Heat shock proteins (HSPs) widely exist in all organisms, the structures of which are usually extraordinarily conservative. They are also well-known stress proteins that are involved in response to physical, chemical and biological stresses. HSP70 is an important member of the HSPs family. In order to study the roles of amphibians HSP70 during infection, the cDNA sequence of Rana amurensis hsp70 family genes were cloned by homologous cloning method. The sequence characteristics, three-dimensional structure and genetic relationship of Ra-hsp70s were analyzed by bioinformatics methods. The expression profiles under bacterial infection were also analyzed by real-time quantitative PCR (qRT-PCR). Expression and localization of HSP70 protein were tested by immunohistochemical techniques. The results showed that three conservative tag sequences of HSP70 family, HSPA5, HSPA8 and HSPA13, were found in HSP70. Phylogenetic tree analysis indicated four members are distributed in four different branches, and members with the same subcellular localization motif are distributed in the same branch. The relative expression levels of the mRNA of four members were all significantly upregulated (P < 0.01) upon infection, but the time for up-regulating the expression levels were diverse in different tissues. The immunohistochemical analysis showed that HSP70 was expressed to different degrees in the cytoplasm of liver, kidney, skin and stomach tissue. The four members of Ra-hsp70 family have ability to respond bacterial infection to varying degrees. Therefore, it was proposed that they are involved in biological processes against pathogen and play different biological functions. The study provides a theoretical basis for functional studies of HSP70 gene in amphibians.
Heat-Shock Proteins/genetics* ; Phylogeny ; Amino Acid Sequence ; HSP70 Heat-Shock Proteins/metabolism* ; Stress, Physiological

OBJECTIVE: Our aim was to explore whether heat stress protein (HSP) 9 preferentially expresses under heat stress and affects the expression of other heat stress proteins as well as to explore the effect of HSPB9 overexpression and knockdown on apoptosis in DF-1. METHODS: We used gene cloning to construct an overexpression vector of the target gene, and synthesized the target gene interference fragment to transfect the chicken fibroblast cell line. Gene and protein expression, as well as apoptosis, were detected by RT-qPCR, Western blot, and flow cytometry. RESULTS: Chicken DF-1 cells showed an early state of apoptosis in the early stages of HSPB9 overexpression. In the later stages, as HSPB9 expression increased, the cells showed inhibition of apoptosis. When the cells were under heat stress, HSPB9 expression was much higher and earlier than the expression of HSPB1 and HSPA2. In addition, high expression of HSPB9 had a negative effect on HSPB1 and HSPA2 expression. This negative feedback decreased the percentage of early stages of apoptotic cells and promoted cell survival. CONCLUSION HSPB9 expression, although rapid, is detrimental to cell survival early during its overexpression. In heat stress, HSPB9 overexpression, while inhibiting the expression of HSPA2 and HSPB1, is beneficial to cell survival.
Animals ; Apoptosis ; genetics ; Avian Proteins ; genetics ; Cell Line ; Chickens ; Heat-Shock Proteins ; genetics ; Heat-Shock Response ; genetics

OBJECTIVEA CAT reporter plasmid (pBLCAT3alpha1) driven by the promoter of hsp90alpha was in vitro assembled into chromatin to investigate the transcription activity of the reporter gene upon heat shock.

METHODSA competitive RT-PCR-based technique was used to quantify the promoter activity of hsp90alpha gene on chromatin or naked DNA templates in vitro.

RESULTSThe in vitro transcription efficiency was first optimized by using different amounts of whole cell extracts from heat shock-treated HeLa cells. In vitro chromatin assembly was carried out with purified components of chromatin assembly associated factors, core histones and CAT reporter gene driven by the promoter of hsp90alpha gene. Results showed that chromatin formation repressed the in vitro transcription of the gene.

CONCLUSIONThe heat shock induced transcription of hsp90alpha gene on chromatin template is more efficient than that on naked DNA in vitro.

Chromatin Assembly and Disassembly ; genetics ; Genes, Reporter ; genetics ; HSP90 Heat-Shock Proteins ; genetics ; Heat-Shock Response ; genetics ; Humans ; Transcription, Genetic

The expression levels of heat shock protein 70 (HSP70) from peripheral lymphocytes of the patients with allergic rhinitis (AR) and the clinical implication were investigated. In the morning, 3 ml of fasting venous blood was taken out. The lymphocytes were isolated by using Ficoll-Hypaque and the expression of HSP70 in the lymphocytes was detected by using Western blot. In the AR patients the HSP70 level (41.49 +/- 15.77 integrated optical density, IOD) were significantly higher than that in the control group (23.89 +/- 10.13 IOD, P < 0.05). Western blot demonstrated that HSP70 bands in AR patients were more intensive than those in the control group. It was concluded that the elevated HSP70 level in peripheral lymphocytes of the AR patients might contribute to the development of AR.
Gene Expression Regulation ; HSP70 Heat-Shock Proteins/*biosynthesis ; HSP70 Heat-Shock Proteins/blood ; HSP70 Heat-Shock Proteins/genetics ; Lymphocytes/*metabolism ; Rhinitis, Allergic, Seasonal/*blood

Female ; Genetic Predisposition to Disease ; HSP70 Heat-Shock Proteins ; genetics ; Humans ; Lung Neoplasms ; genetics ; Male ; Polymorphism, Genetic

HSP21 gene is a key gene to respond high temperature stress in plant and plays an important role in preventing protein denaturation, protecting cell structure and maintaining normal growth and development. Therefore, cloning HSP21 gene is the basis for revealing the molecular mechanism of resistance to high temperature stress in cassava. To obtain cassava HSP21 homologous gene and analyze the properties of predicted protein, electronic cloning technology was used to assemble and derivate new gene in this study, and bioinformatics analysis method was used to analyze the primary to highest structure, hydrophilicity/hydrophobicity, signal peptide, protein homology and phylogenetic evolution of expressed protein. HSP21 gene was 969 bp, its open reading frame was 705 bp, and the predicted protein contains 234 amino acids. The predicted protein is a non-transmembrane protein that is alkaline and hydrophilic, and is mainly localized in the chloroplast. Through multiple sequence alignment and phylogenetic analysis, it was found that the cassava HSP21 protein has high homology with other plants such as Hevea brasiliensis, Ricinus communis, and Jatropha curcas. The results could provide reference for the study of cloning and transformation of this gene.
Chloroplasts ; Cloning, Molecular ; Computational Biology ; Computer Simulation ; Evolution, Molecular ; Heat-Shock Proteins ; genetics ; Manihot ; genetics ; Phylogeny

OBJECTIVE: To explore the clinical feature and genetic variant of a child with autosomal recessive Charlevoix-Saguenay type spastic ataxia (ARSACS). METHODS: Clinical data of a child who was admitted to the West China Second Hospital of Sichuan University on April 30, 2021 was collected. Whole exome sequencing (WES) was carried out for the child and his parents. Candidate variants were verified by Sanger sequencing and bioinformatic analysis based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). RESULTS: The child, a 3-year-and-3-month-old female, had a complain of "walking instability for over a year". Physical and laboratory examination revealed progressive and aggravated gait instability, increased muscle tone of the right limbs, peripheral neuropathy of the lower limbs, and thickening of retinal nerve fiber layer. The results of WES revealed that she has harbored a maternally derived heterozygous deletion of exons 1 to 10 of the SACS gene, in addition with a de novo heterozygous c.3328dupA variant in exon 10 of the SACS gene. Based on the ACMG guidelines, the exons 1-10 deletion was rated as likely pathogenic (PVS1+PM2_Supporting), and the c.3328dupA was rated as a pathogenic variant (PVS1_Strong+PS2+PM2_Supporting). Neither variant was recorded in the human population databases. CONCLUSION The c.3328dupA variant and the deletion of exons 1-10 of the SACS gene probably underlay the ARSACS in this patient.
Female ; Humans ; Heat-Shock Proteins/genetics* ; Muscle Spasticity/genetics* ; Mutation ; Spinocerebellar Ataxias/pathology* ; Child, Preschool

OBJECTIVETo establish a RT-PCR system for detecting mRNA expression level of 4 hsp genes in human cells.

METHODSRT-PCR system was established with gene cloning, gene recombination, in vitro transcription and RT-PCR techniques; The detection of expression level of 4 hsp genes in SW13 cell was carried out with this system.

RESULTSIn SW13 cell, hsp70 and hsp90 alpha were typical heat shock induced genes, while hsp60 and hsp90 beta were efficiently expressed and further induced by heat-shock to various extent.

CONCLUSIONSIn our hands novel RT-PCR system can be used to detect mRNA expression level of 4 human hsp genes.

Chaperonin 60 ; biosynthesis ; genetics ; HSP40 Heat-Shock Proteins ; HSP70 Heat-Shock Proteins ; biosynthesis ; genetics ; HSP90 Heat-Shock Proteins ; biosynthesis ; genetics ; Heat-Shock Proteins ; biosynthesis ; genetics ; Humans ; Leukemia, T-Cell ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Cells, Cultured

OBJECTIVE: To carry out mutation analysis for a Chinese family affected with autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). METHODS: Whole exome sequencing (WES) was used to screen potential mutations within genomic DNA extracted from the proband. Suspected mutation was validated by combining clinical data and results of Sanger sequencing. RESULTS: A homozygous deletional mutation c.3665_3675delGTGCTGTCTTA (p.S1222fs) was found in the proband, for which her parents were both heterozygous carriers. CONCLUSION WES is capable of detecting mutation underlying this disorder and facilitating genetic counseling and prenatal diagnosis for the affected family. A novel pathogenic mutation of the SACS gene was discovered.
Female ; Genes, Recessive ; Heat-Shock Proteins ; genetics ; Humans ; Muscle Spasticity ; Mutation ; Spinocerebellar Ataxias ; congenital

The promoter of mitochondria-localized small heat shock protein gene in Lycopersicon esculentum (Lehsp23.8) is characterized as strongly heat-inducible. In this study, to determine how the expression of Lehsp23.8 is regulated, we conducted five expression vectors carrying the gus gene driven by the 5' deletion products of the Lehsp23.8 promoter. The corresponding transgenic tobacco plants were generated via Agrobacterium tumefaciens-mediated transformation. Transgenic plants were identified by PCR and Southern blotting analysis. GUS activities under heat-shock conditions were characterized in transgenic tobacco plants. After heat shock, obvious GUS staining was detected in the leaves, shoots, roots, flowers and fruits of the transgenic tobacco plants. The result of fluorometric GUS assays in leaves showed that the heat-induced GUS activity of the 565 bp promoter was the strongest, while that of the 255 bp promoter was the lowest. Deletion analysis shows that the smallest promoter fragment (-255 bp to -23 bp) is sufficient for heat induction. It also indicates that the sequences between -255 bp and -565 bp serve as enhancers, while the sequences between -565 bp and -871 bp can repress the heat-induced activity of the Lehsp23.8 promoter.
Gene Expression Regulation, Plant ; genetics ; Genetic Vectors ; Heat-Shock Proteins ; genetics ; Heat-Shock Proteins, Small ; genetics ; metabolism ; Hot Temperature ; Lycopersicon esculentum ; genetics ; Mitochondria ; genetics ; metabolism ; Plants, Genetically Modified ; genetics ; Promoter Regions, Genetic ; genetics ; Tobacco ; genetics