Biochimica et Biophysica Acta, 1130(1092) 90-94 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
90
BBAEXP 90321
Short Sequence-Paper
Cloning of HSP60 (GroEL) operon from Clostridium perfringens using a polymerase chain reaction based approach Emmy Rusanganwa, Bhag Singh and Radhey S. Gupta Department of Biochemistry McMaster Unicersity, Hamilton (Canada) (Received 19 December 19~i)
Key words: HSP60 sequence: GroEL operon: Polymerase chain reaction: (C la'rfringens)
Using degenerate oligonuclcotidc primers for conserved regions of HSP60, a 0.6 kilobasc fragment of ClostrMium perfringe,s DNA was amplified by the polymerase chain reaction. The amplified fragment was used as a probe to isolate a genomic clone containing the C la,rfringens HSP60 operon. The clone contained two open reading frames homologous to the GroES and GroEL (or HSP60) hmily of bacterial and eukar?otic proteins as well as other upstream and downstream sequences. The approach described here, employing this set of degenerate oligonucleotide primers, could be used to clone !1SP60 gene/cDNA from any species.
The clostrodia are large Gram-positive spore-forming anaerobic bacilli, present in soil as well as in intestinal tracts of humans and animals [1]. The infection of open wounds by ciostridia bacilli is responsible for a number of common pathological conditions. Infection by Clostridium tetani is responsible for tetanus, whereas CIostridium perfringens is the causative agent of gas gangrene and many cases of food poisoning (see Ref. I). Studies during the past few years show that the stress or heat shock family of proteins (HSPs), whose synthesis is induced in ~'espo.~ge to a variety of environmental stresses, are major antigenic proteins of numerous pathogenic organisms (see Refs. 2-4). One of these proteins, HSP60 (60 kDa heat shock protein) has been shown to correspond to the bacterial common antigen - a major antigenic protein common to all bacteria [5,6]. in Escherichia coil and other prokaryotic cells, HSP60 gene forms a part of the GroE operon which encodes two different proteins (HSP60 or GroEL and HSPI0 or GroES) [7-II]. in the past few years HSP60 gene/cDNA has been cloned and sequenced from a number of different species [7,9-2 I]. Based upon information regarding conserved sequences among different species, and by using the DNA polymerase chain reaction (PCR), we describe the cloning and complete The sequence data for C ln~rfringens HSP60 have b~,m deposited to the EMBL Databank under the accession number Xo2914. Correspondence: R.S. Gupta. Department of Biochemistry. McMaster University, Hamilton, Ontario, Canada L8N 3Z5.
nudeotide sequence of the GroE operon from C perfringens. Oligonucleotide primers with opposing orientations were custom synthesized for two conserved regions of the HSP60 family of proteins. The forward (5'GGNGAYGGNACNACNACNGCNACNGT-3', where N - A , C, G or T and Y = C or T) and the reverse (5'-TCNCCRAANCCNGGNGCYTTNACNGC-3', where R = A or G) primers were made for the sequences GDGTTATV and AVKAPGFGD. respectively. For these primers to work with DNA of different species, the third codon position in these was degenerate to allow for different codon usage [22,23]. PCR amplification of DNA was carried out essentially as described by Gould et al. [20]. When these primers, were used in the PCR experiments using either E. coli or CIostridium DNA or eDNA from CHO cells, a fragment of about 0.6 kb was specifically amplified (Fig. I). The size of the amplified fragment was in accordance with the length of the amino acid sequence encompassed by the two primers. To confirm their HSP60 relatedness, the amplified 0.6 kb fragments from CHO eDNA and C perfringens DNA were subcloned and their nucleotide sequences determined. The sequence of the 0.6 kb fragment from CHO eDNA showed a perfect match with the published sequence [19] of CHO HSP60 (results not shown). The deduced amino acid sequence of the 0.6 kb fragment from C. perfringens also showed extensive similarity to the HSP60 family of proteins indicating that it corresponded to HSP60.
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zymes (Fig. 2). 'the 3-5 kb fragments from HindllI-digested genomic D N A were excised from an agarose gel and subcloned in the plasmid vector pGEM-7z(f). Upon screening of the recombinant colonies with the 0.6 kb probe, two positive clones were identified. These clones contained an insert of approx. 4.0 kb which hybridized with the 0.6 kb C. perfringens probe (Fig. 2b,c). To sequence the 4.0 kb insert, nested sets of deletions using exonuclease I!1 were made in both orientations, and the nucleotide sequence of both D N A strands was determined (Fig. 3). The D N A insert consisted of 3987 nt and it contained two open reading frames separated by 54 nt. The two open reading frames were 282 and 1617 nt long and they encoded proteins which correspond to the GroES and GroEL proteins of E. coil. The 0.6 kb sequence of the PCR probe matched exactly with a portion (nt 1345-1935; Fig. 3) of the GroEL-like protein. For both GroEL and GmES proteins, a strong preference for codon with either A or T in the third position was observed. This is consistent with the low G + C content (26.9c,,;) of the cloned fragment and of the C pet~'ingens D N A (24-27%) [I]. Upstream of the GroES-like protein, a Shine-Dalgarno sequence as well as sequences showing a reasonable match to - 1 0 and - 3 5 consensus sequences were observed (Fig. 3). These results indicated that the 4.1) kb HindIIl fragment contained the complete GroESL operon of C pelfringens. Figs. 4 and 5 show comparison of the deduced amino acid sequence of GruELand GroES-like proteins from (,2 petfringens with that from other species. As seen, the two amino acid sequences show extensive homology throughout thcir length to the GruEL and ( b y E S homologs from various species. The protein sequences fronl C. pofringens showcd maximal similarity to thc protcin from Grampositive group of bacteria (viz. M. leprae), while the
OKB ,5 KB
Fig. 1. Agarose gel electrophoresis of the PCR reaction products. PCR amplification using degenerate N- and C-oligonucleotide primers and different DNA samples was carried out as described in the text. After 3ll reaction cycles, 1/10th of the reaction products were analyzed on If'; agarose gels. Lane 1, eDNA from CHO cells; hme 2. 1:2,.coil DNA: lane 3, C. pelfringens DNA: hme 4. molecular weight ladder. To isolate a genomic clone from C pofringens, its D N A was digested with several restriction enzymes (viz. BamHl, EcoRl, H i n d l l l and Xhol), blotted and probed with the 0.6 kb PCR fragment from C petfringens. The 0.6 kb probe hybridized to D N A fragments in the range of 4 - 1 0 kb for different restriction en-
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90
BBAEXP 90321
Short Sequence-Paper
Cloning of HSP60 (GroEL) operon from Clostridium perfringens using a polymerase chain reaction based approach Emmy Rusanganwa, Bhag Singh and Radhey S. Gupta Department of Biochemistry McMaster Unicersity, Hamilton (Canada) (Received 19 December 19~i)
Key words: HSP60 sequence: GroEL operon: Polymerase chain reaction: (C la'rfringens)
Using degenerate oligonuclcotidc primers for conserved regions of HSP60, a 0.6 kilobasc fragment of ClostrMium perfringe,s DNA was amplified by the polymerase chain reaction. The amplified fragment was used as a probe to isolate a genomic clone containing the C la,rfringens HSP60 operon. The clone contained two open reading frames homologous to the GroES and GroEL (or HSP60) hmily of bacterial and eukar?otic proteins as well as other upstream and downstream sequences. The approach described here, employing this set of degenerate oligonucleotide primers, could be used to clone !1SP60 gene/cDNA from any species.
The clostrodia are large Gram-positive spore-forming anaerobic bacilli, present in soil as well as in intestinal tracts of humans and animals [1]. The infection of open wounds by ciostridia bacilli is responsible for a number of common pathological conditions. Infection by Clostridium tetani is responsible for tetanus, whereas CIostridium perfringens is the causative agent of gas gangrene and many cases of food poisoning (see Ref. I). Studies during the past few years show that the stress or heat shock family of proteins (HSPs), whose synthesis is induced in ~'espo.~ge to a variety of environmental stresses, are major antigenic proteins of numerous pathogenic organisms (see Refs. 2-4). One of these proteins, HSP60 (60 kDa heat shock protein) has been shown to correspond to the bacterial common antigen - a major antigenic protein common to all bacteria [5,6]. in Escherichia coil and other prokaryotic cells, HSP60 gene forms a part of the GroE operon which encodes two different proteins (HSP60 or GroEL and HSPI0 or GroES) [7-II]. in the past few years HSP60 gene/cDNA has been cloned and sequenced from a number of different species [7,9-2 I]. Based upon information regarding conserved sequences among different species, and by using the DNA polymerase chain reaction (PCR), we describe the cloning and complete The sequence data for C ln~rfringens HSP60 have b~,m deposited to the EMBL Databank under the accession number Xo2914. Correspondence: R.S. Gupta. Department of Biochemistry. McMaster University, Hamilton, Ontario, Canada L8N 3Z5.
nudeotide sequence of the GroE operon from C perfringens. Oligonucleotide primers with opposing orientations were custom synthesized for two conserved regions of the HSP60 family of proteins. The forward (5'GGNGAYGGNACNACNACNGCNACNGT-3', where N - A , C, G or T and Y = C or T) and the reverse (5'-TCNCCRAANCCNGGNGCYTTNACNGC-3', where R = A or G) primers were made for the sequences GDGTTATV and AVKAPGFGD. respectively. For these primers to work with DNA of different species, the third codon position in these was degenerate to allow for different codon usage [22,23]. PCR amplification of DNA was carried out essentially as described by Gould et al. [20]. When these primers, were used in the PCR experiments using either E. coli or CIostridium DNA or eDNA from CHO cells, a fragment of about 0.6 kb was specifically amplified (Fig. I). The size of the amplified fragment was in accordance with the length of the amino acid sequence encompassed by the two primers. To confirm their HSP60 relatedness, the amplified 0.6 kb fragments from CHO eDNA and C perfringens DNA were subcloned and their nucleotide sequences determined. The sequence of the 0.6 kb fragment from CHO eDNA showed a perfect match with the published sequence [19] of CHO HSP60 (results not shown). The deduced amino acid sequence of the 0.6 kb fragment from C. perfringens also showed extensive similarity to the HSP60 family of proteins indicating that it corresponded to HSP60.
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zymes (Fig. 2). 'the 3-5 kb fragments from HindllI-digested genomic D N A were excised from an agarose gel and subcloned in the plasmid vector pGEM-7z(f). Upon screening of the recombinant colonies with the 0.6 kb probe, two positive clones were identified. These clones contained an insert of approx. 4.0 kb which hybridized with the 0.6 kb C. perfringens probe (Fig. 2b,c). To sequence the 4.0 kb insert, nested sets of deletions using exonuclease I!1 were made in both orientations, and the nucleotide sequence of both D N A strands was determined (Fig. 3). The D N A insert consisted of 3987 nt and it contained two open reading frames separated by 54 nt. The two open reading frames were 282 and 1617 nt long and they encoded proteins which correspond to the GroES and GroEL proteins of E. coil. The 0.6 kb sequence of the PCR probe matched exactly with a portion (nt 1345-1935; Fig. 3) of the GroEL-like protein. For both GroEL and GmES proteins, a strong preference for codon with either A or T in the third position was observed. This is consistent with the low G + C content (26.9c,,;) of the cloned fragment and of the C pet~'ingens D N A (24-27%) [I]. Upstream of the GroES-like protein, a Shine-Dalgarno sequence as well as sequences showing a reasonable match to - 1 0 and - 3 5 consensus sequences were observed (Fig. 3). These results indicated that the 4.1) kb HindIIl fragment contained the complete GroESL operon of C pelfringens. Figs. 4 and 5 show comparison of the deduced amino acid sequence of GruELand GroES-like proteins from (,2 petfringens with that from other species. As seen, the two amino acid sequences show extensive homology throughout thcir length to the GruEL and ( b y E S homologs from various species. The protein sequences fronl C. pofringens showcd maximal similarity to thc protcin from Grampositive group of bacteria (viz. M. leprae), while the
OKB ,5 KB
Fig. 1. Agarose gel electrophoresis of the PCR reaction products. PCR amplification using degenerate N- and C-oligonucleotide primers and different DNA samples was carried out as described in the text. After 3ll reaction cycles, 1/10th of the reaction products were analyzed on If'; agarose gels. Lane 1, eDNA from CHO cells; hme 2. 1:2,.coil DNA: lane 3, C. pelfringens DNA: hme 4. molecular weight ladder. To isolate a genomic clone from C pofringens, its D N A was digested with several restriction enzymes (viz. BamHl, EcoRl, H i n d l l l and Xhol), blotted and probed with the 0.6 kb PCR fragment from C petfringens. The 0.6 kb probe hybridized to D N A fragments in the range of 4 - 1 0 kb for different restriction en-
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