Eur. J. Biochcm. 204. 893 - 900 (1997) K, FEBS 1992

Molecular cloning and characterization of a constitutively expressed heat-shock-cognate hsc71 gene from rainbow trout Muhammad ZAFARULLAH, Jan WISNIEWSKI. NicholasW. SHWORAK, Steven SCHIEMAN, Santosh MISRA and Lashitew GEDAMU Department of Biological Sciences. University of Calgary. Canada (Received Septcrnber 30/November 26. 1991) - EJB 91 1301

A rainbow trout major heat-shock-protein-like gene (hsp 70) and corresponding cDNA clones were isolated by hybridization to heterologous hsp70 probes. D N A sequencing revealed that this gene is structurally similar to a mammalian heat-shock-cognate hsc70 gene and consists of eight introns. Northern blot and primer extension analyses showed that the corresponding mRNA is constitutively abundant in different trout tissues and salmonid cell lines. Fragments of the isolated gene containing the -900 - + 30 and -217 - + 58 sequence were linked to a bacterial chloramphenicol acetyltransferase reporter gene and transiently transfected into salmonid cells. The expression pattern of these constructs supports our conclusion that the isolated genomic and cDNA clones correspond to a trout heat-shock-cognate hsc70 gene.

All organisms synthesize a few evolutionary conserved proteins (called heat-shock proteins or HSP) in response to various harmful environmental stresses [l, 21. The most abundant and conserved HSP has a molecular mass of about 70 kDa. In eukaryotes, HSP70 proteins are encoded by a few heat-inducible hsp70 genes and usually their basal expression is negligible. Howcver, even without stress eukaryotic cells contain high levels of structurally related heat-shock-cognate proteins (HSC) encoded by constitutively active hsc70 genes [3, 41. The grp78 gene coding for glucose-regulated protein [5] and two mammalian testis-specific hsp70-related genes [6 - 81 represent other members of that so-called hsp70 multigene family. Major information on the hsp70 gene family slructurc and regulation was accumulated from studies conducted on mammals, fruit fly and yeast. However, little is known about such genes in fish which arc important economically and are affected by such adverse environmental changes as water pollution. Iri vifro studies showed that cultured cells of chinook salmon and rainbow trout synthesize HSP70 and corresponding inRNA in response to hyperthermia, heavy metal ions and sodium arseiiite [9 - 131. So far, two partial cDNA clones were obtained from arsenite-treated rainbow trout gonad (RTG-2) cells which show extensive identity with fruit fly and yeast hsp70 genes [12]. Our studies on salmonid cells revealed a high basal level of the 70-kDa band on SDS protein gels corresponding to the position of HSP70 accumulated after hyperthermia [13]. To obtain a further insight into molecular mechanisms involved in the constitutive and stress-inducible regulation of the rainbow trout (Salmo gairdneri) hsp70 ___ C'orrrsponderirt~to L. Gedaniu. Department of Biological Sciences, University of Calgary, 1500 Univcrsity Dr. N W . Calgary, Alberta. Canada T2N IN4 Ahhrc3viations. CAT, chloramphenicol acetyltransfcrase; HSC, heat-shock-cognate prolein: HSP, heat-shock protein.

multigene family. we decided to clone the corresponding genes. Here. we describe for the first time the complete structure of the fish hsp70-related gene and its cDNA. Expression analyses of the endogenous gene and transfection studies (in fish cells) of constructs containing an upstream region of the cloned gene revealed that the isolated sequence represents a heat shock cognate gene. MATERIALS A N D METHODS Trout DNA isolation and Southern blot analysis Total genomic DNA was isolated from rainbow trout testes by a proteinase K/phenol extraction [14]. Samples coiitaining 10 pg DNA were digested with restriction nucleases, fractionated on 0.8%, agarose gel and capillary transferred onto nitrocellulose [15] or a Zeta Probe membrane [16]. Hybridization was carried out at 60 "Cin 3 x buffer A (1 x buffer A = 0.15 NaC1, 0.3 Tris/HCl, 2 inM EDTA). 10 x Denhardt's solution [17], 0.5% SDS, 0.1 OO/ sodium pyrophosphate, 10 pg/ nil poly(A), 250 pg/ml total yeast R N A and 50 pg/ml shcared, single-stranded Esciirrichin coli DNA. Filters were then washed at 60°C in 1 x buffer A (fruit-fly hsp7O probe) or 0.1 x buffer A (trout probes) containing 0.1 YOSDS and 0.1"/0 sodium pyrophosphate, and exposed to X-ray film at - 70 'C using intensifying screens. Gene library screening, DNA subcloning and sequencing The trout genomic library in a Charon 4A vector [18] and the lambda g t l l cDNA library from trout testes [ 191 were screened [20] under conditions described above. Recombinant phages were plaque-purified and their D N A was isolated by the DEAE-cellulose method [21]. The insert was analyzed by digestion with restriction enzymes and Southern blot hy-

894 bridization. Fragments of interest were subcloned into pGEM-2 or the pUCl3 vector; the plasmid DNA was purified by the alkaline-lysis method [22] and sequenced [23] with T7 DNA polymerase (Pharmacia), [ C ~ - ~ % ] ~ Aand T P universal M13. SP6 and T7 primers or custom synthesized internal primers. Reaction products were fractionated on 6% polyacrylamide, 7 M urea gels and analysed by autoradiography. RNA purification and analysis

Total RNA was extracted from fish cells and tissues by guanidinium isothiocyanate/CsCl centrifugation as described previously [24]. RNA samples were fractionated on 1.2% agarose. 10 mM methylmercury hydroxide gel and electroblotted in 30 mM sodium phosphate, pH 6.8, onto a Zeta Probe membrane. Hybridization was carried out as described above. RN A primer-extension analysis and sequencing

A 20-mer oligonucleotide 5’-dTGGTCCCTTAGACATGTTAC-3’ was 5’-end labelled with [ Y - ~ ~ P J Aand T PT4 polynucleotide kinase. The above primer (4 ng) was annealed to 40 pg total RNA from gills for 45 min at 50°C in lop1 buffer consisting of 0.4 M NaCI, 10 mM Pipes, pH 6.4 and used for primer extension [25]. For sequencing, 2 p1 of the above primer-RNA hybridization solution was mixed with 3.3 p1 reaction buffer containing dNTP and 6 U AMV reverse transcriptase. and transferred into each of four tubes containing 1 p1 corresponding ddNTP (7mM ddATP or 1 mM ddTTP, ddCTP or ddGTP). The sequencing reaction was carried out at SO’ C for 45 min and the products were analysed as described for DNA sequencing. Molecular probes

Antisense cRNA probes were synthesized with T7 RNA polymerase [26] and [ c ~ - ~ ~ P ] Cusing T P the pGEM-1 plasmid containing a 1-kbp PstI fragment from fruit fly hsp70 cDNA [27]. the pGEM-2 plasmid derivatives containing various restriction fragments of the trout gene cloned in this study or the pTZ 19R plasmid containing trout a-tubulin cDNA (a gift of Dr. G. H. Dixon). Plasmid p68/1.8 containing the 5’ half of a rat hsp70 gene [XI was random primed with Klenow polymerase and [LF~~PI~CTP.

trout gonad cells (RTG-2) [33] were grown at 18°C under 5 % C 0 2 in Eagle’s minimal essential medium containing Earl’s salts, 5% fetal calf serum, 0.1% NaHC03 and antibiotic/ antimycotic solution (Gibco). Semiconfluent cells were transfected for 5 h at 22°C with 20 pg supercoiled plasmid DNA co-precipitated with calcium phosphate [34], glycerol-shocked (RTH cells only) and grown in fresh medium for 36 h. Cells were heart shocked at 28 “C for 1 - 6 h or exposed to 150 pM ZnCI,, 20 pM CdC12 or 50 pM sodium arsenite for 6 h, then harvested and sonicated. Samples containing 25 pg (RTH-t49 and RTG-2) or 50 pg (CHSE-214) of protein were used for the enzyme assay [35] using ‘‘C-chloramphenicol as a substrate. Reaction products were identified by autoradiography after thin-layer chromatography on silica gel.

RESULTS Isolation of trout hsp70-related genomic clones

Hybridization of trout genomic DNA to radioactive fruitfly hsp7O cDNA, revealed the presence of a few hsp70-related sequences in the trout genome (unpublished results and [12]). To isolate these putative genes, the trout genomic library (approximately 2 x lo5 plaques) was screened with the above probe at medium stringency, yielding four positive recombinant clones. Preliminary restriction mapping and Southern blot analysis of their DNA showed that they all contain the same hybridizing, 5.4-kb long Hind111 fragment (unpublished results). This segment was later subcloned into the pGEM-2 vector and subjected to detailed restriction mapping. further subcloning and sequencing. Subsequent analysis of the DNA sequence revealed that the 5‘-end of the gene was not present in the 5.4-kb Hind111 region, so an overlapping 2kb AvuI fragment was also analyzed in detail. The restriction map and general structural organization of the entire isolated trout gene is depicted in Fig. 1. The DNA sequence of this gene is also presented in Fig. 2. Isolation of hsp7O-related cDNA clones

The trout testicular cDNA library (5 x lo5 plaques) was independently screened with a radioactive rat hsp70 probe and about 400 positive clones were detected, but only four of them were further analysed. One of these cDNA clones

Construction of chloramphenicol acetyltransferase (CAT) hybrid genes

+

The -90030 region of the cloned trout gene was amplified in vitro using the polymerase chain reaction technique [29] from the @EM-2 plasmid carrying the AvaI - Sa[I region of the trout gene (nucleotides - 900 - 271) using SP6 primcr and a primer 5’-dCCTTTCTTCGCCTTGTGTGTTGTG-3’ complementary to the + 30- + 7 region. The resulting DNA was cleaved with SslI and cloned into the Ssti site of pGEM-2 CAT plasmid [30]. The -217- + 58 region of the trout gene was excised with XJnnl and Sau3Al. endfilled with Klenow polymerase and subcloned into the SsrI SwaI site of the pCEM-2 CAT plasmid. The primary structure of both constructs was confirmed by sequencing.

+

Cell culture, transfection and the CAT assay

The Chinook salmon embryonic cells (CHSE-214) [31], rainbow trout hepatoma cells (RTH-149) [32] and rainbow

Fig. 1. Structure of the isolated trout k 7 1 clones. Various DNA regions arc depicted on the restriction map of the genomic DNA (top) and full-length cDNA clone (bottom) as a thin linc for the uncoding and intcrvening sequences, and hatched and solid boxes for untranslated and translated parts of the exons. respectively. Translational start and termination codons are marked as ATG and TAA. respectively. and A, denotes the cDNA poly(A)-rich tail. Thc sequcncing strategy is illustrated by horizontal arrows.

-900

ccuauqgctagcaacgcaaccaaggqctagcaatgtcagtaaagctagtcacctggaccacgaqttgccgaagattgttcagtgtttcaqagtttaaaaa Ava I

-801

-800

HSE tqtqttttaaataatttaagaactqcaaatgcaatgctcacttataatttaataattacaattttgcttcctatq?cggqcaaggqaqqtctg~ttgtcq -701

-700

HSE gccatqctgqaaatgtqatctcqcqttattgtgccattgtcaatacagtacatctacaccacgatatgaaacgttgatttgaaccttctgactcacttqt -601

-600

qaattagacqgqccccatggtctgatttaaatcqcatgatttctgatgatagttgtcgaatatatcqactgattqcattacctcatccccatactgtatt -501

-500

MRE MRE MRE HSE tatttatttatcttgctcctttgcacctcaqtatctctacttgcacattcatcttctgcacatciaacattccagtgtttaattgctctattqtaattac

HSE

-401

MRE -400

ttcgccaccatggcctatttattgccttaacgctcttatcttac~tcatttgcactcactgcattctttttctactgtattattqactgtatgttttgtt -301

-300

tattccatgtgtaactctgtgttgttgtaaatgtcqaactgctatgatttatcttggccaggtcgcagttqcaaatgauaacttqttctcaactaaccta - 7 0 1 XmnI

MRE -200 cctqgttaaataaagqttaaataaaaaaataaataaataaaaagtgtatccggtgcgaacgcaacccgacqtcttcatgqgaagtqgqagqgaqcaacaq - 1 0 1

CAAT HSE CAAT TATA-box -100 cctqtgtttttattggtattttggaatgtcaagttgaatcctqagctatqattggttgccttgccqtttatatagtaatggtagacttatccttccttct

-1

+ 1 TTCTCTCACAACACACMGGCGAAG~GGGAGGCTTCGCCATTGTTCAACTCCGATCAACATCAGCATCACCTTCGGTC~TAATTTATTCGqtaagt 100

.......................................................................................

C''"'t

101 aagaagtagtttaatttaatagtaatgtaataaagaaagccttttttttttagttcaccgtaatttattttgctgaccacaaqcaggtacctcttatatc 200

2 0 1 cqqaagacaaggctgrJctaacgtaaattaactactacttqqccttgtagttttaactaatatcacctautcuacaaattaaaccttgtaaaacttacctaact

300

6alI 3 0 1 gatctagctttaactcgqaattactgcaatatatttacatgttctatcctatttgatatcatttaattqttaacgttyggcaactagttaqcttttggtt

400

401 ggcctttgcttgctaactaatcggtcatcctttaatgtcqtqqttaatatcacaqctaataattatttgattgacattcgatcqacaqtttcaaacagtt 5 0 0

5 0 1 ttgtcactqcyctaggtaacaacggttgtatgcggtataccagqatatgqcgggcgaccaccctcgtgttcggcctagttctauagactctqcaccccgc 6 0 0

XbaI 601 gtqatqtgacgcacaaaaggacqtccgtagttctttctgaagtgtaataccqqtacataccgttatctqttqctqaaqttgqtqtcgacatgagcacctt 700

701 atcttgaattaaaccqttttattgqttagtcgatactttagttttgctgaccttattctcataqgctgttatatcttaaqatctctttatagcqatcact

800

801 attttqagcqaagqacttaaaataagggaagtttattgtttttctagtcaatggtatcaaatcaattgaattaactttttgqgcgactgacqgtaattcg

900

901 catgcqaqttatagaqctctcattgasaqtcttaaaagcggaqqgtctg~tataggtgcgccatttctatgcttccgggtcacaaucttctttctgcttc 1 0 0 0

Hind111 1 0 0 1 tactttctattttctgaaagtatttttggctttggaaaaaaattcagtagtttcgatgqtqcgaccqqtctaccctatagctgttqcataaactqtattt 1100

1 1 0 1 ggqaaaatatcagttttggcaaccaggtaatgqctgcggtttatgcttagtggctttaaatttaacttattatttcaaaccactaattgdctgactttgq 1 2 0 0

1 2 0 1 ttcaattatttqaqqaatattttcccctqt~agtgcqctcgctqccqtttcttgqqtgaaatcaqatccagcctgaactgtqccatgtagtaggt1300 EstI

1 3 0 1 aqtttgtaacttccaacgqqccaatttcctacacatttqagaacatgtqgtaattaaccacaatgactaattqtttatctacattcatccctqcccctqa 1400

1401

1500

1501

IGOO

1601

1700

1701 tctacaatgtgcayttggqcctagaggqgtaactaatgtttac~aatgacc~atgtattttgatgaccacattqgcttctagtgactgctgatattttta 1800

IB01

AcCI H S K G P A V G I ctgcqtttgttgtattcttqcatagtgtccatttqttacctaacaccatgctgttcttcattqtccagGTAACATGTCT~GGGACCAGCAGTCGGCATC 1 9 0 0

................................

D L G T T Y E C V G V F Q H G K V E I I A N D Q G N R T T P S Y V 1901 GAT~ACCACCTACTCCTGCGTGGGTGTGTTCCAGCATGGCAAGGTTGAAATCATTGCCAACGACCAAGGCAACAGGACCACTCCAAGCTACGTTG2 0 0 0 .................................................. A F T D S E R L I G D A A K N Q V A H N P C N T V F 210u 2001 C C T T C A C T G A C T C T G A G A C G C 1 ' C A T C G G T G A T G C T G C C A C A C A G T A T ~ C G g t a c g t t t t t a c a t t c t g g t a a

..............................................................................

2200 z i n i tgtctcctaaaataqgtaqattgttatttataattttt~~ctttqt.qqcaatgcttgttcactqtgatgaaqtttattcctgccattcgtagtttccacc 2201

agaqgttgaaaqaccaaagatggcccaataccttccttggdtgtctgaqtgatacqccatttaactcacttqactctatcgctqtaqaaaatqctaaatc

2300

D A K R L I G R R F D D G V V Q S D M K H W P F E V I 2400 2 3 0 1 taactggctgtcttgttqcagATGCTAAGAGACTAAGAGA~GATTGGCCGCAGGTTTGA~GATGGAGTTGTTCAA'~CGGACAT~AAGCATTGGCCCTTTGAAG'rTAT

...............................................................................

N D S T R P K L Q V E Y K G E T K S F Y P E E I S S H V L V K H K 2 4 0 1 CAATGATTCTACTCGGCCTAAGCTCCAAGTTGAATAtAAAGGAGAGnCTAAGTCCTTCTACCCAGAAGAAATTTCATCTATGGTTCTGGTCAAGE\TGFIAG

....................................................................................................

2500

E I A E A Y L G K 2 5 0 1 GAGATTGCTGAGGCCTACCTTGGG~gtaagtattgtgtggctgqttctqtaacaqqaaggtgtttttaqtttaccatgactggtctattqg~ttatt~ 260U

...........................

Fig. 2.

2601

T V N N A V V T V P A Y F N D S Q R Q A T K D A 2 7 (I 0 aaatqcatgtacattttttctcectagACTGTCAACAATGcTG'rTG'~'1ACCGTACCTGCCTACTTCAATGACTCCCAGCGCCAGGCAACCAAACA'~GC~G

.........................................................................

G

T

I

S

G

L

N

V

L

R

I

I

N

E

P

T

A

A

A

I

A

Y

G

L

D

K

K 2800

2 / 0 1 GTACCATCTCGGGGCTGAATGTGCTGCGTATCATCAATGAGCCAACTGCI'GCTGCCATTGCCTACGGCCTGGACAAGAAG~tCCattaCttgata

.......' A " . . . . . . ' . . . . . . . . . . " . . . " ' . ' ' . . . . . . . . ' . . . . . . . . . . . . t . ' ' . . ' . . . . . . . . . . . .

HpaI

?no1

2900

2901

3000

JOOI

V G A E R N V L I F D L G G G T F D V S I L T I E D G I F E V X CaqGTCGGTGCTGAAAGGAATGTCCTTATCTTTGATCTGGGTGGCGGCACCTTTGACGTGTCCATCTTGACCATCGAGGATGGCATCTTTGAGGTCAAGGTCAAGT 3 1 0 0

.................................................................................................

S T A G D T H L G G E D F D N R M V N H F I A E F K R K Y K K D I S ? l o 1 CCACTGCTGGAGACACTCATCTGGGTGGAGAAGnCTTTGACC~CAPGGTCAACCACTTCATCGCGGAGTTCAAACGCAAGTACAAGAAAGACATCAG3 2 IJ li ............. " . ' . ' . . ' . . . ' . . ' " . ' . . . . . ' . . ' . . . . . . . ' ' . . . . ' . . . . . . . . . . . A . . . . " . . " . . . . . . . . . . . . . . . " . . D N K R A V R R L R T A C E R A X R T L S S S T Q A S I E I D S L 3300 3 2 0 1 CGACAACAAGAGGGCTGTTCGCCGTCTCCGCACCGCATCTGAGAGGGCAAAGCGCACCCTGTCCTCCAGCACCCAGGCCAGCATCGAGATCGACTCTTTG

...

................................................................................................... Y

E

G

I

D

F

Y

T

S

I

T

R

A

R

F

E

E

L

N

A

D

L

F

R

G

T

L

D

P

V

E

K

S

3 3 0 1 TACGAGGGAATCGACTTCTACACCTCCATCACCAGGGCTCGCTTTGAGGAGCTCAATGCAGACCTTTTCCGTGGCACCCTTGACCCAGTGGAGAAATCCC 3 4 0 0

...e.'... .............................

......................................................... L R D A K M D K A Q V H D I V L V

G

G

S

T

R

I

P

K

I

Q

K

L

L

Q

D

F

F

3 4 0 1 TCCGCGACGCCAAGATGGACAAAGCCCAGGTACACGACATCGTCC'PGGTCGGAGGCTCCACTCGTATCCCCAAGATCCAGATCCAG~CTGCTCCAAGATTTCT'r

..................................................................................

3500

N G K E L N K S I N P D E A V A Y G A 3 5 0 1 CAACGGCAAAGAGCTCAAC~AAGCATCAACCCCGACGAAGCTG'rGGcC~ATGGCGCAG~tgaatcactgataatcttqtttqctctqtqqccaatgqqc 3600 sstI

...........

3601

............................................

3700 ttctataatqagccttaaatcaataqtttcqccgt~atqaaqtttaatcctgccattcqtaqtttccaccaqa~~ttqaaagaccaagatg~cccaata~

A

V

Q

A

A

3701 C t t C C t t g g a t g t C t g a g C g a C a a t g c g t c t a c a 9 t a a t g t t t t t C ~ a t a t a t C t a a C C t c C C a t t C t c a t t t t c a q C T G T C C A C G C A G C C P

...............

3801

3800

I L S G D K S E N V Q D L L L L D V T P L S L G I E T A G G V M T V TACTCTCAGGTGACAAGTCTGAGAATGTCCAGGACCTGCTTTTGCTGGACGTCACACCCCTCTCCCTGGGTATTGAGACCGCTGGAGGTGTCATGACCG? 3 9 0 0

. . . ................................................................................................ L

I

K

R

N

T

T

I

P

T

K

Q

T

Q

T

F

T

T

Y

S

D

N

Q

P

G

V

L

I

Q

3 9 0 1 CCTGATCAAACGTAACACCACCATCCCAACCAAGCAGACTCAGACC'rTCACCACCTACTCAGACAACCAGCCTGGTGTGCTCATTCAGqtqagqqgqgcc 4 0 0 0

........................................................................................

4001 tqcaaqaaatgtccatttcaqggcaatgtttcctcattcctttggccactatctgatqccqtttactcct~caatttgttqtttccaccaqcgqttgaaa4 1 0 0 4101

4200

V Y E G E R A M T K D N N L L G K F E L T G I P P A P R G V P 4 2 0 1 CctccaqGTGTATGAGGGTGAGAGGGCCATGACCAAGGACAACAACCTGTTGGGCAAGTTTGAGCTGACTGGAATCCCCCCTGCACCTCGCGGTGTTCC?

................................................................................

4300

"'t""'"" I T I T

4301

Q I E V T F D I D A N G I M N V S A A D K S T G K E N K N CAGATTGAGGTCACATTTGACATTGATGCTAACGGCATCATCATG~CGTGTCTGCTGCTGAC~GAGCACTGGGAAGGAGMCAAGATCACCATCAC~TG 4400 t................................................................... D K

4401

ACAAGGgtaatgttgqatggcattgtagtttaacactggaaaatg~gctat~~ttactaqccctqqccccaatagtttcqctgt~atgaagtttactcct 4500

................................

......

11501 gccattcgtagtttccaccagaqgtcqaaagaccaaagatq~cccaataccttccttgqatqtctqa~cqacaaatctagtaca~ttaatt~ta~tttaq 4600

G

R

L

S

K

E

D

I

E

R

M

V

Q

E

A

E

K

Y

K

C

E

D

4601 gtCaaacattggttgatttgattattttCCattagGTCGCCTGAGC~GGAGGACATTGAGCGCATGGTCCAGGAGGCTGAG~GTACAAGTGTGAGGAT $-/on

.................................................................

D

V

Q

R

D

K

V

S

S

R

N

S

L

E

S

Y

A

F

N

M

K

S

T

V

E

D

E

K

L

Q

G

K

I

4701 GATGTGCAGCGTGACAAGGTCTCTTCTAAGAACTCCCTAGAGTCCTACGCTTTCAACATGAAGTCTACTGTGGAGGAGGATGAGAAACTGCAGGGGAAAATCA 4 8 0 0

.....a...............................................................................

S

D

E

D

K

T

K

I

L

E

K

C

N

E

V

I

G

W

L

D

K

N

Q

---- ........... Ps+T

4 8 0 1 GTGACGAGGACAAGACTMGATTCTGGAGAAGTGC~CGAGGTCATCGGGTGGCTGGAC~G~CCAGqtaagaatttaattqggqtqacttqtactqtc 4900

............ ................................ . . . . . . . . . . . . . . . . . . . . . . . .

4901

SO00

5001

.lo1

5100

E L E K V C N P I I T K L Y Q G A G G X P G G M P E G M A G G F P GAGTTGGAGAAGGTGTGCAACCCCATCATCACCAAGCTGTACCAGGGTGCTGGTGGGATGCCCGGCGGTATGCCTGAGGGCATGGCTGGCGGATTCCCTG

....................................................................................................

G

A

G

G

A

A

P

S S G P T I E E V DStop 5 2 0 1 GAGCTGGTGGTGCTGCTCCTGGAGGTGGTGGATCATCTGGACCAACCATTGAGGAAGTCGACTAAACATTCCATTGTTTCTGCAACTACCTCCCCCTAAC 0

G

G

.........................................................

5301

5200

G

SalI'

......................................

5300

RAGCAAAGTTTGAAATGTGTTCTACCCTCTTGTGTCTGAGTCTCG'rGACTTGTCAAGGAGTGAAATTTGAAGATCCATAAAGGTTGGGGGATCACATATTTTTC~ 5400

....................................................................................................

................................

5401 CTTTGTTTGC~ACGCAACAGGGAACAGTATTGTC'rGTTAGCACAACTAAGCTGTCAAATTTCACATGTGTATTTTCTCAGTTGTCATCTGCCTTTGTCA510C

T'

..................................................................

poly(A) s i s n a l 550 1

CAATGCCTTTAGGACAARAATAAAATATACGGTGACCTTCCACCTTTGACTTCTaa~tgtttggqtctttacactactttqqaaaaqggaaqa tgttagq 5 6 0 0 ' F O l y ( A ) tail

.....................................................

5700

5701

qtcctqcqtccattcatacctqaccaatttttttttqaaLgtqqta~tqcttcatatctataaccattqgaaacacatcaattgctttdaqtqdct~ccg5 a 0 0

5801 actcattttggtaaattatcctcqtgtgcctaaccaaaqcdda~a~~c~cc~tqqcctatggaaacacaattgqtcagttqatadatacaaqgactqccact 5900 5901 gcatgaqaqtttaataaacaaactt

Hind111

Fig. 2. Nucleotide sequence of the trout hsc71 gene. Lower case lettcrs are used for uncoding and intron regions, and capital letters for exons. The lower row shows the cDNA sequence; (.) nucleotides identical with the genomic sequence; ( - ) deletions; capital and lower case letters denote cxchanges found in all or only in some of the cDNA clones analysed, respectively. Encoded amino acids are listed above the sequence in bold. Putative promoter elements and the polyadenylation signal are bolded and the restriction sites shown in Fig. 1 are underlined.

897 represented a full-length copy of hsp70-like mRNA, while the corresponding 5’-end region was absent in other clones. All four cDNA clones share the same restriction map (Fig. 1) and their sequence is almost identical; only a few bases in the untrdnslated region and degenerated codon positions are substituted. The consensus DNA sequence of the above cDNA is listed in Fig. 2. Structure of the trout hsp70-related gene Comparison of the sequence of genomic and cDNA clones revealed that the isolated trout gene contains eight introns ranging from 100 bp (intron 3 ) to 1774 bp (intron 1 ) (Figs 1 and 2). The size of the nine exons varies from 94 bp (exon 1) to 556 bp (exon 5). A computer search for an open reading frame localized a putative initiator ATG codon 5 bp downstream from intron 1 (position 1874). The protein coding region is composed of651 codons (see below) and ends with a TAA triplet at position 5263 in exon 9. The sequence of two cDNA clones showed a polyadenylation site after nucleotide + 5554 while the third sequence contained a poly(A)-rich tail after nucleotide 5547 (in the fourth clone an EcoR1 linker used for library construction was present after nucleotide 5553). The 3’-untranslated fragment of the trout gene contains a standard polyadenyhtion signal at position 5519. An additional AATAAA sequence was found 5913), but no cDNA clone further downstream (position extends to that region.

+

+

+

+

+

+

Localization of the transcription initiation site To confirm the position of the 5‘-end of the cloned trout hsp70-like gene deduced from a cDNA sequence analysis (Fig. 2), we used reverse-transcriptase-catalysed RNA sequencing. The reaction was performed from an end-labelled 20-mer oligonucleotide primer complementary to the beginning of exon 2; total trout RNA (Fig. 3), as well as poly(A)rich RNA (not shown) were used as templates. The resulting sequence ladder as well as the length of the major primerextension product shown in Fig. 5C, supports our conclusion that transcription of the isolated trout gene starts at nucleotide T marked as + 1 in Fig. 2. Structurc of the putative protein product of the cloned trout gene Fig. 2 also shows the primary structure of the protein encoded by an open reading frame contained in the cloned gene. The calculated molecular mass of this trout HSP70-like protein corresponds to about 71.3 kDa. Comparison of its sequence with the human constitutively synthesized HSC70 and heat-inducible HSP70 proteins is presented in Fig. 4. While the overall similarity between them is very high, the putative trout protein is closest to the human heat-shockcognate HSC70 protein. Constitutive expression of the isolated trout gene

To investigate the expression pattern of the isolated trout gene, Northern blot analyses of total RNA from normal and heat-shocked cells were probed with two different radioactive fragments of the trout hsp70-like gene. While the probe from evolutionary highly conserved exons 6 and 7 (nucleotides + 3784 - + 4266) detected a single RNA band of about 2.2 kb whose intensity increased after heat shock (Fig. 5A), an equal

Fig. 3.5‘-end localization by RNA sequencing. Total RNA from trout gills was used as a template for oligonucleotide-directed sequencing as described in Materidis and Melhods. Products of the standard primer-extension reaction (PE) are shown as a marker. The sequence obtained from a gel (bold, location of a band corresponding to the first nucleotide in each row is marked by an arrowhead on the autoradiogram) is compared to that of genomic DNA (first nucleotide in each row is numbered according to Fig. 2).

intensity of a 2.2-kb band was observed with a more specific probe from the very 3’-end of the gene (nucleotides + 47855262) (Fig. 5B). To elucidate that result further, primer extension experiments were carried out with the 20-mer primer mentioned above. Synthesis of two fragments of about 114 and 116 bases was observed even on RNA templates isolated from uninduced cells and no significant signal increase was observed after heat-shock, heavy metal ions or arsenite treatment (Fig. 5C). To estimate the level of constitutive expression of the cloned gene in different trout tissues, the Northern blot containing total RNA from various trout tissues was cohybridized with the probe derived from exons 8-9 of the isolated trout gene and with the trout a-tubulin probe (Fig. 6A). The detected hsp70-like RNA was found to be very abundant in all tissues examined, exceeding the level of a-tubulin mRNA. A

+

898 t H s C 7 1 M S K G P A V G I D L G T T Y S C V G V F Q H G K V E I I R N D Q G N R T T P S Y V A F T D S E R L I G D A A K N Q V A M N P C N T V F I N D S T R 100 h H S C 7 1 ..............................................T.................................A...........M.V..A G. 1 0 0 h H S P 7 0 . A . A A .........................................T.............L..Q............K.G.P...........Q.... GDK 1 0 0 t H S C 7 1 P K L Q V E Y K G E T K S F Y P E E I S S ~ L ~ E I A E Y L G K T V N N A W T V P A Y F N D S Q R Q A T K D A G T I S G ~ I I N E P T A A A I A Y G L D K K V G A E R N V L I F2D0L 0 V. T..............T........................A................................... 200 hHSC71 V 200 hHSP70 V S......A...........T...........YP.T...I..................V.A.....................RTGKG.........

..

............... ....

.. ..

t H S C 7 1 GGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNHFIAEFKRKYKKDISDNKRAVRRLRTACERAKRTLSSSTQASIEIDSLYEGIDFYTSITRA 300 hHSC71 H. E. 300 hHSP70 D A...............L....VE.....H.....Q..........................L.....F............ 300

................................................ ............ .......

.... ............................................

t H S C 7 1 R F E E L N A D L F R G T L D P V M S L R D ~ ~ Q ~ D I V L V G G S T R I P K I Q K ~ D F F N G K E L N K S I N P D ~ V A Y G ~ V Q ~ I L S G D K S E N V Q D L L L L D 4V0T0P L S hHSC71 A.....L S.1 400 hHSP70 CS S E....A.....L....I..............V..........RD...........G..........M...............A... 400

...................

..... .... ..

.. .....................................................................

t H S C 7 1 LGIETAGG~IKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGE~TKDNNL~KFELTGIPPAPRGVPQI~FDIDRNGINNVSAADKSTGKENK 500 L V... 500 hHSC71 hHSP70 L A.....S........I..............................R...S......-................L..T.T......A.. 499

..................................................................................... .. ........

....

......

tHSC71 I T I T N D K G R L S K E D I E R M V Q E A E K Y K C E D D V Q R D K V S S K N Q T A E K E E Y E H H Q K E L E hHSC71 A..EK.. A N....Q...D....I.N............F..Q..... hHSP70 E A..E E R A..A..........A......K....EA..K.V.D..Q...S...A.TL...D.F..KR....

.......................... ............. ............

................. ...........

... ..

600 600 599

t H S C 7 1 KVCNPIITKLYQGAGGMPGGMPEGMLGGFPGAGG~GGGGSSGPTIEEVD651 hHSC71 S... P.S..A 646 hHSP70 Q SG Q.--.K SG.... 640

---- .....-... ............ -...... ...... . . . . . . . .--. . ---- ...-..

..

.......... .....

Fig. 4. Protein comparison. Deduced amino acid sequence of a protein encoded by the cloned trout gene (tHSC71) is compared to that of human constitutively synthesized HSC71 [39] and human heat-inducible HSP70 [40]. Only different amino acids are indicated and hyphens mark deletions.

high amount of this transcript is also present during different stages of trout testes development (Fig. 6B).

Functional analysis of the upstream region of the isolated trout gene Analysis of the DNA sequence upstream to the hsp70-like coding region revealed the presence of a few putative promoter elements (Fig. 2). A TATA-box was found at position -32 and two inverted CAAT boxes are present at position -50 and -89. Sequences similar to a core of the heat-shock box are localized at -82, -436, -548, -620 and -786, and regions resembling metal responsive elements are present at -147, -350, -444, -459 and -479. To test the functionality of that putative promoter region, DNA fragments containing nucleotides -90030 and -21758 (long and short promoters, respectively) were inserted upstream to the promoterless bacterial CAT reporter gene. The transient enzyme assay in lysates of transfected fish cells showed that both fragments are active as constitutive promoters and treatment of transfected cells with heat shock, Zn2+ ions and arsenite has no significant stimulatory effect on CAT expression (Fig. 7).

+

+

DISCUSSION This paper describes for the first time the complete structure and regulation of a hsp70-related gene from trout. Comparison of its DNA sequence with other known trout genes revealed that its exons correspond to the partial cDNA clone THS70.14 isolated previously from arsenite-treated rainbow trout RTG-2 cells [12]. The only differences are: (a) a different sequence at the very 5'-end of the THS70.34 clone, which probably resulted from a reverse-transcription error, and (b) different order of nucleotides in the 619 - 624 region (+ 2760 - + 2765), possibly caused by compression due to a high GC content of surrounding sequences. Since we obtained

Fig. 5. RNA expression analysis. Northern blot analyses containing 10 pg total R N A isolated from control (C) and heat-shocked (I) CHSE cells were hybridized to a probe derived from exons 6-7 (A) or exons 8 -9 (B) of the trout hsc71 gene. The position of ribosomal R N A is marked by arrowheads. Primer-extension products (C) gencrated on R N A from control (C), heat shocked (28°C) or heavy-metalion exposed (Cd and Zn) RTG or CHSE cells were analyzed along radioactive DNA markers (length of corresponding fragments is indicated in the bases).

the identical result both from analysis of genomic and cDNA sequences, we regard our data as correct. The study of the expression pattern of the cloned trout hsp70-like gene shows that the corresponding mRNA is very abundant in normal cultured cells and trout tissues (Figs 5

899

Fig. 6. Hsc7O RNA level in various trout tissues. Northern blots containing about 10 pg total RNA from uninduced RTG cells and various trout tissues (A), as well as from developing trout testes (B) were cohybridized to a trout hsc71 (exons 8-9) and a-tubulin probes. Corresponding mKNA and ribosomal bands are marked by arrowheads. VE, very early; E, early; M, middle stage of development.

and 6), and its level does not increase significantly after treatment with heat-shock response inducers such as hyperthermia, heavy metal ions and arsenite (Fig. 5). This situation is also reflected by the unusual abundance of corresponding clones in the cDNA library screened in our study (data not shown). Such constitutively h g h expression characterizes the so-called heat-shock-cognate (hsc70) genes identified so far in fruit fly [3, 361, mammals [37-391 and yeast [40]. The predicted sequence of the putative protein encoded by the cloned trout hsp70-like gene is also most similar to the primary structure of mammalian HSC70 proteins. About 94% of its amino acids are identical with the human HSC70 [38], while similarity to the human heat inducible HSP70 [41] amounts to 84.5%. Other members of the mammalian hsp70 family are even more divergent (not shown) with about 83.4% similarity for the product of the murine spermatocyte-specific hsp70.2 gene [6], 72.5% for the protein encoded by the murine spermatidspecific hsc70t gene [7] and only 61.8% of residues identical to the rat GRP78 protein [5]. These structural similarities together with an identical mode of expression leave no doubts that the trout sequences described in the above study represent an HSC gene. The molecular mass of the protein, estimated from DNA sequence data, suggests that the cloned gene encodes a prominent cellular protein of about 71 kDa, characterized previously by electrophoresis [13]. As a result we decided to name this trout gene hsc71. The isolated trout hsc71 gene also shares another important characteristic of HSC genes in that unlike the heat inducible hsp70 genes, its coding sequence is not continuous. Comparison of the trout genomic sequence with cDNA clones purified from the trout testicular cDNA library (this study) and with the THS70.14 clone 1121, revealed the presence of

Fig. 7.Identification of hsc7O promoter. The CAT assay was performed using lysates of cells transiently transfected with constructs containing the -900- + 30 (long) or -21758 (short) region of thc trout hsc71 gene. Cells were grown at physiological conditions (C), heat shocked (28°C) or treated with heavy metal ions (Zn) or arsenite (ARS) prior to assay. Reaction products were analysed by thin-layer chromatography followed by autoradiography. Control lysates: untransfected cells (no enzyme), cells treated by calcium phosphate precipitate without plasmid DNA (mock), cells of cells transfectcd with promoterless CAT gene (pGEM2 CAT).

+

eight introns (Figs 1 and 2). All these introns (despite differences in length) are localized in the same position (relatively to the protein coding region) as introns in mammalian hsc70 genes [38,39]. The above similarity in intron position partially extends also to fruit-fly hsc70 genes [36], the sea urchin hsp70like gene [42] and even the maize hsp70 gene [43]. Transient transfection studies with a bacterial CAT gene linked to a trout hsc7l upstream region showed that all elements necessary for constitutive and stress-uninducible expression (in trout and chinook salmon cells) reside inside the -216- + 30 region. The examination of the genomic hsc71 sequence revealed a TATA motif, two inverted CAAT boxes and regions resembling a heat-shock element and a metal-response element upstream to the transcription start site. As the trout hsc71 gene is active constitutively and no significant further induction was observed after heat shock and other stresses, the functionality and the role of the last two sequence motifs is unclear. However, copies of the heatshock element are present in promoters of mammalian hsc70 genes which also lack significant heat-shock inducibility [38, 391. It is possible that these heat-shock elements are important to support high constitutive transcription of the HSC genes even under adverse conditions and to avoid transcriptional inhibition experienced by other cellular genes after severe heat shock [1, 21. Our study revealed the complete structure of the trout heat-shock-cognate hsc7l gene and characterized its expression pattern in normal tissues and cultured cell lines, as

well as in cells treated with inducers of the heat-shock response. Preliminary transfection analysis allowed us to localize the hsc71 promoter inside short fragment of its upstream sequence. Further elucidation of the importance and function of individual promoter elements in the regulation of the trout hsc71 gene expression, as well as their interaction with transacting factors will be the object of our future investigation. L. G. were supported by a Natural Sciences and Engineering Council of Canada (NSERC) grant, J . W. by a fellowship from the Alberta Heritage Foundation for Medical Research (AHFMR), N. W. S. by a fellowship from the Medical Research Council of Canada, S. S. by an AHFMR summer studentship and S. M. by a NSERC fellowship. We thank Dr. S.-L. Wong for assistance in the in vitro DNA amplification.

REFERENCES 1. Lindquist, S. & Craig, E. A. (1988) Annu. Rev. Genet. 22, 631677. 2. Morimoto, R. I., Tissieres, A. & Georgopoulos, C. (1990) Stress protein in biology andmedicine, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 3. Ingolia, T. D. & Craig, E. A. (1982) Proc. Nut1 Acad. Sci. U S A 79, 525 - 529. 4. Kelley, P. M. & Schlesinger, M. J. (1982) Mol. Cell. Biol. 2,267 274. 5. Munro, S. & Pelham, H. R. B. (1986) CeN46,291-300. 6. Zakeri. Z. F., Wolgemuth, D. J . & Hunt, C. R. (1988) Mol. Cell. Biol. 8, 2925 - 2932. 7. Matsumoto, M. & Fujimoto, H. (1990) Biochim. Biophys. Res. Commun. I66,43 -49, 8. Wisniewski, J., Kordula, T. & Krawczyk, Z. (1990) Biochim. Biophys. Acta 1048, 93 -99. 9. Heikkila. J . J., Schultz, G. A., Iatrou, K. & Gedamu, L. (1982) J . Biol. Chem. 257, 12000-12005. 10. Gedamu, L., Culham, B. & Heikkila, J. J. (1983) Biosci. Rep. 3, 647 - 658. 1 1 . Kothary, R. K., Burgess, E. A. & Candido, E. P. M. (1984) Biochim. Biophys. Acta 783, 137- 143. 12. Kothary. R. K., Jones, D. & Candido, E. P. M. (1984) Mol. Cell. B i d . 4, 1785 - 1791. 13. Misra, S., Zafarullah, M., Price-Haughey, J. & Gedamu, L. (1989) Biochim. Biophys. Acta 1007, 325 - 333. 14. Blin, N. & Starrod, D. W. (1976) Nucleic Acids Res. 3, 23032308. 15. Southern, E. M. (1975)J. Mol. Biol. 98, 503-517. 16. Recd. K. C. & Mann, D. A. (1985) Nucleic Acids Res. 13, 72077221.

17. Denhardt, D. T. (1966) Biochem. Biophys. Res. Commun. 23, 641 - 646. 18. States, J. C., Connor, W., Wosnick, M. A., Aiken, J. M., Gedamu, L. & Dixon, G . H. (1982) Nucleic Acids Res. 10, 4551 -4563. 19. Garber, A. T., Retief, J. D. & Dixon, G. H. (1989) E M B O J . 8 , 1727-1734. 20. Benton, W. D. &Davis, R. W. (1977) Science lY6,180--182. 21. Helms, C., Graham, M. Y., Dutchik, J. E. &Olson, M. V. (1985) DNA 4, 39-49. 22. Birnboim, H. C. & Doly, J. (1979) Nucleic Acids Res. 7, 15131523. 23. Hattori, M. & Sakaki, Y. (1986) Anal. Biochem. f52,232-238. 24. Sadhu, C. & Gedamu, L. (1988) J . Biol. Chem. 263, 2679-2684. 25. Wiliams, J. B. & Mason, P. J. (1985) in Nucleic acidhybridization. A practical approach (Hames, B. D. & Higgins, S. J., eds) pp. 139 - 160, IRL Press, Oxford. 26. Tabor, S. & Richardson, C . C. (1985) Proc. Nut1 Acad. Sci. USA 82,1074-1078. 27. Livak, K. J., Freund, R., Schweber, M., Wcnsink, P. C. & Meselson, M. (1978) Proc. Natl Acad. Sci. USA 75, 56135617. 28. Lisowska, K., Wisniewski, J. & Krawczyk, Z. (1990) Actu Biochim. Polon. 371, 5 5 - 58. 29. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science 239,487-491. 30. Jahroudi, N., Foster, R., Price-Haughey, J., Beitel, G . & Gedamu, L. (1990) J . B i d . Chem. 265,6506-6511. 31. Fryer, J. L., Yusha. A. & Pilcher, K. S. (1965) Ann. N . Y . Acad. Sci. 126, 566-586. 32. Fryer, J. L., McCain, B. B. & Leong, A. C. (1980) Fish Pathol. 15, 193-200. 33. Wolf, K. & Quimby, M. C. (1962) Science 135, 1065-1066. 34. Graham, F. L. & van der Eb, A. J. (1973) Virologj 52,456-467. 35. Gorman, C. M., Moffat, L. D. & Howard, B. H . (lY82) Mol. Cell. Biol. 2, 1044- 1051. 36. Craig, E. A., Ingolia, T. D. & Maneau, L. J . (1983) Dev. Biol. YY, 41 8 - 426. 37. O’Malley, K., Mauron, A., Barchas, J. D. & Kedes, L. (1985) Mol. Cell. B i d . 5, 3476 - 3483. 38. Dworniczak, B. & Mirault, M.-E. (1987) Nucleic Acid.s Res. 15. 5181 -5197. 39. Sorger, P. K. & Pelham, H. R. B. (1987) EMBO J . 6 , 993-998. 40. Ellwood, M. & Craig, E. A. (1984) Mol. Cell. Biol. 4 , 1454- 1459. 41. Hunt, C. & Morimoto, R. I. (1985) Proc. Natl Acud. Sci. USA 82,6455-6459. 42. La Rosa, M., Sconxo, G., Giudice, G., Roccheri, M . C. & Di Carlo, M. (1990) Gene 96,295-300. 43. Rochester, D. E., Winer, J. A. & Sah, D. M. (1986) EMBO J . 5 , 451 -458.