Neurological Research A Journal of Progress in Neurosurgery, Neurology and Neurosciences
ISSN: 0161-6412 (Print) 1743-1328 (Online) Journal homepage: http://www.tandfonline.com/loi/yner20
Isolation of complementary DNAs for heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 genes and the expressions in postischaemic gerbil brain Shigeru Sato, Koji Abe, Jun-ichi Kawagoe, Masashi Aoki & Kyuya Kogure To cite this article: Shigeru Sato, Koji Abe, Jun-ichi Kawagoe, Masashi Aoki & Kyuya Kogure (1992) Isolation of complementary DNAs for heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 genes and the expressions in post-ischaemic gerbil brain, Neurological Research, 14:5, 375-380, DOI: 10.1080/01616412.1992.11740088 To link to this article: http://dx.doi.org/10.1080/01616412.1992.11740088
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Isolation of complementary DNAs for heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 genes and the expressions in post-ischaemic gerbil brain Shigeru Sato, Koji Abe, jun-ichi Kawagoe, Masashi Aoki and Kyuya Kogure
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Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai, Japan
A complementary DNA (eDNA) library was constructed with a plasmid vector from cerebral cortices of gerbils at 8 h of reperfusion after 10 min of bilateral common carotid ligation. After the 3rd screening of this eDNA library with a human genomic DNA probe for HSP70 (pH2.3 ), 4 eDNA clones were isolated (named pCAy pCB,, pCD3 and pCE., respectively). Southern and Northern blot analysis, and partial nucleotide sequence analysis indicated that pCA3 and pC£4 were the HSP70 eDNA clones, and that pCB, and pCD3 were the HSC70 eDNA clones, which selectively recognized HSP70 or HSC70 mRNA, respectively. HSP70 mRNA is present in a very small amount in normal controls, and is greatly induced after the transient ischaemia. HSC70 mRNA is constitutively expressed in a normal gerbil brain, but is still inducible.ln situ hybridization study demonstrated that the HSP70 mRNA was present in a very small amount in the hippocampal pyramidal and dentate granule cells in the sham control, and that the mRNA was greatly induced in the cells of hippocampus, dentate gyrus, medial habenula, ventral thalamic nuclei, caudate putamen, ventromedial and arcuate hypothalamic nuclei, amygdaloid nuclei and cerebral cortex after 8 h of reperfusion. HSC70 mRNA was present in almost the same areas of sham control, and was slightly induced after 8 h of reperfusion. Our results indicate that HSP70 and HSC70 eDNA clones were first isolated from post-ischaemic gerbil brain, and selectively recognize HSP70 or HSC70 mRNA, respectively. A regional difference in the induction of the HSP70 and HSC70 mRNA in post-ischaemic gerbil brain was observed by in situ hybridization. [Neural Res 1992; 14: 000-000 J Keywords: HSP70; HSC70; ischaemia; gerbil
INTRODUCTION Cerebral ischaemia prevents the maJonty of protein syntheses in brain cells, while some kinds of proteins are selectively induced1 • HSP70 is one of such proteins which are induced by cerbral ischaemia. HSP70 is not present in normal conditions, but is induced by hyperthermia, hypoxia, hypoglycemia, alcohol, heavy metals, or other various stresses 2 • This phenomenon is conserved from prokaryote to human. On the other hand, some HSP70 related proteins are expressed in normal non-stressful conditions. These proteins are called heat shock cognate protein 70 (HSC70P. Inductions of HSP70 mRNA and protein have been reported in a gerbil and rat brain after transient ischaemia, and their protective role against an ischaemic neuronal injury has been implicated4 - 9 . Vass et a/. first reported the regional deference of the induction of this protein in the gerbil brain 4 • A 10 min ischaemia greatly induced an immunoreactive HSP70 protein in the hippocampal CA3 cells (resistant to an ischaemic insult), but induced only a minimum amount of the protein in the CA 1 cells (more susceptible to
the ischaemia). Kirino et a/. showed that a 2 min ischaemia strongly induced the immunoreactive HSP70 protein in the CA 1 cells in the gerbil brain and CA1 cells became resistant to the next insult' . Gonzales et a/. reported that the reduced induction of HSP70 protein correlated with the selective vulnerability of rat hippocampal neurons6 • The failure to induce enough HSP70 in the vulnerable neurons may be related to the severe injury causing disruption of protein synthesis. Thus it is suggested that the regional difference in the HSP70 gene expression in the hippocampal formation correlates with regional vulnerability of neuronal cells to the ischaemic injury. A cooperative role of HSP70 and HSC70 in a stressful condition has been suggested. However, the mechanism of the regional difference in the induction of HSP70 gene and the exact roles of the genes are not fully understood. As a first step to understand this mechanism more clearly, we attempted to isolate cDNAs of HSP70 and HSC70 from a post ischaemic gerbil brain, and examined the expressions of these mRNAs after a transient ischaemia.
Correspondence to: -Shigeru Sato, Department of Neurology, Institute of Brain Diseases, Tohoku University School of M edicine, 1-1 Seiryocmachi, Aobaku, Sendai 980, Japan. Accepted for publication january 1992.
Abbreviations used: HSC, heat shock cognate protein; HSP, heat shock protein.
© 1992 Forefront Publishing Group 0161 - 6412/ 92/ 050375-06
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Isolation of eDNA for HSP70 and HSC70 of gerbil: Shigeru Sato et al.
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MATERIALS AND METHODS Animal preparation Male Mongolian gerbils (Meriones unguiculatus) aged 10 weeks and weighting 70-75 g, were lightly anaesthetized by inhalation of a nitrous oxide I oxygen I halothane (69%:30%:1%) mixture. A midline neck incision exposed both common carotid arteries. When the animals began to regain consciousness after the stop of anaesthesia, both common carotid arteries were occluded for 10 min using surgical clips' 0 • Body temperature was monitored in all animals, and was maintained at 37°C using a heat pad during the surgical preparation and the occlusion of the carotid arteries. After the reperfusion, no attempt was made to maintain constant body temperature of animals. The animals recovered for 8 h or 2 days at the ambient temperature (21-23°C), then the animals were decapitated. Their parietal cerebral cortices were dissected, quickly frozen in liquid nitrogen, and stored at - 80°C until extraction of RNA Normal control animals were also decapitated in the same manner without anaesthesia, surgical preparation, or clamping the carotid arteries. For the study of in situ hybridization, the dissected brains were frozen in powdered dry ice and stored at -80°C. Sections (10 J..l.m) at dorsal hippocampal levels were cut out on a cryostat and collected on Histostik (Accurate Chemical and Scientific Corp., Westbury, NY)-coated slides. Sham animals were sacrificed just after exposing the carotid arteries without clamping the vessels. Isolation of RNA and eDNA synthesis Total RNA was extracted from the parietal cortices by the modified method of Chomoczynski and Sacchi (the RNA isolation kit by Stratagene, CA, USA) using guanidine isothiocyanate". Poly(A)+ mRNA was isolated from total RNA of 8 h reperfusion by an affinity chromatography on oligo (dT) cellulose. With this mRNA as a template (5 J..l.g), eDNA was synthesized using an oligo-dT as a primer. After discarding eDNA below 0.5 kb and over 3 kb in size, a eDNA library was constructed with a plasmid vector (pTZ18R, Invitrogen, CA, USA) by the Gubler-Hoffman eDNA synthesis method (The Librarian II, Version 2.4, Invitrogen). These cDNAs were transformed into competent E. coli cells (INV1aF', Invitrogen) to obtain full scale transformants. Screening of the eDNA library Colony filters were made using the standard method for making a replica 12 • Bacterial colonies were grown on a nylon membrane (Hybond N, Amersham, IL, USA) on LB plates which contfil ned ampicillin (50 J..l.gl mD. The membranes were treated with NaOH (0.5 M), neutralized twice with Tris-HCI (1 M, pH 7.5)I NaCI (1.5 M), washed with 2 X SSC (1 X SSC = 150 mM NaCII i5 mM sodium citrate), and then dried in an air for 2 h. The dried membrane filters were baked in a vacuum oven at 80°C for 2 h to fix DNA to the membrane. The baked filters were soaked with NaOH (0.5 M) at 42°C for 30 min to remove cell debris from the filter, neutralized with Tris-HCI (0.5 M, pH 7.5)1 0.1 x SCC / 0.1% SDS (sodium dodecyl sulphate) at 42°C for 30 min, and then were prehybridized with a solution containing formamide (50% formamide, 5 X 5 X Denhardt's solution, 100 J..l.gl ml fish
sse,
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sperm DNA, 50 mM KP0 4 and 0.2% SDS ) at 42°C for over several hours. This eDNA library was screened with a human genomic DNA probe, pH 2.3, for HSP7013' 14 • The insert DNA of pH 2.3 was radiolabelled with et- 32 P-dATP (6000 Ci l mmol, Amersham) with a random primer as reported before9 • The radiolabelled probe was added to the hybridization mixture (the same as the prehybridization solution), and the mixture was kept at 42°C overnight with gentle agitation. Filters were first washed several times with 2 x SSCI 0.1% SDS at room temperature. In order not to lose HSC70 clones, the filters were finally washed with a relatively low stringent condition (1 x SSCI 0.1% SDS at 65° for 30 min). These filters were exposed to X-ray films at - 80°C overnight. Subcloning Subcloning was performed because the nucleotide sequence of pTZ18R was not available. The cDNAs were digested with Xho I (Takara, Kyoto, Japan) and electrophoresed in a 0.7% agarose gel in 1 x TBE (90 mM Tris base, 90 mM boric acid, and 2 mM EDT A ) buffer. The inserts of cDNAs were recovered from an agarose gel by centrifugation tubes with a dural membrane filter (SUPREC™-01 tube, Takara ). The inserts were then subcloned into a plasmid vector pHSG396 (Takara) at Xho I site15 • Subcloning was performed with a T4 DNA ligase (DNA Ligation Kit, Takara) at 16°C for 2 h. These cDNAs were transformed into competent E. coli cells (JM109, Takara). Recombinant clones were selected by a-complementation 16 • On LB agar plates containing 25 J..l.gl ml of chloramphenicol (Sigma, St Louis, MO, USA), 60 J..l.l of a stock solution of 5-bromo-4-chloro-3-indolyl-/3-o-galactoside (X-gal, 20 mgl ml in dimethylformamide, Sigma) and 4 J..l.l of a solution of isopropylthio-/3-o-galactoside (IPTG, 200 mgl ml, Sigma) were spread. The transformed E. coli was inoculated on the plate, and was incubated for 12-16 hat 37°C. Positive clones showed white colonies, while negative clones showed blue colonies. Southern blot analysis Southern blot analysis was performed by the standard method 17 • DNAs were separated in a 1% agarose gel r'n 1 x TBE buffer, and transferred to a nylon membrane (Hybond N, Amersham) and then prehybridized at 42°C for 4 h in a hybridization solution (same as above). The insert of pH 2.3 was radiolabelled with a- 32 P-dATP by a random primer labelling system using Klenow fragment of E. coli DNA polymerase I (Boeringer Mannheim, Mannheim, Germany), and hybridized against Southern blot at 42°C for 20 h in a hybridization solution. After the hybridization, the filter was washed with 2 x SSC I 0.2% SDS at room temperature, and finally washed with 0.2 x SSC I 0.2% SDS at 65°C. The filters were exposed to X-ray films for 20 hat - 80°C. Northern blot analysis Northern blot analysis was performed by the previous method 9 • Inserts of pH 2.3 and eDNA clones, which were isolated in this experiment, were used as the probes for Northern blot analysis. Conditions of labelling, hybridization, washing and autoradiography were the same as Southern blot analysis.
···
Isolation of eDNA for HSP70 and HSC70 of gerbil: Shigeru Sato et al.
A S
oGA3 PGE.:
B oGB,
oGD.>
S
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•·• An agarose gel (A) stained with ethidium bromide (EB) and Southern blot analysi.s (B) using a human genomic HSP70 fragment, pH2.3, as a probe. Southern filter was washed with 1 x SCC/ 0.2% SDS at 65°C. A. Hind Ill was used as a standard size marker (S) Figure 1:
bovine serum albumin, 200 ,ugl ml sonicated, denatured calf thymus DNA, and 1 mgl ml calf liver rRNA. The insert of pGA3 for HSP70 and that of PGD 3 for HSC70 were radiolabelled with et- 35 S dCTP (1000 Ci l mmol, Amersham ) by random primer labelling kit (Boehringer Mannheim ), resulting in a specific activity of about 5 x 108 dpm l ,ug. Hybridization was performed for 20 h at 42°( in the same solution as prehybridization supplemented with 10% dextran sulphate and 10 mM dithiothreitol. The 1.1 kb of pHSG396 fragment digested by Hinf I (Takara) was similarly labelled as a control probe. The sections were washed for 2 h with 2 X sse and following for 1 h with 1 X SSC at 42°( and dehydrated through graded ethanol series containing 0.3 M ammonium acetate. Hybridization signals were visualized by exposure against an X-ray film, then the slides were dipped in a liquid emulsion (NR-M2, Konica Corp. Ltd, Tokyo, Japan ) and further exposed for 3 weeks. Several sections at 8 h of reperfusion were treated with 100 ,ugl ml RNase A and 10 units I ml RNase T1 (Sigma) at 37°( for 30 min prior to prehybridization and were hybridized with the probes.
RESULTS Sequence determination A small scale plasmid preparation was performed by an anion-exchange resin column (Quiagen tip-20, Stratagene). Their nucleotide sequences were determined by the modified dideoxy chain termination method (Sequenase ver. 2.0; United States Biochemical, OH, USA). 2 pmol of plasmid DNA was denatured with NaOH (20 mM), and precipitated with ethanol in the presence of ammonium acetate. The single-strand plasmid DNA (ssDNA template) was annealed with M13 primer M4 (17mer, United States Biochemical) or M13 primer RV (17mer, Takara), then the double stranded DNA was synthesized with T7 DNA polymerase in the presence of an et-32 P-dCTP (3000 Cil mmol, Amersham) or et- 35 S-dCTP (1000 Ci I mmol, Amersham ). The reaction was terminated by an addition of dideoxy nucleotides to the reaction mixture. Each strand was sequenced twice in separate experiments. Sequence data management was performed by using the GENETYX ver. 2 program (Software development Co., Tokyo, Japan). Determined sequences of these clones were compared with some sequences of mouse, rat and human HSP related proteins in the Gen Bank (Cambridge, MA, USA) and the European Molecular Biology Laboratory (EMBL, Heiderberg, Germany).
In situ hybridization In situ hybridization was performed by the method of Yoshioka et a/. with a slight modification 18 • Briefly, the sections were fixed for 10 min in an ethanol I acetic acid (3: 1) mixture, ~transferred to 0.2 M HCI for 20 min, and immersed for 20 min in 0.3 M NaCII 0.03 M Na citrate (2 X SSC, pH 7.0) at 50°C. Then they were digested for 15 min at 37°( with 100 ,ugl ml of proteinase K (Merck & Co. Inc., Rahway, NJ) in 20 mM Tris-HCI buffer (pH 7.4) with 2 mM CaCI 2 and dehydrated through graded ethanol. Slides were prehybridized for 2 h at room temperature in a solution containing 50% formamide, 600 mM NaCI, 10 mM Tris- HCI (pH 7.5), 10 mM EDTA, 0.02% poly( vinyl pyrrolidone), 0.02%
Screening of the eDNA library The size of eDNA library (number of transformants) for the ischaemic cerebral cortices of gerbils was 27,800. After the 3rd screening, 4 eDNA clones were finally isolated from the library (named pGA31 pGB, pGD 3 and pGE., respectively). Sizes of eDNA insert are 1.0 kb (pGA), 1.1 kb (pGB,), 1.4 kb (pGD 3 ), and 0.9 kb (pGE4 ) (Figure 1A ). Southern blot analysis Southern blot analysis revealed that pH 2.3 probe hybridized the inserts of pGA3, pGB,, pGD 3 and pGE4, but did not hybridize the vector DNA in 1 x SSC at 65°( (Figure 18). But in 0.2 X sse at 65°( , the radiolabelled pH 2.3 probe was almost washed out from the insert of pGB, and pGD 3 although this probe still hybridized to the insert of pGA3 and pGE4 (data not shown). Northern blot analysis Northern blot analysis showed that pH 2.3 probe hybridized 2 different sizes of mRNA in the gerbil brain (Figure 2). One was a 2.8 kb size, and another was a 2.4 kb. The 2.8 kb mRNA was present in a very small amount in a normal condition, and was greatly induced after 8 h of reperfusion. The 2.4 kb mRNA was expressed in a considerable amount in a normal gerbil brain, but was still inducible. When the filter was washed with 1 X sse at 65° ( , 2 different sizes of the mRNA were detected (Figure 2A). However, only the 2.8 kb mRNA was detected after the washing with a more stringent condition such as 0.2 x sse at 65°( (Figure 28). Northern blot analysis showed that inserts of pGA 3 and pGE4 hybridized only the 2.8 kb mRNA, and that the inserts of pGB, and pGD 3 hybridized only the 2.4 kb of mRNA both in a low stringent washing condition (1 X SSC, room temperature) and in a high stringent washing condition (0.2 X SSC, 65°() (Figure 3).
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8
A •
•
28S--
185-
185--
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•.
285--
.•
Northern blot analysis using a human genomic HSP70 fragment, pH2.3, as a probe. e , and e, represent the cases of normal control. 8h, 8h, 2d,, and 2d, represent the cases of 8 h or 2 days reperfusion after 10 min of common carotid artery occlusion, respectively. Arrow show positions of 18s and 28s ribosomal RNA (rRNA ). Two mRNAs (2.8 kb and 2.4 kb) are detected with a washing condition of 1 x SSe/ 0.2% SDS at 65°e (A). Only one mRNA (2.8 kb) is detected with that of 0.2 x SSe/0.2% SDS at 65°e (B) Figure 2:
8
A
• 28S--
28S-
185-
185--
Northern blot analysis using gerbil eDNA probes. The pGA, insert detects only the 2.8 kb size mRNA (A). The pGE, insert also hybridizes the same transcript (data not shown). The pGD, insert detects only the 2.4 kb size mRNA (B ). The pGB, insert also hybridize~ the same transcript (data not shown). Arrow show positions of 18s and 28s rRNA. Washing condition; 1 x SSe, 0.2% SDS at 65°C. When the wash condition was changed to higher stringency (0.2 X SSe at 65°e), these results were not changed Figure 3:
" Sequence determination
.
About 350 bp nucleotide seque nces at both sides of the insert of each clone were determined (Figure 4 ). From the analysis of nucleotide sequences, an identity of 126 nucleotides of the 5' end of the pGE, insert with 76-201 nucleotides from the 5' end of the pGA3 insert was found . An identity of 74 nucleotides of the 5' end of the pGB, insert with 297-370 nucleotides from the 5' end of the pG0 3 insert was also found. The 3' e nd of
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the pGA 3 insert had the same sequence as that of the pGE, insert, and the 3' of the pGB, insert had the same sequence as that of the pGD 3 insert. A terminal codon was found in the insert of each clone and the open reading frame and predicted amino acid sequence were determined. HSP70 in a gerbil has about 700 bp of 3' untranslated region and HSC70 had 245 bp of that. The polyadenylation signa l AATAAA
Isolation of eDNA for HSP70 and HSC70 of gerbil: Shigeru Sato et al.
Table 1: Homologies of nucleotide sequences of coding region between gerbil and mouse, rat or human Mouse Gerbil
HSP70"
HSP70 HSC70
Rat
HSC70"
Human
HSC70"
87.9 (86.5) 68.3 (64.8) 70.0 (64.8) 74.5 (88.4) 94.2 (99.1) 92.8 (98.6)
HSP70"
HSC70"
86.2 (90.1) 66.7 (62.9) 70.0 (86.7) 93.9 (98.7)
Homologies of nucleotide sequences of coding region are shown by percentage. A homology of predicted amino acids are shown in parentheses.
HSP70 : l.M kb
s·
()40bpJ
( ~22bp)
- - - - - - - - - - - - - - - 3'(polyA site) pGAJ ( J.Okb)
pGE4 (0.9kbl
HSC70 : 2.4 kb (63Rbpl
CJJObpl
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s·
)' {polyA site )
pGBI ( I.Jkb) pGB3 ( 1.4kbl
-
coding region
-
J' non coding region
~
region where nucleotide .o;e4uencc is detcrmin~d
f-l lkb
Schematic view of isolated clones. About 200-350 bp nucleotide sequences at both sides of the insert of each clone were determined using two primers, M13RV and M13m4. The bold bars represent open reading frames, and the thin bars represent 3' untranslated sequences Figure 4:
and poly (A) were included in these regions of both HSP70 and HSC70. Results of homology analysis between these clones and HSP70 or HSC70 of a mouse, rat and human are summarized in Table ?1 4·' 9 - 22 • Nucleotide sequence comparisons of the coding sequence of HSP70 genes revealed a homology of 87.9% between gerbil and mouse, and of 86.2% between gerbil and human. At the protein level, there was a homology of 86.5% between gerbil and mouse and of 90.1% between gerbil and human. The search for HSC70 revealed a homology of 94.2% between gerbil and mouse, of 92.8% between gerbil and .rat, and of 93.9% between gerbil and human at the nucleotide level. At the protein level, there were two or three amino acid differences between gerbil and ·mouse, rat or human. In contrast, there was a homology of about 70% between the gerbil HSP70 and other HSC70 genes or between gerbil HSC70 and other HSP70 genes. There was no homology in the 3' untranslated region between the gerbil HSP70 genes and other HSC70 genes, or between gerbil HSC70 genes and other HSP70 genes.
In situ hybridization HSP70 mRNA was present in a very small amount in the hippocampal pyramidal and dentate granule cells of the sham control brain. The amounts of mRNA was greatly induced in the cells of hippocampus and dentate gyrus after 8 h of recirculation . In addition, the HSP70 mRNA was newly induced in cells of medial habenula, ventral thalamic ·nuclei, ventromedial and arcuate hypothalamic nuclei, caudate putamen, amygdaloid nuclei and cerebral cortex after 8 h of recirculation
(Figure 5 ). On the other hand, HSC70 mRNA was present in the cells of these regions of the sham control, and was slightly induced after 8 h of recirculation in these regions (data not shown). No detective signal was found after the treatment of RNases and in the case with pHSG396 (plasmid vector) as the probe (data not shown). DISCUSSION Four clones were first isolated as HSP70 related genes from the postischaemic gerbil brain. The results suggested that pGA3 and pGE4 are the HSP70 eDNA clones and pGB, and pGD 3 are the HSC70 eDNA clones because: (1 ) Southern blot analysis showed that each insert hybridized the human genomic HSP70 probe; (2) Northern blot analysis showed that the human genomic HSP70 probe detected both 2.8 and 2.4 kb mRNA in the low stringent washing condition. The inserts of pGA 3 and pGE. hybridized only the 2.8 kb mRNA, and the inserts of pGB, and pGD3 hybridized only the 2.4 kb mRNA; (3 ) a nucleotide sequence analysis suggested that pGE4 was a part of pGA3, and that pGB, was a part of pGD 3; (4) the nucleotide sequence of pGA3 and pGE. has a homology of over 85% with that of mouse and human HSP70 genes. The nucleotide of sequence of pGB, and pGD 3 has a homology of over 90% with that of mouse, rat and human HSC70. An about 70% of the insert of pGA 3 (total insert size is 1.0 kb ) is the 3' non-coding region including a polyadenylation signal and a poly(A) sequence and the coding region of the inset is only 306 bp (Figure 4). This untranslational region has less homology with that of HSC70 genes. On the other hand, the insert of pGD 3
HSP70
s Bh
..
_., ;:_·~--~-~~"·:.
Figure 5:
In situ hybridization using the insert of pGA, fo r HSP70 as a probe. Sections were prepared from the sham control (5 ) and 8 h reperfusion after 10 min of ischaemia in the gerbil brains (8 h)
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(total insert size is 1.4 kb) has the 245 bp of 3' non-coding region, which has no homology with that of HSP70 genes. In general, the coding regions of HSP70 and HSC70 show a relative high homology between species. However, our clones have a highly selective sequence for HSP70 or HSC70 mRNA of gerbil, and, therefore, selectively detects HSP70 or HSC70 mRNA
4
5
6
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(Figure 3). Northern blot analysis showed that HSP70 mRNA was present in a very small amount in a normal condition, but was greatly induced after 8 h reperfusion. On the other hand, HSC70 mRNA was present in a considerable amount in a normal gerbil brain, but was still inducible after 8 h. The time course of the induction of HSP70 and HSC70 of mRNA is reasonable as compared to previous reports9•23 • The results of in situ hybridization indicated that the insert of pGA3 and pGD 3 selectively recognized mRNA for HSP70 or HSC70, respectively, because no signal was detecte~ after treating RNases and the non-sense probe did not show any signal. HSP70 mRNA was induced in the cells of hippocampus, dentate gyrus, medial habenula, ventral thalamic nuclei, caudate putamen, ventromedial and arculate hypothalamic nuclei, amygdaloid nuclei and cerebral cortex after 8 h of reperfusion. HSC70 mRNA was present in almost the same areas of the sham control. It is interesting that these regions are consistent with the regions at which the protein synthesis is active in normal conditions24·25 • The cooperative induction of HSP70 and HSC70 mRNA after a transient ischaemia suggests the cooperative role of these proteins as molecular chaperoning26 • Because of a relatively high homology between the HSP70 and HSC70 gene, it should be better to use probes selectively recognize each mRNA species especially in cases of in situ hybridization study. Our clones are constituted from 3' part of the mRNAs, so that the specificity is very high to recognize each species of the mRNA. Thus, our results are the first of isolating HSP70 and HSC70 eDNA dones from the postischemic gerbil brain, which can selectively recognize HSP70 or HSC70 mRNA. Although these eDNA clones are not full length, they provide good probes to study the selective changes of these genes after ischemia for in situ hybridization study.
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ACKNOWLEDGEMENTS This work was partly supported by Monbusho grant 01044018. The authors would like to thank Dr Sugawara A for many advices for molecular biology strategies, and to Mrs Matsumoto M. for her excellent technical assistance. REFERENCES Jacewicz M, Kiessling M, Pulsinelli WA. Selective gene . expression in focal cerebral isc;,f;lemia. 1Cereb Blood Flow Metab 1986; 6: 263-272 ' 2 Morimoto Rl, Tissieres A, Georgopoulos C. The stress response, function of the proteins, and perspectives. In: Morimoto Rl, Tissieres A, Georgopoulos C, eds. Stress Proteins in Biology and Medicine. New York: Cold Spring Harbor Laboratory, 1990: pp. 1-36 3 Flaherty KM, Flaherty CD, McKay DB. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature 1990; 346: 623-628
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Vass K, Welch WJ, Nowak TS Jr. Localization of 70-kDa stress protein induction in gerbil brain after ischemia. Acta Neuropatho/1988; 77: 128-135 Kirino T, Tsujita Y, Tamura A. Induced tolerance to ischemia in gerbil hippocampal neurons. 1Cereb Blood Flow Metab 1991; 11: 299-307 Gonzalez MF, Lowenstein D, Fernyak S, Hisanaga K, Simon R, Sharp FR. Induction of heat shock protein 72-Like immunoreactivity in the hippocampal formation following transient global ischemia. Brain Res Bull1991; 26: 241-250 Nowak TS Jr. Localization of 70 kDa stress protein mRNA induction in gerbil brain after ischemia. 1 Cereb Blood Flow Metab 1991; 11: 432-439 Nowak TS Jr and Osborne OC. Threshold ischemic duration for stress protein induction in gerbil brain (abstract). Stroke 1991; 22: 131 Abe K, Tanzi RE, Kogure K. Induction of HSP70 mRNA after transient ischemia in gerbil brain. Neurosci lett 1991; 125: 166-168 Abe K, Yoshidomi M, Kogure K. Arachidonic acid metabolism in ischemic neuronal damage. Ann NY Acad Sci 1989; 559: 259-268 Chmoczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidine thiocyanate-phenol-chloroform extraction. Anal Biochem 1983; 162: 156-159 Ausubel FM, Brant R, Kingston RE, Moore DD, Sederman JG, Smith JA, Struhl K, eds. Current Protocols in Molecular Biology. New York: Green Publishing Associates and Wiley-lnterscience, 1986: pp. 6.2.1-6.2.3 Wu B, Hunt C, Morimoto R. Structure and expression of the human gene encoding major heat shock protein HSP70. Mol Cell Bio/1985; 5: 330-341 Hunt C and Morimoto Rl. Conserved features of eukaryot ic hsp70 genes revealed by comparison with the nucleotide sequence of hsp70. Proc Nat/ Acad Sci USA 1985; 82: 6455-6459 Takeshita S, Sato M, Toba M, Masahashi W , Gotoh TH. High-copy-number and low-copy-number plasmid vectors for lacZIX-complimentation and chloramphenicol- or kanamycinresistance selection. Gene 1987; 61 : 63-74 Sambrook J, Fritch EF, Maniatis T. Molecular Cloning. New York: Cold Spring Harbor Laboratory, 1989: pp. 1.85- 1.86 Sambrook J, Fritch EF, Maniatis T. Molecular Cloning. New York: Cold spring Harbor Laboratory, 1989: pp. 9.31-9.58 Yoshioka M, Nagano I, Nakamura S, lmaizumi M, Kimura N. Detection of vasoactive intestinal polypeptide messenger RNA in ganglia neuroblastoma by an in situ hybridization. Endocr Patho/1990; 1: 51-57 Hunt C and Calderwood S. Characterization and sequence of a mouse hsp70 gene and its expression in mouse cell lines. Gene 1990; 87: 199-204 Giebel LB, Dworniczak BP, Bautz EKF. Developmental regulation of a constitutively expression mouse mRNA encoding a 72-kDa heat shock-like protein. Dev Bio/1988; 125: 200-207 O'Malley K, Mauron A, Barchas JD, Kedes L. Constitutively expressed rat mRNA encoding a 70-kilodalton heat-shock-like protein. Mol Cell Bio/1985; 5: 3476- 3483 Dworniczak B, Mirault ME. Structure and expression of a human gene coding for a 71 kd heat shock 'cognate' protein. Nucleic Acids Res 1987; 15: 5181-5197 Nowak TS Jr, Bond U, Schlesinger MJ. Heat shock RNA levels in brain and other tissues after hyperthermia and transient ischemia. 1 Neurochem 1990; 54: 451-458 Araki T, Kao H, Inoue T, Kogure K. Regional impairment of protein synthesis following brief cerebral ischemia in the gerbil. Acta Neuropatho/1990; 79: 501 - 505 Yoshidomi M, Hayashi T, Abe K, Kogure K. Effects of a new calcium channel blocker, KB-2796, on protein synthesis of the CA1 pyramidal cell and delayed neuronal death following transient forebrain ischemia. 1Neurochem 1989; 53: 1589-1594 Hightower LE. Heat shock, stress proteins, chaperones, and proteotoxicity. Cell1991; 66: 191-197
ISSN: 0161-6412 (Print) 1743-1328 (Online) Journal homepage: http://www.tandfonline.com/loi/yner20
Isolation of complementary DNAs for heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 genes and the expressions in postischaemic gerbil brain Shigeru Sato, Koji Abe, Jun-ichi Kawagoe, Masashi Aoki & Kyuya Kogure To cite this article: Shigeru Sato, Koji Abe, Jun-ichi Kawagoe, Masashi Aoki & Kyuya Kogure (1992) Isolation of complementary DNAs for heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 genes and the expressions in post-ischaemic gerbil brain, Neurological Research, 14:5, 375-380, DOI: 10.1080/01616412.1992.11740088 To link to this article: http://dx.doi.org/10.1080/01616412.1992.11740088
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Isolation of complementary DNAs for heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 genes and the expressions in post-ischaemic gerbil brain Shigeru Sato, Koji Abe, jun-ichi Kawagoe, Masashi Aoki and Kyuya Kogure
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Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai, Japan
A complementary DNA (eDNA) library was constructed with a plasmid vector from cerebral cortices of gerbils at 8 h of reperfusion after 10 min of bilateral common carotid ligation. After the 3rd screening of this eDNA library with a human genomic DNA probe for HSP70 (pH2.3 ), 4 eDNA clones were isolated (named pCAy pCB,, pCD3 and pCE., respectively). Southern and Northern blot analysis, and partial nucleotide sequence analysis indicated that pCA3 and pC£4 were the HSP70 eDNA clones, and that pCB, and pCD3 were the HSC70 eDNA clones, which selectively recognized HSP70 or HSC70 mRNA, respectively. HSP70 mRNA is present in a very small amount in normal controls, and is greatly induced after the transient ischaemia. HSC70 mRNA is constitutively expressed in a normal gerbil brain, but is still inducible.ln situ hybridization study demonstrated that the HSP70 mRNA was present in a very small amount in the hippocampal pyramidal and dentate granule cells in the sham control, and that the mRNA was greatly induced in the cells of hippocampus, dentate gyrus, medial habenula, ventral thalamic nuclei, caudate putamen, ventromedial and arcuate hypothalamic nuclei, amygdaloid nuclei and cerebral cortex after 8 h of reperfusion. HSC70 mRNA was present in almost the same areas of sham control, and was slightly induced after 8 h of reperfusion. Our results indicate that HSP70 and HSC70 eDNA clones were first isolated from post-ischaemic gerbil brain, and selectively recognize HSP70 or HSC70 mRNA, respectively. A regional difference in the induction of the HSP70 and HSC70 mRNA in post-ischaemic gerbil brain was observed by in situ hybridization. [Neural Res 1992; 14: 000-000 J Keywords: HSP70; HSC70; ischaemia; gerbil
INTRODUCTION Cerebral ischaemia prevents the maJonty of protein syntheses in brain cells, while some kinds of proteins are selectively induced1 • HSP70 is one of such proteins which are induced by cerbral ischaemia. HSP70 is not present in normal conditions, but is induced by hyperthermia, hypoxia, hypoglycemia, alcohol, heavy metals, or other various stresses 2 • This phenomenon is conserved from prokaryote to human. On the other hand, some HSP70 related proteins are expressed in normal non-stressful conditions. These proteins are called heat shock cognate protein 70 (HSC70P. Inductions of HSP70 mRNA and protein have been reported in a gerbil and rat brain after transient ischaemia, and their protective role against an ischaemic neuronal injury has been implicated4 - 9 . Vass et a/. first reported the regional deference of the induction of this protein in the gerbil brain 4 • A 10 min ischaemia greatly induced an immunoreactive HSP70 protein in the hippocampal CA3 cells (resistant to an ischaemic insult), but induced only a minimum amount of the protein in the CA 1 cells (more susceptible to
the ischaemia). Kirino et a/. showed that a 2 min ischaemia strongly induced the immunoreactive HSP70 protein in the CA 1 cells in the gerbil brain and CA1 cells became resistant to the next insult' . Gonzales et a/. reported that the reduced induction of HSP70 protein correlated with the selective vulnerability of rat hippocampal neurons6 • The failure to induce enough HSP70 in the vulnerable neurons may be related to the severe injury causing disruption of protein synthesis. Thus it is suggested that the regional difference in the HSP70 gene expression in the hippocampal formation correlates with regional vulnerability of neuronal cells to the ischaemic injury. A cooperative role of HSP70 and HSC70 in a stressful condition has been suggested. However, the mechanism of the regional difference in the induction of HSP70 gene and the exact roles of the genes are not fully understood. As a first step to understand this mechanism more clearly, we attempted to isolate cDNAs of HSP70 and HSC70 from a post ischaemic gerbil brain, and examined the expressions of these mRNAs after a transient ischaemia.
Correspondence to: -Shigeru Sato, Department of Neurology, Institute of Brain Diseases, Tohoku University School of M edicine, 1-1 Seiryocmachi, Aobaku, Sendai 980, Japan. Accepted for publication january 1992.
Abbreviations used: HSC, heat shock cognate protein; HSP, heat shock protein.
© 1992 Forefront Publishing Group 0161 - 6412/ 92/ 050375-06
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MATERIALS AND METHODS Animal preparation Male Mongolian gerbils (Meriones unguiculatus) aged 10 weeks and weighting 70-75 g, were lightly anaesthetized by inhalation of a nitrous oxide I oxygen I halothane (69%:30%:1%) mixture. A midline neck incision exposed both common carotid arteries. When the animals began to regain consciousness after the stop of anaesthesia, both common carotid arteries were occluded for 10 min using surgical clips' 0 • Body temperature was monitored in all animals, and was maintained at 37°C using a heat pad during the surgical preparation and the occlusion of the carotid arteries. After the reperfusion, no attempt was made to maintain constant body temperature of animals. The animals recovered for 8 h or 2 days at the ambient temperature (21-23°C), then the animals were decapitated. Their parietal cerebral cortices were dissected, quickly frozen in liquid nitrogen, and stored at - 80°C until extraction of RNA Normal control animals were also decapitated in the same manner without anaesthesia, surgical preparation, or clamping the carotid arteries. For the study of in situ hybridization, the dissected brains were frozen in powdered dry ice and stored at -80°C. Sections (10 J..l.m) at dorsal hippocampal levels were cut out on a cryostat and collected on Histostik (Accurate Chemical and Scientific Corp., Westbury, NY)-coated slides. Sham animals were sacrificed just after exposing the carotid arteries without clamping the vessels. Isolation of RNA and eDNA synthesis Total RNA was extracted from the parietal cortices by the modified method of Chomoczynski and Sacchi (the RNA isolation kit by Stratagene, CA, USA) using guanidine isothiocyanate". Poly(A)+ mRNA was isolated from total RNA of 8 h reperfusion by an affinity chromatography on oligo (dT) cellulose. With this mRNA as a template (5 J..l.g), eDNA was synthesized using an oligo-dT as a primer. After discarding eDNA below 0.5 kb and over 3 kb in size, a eDNA library was constructed with a plasmid vector (pTZ18R, Invitrogen, CA, USA) by the Gubler-Hoffman eDNA synthesis method (The Librarian II, Version 2.4, Invitrogen). These cDNAs were transformed into competent E. coli cells (INV1aF', Invitrogen) to obtain full scale transformants. Screening of the eDNA library Colony filters were made using the standard method for making a replica 12 • Bacterial colonies were grown on a nylon membrane (Hybond N, Amersham, IL, USA) on LB plates which contfil ned ampicillin (50 J..l.gl mD. The membranes were treated with NaOH (0.5 M), neutralized twice with Tris-HCI (1 M, pH 7.5)I NaCI (1.5 M), washed with 2 X SSC (1 X SSC = 150 mM NaCII i5 mM sodium citrate), and then dried in an air for 2 h. The dried membrane filters were baked in a vacuum oven at 80°C for 2 h to fix DNA to the membrane. The baked filters were soaked with NaOH (0.5 M) at 42°C for 30 min to remove cell debris from the filter, neutralized with Tris-HCI (0.5 M, pH 7.5)1 0.1 x SCC / 0.1% SDS (sodium dodecyl sulphate) at 42°C for 30 min, and then were prehybridized with a solution containing formamide (50% formamide, 5 X 5 X Denhardt's solution, 100 J..l.gl ml fish
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sperm DNA, 50 mM KP0 4 and 0.2% SDS ) at 42°C for over several hours. This eDNA library was screened with a human genomic DNA probe, pH 2.3, for HSP7013' 14 • The insert DNA of pH 2.3 was radiolabelled with et- 32 P-dATP (6000 Ci l mmol, Amersham) with a random primer as reported before9 • The radiolabelled probe was added to the hybridization mixture (the same as the prehybridization solution), and the mixture was kept at 42°C overnight with gentle agitation. Filters were first washed several times with 2 x SSCI 0.1% SDS at room temperature. In order not to lose HSC70 clones, the filters were finally washed with a relatively low stringent condition (1 x SSCI 0.1% SDS at 65° for 30 min). These filters were exposed to X-ray films at - 80°C overnight. Subcloning Subcloning was performed because the nucleotide sequence of pTZ18R was not available. The cDNAs were digested with Xho I (Takara, Kyoto, Japan) and electrophoresed in a 0.7% agarose gel in 1 x TBE (90 mM Tris base, 90 mM boric acid, and 2 mM EDT A ) buffer. The inserts of cDNAs were recovered from an agarose gel by centrifugation tubes with a dural membrane filter (SUPREC™-01 tube, Takara ). The inserts were then subcloned into a plasmid vector pHSG396 (Takara) at Xho I site15 • Subcloning was performed with a T4 DNA ligase (DNA Ligation Kit, Takara) at 16°C for 2 h. These cDNAs were transformed into competent E. coli cells (JM109, Takara). Recombinant clones were selected by a-complementation 16 • On LB agar plates containing 25 J..l.gl ml of chloramphenicol (Sigma, St Louis, MO, USA), 60 J..l.l of a stock solution of 5-bromo-4-chloro-3-indolyl-/3-o-galactoside (X-gal, 20 mgl ml in dimethylformamide, Sigma) and 4 J..l.l of a solution of isopropylthio-/3-o-galactoside (IPTG, 200 mgl ml, Sigma) were spread. The transformed E. coli was inoculated on the plate, and was incubated for 12-16 hat 37°C. Positive clones showed white colonies, while negative clones showed blue colonies. Southern blot analysis Southern blot analysis was performed by the standard method 17 • DNAs were separated in a 1% agarose gel r'n 1 x TBE buffer, and transferred to a nylon membrane (Hybond N, Amersham) and then prehybridized at 42°C for 4 h in a hybridization solution (same as above). The insert of pH 2.3 was radiolabelled with a- 32 P-dATP by a random primer labelling system using Klenow fragment of E. coli DNA polymerase I (Boeringer Mannheim, Mannheim, Germany), and hybridized against Southern blot at 42°C for 20 h in a hybridization solution. After the hybridization, the filter was washed with 2 x SSC I 0.2% SDS at room temperature, and finally washed with 0.2 x SSC I 0.2% SDS at 65°C. The filters were exposed to X-ray films for 20 hat - 80°C. Northern blot analysis Northern blot analysis was performed by the previous method 9 • Inserts of pH 2.3 and eDNA clones, which were isolated in this experiment, were used as the probes for Northern blot analysis. Conditions of labelling, hybridization, washing and autoradiography were the same as Southern blot analysis.
···
Isolation of eDNA for HSP70 and HSC70 of gerbil: Shigeru Sato et al.
A S
oGA3 PGE.:
B oGB,
oGD.>
S
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•·• An agarose gel (A) stained with ethidium bromide (EB) and Southern blot analysi.s (B) using a human genomic HSP70 fragment, pH2.3, as a probe. Southern filter was washed with 1 x SCC/ 0.2% SDS at 65°C. A. Hind Ill was used as a standard size marker (S) Figure 1:
bovine serum albumin, 200 ,ugl ml sonicated, denatured calf thymus DNA, and 1 mgl ml calf liver rRNA. The insert of pGA3 for HSP70 and that of PGD 3 for HSC70 were radiolabelled with et- 35 S dCTP (1000 Ci l mmol, Amersham ) by random primer labelling kit (Boehringer Mannheim ), resulting in a specific activity of about 5 x 108 dpm l ,ug. Hybridization was performed for 20 h at 42°( in the same solution as prehybridization supplemented with 10% dextran sulphate and 10 mM dithiothreitol. The 1.1 kb of pHSG396 fragment digested by Hinf I (Takara) was similarly labelled as a control probe. The sections were washed for 2 h with 2 X sse and following for 1 h with 1 X SSC at 42°( and dehydrated through graded ethanol series containing 0.3 M ammonium acetate. Hybridization signals were visualized by exposure against an X-ray film, then the slides were dipped in a liquid emulsion (NR-M2, Konica Corp. Ltd, Tokyo, Japan ) and further exposed for 3 weeks. Several sections at 8 h of reperfusion were treated with 100 ,ugl ml RNase A and 10 units I ml RNase T1 (Sigma) at 37°( for 30 min prior to prehybridization and were hybridized with the probes.
RESULTS Sequence determination A small scale plasmid preparation was performed by an anion-exchange resin column (Quiagen tip-20, Stratagene). Their nucleotide sequences were determined by the modified dideoxy chain termination method (Sequenase ver. 2.0; United States Biochemical, OH, USA). 2 pmol of plasmid DNA was denatured with NaOH (20 mM), and precipitated with ethanol in the presence of ammonium acetate. The single-strand plasmid DNA (ssDNA template) was annealed with M13 primer M4 (17mer, United States Biochemical) or M13 primer RV (17mer, Takara), then the double stranded DNA was synthesized with T7 DNA polymerase in the presence of an et-32 P-dCTP (3000 Cil mmol, Amersham) or et- 35 S-dCTP (1000 Ci I mmol, Amersham ). The reaction was terminated by an addition of dideoxy nucleotides to the reaction mixture. Each strand was sequenced twice in separate experiments. Sequence data management was performed by using the GENETYX ver. 2 program (Software development Co., Tokyo, Japan). Determined sequences of these clones were compared with some sequences of mouse, rat and human HSP related proteins in the Gen Bank (Cambridge, MA, USA) and the European Molecular Biology Laboratory (EMBL, Heiderberg, Germany).
In situ hybridization In situ hybridization was performed by the method of Yoshioka et a/. with a slight modification 18 • Briefly, the sections were fixed for 10 min in an ethanol I acetic acid (3: 1) mixture, ~transferred to 0.2 M HCI for 20 min, and immersed for 20 min in 0.3 M NaCII 0.03 M Na citrate (2 X SSC, pH 7.0) at 50°C. Then they were digested for 15 min at 37°( with 100 ,ugl ml of proteinase K (Merck & Co. Inc., Rahway, NJ) in 20 mM Tris-HCI buffer (pH 7.4) with 2 mM CaCI 2 and dehydrated through graded ethanol. Slides were prehybridized for 2 h at room temperature in a solution containing 50% formamide, 600 mM NaCI, 10 mM Tris- HCI (pH 7.5), 10 mM EDTA, 0.02% poly( vinyl pyrrolidone), 0.02%
Screening of the eDNA library The size of eDNA library (number of transformants) for the ischaemic cerebral cortices of gerbils was 27,800. After the 3rd screening, 4 eDNA clones were finally isolated from the library (named pGA31 pGB, pGD 3 and pGE., respectively). Sizes of eDNA insert are 1.0 kb (pGA), 1.1 kb (pGB,), 1.4 kb (pGD 3 ), and 0.9 kb (pGE4 ) (Figure 1A ). Southern blot analysis Southern blot analysis revealed that pH 2.3 probe hybridized the inserts of pGA3, pGB,, pGD 3 and pGE4, but did not hybridize the vector DNA in 1 x SSC at 65°( (Figure 18). But in 0.2 X sse at 65°( , the radiolabelled pH 2.3 probe was almost washed out from the insert of pGB, and pGD 3 although this probe still hybridized to the insert of pGA3 and pGE4 (data not shown). Northern blot analysis Northern blot analysis showed that pH 2.3 probe hybridized 2 different sizes of mRNA in the gerbil brain (Figure 2). One was a 2.8 kb size, and another was a 2.4 kb. The 2.8 kb mRNA was present in a very small amount in a normal condition, and was greatly induced after 8 h of reperfusion. The 2.4 kb mRNA was expressed in a considerable amount in a normal gerbil brain, but was still inducible. When the filter was washed with 1 X sse at 65° ( , 2 different sizes of the mRNA were detected (Figure 2A). However, only the 2.8 kb mRNA was detected after the washing with a more stringent condition such as 0.2 x sse at 65°( (Figure 28). Northern blot analysis showed that inserts of pGA 3 and pGE4 hybridized only the 2.8 kb mRNA, and that the inserts of pGB, and pGD 3 hybridized only the 2.4 kb of mRNA both in a low stringent washing condition (1 X SSC, room temperature) and in a high stringent washing condition (0.2 X SSC, 65°() (Figure 3).
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8
A •
•
28S--
185-
185--
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•.
285--
.•
Northern blot analysis using a human genomic HSP70 fragment, pH2.3, as a probe. e , and e, represent the cases of normal control. 8h, 8h, 2d,, and 2d, represent the cases of 8 h or 2 days reperfusion after 10 min of common carotid artery occlusion, respectively. Arrow show positions of 18s and 28s ribosomal RNA (rRNA ). Two mRNAs (2.8 kb and 2.4 kb) are detected with a washing condition of 1 x SSe/ 0.2% SDS at 65°e (A). Only one mRNA (2.8 kb) is detected with that of 0.2 x SSe/0.2% SDS at 65°e (B) Figure 2:
8
A
• 28S--
28S-
185-
185--
Northern blot analysis using gerbil eDNA probes. The pGA, insert detects only the 2.8 kb size mRNA (A). The pGE, insert also hybridizes the same transcript (data not shown). The pGD, insert detects only the 2.4 kb size mRNA (B ). The pGB, insert also hybridize~ the same transcript (data not shown). Arrow show positions of 18s and 28s rRNA. Washing condition; 1 x SSe, 0.2% SDS at 65°C. When the wash condition was changed to higher stringency (0.2 X SSe at 65°e), these results were not changed Figure 3:
" Sequence determination
.
About 350 bp nucleotide seque nces at both sides of the insert of each clone were determined (Figure 4 ). From the analysis of nucleotide sequences, an identity of 126 nucleotides of the 5' end of the pGE, insert with 76-201 nucleotides from the 5' end of the pGA3 insert was found . An identity of 74 nucleotides of the 5' end of the pGB, insert with 297-370 nucleotides from the 5' end of the pG0 3 insert was also found. The 3' e nd of
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the pGA 3 insert had the same sequence as that of the pGE, insert, and the 3' of the pGB, insert had the same sequence as that of the pGD 3 insert. A terminal codon was found in the insert of each clone and the open reading frame and predicted amino acid sequence were determined. HSP70 in a gerbil has about 700 bp of 3' untranslated region and HSC70 had 245 bp of that. The polyadenylation signa l AATAAA
Isolation of eDNA for HSP70 and HSC70 of gerbil: Shigeru Sato et al.
Table 1: Homologies of nucleotide sequences of coding region between gerbil and mouse, rat or human Mouse Gerbil
HSP70"
HSP70 HSC70
Rat
HSC70"
Human
HSC70"
87.9 (86.5) 68.3 (64.8) 70.0 (64.8) 74.5 (88.4) 94.2 (99.1) 92.8 (98.6)
HSP70"
HSC70"
86.2 (90.1) 66.7 (62.9) 70.0 (86.7) 93.9 (98.7)
Homologies of nucleotide sequences of coding region are shown by percentage. A homology of predicted amino acids are shown in parentheses.
HSP70 : l.M kb
s·
()40bpJ
( ~22bp)
- - - - - - - - - - - - - - - 3'(polyA site) pGAJ ( J.Okb)
pGE4 (0.9kbl
HSC70 : 2.4 kb (63Rbpl
CJJObpl
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s·
)' {polyA site )
pGBI ( I.Jkb) pGB3 ( 1.4kbl
-
coding region
-
J' non coding region
~
region where nucleotide .o;e4uencc is detcrmin~d
f-l lkb
Schematic view of isolated clones. About 200-350 bp nucleotide sequences at both sides of the insert of each clone were determined using two primers, M13RV and M13m4. The bold bars represent open reading frames, and the thin bars represent 3' untranslated sequences Figure 4:
and poly (A) were included in these regions of both HSP70 and HSC70. Results of homology analysis between these clones and HSP70 or HSC70 of a mouse, rat and human are summarized in Table ?1 4·' 9 - 22 • Nucleotide sequence comparisons of the coding sequence of HSP70 genes revealed a homology of 87.9% between gerbil and mouse, and of 86.2% between gerbil and human. At the protein level, there was a homology of 86.5% between gerbil and mouse and of 90.1% between gerbil and human. The search for HSC70 revealed a homology of 94.2% between gerbil and mouse, of 92.8% between gerbil and .rat, and of 93.9% between gerbil and human at the nucleotide level. At the protein level, there were two or three amino acid differences between gerbil and ·mouse, rat or human. In contrast, there was a homology of about 70% between the gerbil HSP70 and other HSC70 genes or between gerbil HSC70 and other HSP70 genes. There was no homology in the 3' untranslated region between the gerbil HSP70 genes and other HSC70 genes, or between gerbil HSC70 genes and other HSP70 genes.
In situ hybridization HSP70 mRNA was present in a very small amount in the hippocampal pyramidal and dentate granule cells of the sham control brain. The amounts of mRNA was greatly induced in the cells of hippocampus and dentate gyrus after 8 h of recirculation . In addition, the HSP70 mRNA was newly induced in cells of medial habenula, ventral thalamic ·nuclei, ventromedial and arcuate hypothalamic nuclei, caudate putamen, amygdaloid nuclei and cerebral cortex after 8 h of recirculation
(Figure 5 ). On the other hand, HSC70 mRNA was present in the cells of these regions of the sham control, and was slightly induced after 8 h of recirculation in these regions (data not shown). No detective signal was found after the treatment of RNases and in the case with pHSG396 (plasmid vector) as the probe (data not shown). DISCUSSION Four clones were first isolated as HSP70 related genes from the postischaemic gerbil brain. The results suggested that pGA3 and pGE4 are the HSP70 eDNA clones and pGB, and pGD 3 are the HSC70 eDNA clones because: (1 ) Southern blot analysis showed that each insert hybridized the human genomic HSP70 probe; (2) Northern blot analysis showed that the human genomic HSP70 probe detected both 2.8 and 2.4 kb mRNA in the low stringent washing condition. The inserts of pGA 3 and pGE. hybridized only the 2.8 kb mRNA, and the inserts of pGB, and pGD3 hybridized only the 2.4 kb mRNA; (3 ) a nucleotide sequence analysis suggested that pGE4 was a part of pGA3, and that pGB, was a part of pGD 3; (4) the nucleotide sequence of pGA3 and pGE. has a homology of over 85% with that of mouse and human HSP70 genes. The nucleotide of sequence of pGB, and pGD 3 has a homology of over 90% with that of mouse, rat and human HSC70. An about 70% of the insert of pGA 3 (total insert size is 1.0 kb ) is the 3' non-coding region including a polyadenylation signal and a poly(A) sequence and the coding region of the inset is only 306 bp (Figure 4). This untranslational region has less homology with that of HSC70 genes. On the other hand, the insert of pGD 3
HSP70
s Bh
..
_., ;:_·~--~-~~"·:.
Figure 5:
In situ hybridization using the insert of pGA, fo r HSP70 as a probe. Sections were prepared from the sham control (5 ) and 8 h reperfusion after 10 min of ischaemia in the gerbil brains (8 h)
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(total insert size is 1.4 kb) has the 245 bp of 3' non-coding region, which has no homology with that of HSP70 genes. In general, the coding regions of HSP70 and HSC70 show a relative high homology between species. However, our clones have a highly selective sequence for HSP70 or HSC70 mRNA of gerbil, and, therefore, selectively detects HSP70 or HSC70 mRNA
4
5
6
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(Figure 3). Northern blot analysis showed that HSP70 mRNA was present in a very small amount in a normal condition, but was greatly induced after 8 h reperfusion. On the other hand, HSC70 mRNA was present in a considerable amount in a normal gerbil brain, but was still inducible after 8 h. The time course of the induction of HSP70 and HSC70 of mRNA is reasonable as compared to previous reports9•23 • The results of in situ hybridization indicated that the insert of pGA3 and pGD 3 selectively recognized mRNA for HSP70 or HSC70, respectively, because no signal was detecte~ after treating RNases and the non-sense probe did not show any signal. HSP70 mRNA was induced in the cells of hippocampus, dentate gyrus, medial habenula, ventral thalamic nuclei, caudate putamen, ventromedial and arculate hypothalamic nuclei, amygdaloid nuclei and cerebral cortex after 8 h of reperfusion. HSC70 mRNA was present in almost the same areas of the sham control. It is interesting that these regions are consistent with the regions at which the protein synthesis is active in normal conditions24·25 • The cooperative induction of HSP70 and HSC70 mRNA after a transient ischaemia suggests the cooperative role of these proteins as molecular chaperoning26 • Because of a relatively high homology between the HSP70 and HSC70 gene, it should be better to use probes selectively recognize each mRNA species especially in cases of in situ hybridization study. Our clones are constituted from 3' part of the mRNAs, so that the specificity is very high to recognize each species of the mRNA. Thus, our results are the first of isolating HSP70 and HSC70 eDNA dones from the postischemic gerbil brain, which can selectively recognize HSP70 or HSC70 mRNA. Although these eDNA clones are not full length, they provide good probes to study the selective changes of these genes after ischemia for in situ hybridization study.
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ACKNOWLEDGEMENTS This work was partly supported by Monbusho grant 01044018. The authors would like to thank Dr Sugawara A for many advices for molecular biology strategies, and to Mrs Matsumoto M. for her excellent technical assistance. REFERENCES Jacewicz M, Kiessling M, Pulsinelli WA. Selective gene . expression in focal cerebral isc;,f;lemia. 1Cereb Blood Flow Metab 1986; 6: 263-272 ' 2 Morimoto Rl, Tissieres A, Georgopoulos C. The stress response, function of the proteins, and perspectives. In: Morimoto Rl, Tissieres A, Georgopoulos C, eds. Stress Proteins in Biology and Medicine. New York: Cold Spring Harbor Laboratory, 1990: pp. 1-36 3 Flaherty KM, Flaherty CD, McKay DB. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature 1990; 346: 623-628
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