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The heat shock protein 70 ( HSP70 ) gene family perform a fundamental role in protecting plants against biotic and abiotic stresses. Aquilaria sinensis is a classic stress-induced medicinal plant, producing a valuable dark resin in a wood matrix, known as agarwood, in response to environmental stresses. The HSP70 gene family has been systematic identified in many plants, but there is no comprehensive analysis at the genomic level in A. sinensis . In this study, 15 putative HSP70 genes were identified in A. sinensis through genome-wide bioinformatics analysis. Based on their phylogenetic relationships, the 15 AsHSP70 were grouped into six sub-families that with the conserved motifs and gene structures, and the genes were mapped onto six separate linkage groups. A qRT-PCR analysis showed that the relative expression levels of all the AsHSP70 genes were up-regulated by heat stress. Subcellular localization of all HSP70s was predicted, and three were verified by transiently expressed in Arabidopsis protoplasts. Based on the expression profiles in different tissues and different layers treated with Agar-Wit, we predict AsHSP70 genes are involved in different stages of agarwood formation. The systematic identification and expression analysis of HSP70s gene family imply some of them may play important roles in the formation of agarwood. Our findings not only provide a foundation for further study their biological function in the later research in A. sinensis , but also provides a reference for the analysis of HSPs in other species.

Ubiquitin (Ub) and Ub-like proteins (Ubls) such as NEDD8 are best known for their function as covalent modifiers of other proteins but they are also themselves subject to post-translational modifications including phosphorylation. While functions of phosphorylated Ub (pUb) have been characterized, the consequences of Ubl phosphorylation remain unclear. Here we report that NEDD8 can be phosphorylated at S65 - the same site as Ub - and that S65 phosphorylation affects the structural dynamics of NEDD8 and Ub in a similar manner. While both pUb and phosphorylated NEDD8 (pNEDD8) can allosterically activate the Ub ligase Parkin, they have different protein interactomes that in turn are distinct from those of unmodified Ub and NEDD8. Among the preferential pNEDD8 interactors are HSP70 family members and we show that pNEDD8 stimulates HSP70 ATPase activity more pronouncedly than unmodified NEDD8. Our findings highlight the general importance of Ub/NEDD8 phosphorylation and support the notion that the function of pUb/pNEDD8 does not require their covalent attachment to other proteins., (© 2021. The Author(s).)

Heat shock proteins (HSPs) are important elements of the cellular group of molecular chaperones. Specifically, HSP70 proteins protect cells from being damaged when plants are exposed to environmental stresses. These proteins are catalysts that manage the correct folding of other proteins, and they play a key role in the development of tolerance against biotic and abiotic stresses. In the present study, 113 HSP70 genes were retrieved from the available genome assemblies of four cotton species, including Gossypium hirsutum, G. barbadense, G. arboreum, and G. raimondii. The HSP70 genes were clustered into 11 subfamilies based on phylogeny. One hundred and nine (109) gene duplications were found across these four species. Localization of genes revealed that several HSP70 genes reside in the cytoplasm. Synonymous and non-synonymous substitution rates revealed that functional segregation of HSP70 genes in cotton is due to purifying selection. Furthermore, HSP70 genes in cotton are expressed constitutively during developmental stages. These findings are valuable to understand the complex mechanism of HSP70 gene regulation that occurs in signaling pathways in response to plant stress., (Copyright © 2020. Published by Elsevier Inc.)

Amoebiasis is a human intestinal disease caused by the parasite Entamoeba histolytica. It has been previously demonstrated that E. histolytica heat shock protein 70 (EhHSP70) plays an important role in amoebic pathogenicity by protecting the parasite from the dangerous effects of oxidative and nitrosative stresses. Despite its relevance, this protein has not yet been characterized. In this study, the EhHSP70 genes were cloned, and the two recombinant EhHSP70 proteins were expressed, purifying and biochemically characterized. Additionally, after being subjected to some host stressors, the intracellular distribution of the proteins in the parasite was documented. Two amoebic HSP70 isoforms, EhHSP70-A and EhHSP70-B, with 637 and 656 amino acids, respectively, were identified. Kinetic parameters of ATP hydrolysis showed low rates, which were in accordance with those of the HSP70 family members. Circular dichroism analysis showed differences in their secondary structures but similarities in their thermal stability. Immunocytochemistry in trophozoites detected EhHSP70 in the nuclei and cytoplasm as well as a slight overexpression when the parasites were subjected to oxidants and heat. The structural differences of amoebic HSP70s with their human counterparts may be used to design specific inhibitors to treat human amoebiasis.

This study was aimed to genetic profiling of heat shock protein 70 (Hsp70) gene in Murrah buffalo investigating 50 unrelated adult animals at ICAR-Research Complex for Eastern Region, Patna (India) in winter, spring, and summer. PCR ready genomic DNA samples and season-wise total RNA samples were prepared. The PCR products of Hsp70 eluted from agarose gel were sequenced and analyzed. The first-strand cDNA was synthesized and concentration was equalized to 25 ng/μl. Expression kinetics of mRNA transcripts in different seasons was studied using Brilliant SYBR Green QPCR technique and the data retrieved was analyzed by least-squares ANOVA. DNA sequencing by primer walking revealed four allelic variants of Hsp70 gene. Alignment study revealed one substitution in 5'UTR, six substitutions in coding region, and one addition in 3'UTR. The highest percent identity and negligible phylogenetic distance were found among the alleles and reference bovine sequences. The relative mRNA expression was significantly higher in summer when THI ≥ 84 than the spring and winter; fold change increased by 4.5 times in summer than the spring whereas found nearly half in winter. These findings can be useful for heat stress management in buffaloes and help in understanding the mechanism of thermo-regulation well.

Heat shock proteins 70 (Hsp70) constitute a highly conserved protein family of cellular chaperones widely distributed in plants, where they play a fundamental role in response to biotic and abiotic stress. Until now, genome-wide analyses of the Hsp70 gene family have been conducted for some species. However, reports about Hsp70 genes in Nicotiana tabacum are scarce. In this study, we systematically conducted genome-wide identification and expression analysis of the Hsp70 gene family in tobacco, including gene structure, classification, evolutionary relationships, promoters, and transcript levels in response to abiotic stress treatments. In all, 61 Hsp70 members were identified and classified into six groups that were mapped onto 18 chromosomes, where most were distributed on both ends of the chromosome. The conserved structures and motifs of NtHsp70 proteins in the same subfamily were highly consistent. At least 15 pairs of NtHsp70 genes underwent gene duplication by segment and tandem duplications. Most NtHsp70 proteins contained N-terminal hexokinase conserved motifs. Phylogenetic analysis showed that most species expanded according to their own species-specific approach during the evolution of Hsp70s. Tissue-specific expression analysis indicated that all NtHsp70 genes were involved in at least one or more abiotic stress responses, highlighting the wide participation of NtHsp70 genes in environmental adaptation. This is the first genome-wide analysis of Hsp70 in N. tabacum. These results indicate that each NtHsp70 member fulfilled distinct functions in response to various abiotic stresses.

Proctophyllodid feather mites (400+ species) are permanent (full-time) symbionts commonly occurring on passerine birds. Phenotypic evolution of these mites appears to be greatly influenced by characters related to reproduction (>87.5% of a total of 32 taxonomically important discrete characters) and male genitalic characters (21.9%). Because sexual selection could the major evolutionary driver in this system, we test the theoretical expectation that genitalic or sexually dimorphic characters should evolve more rapidly and divergently then other characters. We inferred a time-calibrated molecular phylogeny (6 genes, 8571 nt aligned, no missing data) for 133 taxa of proctophyllodid mites and 40 outgroups. Comparisons of the average number of character state changes inferred on 10,696 Bayesian stationary trees indicate that male genitalic or sexually dimorphic characters do not evolve significantly faster than other characters (p=0.537 and p=0.819, respectively). However, among the male genitalic characters, a trait related to the relative length of the aedeagus experienced extremely fast rates of evolution and was detected as a statistical outlier. In this character, the transitions between short, long, and several intermediate states occurred in both directions. In contrast, the evolution of extremely long aedeagi (nearly as long as the body) occurred unidirectionally and irreversibly. This surprising result may be due to constraints imposed by the female spermathecal canal, which, in species where males have extremely long aedeagi, is also very long and may impede pumping sperm by short aedeagi. In proctophyllodid mites, extremely long aedeagi evolved independently five times in five different monophyletic lineages. Several of these lineages were lumped together by taxonomists to form easy-to-distinguish but apparently artificial species-groups. Male genitalic characters, thus, can introduce false synapomorphies that could affect morphology-based phylogenetic inference. For the most species-rich genus, Proctophyllodes, we develop a predictive classification of species-groups that reconciles molecular and morphological data., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Recent studies have indicated a metabolic temperature sensitivity in both the arcto-boreal krill species Thysanoessa inermis and Thysanoessa raschii that may determine these species' abundance and population persistence at lower latitudes (up to 40° N). T. inermis currently dominates the krill community in the Barents Sea and in the high Arctic Kongsfjord. We aimed to increase the knowledge on the upper thermal limit found in the latter species by estimating the CT 50 value (19.7 °C) (critical temperature at which 50 % of animals are reactive) and by linking metabolic rate measurements with molecular approaches. Optical oxygen sensors were used to measure respiration rates in steps of 2 °C (from 0 to 16 °C). To follow the temperature-mediated mechanisms of passive response, i.e., as a proxy for molecular stress, molecular chaperone heat shock protein 70 (Hsp70) sequences were extracted from a transcriptome assembly, and the gene expression kinetics were monitored during an acute temperature exposure to 6 or 10 °C with subsequent recovery at 4 °C. Our results showed upregulation of hsp70 genes, especially the structurally constitutive and mitochondrial isoforms. These findings confirmed the temperature sensitivity of T. inermis and showed that the thermal stress took place before reaching the upper temperature limit estimated by respirometry at 12 °C. This study provides a baseline for further investigations into the thermal tolerances of arcto-boreal Thysanoessa spp. and comparisons with other krill species under different climatic regimes, especially Antarctica.

The cytosolic 70KDa heat shock proteins (Hsp70s) are widely used as biomarkers of environmental stress in ecological and toxicological studies in fish. Here we analyze teleost genome sequences to show that two genes encoding inducible hsp70s (hsp70-1 and hsp70-2) are likely present in all teleost fish. Phylogenetic and synteny analyses indicate that hsp70-1 and hsp70-2 are distinct paralogs that originated prior to the diversification of the teleosts. The promoters of both genes contain a TATA box and conserved heat shock elements (HSEs), but unlike mammalian HSP70s, both genes contain an intron in the 5' UTR. The hsp70-2 gene has undergone tandem duplication in several species. In addition, many other teleost genome assemblies have multiple copies of hsp70-2 present on separate, small, genomic scaffolds. To verify that these represent poorly assembled tandem duplicates, we cloned the genomic region surrounding hsp70-2 in Fundulus heteroclitus and showed that the hsp70-2 gene copies that are on separate scaffolds in the genome assembly are arranged as tandem duplicates. Real-time quantitative PCR of F. heteroclitus genomic DNA indicates that four copies of the hsp70-2 gene are likely present in the F. heteroclitus genome. Comparison of expression patterns in F. heteroclitus and Gasterosteus aculeatus demonstrates that hsp70-2 has a higher fold increase than hsp70-1 following heat shock in gill but not in muscle tissue, revealing a conserved difference in expression patterns between isoforms and tissues. These data indicate that ecological and toxicological studies using hsp70 as a biomarker in teleosts should take this complexity into account., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The molecular chaperone heat shock protein A2 (HSPA2), a member of the 70 kDa heat shock protein (HSP70) family, plays an important role in spermatogenesis and male fertility. Although HSPA2 is evolutionarily highly conserved across the metazoan lineages, the observation of striking differences in temperature-sensitive expressions, testicular physiology, spermatogenesis, as well as its role in male fertility indicates that avian and mammalian HSPA2 may exhibit distinct evolutionary trajectory. The present study reports that while mammalian HSPA2 is constrained by intense purifying selection, avian HSPA2 has been subjected to positive selection. The majority of the positively selected amino acid residues fall on the α-helix and β-sheets of the peptide-binding domain located at the carboxyl-terminal region of the avian HSPA2. The detection of positively selected sites at the helix and β-sheets, which are less tolerant to molecular adaptation, indicates an important functional consequence and contribution to the structural and functional diversification of the avian HSPA2. Collectively, avian HSPA2 may have an adaptive advantage over the mammals in response to heat stress, and therefore, mammals with testicular descent may be at a greater risk in the event of scrotal temperature rise.

The gene encoding heat shock protein 70 (HSP70) was identified in Octopus tankahkeei by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length cDNA (2471 bp) consists of a 5'-untranslated region (UTR) (89 bp), a 3'-UTR (426 bp), and an open reading frame (1956 bp) that encodes 651 amino acid residues with a predicted molecular mass of 71.8 kDa and an isoelectric point of 5.34. Based on the amino acid sequence analysis and multiple sequence alignment, this cDNA is a member of cytoplasmic hsp70 subfamily of the hsp70 family and was designated as ot-hsp70. Tissue expression analysis showed that HSP70 expression is highest in the testes when all examined organs were compared. Immunohistochemistry analysis, together with hematoxylin-eosin staining, revealed that the HSP70 protein was expressed in all spermatogenic cells, but not in fibroblasts. In addition, O. tankahkeei were heat challenged by exposure to 32 °C seawater for 2 h, then returned to 13 °C for various recovery time (0-24 h). Relative expression of ot-hsp70 mRNA in the testes was measured at different time points post-challenge by quantitative real-time PCR. A clear time-dependent mRNA expression of ot-hsp70 after thermal stress indicates that the HSP70 gene is inducible. Ultrastructural changes of the heat-stressed testis were observed by transmission electron microscopy. We suggest that HSP70 plays an important role in spermatogenesis and testis protection against thermal stress in O. tankahkeei., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Hsp70s (heat shock protein 70s) are a class of molecular chaperones that are highly conserved and ubiquitous in organisms ranging from microorganisms to plants and humans. Most research on Hsp70s has focused on the mechanisms of their functions as molecular chaperones, but recently, studies on stress responses are coming to the forefront. Hsp70s play key roles in cellular development and protecting living organisms from environmental stresses such as heat, drought, salinity, acidity, and cold. Moreover, functions of human Hsp70s are related to diseases including neurological disorders, cancer, and virus infection. In this review, we provide an overview of the specific roles of Hsp70s in response to stress, particularly abiotic stress, in all living organisms.

In this study, a full-length HSP70 cDNA from Paphia undulata was cloned using reverse transcriptase polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends (RACE). The full-length cDNA is 2,351 bp, consisting of a 5'-untranslated region (UTR) of 83 bp, a 3'-UTR of 315 bp, and an open reading frame (ORF) of 1,953 bp. This cDNA encodes 650 amino acids with an estimated molecular weight of 71.3 kDa and an isoelectric point of 5.51. Based on the amino acid sequence analysis and phylogenetic analysis, this HSP70 gene was identified as a member of the cytoplasmic HSP70 family, being the constitutive expression, and it was designated as PuHSC70. The distribution of PuHSC70 mRNA in the mantle, digestive gland, adductor muscle, gonad, gill, heart, and hemocytes suggested that PuHSC70 is ubiquitously expressed. The mRNA levels of PuHSC70 under high temperature and high salinity stresses were analyzed by real-time PCR. Under high temperature stress of 32°C, PuHSC70 mRNA in the mantle, digestive gland, gill, and heart was significantly up-regulated at 1h and 2h, and it was then progressively down-regulated. In the adductor muscle, the level of PuHSC70 mRNA gradually increased throughout the study period; the mRNA levels in the gonad and hemocytes increased significantly at 4h and 8h (P<0.05) and then decreased at 8h and 14 h, respectively, however they increased again afterwards, reaching the highest levels at 50h. Under high salinity (32 ‰) stress, the mRNA levels of PuHSC70 in the mantle and gonad were increased significantly only at 24h and 48 h (P<0.05), and at the rest of the study period they were slightly elevated. Compared with the pretreatment level, the levels of expression in the digestive gland and gill were unchanged or reduced throughout the study period. The levels of PuHSC70 mRNA in the adductor muscle, hemocytes, and heart were significantly increased, reaching a maximum at 24h, and then they gradually decreased; moreover, in the heart, the mRNA expression recovered to the pretreatment level at 50h; while in the adductor muscle and hemocytes, the expression level remained higher than that of the control. The cloning and expression analyses of PuHSC70 provide theoretical basis to further study the mechanism of physiological response to thermal and high salinity stresses., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Gonad differentiation is one of the most important developmental events in vertebrates. Some heat shock proteins are associated with gonad development. Heat shock protein 70 (Hsp70) in the teleost fish and its roles in sex differentiation are poorly understood., Methods and Findings: We have identified a testis-enriched heat shock protein Hspa8b2 in the swamp eel using Western blot analysis and Mass Spectrometry (MS). Fourteen Hsp70 family genes were further identified in this species based on transcriptome information. The phylogenetic tree of Hsp70 family was constructed using the Maximum Likelihood method and their expression patterns in the swamp eel gonads were analyzed by reverse transcription-polymerase chain reaction (RT-PCR)., Conclusion: There are fourteen gene members in the Hsp70 family in the swamp eel genome. Hsp70 family, particularly Hspa8, has expanded in the species. One of the family members Hspa8b2 is predominantly expressed in testis of the swamp eel.

Hsp70 molecular chaperones are ubiquitous. By preventing aggregation, promoting folding, and regulating degradation, Hsp70s are major factors in the ability of cells to maintain proteostasis. Despite a wealth of functional information, little is understood about the evolutionary dynamics of Hsp70s. We undertook an analysis of Hsp70s in the fungal clade Ascomycota. Using the well-characterized 14 Hsp70s of Saccharomyces cerevisiae, we identified 491 orthologs from 53 genomes. Saccharomyces cerevisiae Hsp70s fall into seven subfamilies: four canonical-type Hsp70 chaperones (SSA, SSB, KAR, and SSC) and three atypical Hsp70s (SSE, SSZ, and LHS) that play regulatory roles, modulating the activity of canonical Hsp70 partners. Each of the 53 surveyed genomes harbored at least one member of each subfamily, and thus establishing these seven Hsp70s as units of function and evolution. Genomes of some species contained only one member of each subfamily that is only seven Hsp70s. Overall, members of each subfamily formed a monophyletic group, suggesting that each diversified from their corresponding ancestral gene present in the common ancestor of all surveyed species. However, the pattern of evolution varied across subfamilies. At one extreme, members of the SSB subfamily evolved under concerted evolution. At the other extreme, SSA and SSC subfamilies exhibited a high degree of copy number dynamics, consistent with a birth-death mode of evolution. KAR, SSE, SSZ, and LHS subfamilies evolved in a simple divergent mode with little copy number dynamics. Together, our data revealed that the evolutionary history of this highly conserved and ubiquitous protein family was surprising complex and dynamic.

An increasing amount of data has revealed the importance of inducible promoters in ciliate research and in ciliate-related industries. However, knowledge about these promoters and related genes is relatively sparse. Here we report a novel inducible promoter from a Tetrahymena cytoplasmic Hsp70 gene member, HSP70-2. The reported promoter was able to induce the endogenous gene up to ∼9000-fold after a short heat shock treatment and this remarkable feature has been retained when a relatively short region of the promoter was introduced into a reporter construct followed by transformation. During the recovery period following a short heat shock, both the mRNA and protein levels of the reporter gene were maintained high up to two hours. A constant heat shock treatment to the transformed cells led to a stabilization of the reporter mRNA up to at least six hours and the reporter protein continued to accumulate up to around three hours. The promoter strength appears to be similar to that of the cadmium-induced metallothionein gene (MTT1) promoter. Therefore, the HSP70-2 promoter represents an attractive alternative for the over-expression of proteins in Tetrahymena, and the promoter-reporter gene construct used in this study is an ideal tool to help in understanding the regulation mechanisms of heat shock genes in ciliates., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

In this study, we identified and functionally characterized the mitochondrial heat shock protein 70 (mtHsp70). Over-expression of mtHsp70 suppressed heat- and H(2)O(2)-induced programmed cell death (PCD) in rice protoplasts, as reflected by higher cell viability, decreased DNA laddering and chromatin condensation. Mitochondrial membrane potential (Δψ(m)) after heat shock was destroyed gradually in protoplasts, but mtHsp70 over-expression showed higher Δψ(m) relative to the vector control cells, and partially inhibited cytochrome c release from mitochondria to cytosol. Heat treatment also significantly increased reactive oxygen species (ROS) generation, a phenomenon not observed in protoplasts over-expressing mtHsp70. Together, these results suggest that mtHsp70 may suppress PCD in rice protoplasts by maintaining mitochondrial Δψ(m) and inhibiting the amplification of ROS., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Unicellular photosynthetic dinoflagellates of the genus Symbiodinium are the most common endosymbionts of reef-building scleractinian corals, living in a symbiotic partnership known to be highly susceptible to environmental changes such as hyperthermic stress. In this study, we identified members of two major heat shock proteins (HSPs) families, Hsp70 and Hsp90, in Symbiodinium sp. (clade C) with full-length sequences that showed the highest similarity and evolutionary relationship with other known HSPs from dinoflagellate protists. Regulation of HSPs gene expression was examined in samples of the scleractinian coral Acropora millepora subjected to elevated temperatures progressively over 18 h (fast) and 120 h (gradual thermal stress). Moderate to severe heat stress at 26°C and 29°C (+3°C and +6°C above average sea temperature) resulted in an increase in algal Hsp70 gene expression from 39% to 57%, while extreme heat stress (+9°C) reduced Hsp70 transcript abundance by 60% (after 18 h) and 70% (after 120 h). Elevated temperatures decreased an Hsp90 expression under both rapid and gradual heat stress scenarios. Comparable Hsp70 and Hsp90 gene expression patterns were observed in Symbiodinium cultures and in hospite, indicating their independent regulation from the host. Differential gene expression profiles observed for Hsp70 and Hsp90 suggests diverse roles of these molecular chaperones during heat stress response. Reduced expression of the Hsp90 gene under heat stress can indicate a reduced role in inhibiting the heat shock transcription factor which may lead to activation of heat-inducible genes and heat acclimation.

Whereas the consequences of global warming at population or community levels are well documented, studies at the cellular level are still scarce. The study of the physiological or metabolic effects of such small increases in temperature (between +2 degrees C and +6 degrees C) is difficult because they are below the amplitude of the daily or seasonal thermal variations occurring in most environments. In contrast, subterranean biotopes are highly thermally buffered (+/-1 degrees C within a year), and underground water organisms could thus be particularly well suited to characterise cellular responses of global warming. To this purpose, we studied genes encoding chaperone proteins of the HSP70 family in amphipod crustaceans belonging to the ubiquitous subterranean genus Niphargus. An HSP70 sequence was identified in eight populations of two complexes of species of the Niphargus genus (Niphargus rhenorhodanensis and Niphargus virei complexes). Expression profiles were determined for one of these by reverse transcription and quantitative polymerase chain reaction, confirming the inducible nature of this gene. An increase in temperature of 2 degrees C seemed to be without effect on N. rhenorhodanensis physiology, whereas a heat shock of +6 degrees C represented an important thermal stress for these individuals. Thus, this study shows that although Niphargus individuals do not undergo any daily or seasonal thermal variations in underground water, they display an inducible HSP70 heat shock response. This controlled laboratory-based physiological experiment constitutes a first step towards field investigations of the cellular consequences of global warming on subterranean organisms.

The heat shock protein 70 (Hsp70) family is widely expressed in eukaryotic cells as the major chaperone protein. In this study, the full-length complementary DNA (cDNA) of a novel inducible cytosolic Hsp70 family member (FcHsp70) was cloned from Fenneropenaeus chinensis. FcHsp70 full-length cDNA consists of 2,511 bp with a 1,890-bp open reading frame encoding 629 amino acids. Three Hsp70 protein family signatures, IDLGTTYS, IIDLGGGTFDVSIL, and IVLVGGSTRIPKVQK, were found in the predicted FcHsp70 amino acid sequence. Phylogenetic analysis showed that FcHsp70 was categorized together with the inducible HSP70s reported in other crustaceans. Compared to the previously identified cognate Hsp70 (FcHsc70) in F. chinensis, the expression of FcHsp70 showed quite different expression profiles when the shrimp were subjected to different stresses including heat shock and heavy metal treatments. Under heat shock treatment, the expression of FcHsp70 showed much higher up-regulation than FcHsc70. Copper treatment also induced higher up-regulation of FcHsp70 than FcHsc70. Cadmium treatment did not induce the expression of FcHsp70, but caused down-regulation of FcHsc70. The different expression profiles of FcHsp70 and FcHsc70 in shrimp may indicate their different reactions to different stresses. Therefore, Hsp70 or Hsc70 could be developed as a biomarker to indicate different stresses in shrimp.

Mounting evidence suggests that all organisms at the cellular level respond to stress by synthesizing heat shock proteins at the expense of other proteins, and the ability of human cells to respond to heat stress decreases with aging. We thus investigate the association of 3 variants (A1267G in HSPA1B, G190C in HSPA1A, and T2437C in HSPA1L) in the heat shock protein 70 (Hsp70) family with natural longevity in a Xinjiang Hetian Uygur population. A case-control study was conducted in 191 healthy individuals greater than 90 years of age, and 53 naturally died persons 65-70 years of age. Promoter activity was evaluated by luciferase reporter assays. The data were analyzed using an EH/EH+ program for haplotype prediction and MDR software for gene-gene interaction. All studied variants satisfied the Hardy-Weinberg equilibrium in each group. In single-locus analysis, no significant differences were found between long-lived people and short-lived people in the genotype/allele distributions of all variants. In contrast, haplotype analysis indicated that haplotypes A-G-C and A-C-T were more prevalent in long-lived people than short-lived people (P=0.026 and 0.017), and the analysis conferred a 3.46- and 4.51-fold increased tendency for longevity, respectively (P=0.025 and 0.016). The haplotype results were strengthened by interaction analysis, which suggests an optimal model in which G190C and T2437C exert an interacting effect on longevity. No functional significance was observed between 190G and 190C alleles in both control and heat-inducible A549 cells (P>0.05). Taken together, our findings suggested that common genetic variants in Hsp70 family might contribute interactively to longevity the Xinjiang Hetian Uygur population.

Experimental evidence suggesting that heat shock protein 70 (Hsp70) gene or associated genes are responsible for the pathophysiology of hypertension is accumulating. In this study, we focused on five polymorphisms in three genes (HSPA1A, HSPA1B, and HSPA1L) of Hsp70 family to explore the genetic contribution, alone and in combination, of these polymorphisms to essential hypertension risk in a Uygur population. Genotyping was performed using PCR-RFLP and direct sequencing techniques. Data were analyzed using haplotype and multifactor dimensionality reduction (MDR) methods. Genotype distributions of all the polymorphisms satisfied the Hardy-Weinberg proportions in cases and controls. Statistical significance was only observed in the genotype (P = 0.0028) and (P = 0.0146) allele distributions of -110A/C polymorphism, with the -110C allele conferring a 1.45- and 2.83-fold of relative risk, assuming the additive and recessive models, respectively, and in 1267A/G genotype distribution (P = 0.0106) with the 1267G allele conferring a 44% reduced risk. The interaction information analysis indicated that polymorphisms -110A/C and 1267A/G had a strong synergistic effect, while polymorphisms 2074G/C and 2437T/C had a moderate synergistic effect. Haplotype analyses further strengthened the interaction information. Using the haplotype H(1) as a reference, haplotype H(4) had a 40% reduced risk, while haplotypes H(5) and H(8) had a significantly 5.00- and 3.75-fold increased risk for essential hypertension, respectively. Taken together, our results supported strong genetic interaction of the studied polymorphisms with the risk of having essential hypertension in Uygur ethnicity. Functional studies are warranted to confirm or refute these findings. This is the first study to evaluate the genetic interaction information of the Hsp70 in Uygur ethnicity, which represents one of the major nationalities in China with high homogeneity and unique lifestyles. Moreover, we employed the haplotype and MDR methods to explore the potential interaction of Hsp70 genetic polymorphisms in the pathogenesis of essential hypertension in Uygur.

The expanding number of members in the various human heat shock protein (HSP) families and the inconsistencies in their nomenclature have often led to confusion. Here, we propose new guidelines for the nomenclature of the human HSP families, HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), DNAJ (HSP40), and HSPB (small HSP) as well as for the human chaperonin families HSPD/E (HSP60/HSP10) and CCT (TRiC). The nomenclature is based largely on the more consistent nomenclature assigned by the HUGO Gene Nomenclature Committee and used in the National Center of Biotechnology Information Entrez Gene database for the heat shock genes. In addition to this nomenclature, we provide a list of the human Entrez Gene IDs and the corresponding Entrez Gene IDs for the mouse orthologs.

Aim: To explore the expression and significance of heat shock protein 70 (Hsp70), glucose regulated protein 94 (Grp94) and immunoglobulin G (IgG) in human lung carcinoma., Methods: The expression of Hsp70, Grp94 and IgG in 40 human lung carcinomas was studied using immunohistochemical technique and image analysis. The localization among Hsp70, Grp94 and IgG was analyzed by double labeling immunofluorescent staining and laser scanning confocal microscopy., Results: Hsp70, Grp94 and IgG in Human lung carcinomas showed high expression. The positive rate of Hsp70, Grp94 and IgG was 65% (26/40), 45% (18/40), and 82.5% (33/40), respectively. The average value of optical density was 5.10 +/- 0.32, 3.52 +/- 0.35, and 8.12 +/- 0.31, respectively. Hsp70 was localized in nucleus and cellular cytoplasm while Grp94 and IgG were mainly localized in cellular cytoplasm. Ten cases showed Hsp70 was co-localized with IgG and Eighteen cases showed Grp94 was co-localized with IgG., Conclusion: High expression of Hsp70, Grp94 and IgG in Human lung carcinomas suggested that Hsp70, Grp94 and IgG might play an important role in the development of human lung carcinoma. IgG is co-localized with Hsp70 or Grp94. The study will lay a theorical basis for further research on anti-tumor immunotherapy.

The coding region of the hsp68 gene has been amplified, cloned, and sequenced from 10 Drosophila species, 5 from the melanogaster subgroup and 5 from the montium subgroup. When the predicted amino acid sequences are compared with available Hsp70 sequences, patterns of conservation suggest that the C-terminal region should be subdivided according to predominant secondary structure. Conservation levels between Hsp68 and Hsp70 proteins were high in the N-terminal ATPase and adjacent beta-sheet domains, medium in the alpha-helix domain, and low in the C-terminal mobile domain (78%, 72%, 41%, and 21% identity, respectively). A number of amino acid sites were found to be "diagnostic" for Hsp68 (28 of approximately 635 residues). A few of these occur in the ATPase domain (385 residues) but most (75%) are concentrated in the beta-sheet and alpha-helix domains (34% of the protein) with none in the short mobile domain. Five of the diagnostic sites in the beta-sheet domain are clustered around, but not coincident with, functional sites known to be involved in substrate binding. Nearly all of the Hsp70 family length variation occurs in the mobile domain. Within montium subgroup species, 2 nearly identical hsp68 PCR products that differed in length are either different alleles or products of an ancestral hsp68 duplication.

In order to identify neuroendocrine tumour-specific protein expression, we generated monoclonal antibodies (mAbs) with a tumour-related reaction pattern using a human insulinoma as immunogen. One of the generated mAbs (mAb 1D4) exhibited striking immunoreactivity against various neuroendocrine tumours without staining pancreatic islets of Langerhans. Furthermore, mAb 1D4 immunostained a characteristic subtype of hypothalamic neurones. Using two-dimensional (2-D) gel electrophoresis, mAb 1D4 immunoblotting and mass spectrometry, heat shock protein 70 (Hsp70) isoforms were identified as the mAb 1D4-specific antigen. In hypothalamic tissue, the presence of two different Hsp70 isoforms (Hsp70-8 and Hsp70-1) was revealed by 2-D gel immunoblots and consecutive mass spectrometric peptide analysis. In contrast, insulinoma and other neuroendocrine tumours displayed solely Hsp70-8 expression. Moreover, the tumour-specific presence of an additional mAb 1D4 immunoreactive protein of 40 kDa was observed in eight out of eight tested neuroendocrine tumours. For this variant, exclusively, peptides derived from the C terminus excluding the 299 amino-terminal residues were detected. In cultured tumour-derived fibroblasts, expression of the truncated Hsp70-8 subtype was not present. In conclusion, we have demonstrated a neuroendocrine tumour-specific expression pattern of Hsp70 isoforms and identified an as yet unknown N-terminally truncated Hsp70-8 variant., (Copyright 2004 Society for Endocrinology)

Two genes expressing 70 kDa heat shock proteins were identified in Cyprinus carpio. The sequence similarities and the intron-interrupted structure of the coding regions indicate that carp Hsc70-1 and Hsc70-2 belong to the Hsp70 cognate subfamily. The expressions of the two hsc70 genes were followed by semi-quantitative RT-PCR. Both genes are expressed under unstressed conditions in a characteristic tissue-specific manner. Inducibility of the response to elevated temperature, cold shock, and Cd treatment was investigated in the liver and muscle, in whole-animal experiments. Both genes were insensitive to or only weakly induced by the stressors, with two exceptions: Cd treatment resulted in an 11-13-fold enhanced induction of hsc70-1 in the liver and cold shock enhanced induction of hsc70-2 in the muscle by 7.5-10-fold.

A member of the multi-gene family, encoding 70 kD stress proteins, was identified from the common carp (Cyprimus carpio). Homologies, observed at both nucleic acid and amino acid levels, and also the intronless structure of this gene, strongly suggest that it corresponds to a heat-inducible hsp70 gene in carp. Gene-specific primers were selected and used in RT-PCR reactions to measure the basal hsp70 mRNA levels and to follow the inducer-specific expression of this gene in different tissues during in vivo studies. Carp hsp70 mRNA is not detectable in the brain and muscle, and its concentration is around the limit of detection in the kidney and liver of unstressed animals. The expression of hsp70 is induced by elevated temperature and it responds to Cd treatment in a tissue and time-dependent manner.

Plasmodesmata establish a pathway for the trafficking of non-cell-autonomously acting proteins and ribonucleoprotein complexes. Plasmodesmal enriched cell fractions and the contents of enucleate sieve elements, in the form of phloem sap, were used to isolate and characterize heat shock cognate 70 (Hsc70) chaperones associated with this cell-to-cell transport pathway. Three Cucurbita maxima Hsc70 chaperones were cloned and functional and sequence analysis led to the identification of a previously uncharacterized subclass of non-cell-autonomous chaperones. The highly conserved nature of the heat shock protein 70 (Hsp70) family, in conjunction with mutant analysis, permitted the characterization of a motif that allows these Hsc70 chaperones to engage the plasmodesmal non-cell-autonomous translocation machinery. Proof of concept that this motif is necessary for Hsp70 gain-of-movement function was obtained through the engineering of a human Hsp70 that acquired the capacity to traffic through plasmodesmata. These results are discussed in terms of the roles likely played by this subclass of Hsc70 chaperones in the trafficking of non-cell-autonomous proteins.

The Arabidopsis genome contains at least 18 genes encoding members of the 70-kilodalton heat shock protein (Hsp70) family, 14 in the DnaK subfamily and 4 in the Hsp110/SSE subfamily. While the Hsp70s are highly conserved, a phylogenetic analysis including all members of this family in Arabidopsis and in yeast indicates the homology of Hsp70s in the subgroups, such as those predicted to localize in the same subcellular compartment and those similar to the mammalian Hsp110 and Grp170. Gene structure and genome organization suggest duplication in the origin of some genes. The Arabidopsis hsp70s exhibit distinct expression profiles; representative genes of the subgroups are expressed at relatively high levels during specific developmental stages and under thermal stress.

A polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of a 587-bp region of the Cryptosporidium parvum 70-kDa heat shock protein (HSP70) gene was developed for the detection and discrimination of the two major genotypes of C. parvum, genotype 1 and genotype 2. Ten Cryptosporidium isolates from non-immunocompromised people were identified as genotypes 1 and 2 (five each) by DNA sequencing of the 587-bp PCR product. This distinction was also achieved with the combination of two endonucleases, HinfI and ScaI, which generated a specific pattern for each genotype. A thorough screening of published sequences showed that this combination of enzymes could also be used for the discrimination of other species/genotypes of Cryptosporidium, especially Cryptosporidium meleagridis and the 'dog' genotype of C. parvum, both of which are infectious in humans. The PCR, conducted on genotypes 1 and 2 of C. parvum, could detect one oocyst per reaction. This new and sensitive genotyping procedure should be of particular interest when applied to the monitoring of water resources in which low concentrations of parasites usually occur.

We identified a novel gene encoding molecular chaperone HSP70 in the amitochondriate parasite Giardia lamblia. The predicted protein is similar to bacterial DnaK and mitochondrial HSP70s. The gene is transcribed and translated at a constant level during trophozoite growth and encystation. Alignment of the sequence with a data set of cytosolic, endoplasmic reticulum (ER), mitochondrial, and DnaK HSP70 homologs indicated that the sequence was extremely divergent and contained insertions unique to giardial HSP70s. Phylogenetic analyses demonstrated that this sequence was distinct from the cytosolic and ER forms and was most similar to proteobacterial and mitochondrial DnaKs. However, a specific relationship with the alpha proteobacterial and mitochondrial sequences was not strongly supported by phylogenetic analyses of this data set, in contrast to similar analyses of cpn60. These data neither confirm nor reject the possibility that this gene is a relic of secondary mitochondrial loss; they leave open the possibility that it was acquired in a separate endosymbiotic event.

The endoplasmic reticulum (ER) molecular chaperone, BiP, plays a role in the cotranslational translocation and subsequent folding and assembly of newly synthesized proteins targeted to the ER and secretory pathway. The sequence encoding a Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) BiP homologue (PmBiP) was identified by differential screening of a seedling cDNA library. Southern blotting indicated that PmBiP is most likely present as a single copy. The deduced amino acid sequence of PmBiP contains an HEEL tetrapeptide sequence which functions to retain PmBiP in the ER and is different from HDEL commonly found in angiosperm plant BiPs. Amino acid sequence alignment and phylogenetic analysis show that PmBiP is highly similar to other plant BiPs yet forms a distinct phylogenetic subgroup which is separate from the angiosperm BiPs. Northern and western blotting revealed that PmBiP is subject to developmental regulation during seed development, germination, and early seedling growth and is seasonally regulated in needles of young seedlings.

Protein folding and assembly in the cell requires the assistance of molecular chaperones. These components prevent off-pathway folding reactions that lead to aggregation. They are also critical factors in organismal stress physiology, protecting cells against heat shock and providing thermotolerance. Among this important protein family are chaperonins. They form large cylindrical double ring complexes with a central cavity where protein binding and folding takes place in an ATP-dependent manner. Recently, components functionally related to the eubacterial and organellar chaperonins have been found in the cytosol of archaebacteria and of eukaryotic cells. Based on their sequences and structural features, they have been classified as group II chaperonins, to distinguish them from the group I chaperonins occurring in bacteria. Of particular interest in the group II family is the eukaryotic CCT complex, whose function in protein folding and assembly has been demonstrated mainly for the cytoskeletal proteins tubulin and actin. Together with the Hsp70 chaperone system, it can be considered as an essential helper factor to facilitate the folding of native proteins in the eukaryotic cytosol. Recent structural data have opened the path to a molecular understanding of group II chaperonins and have helped to define their role in cellular protein folding.

Twelve cosmids containing sequences resembling genes encoding members of the 70-kDa heat-shock protein family, HSP70. have been isolated from Fugu rubripes. They can be broadly divided into three groups of overlapping cosmids. Restriction analysis and sequencing of one set of five cosmids have revealed five intronless Fugu HSP70 genes spanning 42 kb, arranged in a combined head-to-head, tail-to-tail and head-to-tail orientation. The levels of DNA and amino acid identity are very high with respect to one another, and are most similar to HSP70 sequences linked to the major histocompatibility complex (MHC) region in other species. Putative heat-shock consensus elements are identified. Non-HSP70 sequences with homology to known genes have been found physically linked to this Fugu HSP70 cluster: the Drosophila melanogaster SOL gene, the Drosophila melanogaster nemo gene, the Caenorhabditis elegans T17E9.1 gene and the sequence encoding the serine protease domain. The linkage relationships described here so far bear no resemblance to those of HSP70 in other organisms. Convergence of mammalian HSP70 and MHC class I and II loci probably occurred after fish had diverged.

We describe here the cloning and sequencing of human and mouse cDNAs encoding a putative GTP binding protein. Sequence comparison shows that these cDNAs (named eRFS) are likely to represent the orthologues of the yeast Saccharomyces cerevisiae HBS1 gene and that the C-terminal domains of the encoded proteins share structural features with eukaryotic elongation factor eEF-1A and release factor 3 (eRF3) families. The phylogenetic analysis suggests that eRFS proteins and Hbs1p form a cluster of orthologous sequences branching with the eRF3 family. Nevertheless, in yeast, the human eRFS protein and Hbs1p do not complement eRF3/Sup35p thermosensitive mutation and do not interact with eRF1.

Pneumocystis carinii, a major opportunistic lung pathogen of AIDS patients, is found in a number of mammals and is proposed to be a member of the fungi. In this work, several members of the highly conserved HSP70 multigene family were characterized from rat-derived P. carinii. Previously, we reported characterization of the ER resident HSP70 homolog known as BiP from prototype (P.c. carinii) and variant (P. c. rattus) strains of the organism. We report here, from P. c. carinii, characterization of Pcsa1, an HSP70 homolog that encodes a cognate/stress-induced HSP70 homolog of the SSA subfamily in Saccharomyces cerevisiae. We also identify, from both rat strains and from a human isolate of P. carinii (P.c. hominis), a third set of HSP70 homologs that apparently encode a ribosome-associated cytoplasmic HSP70 homologous to the S. cerevisiae SSB subfamily. Our data indicate that Pcsal mRNA, like Pcbip mRNA, bears an intron in the 5' untranslated region, is induced by heat shock, and suggest that this gene undergoes alternative transcription and splicing. The SSB homologs display significant sequence heterogeneity between P. carinii source strains, supporting the genetic divergence and likely speciation of P. carinii isolates within and between host species. Phylogenetic analysis with the PcSA1 protein supports inclusion of P. carinii among the higher fungi.

Microsporidia are small (1-20 micron) obligate intracellular parasites of a variety of eukaryotes, and they are serious opportunistic pathogens of immunocompromised patients [1]. Microsporidia are often assigned to the first branch in gene trees of eukaryotes [2,3], and are reported to lack mitochondria [2,4]. Like diplomonads and trichomonads, microsporidia are hypothesised to have diverged from the main eukaryotic stock prior to the event that led to the mitochondrion endosymbiosis [2,4]. They have thus assumed importance as putative relics of premitochondrion eukaryote evolution. Recent data have now revealed that diplomonads and trichomonads contain genes that probably originated from the mitochondrion endosymbiont [5-9], leaving microsporidia as chief candidates for an extant primitively amitochondriate eukaryote group. We have now identified a gene in the microsporidium Vairimorpha necatrix that appears to be orthologous to the eukaryotic (symbiont-derived) Hsp70 gene, the protein product of which normally functions in mitochondria. The simplest interpretation of our data is that microporidia have lost mitochondria while retaining genetic evidence of their past presence. This strongly suggests that microsporidia are not primitively amitochondriate and makes feasible an evolutionary scenario whereby all extant eukaryotes share a common ancestor which contained mitochondria.

The 70-kDa heat shock protein (hsp70) sequences define one of the most conserved proteins known to date. The hsp70 genes from Deinococcus proteolyticus and Thermomicrobium roseum, which were chosen as representatives of two of the most deeply branching divisions in the 16S rRNA trees, were cloned and sequenced. hsp70 from both these species as well as Thermus aquaticus contained a large insert in the N-terminal quadrant, which has been observed before as a unique characteristic of gram-negative eubacteria and eukaryotes and is not found in any gram-positive bacteria or archaebacteria. Phylogenetic analysis of hsp70 sequences shows that all of the gram-negative eubacterial species examined to date (which includes members from the genera Deinococcus and Thermus, green nonsulfur bacteria, cyanobacteria, chlamydiae, spirochetes, and alpha-, beta-, and gamma-subdivisions of proteobacteria) form a monophyletic group (excluding eukaryotic homologs which are derived from this group via endosybitic means) strongly supported by the bootstrap scores. A closer affinity of the Deinococcus and Thermus species to the cyanobacteria than to the other available gram-negative sequences is also observed in the present work. In the hsp7O trees, D. proteolyticus and T. aquaticus were found to be the most deeply branching species within the gram-negative eubacteria. The hsp70 homologs from gram-positive bacteria branched separately from gram-negative bacteria and exhibited a closer relationship to and shared sequence signatures with the archaebacteria. A polyphyletic branching of archaebacteria within gram-positive bacteria is strongly favored by different phylogenetic methods. These observations differ from the rRNA-based phylogenies where both gram-negative and gram-positive species are indicated to be polyphyletic. While it remains unclear whether parts of the genome may have variant evolutionary histories, these results call into question the general validity of the currently favored three-domain dogma.

Folding of newly synthesized polypeptides in the crowded cellular environment requires the assistance of so-called molecular chaperone proteins. Chaperones of the Hsp70 class and their partner proteins interact with nascent polypeptide chains on ribosomes and prevent their premature (mis)folding at least until a domain capable of forming a stable structure is synthesized. For many proteins, completion of folding requires the subsequent interaction with one of the large oligomeric ring-shaped proteins of the chaperonin family, which is composed of the GroEL-like proteins in eubacteria, mitochondria, and chloroplasts, and the TRiC family in eukaryotic cytosol and archaea. These proteins bind partially folded polypeptide in their central cavity and promote folding by ATP-dependent cycles of release and rebinding. In these reactions, molecular chaperones interact predominantly with the hydrophobic surfaces exposed by nonnative polypeptides, thereby preventing incorrect folding and aggregation.

A major mammalian heat shock protein of 110 kDa (hsp110) has long been observed, but has not been cloned. We have cloned the hamster cDNA for hsp110 and show that it hybridizes on a Northern blot to a 3.5-kilobase heat-inducible message in hamster and mouse. The hsp110 sequence was found to share an approximately 30-33% amino acid identity with members of the hsp70 family, most of which occurs in the conserved ATP-binding domain of these molecules. In addition, five sequences were found to be highly similar to hsp110. These are the sea urchin egg receptor for sperm (Foltz, K.R., Partin, J. S., and Lennarz, W.J. (1993) Science 259, 1421-1425) and additional sequences from human and Caenorhaditis elegans and two from yeast. The carboxyl-terminal two-thirds of hsp110 and these five related proteins contain a pattern of highly conserved regions of sequence unique to this group. A probe containing these conserved sequences was found to strongly cross-react on a Southern blot with genomic sequences from yeast to man. A Western blot analysis of several murine tissues indicates that hsp110 is constitutively expressed in all mouse tissues and is highly expressed in brain. Therefore, hsp110 belongs to a new category of large and structurally unique stress proteins that are the most distantly related known members of the hsp70 family.

Background: The evolutionary relationships between archaebacteria, eubacteria and eukaryotic cells are of central importance in biology. The current view is that each of these three groups of organisms constitutes a monophyletic domain, and that eukaryotic cells have evolved fom an archaebacterial ancestor. Recent studies on a number of highly conserved protein sequences do not, however, support this view and raise important questions concerning the evolutionary relationships between all extant organisms, particularly regarding the origin of eukaryotic cells., Results: We have used sequences of 70 kD heat shock protein (hsp70)--the most conserved protein found to date in all species--to examine the evolutionary relationship between various species. We have obtained two new archaebacterial hsp70 sequences from the species, Thermoplasma acidophilum and Halobacterium cutirubrum. A global comparison of hsp70 sequences, including our two new sequences, shows that all known archaebacterial homologs share a number of sequence signatures with the Gram-positive group of bacteria that are not found in any other prokaryotic or eukaryotic species. In contrast, the eukaryotic homologs are shown to share a number of unique sequence features with the Gram-negative bacteria that are not present in any archaebacteria. Detailed phylogenetic analyses of hsp70 sequences strongly support a specific evolutionary relationship between archaebacteria and Gram-positive bacteria on the one hand, and Gram-negative bacteria and eukaryotes on the other. The phylogenetic analyses also indicate a polyphyletic branching of archaebacteria within the Gram-positive species. The possibility that the observed relationships are due to horizontal gene transfers can be excluded on the basis of sequence characteristics of different groups of homologs., Conclusions: Our results do not support the view that archaebacteria constitute a monophyletic domain, but instead suggest a close evolutionary linkage between archaebacteria and Gram-positive bacteria. Furthermore, in contrast to the presently accepted view, eukaryotic hsp70s show a close and specific relationship to those from Gram-negative species. To explain the phylogenies based on different gene sequences, a chimeric model for the origin of the eukaryotic cell nucleus involving fusion between an archaebacterium and a Gram-negative eubacterium is proposed. Several predictions from the chimeric model are discussed.