Cloning, nucleotide sequence and expression of rat heat inducible hsp70 gene

ELSEVIER Biochimica et Biophysica Acta 1219 (1994) 64-72

BB Biochi~ic~a et Biophysica Acta

Cloning, nucleotide sequence and expression of rat heat inducible hsp70 gene

Katarzyna Lisowska, Zdzislaw Krawczyk *, Wies~awa Widtak, Piotr Wolniczek, Jan Wigniewski Department of Tumor Biology, Institute of Oncology, ul. Wybrze2e Armii Krajowej 15, 44-100 Gliwice, Poland

(Received 14 October 1993)

Abstract

In rat cells hyperthermia induces two hsp70 transcripts of 2.5 kb and 2.7 kb. We have cloned and determined the nucleotide sequence of a gene (named hsp70.1) encoding the 2.5 kb transcript as shown by Northern blot analysis using the 5' end and 3' end specific hybridization probes. It contains an uninterrupted open reading frame of 1926 bp, it encodes a protein of approx. 70100 Da and the predicted amino acid sequence of its product shows 98% similarity to the mouse hsp70.1 protein. The transcription start site was localized 224 bp upstream the ATG codon by RNase protection and primer extension mapping. Upstream the transcription initiation site several potential regulatory motifs including a TATA box, two Spl binding sites, one inverted and one direct CCAAT box and three HSEs (heat shock elements) were found. Transfection experiments with constructs in which the CAT reporter gene was fused to fragments of the 5' end flanking sequences of the isolated gene confirmed that the promoter of the rat hsp70.1 gene is functional and heat inducible.

Key words: Nucleotide sequence; Heat-inducible hsp70 gene; Promoter; Cloning; (Rat)

1. Introduction

Each organism studied so far contains so called heat shock genes (hsp genes), transcription of which is induced or activated in response to tempera ture increase as well as to many other environmental factors and endogenous stimuli [1,2]. Beside the strictly heat inducible genes there are also several types of related genes which are constitutively expressed and not induced by heat shock [1,3].

On the basis of nucleotide sequence similarity and molecular weight of encoded proteins the heat shock and related genes are divided into several groups. The most prominent and with the highest evolutionary con- servation is the hsp70 multigene family. In eukaryotes the genes which belong to the hsp70 family are differ- entially regulated and the heat shock proteins they code for (HSP70 proteins of approx. 70 000 Da) reside in different subcellular compartments [1-4].

* Corresponding author. Fax: +48 32 313512. 1 Present address: Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Building 37, Rm. 4C-09, Bethesda, MD 20892, USA.

0167-4781/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4781(94)00055-8

Functionally the HSP70 proteins belong to a class of proteins known as molecular chaperons [5,6]. At nor- mal physiological conditions the HSP70 proteins per- form the chaperon function by controlling the conformation of other cellular proteins [3,7]. In cells subjected to environmental stress (e.g., heat shock) the HSP70 proteins seem to recognize nonspecifically ag- gregated proteins (in particular the preribosomal struc- tures in the nucleoli [8]) and help them to regain the native conformation [6].

The HSP70 proteins may have many particular func- tions, e.g., dissociation of clatrin triscelia from coated vesicles (HSC70 [9]), involvement in antigen processing (PBP72/74 [10]), involvement in formation of certain hormon-receptor complexes (HSP70, HSC70 [1 l]), tar- geting proteins destined to be degraded into lysosomes (prp73 [12]) or complexing the oncogenic, mutated forms of p53 protein (HSC70 [13]). Various aspects of the heat shock response, structure of heat shock genes and function of corresponding proteins have recently been extensively reviewed [14,15].

In mammals the best characterized families of the hsp70 genes are those of man, mouse and rat. Eight members of the human hsp70 multigene family have been described so far. Four of them are heat inducible:

K. Lisowska et al. / Biochimica et Biophysica Acta 1219 (1994) 64-72 65

hsp70/hsp70.1 [16], hsp70B' [17], hsp70B ([18], the promoter region and only part of the transcription unit was sequenced), and hsp70.2 [19]. The sequence of proteins coded by the hsp70.1 and hsp70.2 are virtually identical, however the corresponding genes contain divergent promoters. In contrast to hsp70.2, the hsp70.1 gene is expressed also in the absence of stress, it is serum inducible and its transcription changes in a cell-cycle-dependent manner [20,21]. The other four known hsp70-related genes of the human family are not heat inducible. The grp78 gene codes for glucose regulated protein localized in ER [22], the hsc70 gene codes for constitutively synthesized, abundant cytoplas- mic protein [23], the hsp70-Hom is a homolog of mouse spermatid-specific hsc70t gene [19], and PBP74 is a novel, recently described gene which codes for an alleged antigen processing protein [24].

The mouse hsp70 gene family consists of at least six known genes, cDNAs of three transcripts (clones pMHS213, pMHS243, and pMHS214) derived from two (or three) different heat inducible genes have been cloned [25]. One of these genes named hsp70.1 with the nucleotide sequence most similar to the pMHS214 cDNA was recently isolated and sequenced [26].

Four constitutively expressed, non heat inducible hsp70-related genes have been characterized in mouse. Two of them are shown to be testis specific; the hsp70.2 is transcribed in pachytene spermatocytes [27], while the hsc70t is transcribed in round spermatids [28]. Two other cognates are the hsc70 gene [29] and PBP74 gene [24].

The rat hsp70 gene family is less extensively characterized than those of man and mouse. So far only constitutively expressed, non heat-inducible genes have been isolated and fully sequenced. These are the grp78 gene [30], the hsc70 gene [31], and the testis-specific hst70 gene which is expressed in late pachytene spermatocytes [32].

The rat hsp70 multigene family seems to contain at least two strictly heat inducible genes, expression of which gives rise to two transcripts of 2.5 kb and 2.7 kb, respectively [33,34]. Preliminary characterization of the gene from which the 2.5 transcript is derived was recently published [35]. Here we present the complete nucleotide sequnce of that gene, together with a functional analysis of its expression.

scribed in details earlier [33,34]. Excised tissues were immediately frozen, pulverized in the liquid nitrogen, and stored at -70°C.

2.2. RNA isolation and analysis

Total RNA was isolated from pulverized tissues by acidic phenol extraction [36] and poly(A) + RNA was selected through two cycles of oligo(dT)-cellulose (Boehringer) chromatography [37]. Equalized RNA samples were fractionated on 1.2% agarose-2.2 M formaldehyde gel [37], blotted onto Hybond-N membrane (Amersham) and fixed to the membrane by ultraviolet irradiation. Northern blot hybridization was performed under stringent conditions essentially as described earlier [34,35]. Briefly, the hybridization solution contained 20 mM sodium phosphate (pH 6.5), 50% for- mamide, 5 x SSC, 5 x Denhardt's solution, 250/xg/ml denatured salmon sperm DNA and radioactive probe. Filters were probed with fragments of the rat hsp70.1 gene (Fig. 6). To check the uniformity of RNA loading and transfer blots were rehybridized with the plasmid pRGAPDH [38] containing the rat GAPDH (glycer- aldehyde-3-phosphate dehydrogenase) cDNA (gift of Dr. P.P. Fort). Probes were labelled with [a-32p]dCTP by random priming (Multiprime kit, Amersham) to a specific activity of about 108-109 cpm//zg of DNA.

2.3. Nucleotide sequence analysis

DNA sequencing was performed by dideoxy-chain termination method [39] using [a-35S]dATP and T7 DNA polymerase (Pharmacia) or Klenow fragment of DNA polymerase I (Boehringer) according to the man- ufacturer's instructions. Routinely the double stranded templates were used which were subclones of the pR68/1.7 and pR68/4.1 clones (see the restriction map in the Fig. 1) in pUC19, pBluescript SK(+) or KS(+) plasmids. Unidirectional deletion clones were prepared by exonuclese III /mung bean nuclease (Stratagene) digestion. Some regions of the gene were sequenced using internal primers. The details of the sequencing procedure and strategy is available on request. The analysis of the various features of the DNA sequence of the hsp70.1 gene was performed using the PC/Gene 6.50 computer programs (IntelliGenetics).

2. Materials and methods

2.1. Animals and hyperthermia

2-2.5-Month-old male albino Wistar rats were used. Rats were starved for 18 h before killing by decapita- tion. Where indicated rats were subjected to hyperthermia (42°C, duration indicated in the text) as de-

2.4. RNase protection and primer extension

For RNase protection analysis 5 /zg of poly(A) + RNA was hybridized with 1.8.105 cpm of the RNA probe (Fig. 3) and the reaction mixture was processed as described in Ref. 40. RNA probe was labeled with [a-32P]UTP using T7 RNA polymerase (Promega) as described in Ref. 40. For primer extension analysis 1.105 cpm of the 5' end-labeled synthetic 30-mer

66 K. Lisowska et al. /Biochimica et Biophysica Acta 1219 (1994) 64-72

primer (5'-GGTTCTGGAACGCGCCGCTTGCTC- CGCTI'C-3'; complementary to nucleotides 494-523 in Fig. 2) were hybridized with 3.5/xg of poly(A) + RNA, extended by AMV reverse transcriptase (Promega) and processed as described in Ref. 40. The same oligo- nucleotide was used to generate a sequencing ladder. Oligonucleotides and size markers (DNA restriction fragments generated from pBluescript SK cut with MspI) were 5' end-labeled with [y-32p]ATP and T4 polynucleotide kinase (Promega). Oligonucleotides were synthesized with a PCR-Mate 39l DNA Synthe- sizer (Applied Biosystems).

2.5. Expression vectors used for transient transfection assay

All manipulations with plasmid DNA, restrictions, cloning etc. were performed according to standard procedures [37,40]. The coordinates of the restriction sites of the rat hsp70.1 gene are according to the numbering of nucleotides as in the Fig. 2. The plasmid phsp70(950)-CAT6 was constructed by inserting the PstI-AvaI (blunted with Klenow) fragment (see map in Fig. 6) derived from the clone A 68 (Fig. 1) in front of the CAT (chloramphenicol acetyltransferase) gene in MCS (multiple cloning site) of the plasmid pBL-CAT6. The above PstI-AvaI restriction fragment contains 86 bp of leader sequnces and approx. 950 bp of the 5' end flanking sequences of the rat hsp70.1 gene. The plasmid pBL-CAT6 (M. Boshart etal . , unpublished data, see also Refs. 41 and 42) is a modified version of the plasmid pBLCAT3 [43]. The plasmid pBL-CAT6 contains a promoterless CAT reporter gene preceded by a convenient MCS and two SV40 polyadenylation signals to stop unspecific initiations from the vector sequences. The plasmid phsp70(216)-CAT5 (Fig. 5) was constructed as follows: first, the NcoI-NcoI restriction fragment (nucleotides 156 to 372) of the rat hsp70.1 gene promoter (Fig. 4) was inserted into the NcoI site of the pLH1 vector [44] and the clones of the proper 5'-3' orientation were selected. Then the insert was cut off using HindII! and EcoRI restrictases from the resulting plasmid and inserted.into MCS in the plasmid pBL-CAT5 in front of the HSV-TK promoter. The plasmid pBL-CAT5 (unpublished data, the gift of M. Boshart is a modified version of the plasmid pBLCAT2 [43]). The plasmid pBL-CAT5 contains a fragment of the HSV-TK promoter (spanning from -105 to + 5 [45]) ligated to the CAT gene. The details of the construction of the phsp70-CAT plasmids described above are available on request.

2. 6. Cells, transfections and CA T assay

Rat hepatoma Fe33 cells [46] (gift of W. Schmid) were grown on 1:1 DMEM/Ham medium (Gibco)

supplemented with 5% fetal calf serum (Gibco). Cells were transfected with plasmids phsp70(950)-CAT6 and phsp70(216)-CAT5 using the DEAE-dextran (Phar- macia) procedure essentially as described in Ref. 47. Cells were plated at a density of 1 • l06 per 9 cm tissue culture plate. After 24 h cells were washed with TBS [48] and covered with 1 ml of TBS containing 0.5 /zg DEAE-Dextran and 10 /zg of plasmid DNA. After 30 rain at room temperature cells were washed with TBS, shocked by 2 rain incubation at room temperature in culture medium containing 10% DMSO (without serum). Then cells were washed with full medium and allowed to grow in full medium for an additional 48 h. Where indicated transfected cells were heat shocked at 42°C and allowed to recover at 37°C as described in the text. Cell extracts were prepared and CAT assay performed as described earlier [42]. Protein concentration in cell extracts was determined by the method of Brad- ford [49].

3. Results

3.1. Isolation and sequencing of the rat hsp70.1 gene

Previously, by the screening of a rat DNA library with the mouse and human hsp70 gene probes we isolated 20 genomic regions encompassing hsp70-re- fated sequences [50]. One of these regions contained the spermatocyte-specific hst70 gene [32]. Another genomic region (cloned in a phage A 68) contained sequences which hybridized with the human hsp70/ hsp70.1 gene under stringent conditions [50]. Prelimi- nary characterization of the clone strongly indicated that it contains a heat inducible, hsp70 gene [35].

The structure of the genomic region cloned in the phage A 68 is shown in Fig. 1. The 4.1 kbp KpnI-EcoRI fragment contains the entire transcription unit of the

E K E E R 7 0 - I I , 1 ~ , ~ 8 ) I I i ~ I .... I

E P N K pn68/,.7 t I I J

pR68/4. I

Kn B~ n ps I l l

5 ' - - ~3'

$ B . B E ', J l " , i

Fig. 1. Structure of the rat genomic region containing the hsp70.1 gene. The genomic fragment termed RT0-11 which contains the heat inducible hsp70 gene (open bar) was isolated from rat genomic library as described previously [32,50]. Initial characterization of the A 68 clone was reported earlier [35]. Subclones pR68/4.1 and pR68/1.7 were constructed by inserting indicated DNA fragments into KpnI and EcoRI restriction sites of the pUC19, pR68/4.1 contains the entire transcription unit of the hsp70.1 gene (2480 bp from the 'cap' site to pA site), 176 bp of the 5' end flanking sequences and approx. 1400 bp of the 3' end flanking sequences. The arrow shows the direction of transcription. Only selected restriction sites are shown; B, Ba, E, K, N, P, S correspond to BamHl, Bali, EcoRI, KpnI, NcoI, PstI and SmaI, respectively.


CGTTTTGAGA AAATTTCTGC GTCCGCCATC

CTGACCCTTC TCAGCTTCAC ATACAGAGAC 250

CTGTCCAGCG AAGCCCAGAT CCGTCTGGAG 350 A

AGATTCCTGG CCCCAAGGCC TCCTCCCGCT 45O

CCTCCTCCTA ATCTGACAGA ACCAGTTTCT

ATCCCCACCG CGAAGCGCAA CCTTCTCCAG

661 ATG Gee AAG AAA ACA GCG ATC M A K K T A I

745 ATC GCC AAC GAC CAG GGC AAC I A N D Q G N

829 AAC CAG GTG GCG CTG AAC CCG N Q V A L N P

913 TCG GAC ATG AAG CAC TGG CCC S D M K H W P

997 TCG TTC TAC CCG GAG GAG ATC S F Y P E E I

1081AAC GCC GTG ATC ACC GTG CCC N A V I T V P

1165 GTG CTG CGG ATC ATC AAC GAG V L R I I N E

1249 ATC ~TC GAC CTG GGG GGC GGC I F D L G G G

1333 GAC ACG CAC CTG GGC GGG GAG D T H L G G E

1417 ATC AGe CAG AAC AAG CGC GCG i S Q N K R A

]501AGC CTG GAG ATC GAC TCT CTG S L E I D S L

1585 CTG TTC CGC GGC ACG CTG GAG L F R G T L E

1669 GTG GGC GGC TCG ACG CGC ATC V G G S T R I

1753 CCG GAC GAG GCG GTG GCC TAC D D E A V A Y

1837 CTG CTG GAC GTG GCG CCG CTG L L D V A P L

1921 CCC ACC AAG CAG A~G CAG ACC P T K Q T Q T

2005 ATG ACG OOC GAC AAC AAC CTG M T R D N N L

2089 ACC TTC GAC ATC GAC GCC AAC T F D I D A N

2173 AAC GAC AAG GGC CGC CTG AGC N D K G R L S

2257 CGC GAG AGG GTG GCT GCC AAG R ~ R V A A K

2341 AAG ATC AGC GAG GCT GAC AAG K i S E A D K

2425 AAA GAG GAG TTC GTG CAC AAG K E E F V H K

2509 CCC GGG GCT GGG GGC TTC GGG P G A G G F G

2600 GGCTCTCAGG GTGTTGGCTA GAGACAGACT

GGTAATTGAT TTGAGTTTGT TACATTTTGT

TTTCCTTCCT GCGAACACCT CAGCACTGCC 2950

5ATGTTACAA GTATGTTTTG TCCGTGTGTA

50 i00 CTGTAGGAAG AATTTGTACA CCTTAAACTC CCTCCCTGGT CTGATTCCCA ATGTCTCTCA CCGCCCAGCA CTTTCAGGAG

150 200 CGCATCCTTG CGTCGCCATG GCAACACTTG TCACAACCGG AACAAGCACT TCCTACCACC CCCCGCCTCA GGAATCCAAT

300 AGTTCTGGAC AAGGGCGGTA CCCTCAACAT GGATTACTCA TGGGAGGCGG AGAAGCTCTA ACAGACCCGA AACTGCTGGA

c 400 • ~ ~i

CGCTGATTGG CCCATGGGAG GGTGGGCGGG GCCGGAGGAG GCTCCTTAAA GGCGCAGGGC GGCGCGCAGG ACACCAGATT n c 500 E 550

GGTTCCACTC GCAGAGAAGC AGAGAAGCGG AGCAAGCGGC GCGTTCCAGA ACCTCGGGCA AGACCAGCCT CTCCCAGAGC 600 650

AGCATACCCC AGCGGAGCGC ACCCTTCCCC AGAGCATCCC CGCCGCCAAG CGCAACCTTC CAGAAGCAGA GAGCGGCGAC

GGC ATC GAC CTG GGC ACC ACC TAC TCG TGC GTG GGC GTG TTC CAG CAC GGC AAG GTG GAG ATC 28 G I D L G T T Y S C V G V F Q H G K V E I

CGC ACG ACC CCC AGC TAC GTG GCC TTC ACC GAC ACC GAG CGG CTC ATC GGG GAC GCC GCC AAG 56 R T T P S Y V A F T D T E R L I G D A A K

CAG AAC ACC GTG TTC GAC GCG AAC GGG CTG ATC GGC CGC AAG TTC GGC GAC CCG GTG GTG CAG 84 Q N T V F D A N G L I G R K F G D P V V Q

TTC CAG GTG GTG AAC GAC GGC GAC AAG CCC AAG GTG CAG GTG AAC TAC AAG GGC GAG AAC CGG 112 F Q V V N D G D K P K V Q V N Y K G E N R

TCG TCC ATG GTG CTG ACC AAG ATG AAG GAG ATC GCC GAG GCC TAC CTG GGC CAC CCG GTG ACC 140 S S M V L T K M K E I A E A Y L G H P V T

GCC TAC TTC AAC GAC TCG CAG CGG CAG GCC ACC AAG GAC GCG GGC GTG ATC GCG GGT CTG AAC 168 A Y F N D S Q R Q A T K D A G V I A G L N

CCC ACG GCG GCC GCC ATC GCC TAC GGG CTG GAC eGG ACC GGC AAG GGC GAG CGC AAC GTG CTC 196 P T A A A I A Y G L D R T G K G E R N V L

ACG TTC GAC GTG TCC ATe CTG ACG ATC GAC GAC GGC ATC TTC GAG GTG AAG GCC ACG GCG GGC 224 T F D V S I L T I D D G I F E V K A T A G

GAC TTC GAC AAC CGG CTG GTG AGC CAC TTC GTG GAG GAG TTC AAG AGG AAG CAC AAG AAG GAC 252 D F D N R L V S H F V E E F K R K H K K D

GTG CGG CGA CTG CGC ACG GCG TGC GAG AGG GCC AAG AGG ACG CTG TCG TCC AGe Ace CAG GCC 280 V R R L R T A C E R A K R T L S S S T Q A

TTC GAG GGC ATe GAC TTC TAC ACG Tee ATC ACG eGG GCG CC, G TTC GAG GAG CTG TGC TCG GAC 308 F E G I D F Y T S I T R A R F E E L C S D

CCC GTG GAG AAG Gee CTG CGC GAC GCC AAG CTG GAC AAG GCG CAG ATC CAC GAC CTG GTG CTG 336 P V E K A L R D A K L D K A Q I H D L V L

CCC AAG GTG CAG AAG CTG CTG CA(; GAC TTC TTC AAC GGG CGC GAC CTG AAC AAG AGe ATC AAT 364 P K V Q K L L Q D F F N G R D L N K S I N

GGG GCG GCG GTG CAG GCG GCC ATC CTG ATG GGG GAC AAG TCG GAG AAC GTG CAG GAC CTG CTG 392 G A A V Q A A I L M G D K S E N V Q D L L

TCG CTG GGT CTG GAG ACC GCG GGG GCC GTG ATG ACG GCG CTC ATC AAG CGC AAC TCC ACC ATC 420 S L G L E T A G A V M T A L I K R N S T I

TTC Ace ACC TAC TOO GAC AAC CAG CCC GGG GTG CTG ATC CAG GTG TAC GAG GGC GAG AGG GCC 448 F T T Y S D N Q P G V L I Q v Y E G E R A

CTG C, GG CGC TTC GAG 'FIG AGC GGC ATC CCG CCG GCT CCC AGG GGC GTG CCC CAG ATC GAG GTG 476 L G R F E L S G I P P A P R G V P Q I E V

GGC ATC CTG AAC GTC AOO GCC ACT GAC AAG AGC ACC GGC AAG GCC AAC AAG ATe Ace ATe ACC 504 G I L N V T A T D K S T G K A N K I T I T

AAG GAG GAG ATC GAG CGC ATG GTG CAG GAG GCC GAG CGC TAC AAG GCG GAG GAC GAG GTG CAG 532 K E E I E R M V Q E A E R Y K A E D E V Q

AAT GCG CTC GAG TCC TAT GCC TTC AAC ATG AAG AGC GCC GTG GAG GAC GAG GGT CTC AAG GGC 560 N A L E S Y A F N M K S A V E D E G L K G

AAG AAG GTG CTG GAC AAG TGT CAG GAG GTC ATC TCC TGG CTG GAC TCT AAC ACG CTG GCT GAG 588 K K V L D K C Q E V I S W L D S N T L A

CGG GAG GAG CTG GAG CGC GTG TGC AAC CCG ATe ATe AGC GGG CTG TAT CAG GGT GCG GGT GCT 616 R E E L E R V C N P I I S G L Y Q G A G A

GCC CAG GCG CCC AAG GGA GGC TCT GGG TCG GGG CCC ACC ATC GAG GAG GTG GAT TAG AGGCTTTTCT A Q A - P K G ~ S G S G P T I E E V D

2650 2700 CTTGATGGCT GCTGGTGCAC GATTCTTATC AAGTTACTCC TTCTCTCCGG AGTTCAGTTT AAAGTTACA~ CCTTTTATAC

2750 280O ATGCTCGTGG GTTTTTTATA TATTCAAATT AAGGTTGCAT GTTCTTTGCG TTTAATCTAA GTAGCTGTGT AAAAATGGTG 2850 2900 ACCCTGTGTA CAGTTTTTTC CTTGCATCCC TACAAACTGA GAAAAAAAGT TATCTTTTGT AACTTAAACA TTCAAAATAA

3000 TGTTGGGAGG GCTAATGGAT TCTGGGTTCA TGTGGATTTC TTAGCTTTGC GATGACGGGG AAATGGGGTT TGGGTACTTT

Fig. 2. Nucleotide sequence of the hsp70.1 gene as determined from the genomic DNA in clones pR68/4.1 and pR68/1.7 (EMBL Data Library accesion number is X74271 R. noruegicus hsp70 gene). Nucleotide sequences potentially relevant to the regulation of transcription are underlined and marked by a capital letter under the sequence: A, HSE like sequences; B, CAAT box; C, Spl binding sites; D, inverted CAAT box; E, TATA box; F, polyadenylation signal. Vertical arrows and an asterisk (*) indicate the main and minor transcription initiation start sites, respectively, as determined by the primer extension method (see Fig. 3). Predicted amino acids are listed in one-letter code below the corresponding codons. Those underlined differ from the amino acids at the same position in the highly homologous mouse HSP70.1 protein [26].

68 K. Lisowska et al. / Biochimica et Biophysica Acta 1219 (1994) 64-72

hsp70.1 gene, about 200 bp of the 5' end flanking sequences and more then 1000 bp of the 3' end flanking sequences. The 1.7 kbp KpnI-EcoRI fragment contains 5' end flanking sequences located upstream the KpnI site.

The complete sequence of the transcription unit of the hsp70.1 gene together with the significant portion of the 5' end and 3' end flanking sequences determined by the dideoxy-nucleotide method is listed in Fig. 2. An uninterrupted open reading frame of 1926 nt codes for a protein of 641 amino acids with a molecular weight of approximately 70 100 as estimated on the base of amino acid composition. The predicted amino acid sequence shows the highest similarity (98%) with the mouse hsp70.1 gene [26]. That is why we decided to dubb the rat gene similar to the hsp70.1 gene.

The alignment of the human hsp70/hsp70.1 gene [16] and the rat hsp70.1 gene sequences (not shown) suggested the transcription initiation site of the rat gene to be localized approx. 300 bp upstream the ATG codon. To map the rat hsp70 mRNA start site we used the RNase protection assay. A specific RNA molecular probe was synthesized from a 292 bp Ncol-BalI fragment (nucleotides 372-664) cloned into pBluescript SK( + ) (Fig. 3A).

After hybridization of the radioactive RNA probe with the poly(A) + RNA isolated from liver of rats subjected to hyperthermia the expected fragment of approx. 220 nt was protected from the RNase digestion (Fig. 3B, lane 3). We also used the primer extension method to confirm and map more precisely the position of the mRNA start site. The doublet of main primer extension products (Fig. 3C, lane 6) corresponds to G and A at positions 437 and 438, respectively, and a slightly longer band coincides with A at position 433.

No primer extension products were detected when poly(A) + RNA isolated from control rat liver was used (Fig. 3C, lane 2) but more sensitive RNase protection assay revealed the presence of a very small amount of the hsp70.1 transcript even prior to heat shock (Fig 3B, lane 2). Together, the above data prove that the rat hsp70.1 gene we cloned is functional, heat inducible and its expression at physiological temperature is in- significant.

3.2. Promoter actir, ity of the hsp70 gene

Alignment of the rat hsp70.1 gene 5' flanking sequences to mouse hsp70.1 gene 5' flanking sequences shows a high similarity of DNA sequence and distribu- tion of putative regulatory elements (Fig. 4). The rat hsp70.1 upstream region contains a TATA box localized at position 405 (32 bp upstream the main transcription initiation start site), two Spl binding sites at

A Ncol

Hbal I I i

probe

h sp 70 mRNA

Ball I 1 T7 promoter

:570 nt

protected fragment 221] nt

B

220 nt

C . . . . . .

! ! ~ i i~i~iiii!iiiiiii~!iiiii!ii!i~!!! i ̧ ¸¸~ ~ ~ ~ / ~ ~ ~

iil; ~ii!~iiii!!!ii!!!i!iii~!~!i ii!ii!!!!iiii! ¸̧! ii i ii iii i~ii iiiiii! !ii

1 2 3 4 5 1 2 3 4 5 6

Fig. 3. RNase protection (A,B) and primer extension (C) analysis of the transcription initiation start site of the rat hsp70.1 gene. (A) Schematic diagram of the construct used to generate the specific probe for RNase protection analysis. Open box indicates rat genomic sequences (fragment N~oI-Ball with coordinates as in Fig. 2). Lines correspond to the MCS of the pBluescript SK If( + ). Below the map of the construct the RNase protection probe and expected protected fragment are drawn. (B) Autoradiogram of gel containing products obtained after RNA protection analysis. Lane 1, tRNA; lane 2, poly(A) + RNA from liver of control rats; lane 3, poly(A) ~ RNA from liver of rats subjected to hyperthermia; lane 4, undigested probe; lane 5, molecular size markers (bp). (C) Autoradiogram of gel containing products obtained after primer extension analysis. Lanes 1-4, sequencing ladder; lane 5, poly(A) + RNA from liver of control rats; lane 6, poly(A) + RNA from liver of rats subjected to hyperthermia. Arrows indicate two main products of primer extensi(m. For sequence of the primer used and details of the procedure see Materials and methods.

positions 384 and 263, one inverted and one direct CCAAT box at positions 365 and 216, respectively, as well as three heat shock elements (HSE1 to HSE3) at positions 317, 236 and 96, respectively. The HSE is defined as an array of inverted repeats of the pentamer nGAAn unit with a minimum of three units [51,52]. In the promoter of the rat hsp70.1 gene the HSEI, [cGAAa][cTgCt][gGAAg][aTTCc] is composed of four alternatively oriented nGAAn units with a 11 / 12 match to the consensus sequence. The HSE2,[aGAtc][cgTCt] [gGAga][gTTCt][gGAca] is composed of 5 inverted repeats of the nGAAn pentanucleotide with a 11/15 match to the consensus sequence and HSE3,[aGcAc] [tTTCa][gGAgc] is composed of 3 nGAAn units with a 7 / 9 match to the consensus sequence.


The promoter activity of the rat hsp70.1 gene upstream region was investigated by transfection of the Fe33 rat hepatoma cells with the phsp70(950)-CAT6 plasmid. In this expression vector the 5' end flanking sequences of the hsp70.1 gene (PstI-AeaI fragment, approx. 950 bp) were ligated to the CAT reporter gene (Fig. 5A). Transfected cells contained only trace amounts of CAT activity at 37°C but exhibited high enzyme levels after heat shock followed by recovery (Fig. 5B). These data show that the PstI-AuaI fragment contains a functional heat inducible promoter.

Transcriptional activation of heat shock genes is mediated by the interaction of the specific transcription factor (HSF, heat shock factor) with a specific DNA regulatory sequence (HSE, heat shock element) (for review see, e.g., Ref. 53). As shown above three potential HSEs are localized within 350 bp of the 5' end flanking sequences of the hsp70.1 gene.

Transfection with the plasmid phsp(216)-CAT5 revealed that most if not all regulatory elements which are responsible for heat inducibility of the rat hsp70.1 gene seem to be localized within the 216 bp NcoI-NcoI fragment (nucleotides 156 to 372) of the promoter (Figs. 5B and 4). This fragment contains HSE1 and HSE2, both direct and inverted CAAT boxes and a distal Spl binding site, while it is devoid of a TATA box and proximal Spl binding site. When inserted in front of the TK promoter ligated to CAT reporter gene the NeoI-NcoI fragment confers heat inducibility to this heterologous TK-CAT fusion gene (Fig. 5B).

Ncol 200 R TI'GCGTCGCCATGGCAACACTTGTC RCAACC GGAACAAGC ACTTCCTACCAC CCCCCGCC M TCC G- AT G A C C CCR T C

CAAT HSE Spl R TCAGGARTCCAATCTGTCCRGCGRAGCCCAGATCCGTCTGGAGAGTI'CTGGA CAAGGG CG M GTA C T C AA C

300 HSE II GTAC C CTCAAC ATGGRTTACTCATGGGAGGCGGAGAAGCTCTAACRGRCC~ M A A TCC A G C C G

CARl Ncol Spl II ~ T G G C C C C AA G GC CTC CTC C C GCTC G CTGA'I'fG G C C--C'-m"G-~ AGGGTGG G C M C G GC A

4oo t i t " I j~ II G'GGGCCGGRGGAGGCTCCTTAAAGGEG£AGGGCGGCGCGCRGGRCR£CAGATTCCTCCT NI T A A A G CG TGA A

Fig. 4. Nucleotide alignment of the rat (R) hsp70.l promoter to the mouse (M) hsp70.1 promoter [26]. Nucleotide numbers refer to the sequence of the rat gene (see Fig. 2). The 5' end and 3' end of the mouse hsp70.1 promoter fragment have coordinates 291 and 588 respectively according to nucleotide numbering used by Hunt and Calderwood [26]. Only nucleotides which are different in the sequence of the mouse gene promoter are indicated. Bars indicate the regions which contain sequences known to regulate transcription. Thick and thin vertical arrows indicate the main and minor transcription initiation start sites respectively as determined by primer extension method (see Fig. 3). The promoter fragment contained between the NcoI restriction sites (coordinates 156 and 372, respectively) indicated in the figure confers heat inducibility to a heterologous promoter in the transfection assay (see text and Fig. 5).

A Pstl Ncol Ncol Aual

I ~ ~I~ I I h,p~o ] ~ m A N A

ph~pto(95oj-cete ~ ~ ~1 [ cm

phspTO(21G)-CAT5 ~ ItK I CAT I

B phsp70(950)-CAT6

1 2 3 4 5 6

phspTO(216)-CAT5

1 2 3 4 5 6

Fig. 5. Functional analysis of the rat hsp70.1 gene promoter. (A) Top line shows the structure of the fragment of the hsp70.1 gene with the indication of restriction sites used for construction of the expression vectors shown below. Open box indicates protein coding sequences. Arrow shows the transcription initiation start site and the direction of transcription. Black squares 1, 2 and 3 indicate the TATA box, HSE1 and HSE2, respectively. Construction of the phsp70(950)- CAT6 and phsp70(216)-CAT5 expression vectors is described in Materials and methods. (B) Typical CAT assay with extracts derived from control and heat shocked rat hepatoma Fe33 cells transfected with constructs depicted in part A. Duration of heat shock (HS) at 42°C and the recovery period (R) at 37°C of the transfected cells were as follows: lane 1, HS: 20 min, R: 1 h; lane 2, HS: 20 rain, R: 4 h; lane 3, HS: 45 min, R: 1 h; lane 4, HS: 45 rain, R: 4 h; lane 5, control non HS; lane 6, control HS. Methodological details are described in Materials and methods.

3.3. Identification of the transcript encoded by the rat hsp70.1 gene

Nucleotide sequence similarity between genes which belong to the hsp70 multigene family decreases to- wards the end of the transcription unit and usually the 3' end untranslated regions of various hsp70 genes are highly divergent [54]. This feature allows the determi- nation of the relation between a given transcript and a hsp70 gene under investigation using the 3' end derived hybridization probes.

Heat shock induces in cells of various rat tissues two transcripts the size of which has been estimated by us to be 2.5 kb and 2.7 kb [33,34]. To establish which of these transcripts is encoded by the hsp70.1 gene we hybridized poly(A) + RNA isolated from liver, brain and heart of rats subjected to hyperthermia with two DNA probes. One of these probes, the 650 bp NcoI- NcoI fragment (nucleotides 372 to 1022) derived from the 5' end of the rat hsp70.1 gene, contained 65 bp of

70 K. Lisowska et aL / Biochimica et Biophysica Acta 1219 (1994) 64-72

A

Ncol Nco! i

IT ,l , maN//

5' end probe

0.2 kb

Styl ] Slyl 8

I . i l pA

3' end probe

B Liver Heart Brain

5' end probe

3' end probe

C 15'30'60 ' 30' 60' 30' 60'

Fig. 6. Identification of the RNA species encoded by the cloned rat hsp70.1 gene. (A) Schematic structure of the rat hsp70.1 gene contained in the KpnI (K)-BamHI (B) fragment of the A 68 clone (see also Fig. 1). Open box represents the protein coding sequences, pA indicates the polyadenylation signal, arrow indicates the start and direction of transcription. Bars below the map show the NcoI-NcoI and StyI-Styl restriction fragments used as 5' end and 3' end specific hybridization probes in Northern-blot analysis. (B) Northern-blot analysis of the hsp70 gene expression in liver, heart and brain of rats subjected to hyperthermia. Poly(A) + RNA isolated from indicated tissues was fractionated in 1.2% agarose-2,2 M formaldehyde gel and bloted onto Hybond-N. Each lane was loaded with 3 p~g of RNA. Blot was hybridized first with the 5' end specific probe, then after exposition the probe was stripped off and the filter was hybridized with the 3' end specific probes. Under each lane the duration of hyperthermia is indicated. The C indicates RNA from control rats. Metodological details are described in Materials and methods.

the promoter, 224 bp of the leader and 361 bp of the coding sequences (Fig. 6A). The other probe, the approx. 400 bp StyI-StyI fragment (nucleotides 2540 to approx. 3100) derived from the 3' end of the gene, contained 46 bp of coding sequences and approx. 350 bp of 3' end nontranslated sequences (Fig. 6B). Hy- bridization results presented in Fig. 6B show that the less discriminating, 5' end derived probe, hybridized efficiently to both 2.5 kb and 2.7 kb transcripts. In contrast, only the 2.5 kb mRNA species was detected with specific 3' end derived molecular probe thus iden- tifying it as a product of the rat hsp70.1 gene.

4. Discussion

The rat genome contains multiple hsp70 gene-related sequences [50]. While many of them presumably

correspond to pseudogenes [31], there is no doubt that a significant fraction of these sequences may represent functional genes. However, so far only three functional genes, the hsc70 gene [31], the grp78 gene [30] and the testis specific hst70 gene [32] were isolated. Here we reported the isolation and nucleotide sequence analysis of the heat inducible hsp70.1 gene.

The data presented show that the gene isolated by us has a functional heat shock promoter and that it is expressed at high levels in various rat tissues in response to hyperthermia. This gene is not active (or is transcribed at a very low basal level) at physiological temperature (Figs. 3 and 5), nor is its expression in vitro stimulated by serum (Z. Krawczyk, unpublished results). Our previously published results also show that the hsp70.1 gene is not activated in a cell-cycle-dependent manner at least in vivo during regeneration of rat liver [34].

Two hsp70 transcripts appear in rat tissues after heat shock. These transcripts are about 2.5 kb and 2.7 kb as estimated by us [33,34], while somewhat different estimations for apparently the same transcripts were reported by others (e.g., 3.05 kb and 3.53 kb, respectively [55]). Using 3' end specific probe we showed that a smaller transcript is derived from the hsp70.1 gene we cloned. The hsp70.1 mRNA size calculated from the length of its transcription unit corresponds to approx. 2.49 kb. plus poly(A) tail, so it seems that the real length of the transcript lies presumably between the estimations mentioned above.

The origin of the heat induced transcript of higher size is not known at present but it is reasonable to assume that it is coded by a gene different than the hsp70.1 gene. First, one can expect that similar to the case of the human and mouse hsp70 gene family (see Introduction), the rat genome also contains more than one heat inducible hsp70 gene. Second, our previous Southern analysis of the genomic DNA indicated the existence of two hsp70 genes [50].

Both the hsp70.1 gene and the putative one generat- ing a longer transcript seem to encode an identical or highly similar protein as only one strictly heat inducible protein of 70-72 kDa is usually found in rat cells after heat shock (e.g., Refs. 56-58).

There is the evolutionary interesting question which of the human and mouse heat inducible hsp70 genes are counterparts of the rat hsp70.1 gene.

So far four heat inducible hsp70 genes were isolated from human genome (see Introduction). Three of them show significant differences in the promoter region when compared to the rat hsp70.1 gene. The hsp70/hsp70.1 promoter responds to serum stimula- tion and is activated in a cell-cycle-dependent manner [20,21], while in turn the promoters of the hsp70B and the hsp70B' genes do not contain a TATA box [17,18]. The best candidate for a human counterpart of the rat

K. Lisowska et aL / Biochimica et Biophysica Acta 1219 (1994) 64-72 71

hsp70.1 gene seems to be therefore the hsp70.2 gene. The murine hsp70.1 gene is undoubtedly a direct

counterpart of the rat gene described in this paper. Both genes are 97.2% identical in the coding region. Even more important, the promoter region, the leader sequences as well as the first 38 bp of the 3' end nontranslated sequences of both genes are also highly similar (85% in the promoter, 82% in the leader, and 87% in the 3' end, respectively).

The similarity of the expression pattern and the similarity of the structural features between the rat hsp70.1 gene, the human hsp70.2 gene and the mouse hsp70.1 gene can possibly extend also to their chromo- somal location. Three human genes hsp70.2, hsp70.1 and hsc70t/hsp70-HOM [19,59], and three mouse genes, hsp70.1, hsp70.3 and hsc70t [60,61] were found to map at a corresponding locus within the class III region of the major histocompatibility complex (MHC). Hsp70 gene-related sequences were also found at MHC in the rat [62] and recently this locus was shown to contain three hsp70 genes [63]. It is thus tempting to speculate that the hsp70.1 gene isolated by us is one of two heat inducible hsp70 rat genes localized in MHC.

Acknowledgements

We are grateful to Dr. M. Boshart for gift of plasmids pBLCAT5 and pBLCAT6, to Dr. P. Fort for gift of the plasmid pRGAPDH, to Dr. I. Huhtaniemi and Dr. P. Kraj for providing us with several oligonucleotides, to Dr. W. Schmid for gift of Fe33 cells and to Dr. M. C h o r ~ for critical reading of the manuscript and comments. We thank Dr. I. Huhtaniemi also for a gift of several chemicals and enzymes. We thank Mrs. K. C h o r ~ for skillful technical assistance, and Mrs. M. Krawczyk for maintenance of cell culture. This work was supported by a grant from the Polish National Cancer program CPBR 11.5.

Note added in proof (received 13 April 1994)

Additional recent information on rat heat inducible hsp70 genes has appeared. Cloning of cDNA of a rat heat inducible gene was reported by F.M. Longo, et al. [64]. Also, a gene which corresponds to the one described by us has been recently isolated from rat genomic library and sequenced (E. Gunther, personal communication).

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Documents

Cloning, nucleotide sequence and expression of rat heat inducible hsp70 gene