tissues, and may produce siRNAs that are apparently unable to completely silence its activity. However, at the later stages of colonization Penelope copies may jump into one of the piRNA-clusters, which results in production of homologous piRNAs that are maternally deposited and can silence euchromatic transcriptionally active copies of Penelope in trans and, hence, prevent further amplification of the invader in the host genome. Intact Penelope copies and different classes of Penelope-derived small RNAs were found in most geographical strains of D. virilis collected throughout the world. Importantly, all strains of this species containing full-length Penelope tested do not produce gonadal sterility in dysgenic crosses and, hence, exhibit neutral cytotype. To understand whether RNA interference mechanism able to target Penelope operates in related species of the virilis group, we correlated the presence of full-length and potentially active Penelope with the occurrence of piRNAs homologous to this transposable element in the ovaries of species comprising the group. It was demonstrated that Penelope-derived piRNAs are present in all virilis group species containing full-length but transcriptionally silent copies of this element that probably represent the remnants of its previous invasions taking place in the course of the virilis species divergent evolution.

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

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.

New insights into the induction of the heat shock proteins in baculovirus infected insect cells.

Eight members of the HSP/HSC70 family were identified in Spodoptera frugiperda Sf9 cells infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by 2D electrophoresis followed by mass spectrometry (MALDI/TOF) and a Mascot search. The family includes five HSP70s induced by AcMNPV-infection and three constitutive cognate HSC70s that remained abundant in infected cells. Confocal microscopy revealed dynamic changes in subcellular localization of HSP/HSC70s in the course of infection. At the early stages (4 to 10 hpi), a fraction of HSPs is localized in distinct speckles in cytoplasm. The speckles contained ubiquitinylated proteins suggesting that they may be aggresomes where proteins targeted by ubiquitin are sequestered or processed for proteolysis. S. frugiperda HSP90 was identified in the 2D gels by Western blotting. Its amount was unchanged during infection. A selective inhibitor of HSP90, 17-AAG, decreased the rate of viral DNA synthesis in infected cells suggesting a supportive role of HSP90 in virus replication.

Organization and evolution of hsp70 clusters strikingly differ in two species of Stratiomyidae (Diptera) inhabiting thermally contrasting environments.

BACKGROUND: 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 hsp70 clusters. RESULTS: Although the genomes of both species contain similar numbers of hsp70 genes, the spatial distribution of hsp70 copies differs characteristically. In a population of the eurytopic species Stratiomys singularior, which exists in thermally variable and chemically aggressive (hypersaline) conditions, the hsp70 copies form a tight cluster with approximately equal intergenic distances. In contrast, in a population of the stenotopic Oxycera pardalina that dwells in a stable cold spring, we did not find hsp70 copies in tandem orientation. In this species, the distance between individual hsp70 copies in the genome is very large, if they are linked at all. In O. pardalina we detected the hsp68 gene located next to a hsp70 copy in tandem orientation. Although the hsp70 coding sequences of S. singularior are highly homogenized via conversion, the structure and general arrangement of the hsp70 clusters are highly polymorphic, including gross aberrations, various deletions in intergenic regions, and insertion of incomplete Mariner transposons in close vicinity to the 3'-UTRs. CONCLUSIONS: The hsp70 gene families in S. singularior and O. pardalina evolved quite differently from one another. We demonstrated clear evidence of homogenizing gene conversion in the S. singularior hsp70 genes, which form tight clusters in this species. In the case of the other species, O. pardalina, we found no clear trace of concerted evolution for the dispersed hsp70 genes. Furthermore, in the latter species we detected hsp70 pseudogenes, representing a hallmark of the birth-and-death process.

An important role of the heat shock response in infected cells for replication of baculoviruses.

Baculoviruses serve as a stress factor that can activate both death-inducing and cytoprotective pathways in infected cells. In this report, induction of heat shock proteins (HSPs) of the 70-kDa family (HSP/HSC70) in Sf-9 cells after infection with AcMNPV was monitored by Western blot analysis. Two-dimensional electrophoresis in polyacrylamide gel revealed changes in the cellular pattern of HSP/HSC70s and synthesis of a new member of the HSP/HSC70 family in the infected cells. Although infection with AcMNPV moderately increased the HSP/HSC70 content in cells under standard conditions, the infection potentiated the response to heat shock boosting the HSP/HSC70s content in infected cells several-fold in comparison with uninfected cells. Addition of KNK437, a known inhibitor of inducible HSPs, decreased the rate of viral DNA synthesis in infected cells more than one order of magnitude and markedly suppressed the release of budded viruses indicating the importance of the heat shock response for baculovirus replication.

Alternative transcripts expressed by small bristles, the Drosophila melanogaster nxf1 gene.

The tissue-specific accumulation of small bristles (Dm nxf1) transcripts at different developmental stages of Drosophila melanogaster was analyzed by Northern blots and RT PCR. We identified four distinct transcripts: ubiquitous (3.5kb); ovary and early embryo specific (3.3kb); testis specific (1.9kb and 2.8kb) and nervous system specific (5.1kb). The pattern of Dm nxf1 gene expression in ovaries and early embryos (0-2h) is similar: the sizes of transcripts range from 3.0 to 3.5kb. We propose that this size variability may reflect the different extent of cytoplasmic polyadenylation. In testes, the 2.8-kb transcript originates from alternative termination of transcription and the 1.9-kb transcript is supposed to originate from an alternative transcription start. During ontogenesis, the 5.1-kb transcript can be clearly detected in 10- to 18-h-old embryos, most prominently in the nervous ganglia of larvae, and it represents a major species in imago head extracts. We found that the 5.1-kb transcript, similarly to the nxf1 heavy transcripts in Homo sapiens and Mus musculus, results from the retention of intron 5-6 that corresponds to the intron 10-11 in Hs nxf1 and Mm nxf1 genes.

Location of P element insertions in the proximal promoter region of Hsp70A is consequential for gene expression and correlated with fecundity in Drosophila melanogaster.

We compared a series of Drosophila strains with P element insertions from -28 to -144 nucleotides 5' to the transcription start site of the Hsp70A genes-corresponding to the range of naturally occurring P element insertion sites-to elucidate the consequences of insertion site for Hsp70A gene expression. Although all insertions reduced Hsp70A expression below that of a control strain, the magnitude of the reduction was inversely related to the number of nucleotides between the transcription start site and the insertion site. A pre-existing hypothesis is that naturally occurring transposable element insertions in Hsp promoters may be beneficial in some circumstances, which may account for their retention in natural populations. In the present study, in a control line heat shock reduced fecundity, whereas in lines with P element insertions heat shock typically increased fecundity. Finally, according to cluster-specific quantitative RT-PCR, expression of the Hsp70A cluster genes was typically greater than that of the Hsp70B gene cluster genes, although the latter are more numerous and, in this case, free of P element insertions.