Isolation and sequencing of the 5′ end of the rat microtubule-associated protein (MAP1B)-encoding cDNA

We have isolated and sequenced the 5′ end of the cDNA encoding the rat microtubule-associated protein 1B (MAP1B). We found that this region is highly homologous to the corresponding regions of the human [Lien et al., 22 (1994) 273–280] and mouse [Noble et al., J. Cell Biol. 109 (1989) 3367–3376] MAPIB genes. The combination of the sequence that we are presenting with the previously published sequence [Zauner et al., Eur. J. Cell Biol. 57 (1992) 66–74], represents the complete rat MAP1B cDNA coding sequence.     

Stress proteins and stress tolerance in an Antarctic, psychrophilic yeast, Candida psychrophila

Conditions are described for the heat shock acquisition of thermotolerance, peroxide tolerance and synthesis of heat shock proteins (hsps) in the Antarctic, psychrophilic yeast Candida psychrophila. Cells grown at 15°C and heat shocked at 25°C (3 h) acquired tolerance to heat (35°C) and hydrogen peroxide (100 mM). Novel heat shock inducible proteins at 80 and 110 kDa were observed as well as the presence of hsp 90, 70 and 60. The latter hsps were not significantly heat shock inducible. The absence of hsp 104 was intriguing and it was speculated that the 110 kDa protein may play a role in stress tolerance in psychrophilic yeasts, similar to that of hsp 104 in mesophilic species.     

Expression of hsp70, hsp100 and ubiquitin in Aloe barbadensis Miller under direct heat stress and under temperature acclimation conditions

Key message

The study determined the tolerance of Aloe vera to high temperature, focusing on the expression of hsp70 , hsp100 and ubiquitin genes. These were highly expressed in plants acclimated at 35 °C prior to a heat shock of 45 °C.

Abstract

Aloe barbadensis Miller (Aloe vera), a CAM plant, was introduced into Chile in the semiarid IV and III Regions, which has summer diurnal temperature fluctuations of 25 to 40 °C and annual precipitation of 40 mm (dry years) to 170 mm (rainy years). The aim of this study was to investigate how Aloe vera responds to water and heat stress, focusing on the expression of heat shock genes (hsp70, hsp100) and ubiquitin, which not studied before in Aloe vera. The LT₅₀ of Aloe vera was determined as 53.2 °C. To study gene expression by semi-quantitative RT-PCR, primers were designed against conserved regions of these genes. Sequencing the cDNA fragments for hsp70 and ubiquitin showed a high identity, over 95 %, with the genes from cereals. The protein sequence of hsp70 deduced from the sequence of the cDNA encloses partial domains for binding ATP and the substrate. The protein sequence of ubiquitin deduced from the cDNA encloses a domain for interaction with the enzymes E2, UCH and CUE. The expression increased with temperature and water deficit. Hsp70 expression at 40–45 °C increased 50 % over the controls, while the expression increased by 150 % over the controls under a water deficit of 50 % FC. The expression of all three genes was also studied under 2 h of acclimation at 35 or 40 °C prior to a heat shock at 45 °C. Under these conditions, the plants showed greater expression of all genes than when they were subjected to direct heat stress.     

Molecular cloning and analysis of DNA complementary to three mouse Mr = 68,000 heat shock protein mRNAs

The construction and isolation of three recombinant DNAs complementary to different mouse L-cell Mr = 68,000 heat shock protein (hsp68) mRNAs is described. cDNA libraries derived from heat-shocked mouse L-cell poly(A)+ RNA by the vector-linked primer strategy of cDNA synthesis and cloning of Okayama and Berg (Okayama, H., and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170) were screened first with a Drosophila hsp70 heterologous probe and subsequently with a cDNA probe isolated from the first screening. Positive clones were assigned to one of three sets based on their restriction map, and the largest member of each group was chosen for further analysis. All three cDNAs hybrid-select mRNA for the mouse major heat shock protein (hsp68) as assayed by in vitro translation and hybridize preferentially to two heat shock-induced hsp68 mRNAs on Northern blots. The coding regions of the cDNAs are almost identical and closely resemble other HSP70 genes but the 3' untranslated regions diverge considerably. Differences in the lengths of the untranslated regions are responsible for the two different sized induced hsp68 mRNAs in mouse L-cells. The physical maps of these cDNA clones and the limited number of mouse genomic DNA fragments detected on Southern blots suggest that there are at least three closely related heat shock-inducible members of the mouse HSP70 gene family. None of the cloned cDNAs are derived from the two related cognate genes known to be present in the mouse genome.     

Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein (HSP) genes (hsp70, hsc70, hsp90) were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus (Homoptera: Delphacidae), which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepi‐dopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock (40 °C) upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4°C did not change the expression levels of any hsp in either species.     

Expression of a heat-inducible gene in transfected mosquito cells

The evolutionary conservation of the heat shock response suggests that plasmids containing promoters from Drosophila heat shock protein (hsp) genes will be useful in the development of gene transfer procedures for cell lines representing a variety of insect species. Conditions for induction of endogenous hsp genes and for expression of the chloramphenicol acetyltransferase (CAT) gene regulated by the Drosophila hsp 70 promoter were examined in Aedes albopictus (mosquito) cells. Five hsps, ranging in size from 27,000 to 90,000 D, were induced in A. albopictus cells during incubation at 41°C in medium containing [³⁵S]methionine. Relative synthesis of these proteins at 37 and 41°C indicated that Aedes hsp 66 is homologous to Drosophila hsp 70. Detection of CAT activity in transfected mosquito cells was enhanced 10-fold under heat shock conditions (6 h, 41°C) based on maximal expression of hsp 66, relative to conditions defined for expression of hsp 70 in Drosophila cells. Analysis of the endogenous heat shock response may be essential to the optimal use of plasmids containing the Drosophila hsp 70 promoter with other insect cell types.     

Cloning of trichosanthin cDNA and its expression in Escherichia coli

Several cDNA clones coding for trichosanthin (TCS) have been isolated from a cDNA library prepared from the poly(A)+RNA of the root tuber of Trichosanthes kirilowii Maximowicz. The nucleotide sequence codes for a protein of 289 amino acids (aa) including a putative signal peptide of 23 aa and an extra 19 aa at the C terminus; the latter two have not been found in TCS obtained from the natural product [Collins et al., J. Biol. Chem. 265 (1990) 8665-8669]. Recombinant TCS (reTCS) was synthesized in Escherichia coli, in which the cDNA without the signal sequence was expressed under the control of the trc promoter; reTCS was detected by a rabbit anti-TCS antiserum.     

Geographic variation in thermal tolerance and strategies of heat shock protein expression in the land snail <Emphasis Type="Italic">Theba pisana</Emphasis> in relation to genetic structure

Land snails are exposed to conditions of high ambient temperature and low humidity, and their survival depends on a suite of morphological, behavioral, physiological, and molecular adaptations to the specific microhabitat. We tested in six populations of the land snail Theba pisana whether adaptations to different habitats affect their ability to cope with thermal stress and their strategies of heat shock protein (HSP) expression. Levels of Hsp70 and Hsp90 in the foot tissue were measured in field-collected snails and after acclimation to laboratory conditions. Snails were also exposed to various temperatures (32 up to 54 °C) for 2 h and HSP messenger RNA (mRNA) levels were measured in the foot tissue and survival was determined. To test whether the physiological and molecular data are related to genetic parameters, we analyzed T. pisana populations using partial sequences of nuclear and mitochondrial DNA ribosomal RNA genes. We show that populations collected from warmer habitats were more thermotolerant and had higher constitutive levels of Hsp70 isoforms in the foot tissue. Quantitative real-time polymerase chain reaction (PCR) analysis indicated that hsp70 and hsp90 mRNA levels increased significantly in response to thermal stress, although the increase in hsp70 mRNA was larger compared to hsp90 and its induction continued up to higher temperatures. Generally, warm-adapted populations had higher temperatures of maximal induction of hsp70 mRNA synthesis and higher upper thermal limits to HSP mRNA synthesis. Our study suggests that Hsp70 in the foot tissue of T. pisana snails may have important roles in determining stress resistance, while Hsp90 is more likely implicated in signal transduction processes that are activated by stress. In the phylogenetic analysis, T. pisana haplotypes were principally divided into two major clades largely corresponding to the physiological ability to withstand stress, thus pointing to genetically fixed tolerance.