Volume 6 Number 12 1979
Nucleic Acids Research
5' and 3' terminal nucleotide sequences of the RNA genome segments of influenza virus
James S. Robertson
Division of Virology, Department of Pathology, University of Cambridge, Laboratories Block, Addenbrooke's Hospital, Hills Road, Cambridge, UK Received 18 June 1979 ABSTRACT
Extensive nucleotide sequence analysis of the 5' and the 3' termini of the RNA segments of the genome of fowl plague virus, an avian strain of influenza virus, confirms the presence of a common sequence at the 51 terminus of each segment and a common sequence at the 31 terminus of each segment. Between the ends of each individual segment there is a complementary sequence which may be important in the control of transcription and replication of the genome. In addition, the probable sites of initiation of translation of fowl plague virus mRNA are indicated along with the corresponding NH 2-terminal amino acid sequences of the virus polypeptides.
INTRODUCTION The terminal nucleotide sequences of the individual segments of a virus with a segmented genome such as influenza virus are of considerable interest because of their common involvement in the virus specific processes of transcription, translation and replication. The genome of influenza virus, a negative-strand virus, consists of eight discrete single-stranded RNA segments each of which codes for unique polypeptide species (1,2,3). The eight RNA segments are transcribed in vivo into two classes of complementary plus strand RNA (cRNA), ome of which consists of full length copies of the genome, the other consisting of copies which lack a region complementary to the 51 end of genome RNA and which also contain polyadenylate sequences (4). This latter class of cRNA can be translated in vitro into virus specific polypeptides and apparently contains host cell modifications at the 5' terminus (5). The nucleotide sequence at the terminal regions of the individual segments of the influenza virus genome are thus of interest not only for recognition by the enzymes involved in replication, but also because of the apparent interaction of the host cell with the 3' end of the
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
3745
Nucleic Acids Research genome segments during transcription and because of premature termination of transcription and polyadenylation.
Recently, limited nucleotide sequence analysis of the 5t termini of the genome of several A & B strains of influenza virus has indicated a common region of 13 nucleotides (6). In addition, it was shown that the 5' termini of in vitro transcripts of the genome contain a common region of 12 nucleotides. This report describes extensive nucleotide sequences derived from both the 5' termini and the 31 termini of all eight segments of the genome of fowl plague virus, an avian strain of influenza virus. The analysis confirms the presence of a common region of 13 nucleotides at the 5' terminus of each segment. At the 3' terminus of each segment there is a common region of 12 nucleotides which is complementary to the sequence derived from the 5t termini of in vitro transcripts by Skehel & Hay (6). Additional specific features of the terminal regions of the influenza virus genome are described including complementary sequences and the probable sites of protein synthesis initiation in the complement of each segment with the corresponding NH 2-terminal amino acid sequence of each virus polypeptide.
MATERIALS AND METHODS
Purification of virus RNA. Fowl plague virus (Rostock strain) was grown in fertile hen eggs and purified by sucrose gradient velocity sedimentation as described by Inglis et al. (7). Purified virions were disrupted in 0.1M tris-HCl, pH 7.5, 0.05M NaCl, 0.O1M EDTA, 0.5% SDS and digested with 0.5 mg/ml Pronase (Calbiochem) for lh at 370C. Total RNA was extracted with phenol/chloroform (1:1) and the aqueous layer was precipitated twice with ethanol. The purified virus RNA was resuspended in 1 ml H20 and quantitated by UV absorbance (1.0 OD26 40 Atg/ml).
End-labelling and gel electrophoresis of RNA. Prior to labelling of the 5' termini of virus RNA, the RNA was dephosphorylated with calf intestinal phosphatase (Boehringer) purified as described by Lockard et al. (8) and the phosphatase removed by phenol extraction. After ethanol precipitation, the RNA was resuspended in a mixture containing polynucleotide kinase (Boehringer) and y 32P]ATP (The Radiochemical Centre) and incubated at 370C for 30 min(9). Virus RNA was radiolabelled at the 3' termini in a mixture containing RNA ligase (P-L. Biochemicals) and 5'[32P]pCp (New England Nuclear) incubated at 370C for 30 min (10). The reactions were terminated by the addition of an 3746
Nucleic Acids Research equal volume of formamide/dye mixture (11), heated at 100°C for 30 sec, and applied to a 2.5% polyacrylamide gel (40 x 20 x 0.15 cm) containing 7M
urea,
2.5 mM EDTA and either 90 mM tris-borate, pH 8.3 (11)
36 mM
or
tris-phosphate, pH 7.8 (1). Nucleotide virus RNA
sequence analysis.
were
Individual segments of end-labelled
eluted from polyacrylamide gels by soaking the
correspon-
ding gel slice in 0.5M NH4 acetate, 5 mM Mg acetate, 1% SDS for 16h 370C, and the RNA recovered by ethanol precipitation with 100 9tg carrier tRNA (9).
Aliquots of individual segments
were
ially digested with T1 RNase (12), U2 RNase (12), B. or
Phy I RNase (9),
or
they
were
at
either part-
cereus
RNase (8)
partially degraded with hot formamide
containing 1 mM MgCl2 (9).
The products of the digestions along with
the formamide 'ladder'
analysed
were
on a
20% 'sequencing' gel (13).
In addition, aliquots of formamide degraded RNA dimensional gel electrophoresis (8) using 0.35
were
analysed by two-
thick gels.
mm
RESULTS
Fowl plague virus
genome
RNA
was
terminally-labelled and the
individual segments, ranging in size from 900 to 2,400 nucleotides (14), separated by polyacrylamide gel electrophoresis (Fig. 1). Adequate separation of segments 3-8
was
obtained in tris-borate buffered gels
(Fig. la,b) whilst separation of segments 1, 2 and 3 tris-phosphate buffered gels (Fig. lc).
was
achieved in
The different relative
mobility of the segments in tris-borate buffered gels, especially noticeable with segment 4, arises from differences in the extent of denaturation of the RNA segments during different electrophoretic runs. In general, using either labelling technique, there was a decrease in the amount of radiolabel incorporated into each segment with increasing
size.
The nucleotide sequences presented in Table
1
were obtained
by
direct analysis of terminally-labelled fowl plague virus genome RNA.
Initially the sequence analyses were performed by partial digestion of individual segments with specific endoribonucleases (8,9,12) and analysis of the products on polyacrylamide 'sequencing' gels (13) (Figs. 2a,b & 3a,b). This method located the position of each adenine,
guanine and pyrimidine from the ends of each segment; however the use of Phy I RNase (9) which cleaves specifically at A, G and U residues
3747
Nucleic Acids Research Figure 1.
a
b
Polyacrylamide gel electrophoresis of end-labelled fowl plague virus genome RNA. a) 5' end-labelled RNA in a tris-borate buffered gel, 16h at 400V. b) 3' end-labelled RNA in a tris-borate buffered gel, 16h at 400V. c) 3' end-labelled RNA in a trisphosphate buffered gel, 40h at 200V.
c
*
4 __2 -3 5 _
6
_
_ 4
7
4
_ 6
8
so
_
did not accurately distinguish between the pyrimidines and an alternative method involving a two-dimensional gel electrophoresis wandering spot analysis (8) provided a highly satisfactory method for discriminating between U and C residues. In addition, this technique confirmed the sequences obtained by the enzymatic method by distinguishing A and C residues from U and G residues (Figs. 2c & 3c). Indeed, because of the greater mobility shift of a G residue compared with a U residue, the U and G residues could also be distinguished from each other. The resolution of oligonucleotides in the wandering spot analyses was the limiting factor in accurately determining the terminal nucleotide sequence of each segment. The sequencing techniques employed cannot identify the 3' terminal nucleotide and nearest neighbour analysis of a total alkali digest of 3' terminally-labelled RNA was employed to establish the identity of the terminal nucleotide (not shown). In addition, because of a failure to obtain an adequate nucleotide sequence for the 31 end of segment 1 from a sequencing gel, the sequence was determined in the following way. A wandering spot analysis of the 3' end of segment 1 established the positions of A/C, U and G residues. Comparison of a sequencing gel of segment 2 with that of a mixture of segments 1 and 2 indicated
3748
Nucleic Acids Research Segment
5,
25
50
75
8
UU UUJAA GCUGAAACGAGAAAGUCWUCUCWGCUCCA
7
U
6
UG
5
U
4
CU
3
.
3JGACAAAAUGACCAUCGUCAACAACCACAGCACUCUGCUGWCCUGCCGA UCCAGCUCU
G(UA C
3AC
CAAAgTCUA CWGUGAAUGGCAACUCAGCACCGUCUGG
CUUAUUGUAUACUCCUCGCAUUGUCUCCGAAGAAAUAAGAUCCCAA UUUUUUCCAAACUUAUAUACAAAUAGUGCACCGCAUGUUUCCGGUUCUJrCACACAUAUGAAAA CAGUAUGGAUAGCAAAUAGUAGCAUUGCCACAACUAUCAGUGCAUG - 2 CUGAAGGACAAGCUAAAWUCACAUUUACUGCCGUCUGAGCUCUU
WUWAAACAAUUCGACAWAAUUGAUGGCCAUCCGAAUUCUWUWJGG
1
75
50
25
3,
AAAGAAAGCAGUCUACCUGAAAGCWGACACGUGUUGGAAUCA AJAUGUUUUWG -
8
GACGGGACGACAGAGAGAACGUACGUUCCAACCUCGGUUAGAAGACUGACWUUUAAAUALFLA GACCCAAUGGUUAUUAUUUUCUGAUUUGGAUU
7
C
GAGACGCCAUGAUATX3GA GCCACUCAGUGAGUGAUA
GCCGCAAGGGCGAAAACCAGGAUUUGAGUGUUCA
6
U
UUGUAA
5
C
4
C
2
GGAUUGAAGCAUWXGACGCACAAAUUCUUCCA
GCGCUGGAACUUUCAAGAAAAGUAAAGUCGGGUUGACAUCcUCAAA1JGG UUAGUUCUUUUAUUCUCUC
Segment
3
UGAAUAUA
1
Table 1. Nucleotide sequences derived from the 5' and 3' terminal regions of all eight segments of the fowl plague virus genome. The large boxes indicate the 5' and 3' common regions. The smaller boxes indicate regions of complementarity which exist between the terminal regions of individual segments. The 5' terminal regions of the genome RNA correspond, in part, to the 3' end of influenza mRNA and triplets complementary to termination codons are underlined in the 5' sequences. Similarly, triplets complementary to potential initiation codons at the 5' end of mRNA are underlined in the 3' sequences.
the bands in the mixed sequencing gel which were derived from segment 1. This comparison confirmed the location of A, G and pyrimidine residues as obtained from the wandering spot analysis in addition to
discriminating between A and C residues, resulting in sequence for the 3' end of segment lo
a
complete
In the two-dimensional wandering spot analyses, 5 end-labelled products less than eight nucleotides in size were not resolved (Fig. 2c), whilst although the smallest 3' end-labelled products were
resolved they migrated in the second dimension with a buffer front and the nucleotide sequence could not be deduced from their relative positions. The sequence of the first few nucleotides at the ends of the genome segments was determined entirely by the enzymatic method, including the use of Phy I RNase.
Usually, the sequencing gels produced an unambiguous sequence of A, G and pyrimidine residues. However, in the analysis of 3' end-
3749
Nucleic Acids Research b
a
c G A C
_A_
I;* e
9U * u
*Y
.0 , c(. -.
C-..
3O
-
*
A
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*
I,
-61)
*A
0
G
G
s5AA A
r *O*A
2 A
-
SY
(tAX J.
C
.1
Figure 2. Sequence analysis of the 51 terminal region of fowl plague virus genome segment 7. a,b) sequencing gels of partially degraded end-labelled RNA. Left to right, tracks are: control; T1 RNase specific for G residues; U2 RNase specific for A residues; B. cereus RNase specific for pyrimidines; Phy I RNase specific for A, G and U residues (a only); formamide ladder. c) two-dimensional gel electrophoresis wandering spot analysis of formamide degraded RNA.
labelled segments weak bands in the U2 RNase track often comigrated with weak bands in the B. cereus RNase track approximately 15-20 nucleotides from the 31 end. Several features indicate that the weak U2 RNase bands are contaminants. Firstly, U2 RNase generally produces bands of relatively similar intensity whilst B. cereus RNase produces bands which vary considerably in intensity, those at the 51 end of a 3750
Nucleic Acids Research a
b
C GA U
G AC
C
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2
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Nucleic Acids Research
5' and 3' terminal nucleotide sequences of the RNA genome segments of influenza virus
James S. Robertson
Division of Virology, Department of Pathology, University of Cambridge, Laboratories Block, Addenbrooke's Hospital, Hills Road, Cambridge, UK Received 18 June 1979 ABSTRACT
Extensive nucleotide sequence analysis of the 5' and the 3' termini of the RNA segments of the genome of fowl plague virus, an avian strain of influenza virus, confirms the presence of a common sequence at the 51 terminus of each segment and a common sequence at the 31 terminus of each segment. Between the ends of each individual segment there is a complementary sequence which may be important in the control of transcription and replication of the genome. In addition, the probable sites of initiation of translation of fowl plague virus mRNA are indicated along with the corresponding NH 2-terminal amino acid sequences of the virus polypeptides.
INTRODUCTION The terminal nucleotide sequences of the individual segments of a virus with a segmented genome such as influenza virus are of considerable interest because of their common involvement in the virus specific processes of transcription, translation and replication. The genome of influenza virus, a negative-strand virus, consists of eight discrete single-stranded RNA segments each of which codes for unique polypeptide species (1,2,3). The eight RNA segments are transcribed in vivo into two classes of complementary plus strand RNA (cRNA), ome of which consists of full length copies of the genome, the other consisting of copies which lack a region complementary to the 51 end of genome RNA and which also contain polyadenylate sequences (4). This latter class of cRNA can be translated in vitro into virus specific polypeptides and apparently contains host cell modifications at the 5' terminus (5). The nucleotide sequence at the terminal regions of the individual segments of the influenza virus genome are thus of interest not only for recognition by the enzymes involved in replication, but also because of the apparent interaction of the host cell with the 3' end of the
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
3745
Nucleic Acids Research genome segments during transcription and because of premature termination of transcription and polyadenylation.
Recently, limited nucleotide sequence analysis of the 5t termini of the genome of several A & B strains of influenza virus has indicated a common region of 13 nucleotides (6). In addition, it was shown that the 5' termini of in vitro transcripts of the genome contain a common region of 12 nucleotides. This report describes extensive nucleotide sequences derived from both the 5' termini and the 31 termini of all eight segments of the genome of fowl plague virus, an avian strain of influenza virus. The analysis confirms the presence of a common region of 13 nucleotides at the 5' terminus of each segment. At the 3' terminus of each segment there is a common region of 12 nucleotides which is complementary to the sequence derived from the 5t termini of in vitro transcripts by Skehel & Hay (6). Additional specific features of the terminal regions of the influenza virus genome are described including complementary sequences and the probable sites of protein synthesis initiation in the complement of each segment with the corresponding NH 2-terminal amino acid sequence of each virus polypeptide.
MATERIALS AND METHODS
Purification of virus RNA. Fowl plague virus (Rostock strain) was grown in fertile hen eggs and purified by sucrose gradient velocity sedimentation as described by Inglis et al. (7). Purified virions were disrupted in 0.1M tris-HCl, pH 7.5, 0.05M NaCl, 0.O1M EDTA, 0.5% SDS and digested with 0.5 mg/ml Pronase (Calbiochem) for lh at 370C. Total RNA was extracted with phenol/chloroform (1:1) and the aqueous layer was precipitated twice with ethanol. The purified virus RNA was resuspended in 1 ml H20 and quantitated by UV absorbance (1.0 OD26 40 Atg/ml).
End-labelling and gel electrophoresis of RNA. Prior to labelling of the 5' termini of virus RNA, the RNA was dephosphorylated with calf intestinal phosphatase (Boehringer) purified as described by Lockard et al. (8) and the phosphatase removed by phenol extraction. After ethanol precipitation, the RNA was resuspended in a mixture containing polynucleotide kinase (Boehringer) and y 32P]ATP (The Radiochemical Centre) and incubated at 370C for 30 min(9). Virus RNA was radiolabelled at the 3' termini in a mixture containing RNA ligase (P-L. Biochemicals) and 5'[32P]pCp (New England Nuclear) incubated at 370C for 30 min (10). The reactions were terminated by the addition of an 3746
Nucleic Acids Research equal volume of formamide/dye mixture (11), heated at 100°C for 30 sec, and applied to a 2.5% polyacrylamide gel (40 x 20 x 0.15 cm) containing 7M
urea,
2.5 mM EDTA and either 90 mM tris-borate, pH 8.3 (11)
36 mM
or
tris-phosphate, pH 7.8 (1). Nucleotide virus RNA
sequence analysis.
were
Individual segments of end-labelled
eluted from polyacrylamide gels by soaking the
correspon-
ding gel slice in 0.5M NH4 acetate, 5 mM Mg acetate, 1% SDS for 16h 370C, and the RNA recovered by ethanol precipitation with 100 9tg carrier tRNA (9).
Aliquots of individual segments
were
ially digested with T1 RNase (12), U2 RNase (12), B. or
Phy I RNase (9),
or
they
were
at
either part-
cereus
RNase (8)
partially degraded with hot formamide
containing 1 mM MgCl2 (9).
The products of the digestions along with
the formamide 'ladder'
analysed
were
on a
20% 'sequencing' gel (13).
In addition, aliquots of formamide degraded RNA dimensional gel electrophoresis (8) using 0.35
were
analysed by two-
thick gels.
mm
RESULTS
Fowl plague virus
genome
RNA
was
terminally-labelled and the
individual segments, ranging in size from 900 to 2,400 nucleotides (14), separated by polyacrylamide gel electrophoresis (Fig. 1). Adequate separation of segments 3-8
was
obtained in tris-borate buffered gels
(Fig. la,b) whilst separation of segments 1, 2 and 3 tris-phosphate buffered gels (Fig. lc).
was
achieved in
The different relative
mobility of the segments in tris-borate buffered gels, especially noticeable with segment 4, arises from differences in the extent of denaturation of the RNA segments during different electrophoretic runs. In general, using either labelling technique, there was a decrease in the amount of radiolabel incorporated into each segment with increasing
size.
The nucleotide sequences presented in Table
1
were obtained
by
direct analysis of terminally-labelled fowl plague virus genome RNA.
Initially the sequence analyses were performed by partial digestion of individual segments with specific endoribonucleases (8,9,12) and analysis of the products on polyacrylamide 'sequencing' gels (13) (Figs. 2a,b & 3a,b). This method located the position of each adenine,
guanine and pyrimidine from the ends of each segment; however the use of Phy I RNase (9) which cleaves specifically at A, G and U residues
3747
Nucleic Acids Research Figure 1.
a
b
Polyacrylamide gel electrophoresis of end-labelled fowl plague virus genome RNA. a) 5' end-labelled RNA in a tris-borate buffered gel, 16h at 400V. b) 3' end-labelled RNA in a tris-borate buffered gel, 16h at 400V. c) 3' end-labelled RNA in a trisphosphate buffered gel, 40h at 200V.
c
*
4 __2 -3 5 _
6
_
_ 4
7
4
_ 6
8
so
_
did not accurately distinguish between the pyrimidines and an alternative method involving a two-dimensional gel electrophoresis wandering spot analysis (8) provided a highly satisfactory method for discriminating between U and C residues. In addition, this technique confirmed the sequences obtained by the enzymatic method by distinguishing A and C residues from U and G residues (Figs. 2c & 3c). Indeed, because of the greater mobility shift of a G residue compared with a U residue, the U and G residues could also be distinguished from each other. The resolution of oligonucleotides in the wandering spot analyses was the limiting factor in accurately determining the terminal nucleotide sequence of each segment. The sequencing techniques employed cannot identify the 3' terminal nucleotide and nearest neighbour analysis of a total alkali digest of 3' terminally-labelled RNA was employed to establish the identity of the terminal nucleotide (not shown). In addition, because of a failure to obtain an adequate nucleotide sequence for the 31 end of segment 1 from a sequencing gel, the sequence was determined in the following way. A wandering spot analysis of the 3' end of segment 1 established the positions of A/C, U and G residues. Comparison of a sequencing gel of segment 2 with that of a mixture of segments 1 and 2 indicated
3748
Nucleic Acids Research Segment
5,
25
50
75
8
UU UUJAA GCUGAAACGAGAAAGUCWUCUCWGCUCCA
7
U
6
UG
5
U
4
CU
3
.
3JGACAAAAUGACCAUCGUCAACAACCACAGCACUCUGCUGWCCUGCCGA UCCAGCUCU
G(UA C
3AC
CAAAgTCUA CWGUGAAUGGCAACUCAGCACCGUCUGG
CUUAUUGUAUACUCCUCGCAUUGUCUCCGAAGAAAUAAGAUCCCAA UUUUUUCCAAACUUAUAUACAAAUAGUGCACCGCAUGUUUCCGGUUCUJrCACACAUAUGAAAA CAGUAUGGAUAGCAAAUAGUAGCAUUGCCACAACUAUCAGUGCAUG - 2 CUGAAGGACAAGCUAAAWUCACAUUUACUGCCGUCUGAGCUCUU
WUWAAACAAUUCGACAWAAUUGAUGGCCAUCCGAAUUCUWUWJGG
1
75
50
25
3,
AAAGAAAGCAGUCUACCUGAAAGCWGACACGUGUUGGAAUCA AJAUGUUUUWG -
8
GACGGGACGACAGAGAGAACGUACGUUCCAACCUCGGUUAGAAGACUGACWUUUAAAUALFLA GACCCAAUGGUUAUUAUUUUCUGAUUUGGAUU
7
C
GAGACGCCAUGAUATX3GA GCCACUCAGUGAGUGAUA
GCCGCAAGGGCGAAAACCAGGAUUUGAGUGUUCA
6
U
UUGUAA
5
C
4
C
2
GGAUUGAAGCAUWXGACGCACAAAUUCUUCCA
GCGCUGGAACUUUCAAGAAAAGUAAAGUCGGGUUGACAUCcUCAAA1JGG UUAGUUCUUUUAUUCUCUC
Segment
3
UGAAUAUA
1
Table 1. Nucleotide sequences derived from the 5' and 3' terminal regions of all eight segments of the fowl plague virus genome. The large boxes indicate the 5' and 3' common regions. The smaller boxes indicate regions of complementarity which exist between the terminal regions of individual segments. The 5' terminal regions of the genome RNA correspond, in part, to the 3' end of influenza mRNA and triplets complementary to termination codons are underlined in the 5' sequences. Similarly, triplets complementary to potential initiation codons at the 5' end of mRNA are underlined in the 3' sequences.
the bands in the mixed sequencing gel which were derived from segment 1. This comparison confirmed the location of A, G and pyrimidine residues as obtained from the wandering spot analysis in addition to
discriminating between A and C residues, resulting in sequence for the 3' end of segment lo
a
complete
In the two-dimensional wandering spot analyses, 5 end-labelled products less than eight nucleotides in size were not resolved (Fig. 2c), whilst although the smallest 3' end-labelled products were
resolved they migrated in the second dimension with a buffer front and the nucleotide sequence could not be deduced from their relative positions. The sequence of the first few nucleotides at the ends of the genome segments was determined entirely by the enzymatic method, including the use of Phy I RNase.
Usually, the sequencing gels produced an unambiguous sequence of A, G and pyrimidine residues. However, in the analysis of 3' end-
3749
Nucleic Acids Research b
a
c G A C
_A_
I;* e
9U * u
*Y
.0 , c(. -.
C-..
3O
-
*
A
-10
U-A
-9l
-G
-A
pA
-7
A _ I&
*
I,
-61)
*A
0
G
G
s5AA A
r *O*A
2 A
-
SY
(tAX J.
C
.1
Figure 2. Sequence analysis of the 51 terminal region of fowl plague virus genome segment 7. a,b) sequencing gels of partially degraded end-labelled RNA. Left to right, tracks are: control; T1 RNase specific for G residues; U2 RNase specific for A residues; B. cereus RNase specific for pyrimidines; Phy I RNase specific for A, G and U residues (a only); formamide ladder. c) two-dimensional gel electrophoresis wandering spot analysis of formamide degraded RNA.
labelled segments weak bands in the U2 RNase track often comigrated with weak bands in the B. cereus RNase track approximately 15-20 nucleotides from the 31 end. Several features indicate that the weak U2 RNase bands are contaminants. Firstly, U2 RNase generally produces bands of relatively similar intensity whilst B. cereus RNase produces bands which vary considerably in intensity, those at the 51 end of a 3750
Nucleic Acids Research a
b
C GA U
G AC
C
I!
3.
2
'-,U)
_*.
U
aa em
-c
. 40
_*
-ww - C
* S W**
30
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*
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-20 Y
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