Volume 3 no.10 October 1976
Nucleic Acids Research
The nucleotide sequence of a DNA fragment, 71 base pairs in length, near the origin of DNA replication of bacteriophage OX174 A. D. M. van Mansfeld, J. M. Vereijken and H. S. Jans z Institute for Molecular Biology and Laboratory for Physiological Chemistry, State University, Utrecht, The Netherlands
Received 23 August 1976 ABSTRACT Part of the nucleotide sequence of a restriction fragment covering the origin of *X174 DNA replication 1 has been determined. The fragment A7c was obtained by digestion of *X174 RF DNA by the restriction enzyme from Arthrobacter luteus, Alu I. It was further cleaved into two fragments, one l-argeand one smal l, by the action of the restriction enzyme from Haemophilus aegyptius, Hae II . The nucleotide sequence of the small fragment has been determined by analysis of the transcription products obtained by the action of Escherichia coli DNA-dependent RNA polymerase on denaturated template under conditions7of low salt. Transcripts longer than the template were found. The whole sequence of 71 nucleotide pairs could be derived from complementary oligonucleotides, obtained after digestion of the transcripts with Ti or pancreatic RNAase. The sequence suggests that at least 4 of the 5 amber mutants 2 that have been mapped on this fragment are identical. On account of this and other evidence a reading frame is proposed.
INTRODUCTION
*X174 is a small single stranded DNA phage (recently reviewed by Denhardt 3). Several investigators have determined nucleotide sequences from different regions of the genome by direct or indirect sequence methods 4,5,6,7. The limited number of possibilities to isolate specific DNA fragments has hampered further sequence analysis considerably. However, thanks to the discovery of the restriction enzymes, the whole double stranded *X174 replicative form (RF) DNA now can be cleaved in specific fragments not longer than 250 base pairs 2,8t9j10,11 which are accessible to the different ways of sequence analysis, or can be used as specific primers in the method for sequence analysis described by Sanger and Coulson 12 Using the standard methods for RNA sequencing 13, the sequence analysis of RNA obtained by asymmetric transcription of native template in vitro has been described by several authors 14,15,16 Air et al. 17 and Sugimoto o Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
2827
Nucleic Acids Research
i8used
denaturated DNA restriction fragments as templates for the transcription. This latter method proves to be very powerful since the overlapping sequences can often be derived from complementary oligonucleotides. By this method part of the sequence of A7c has been determined. This fragment was obtained by digestion of OX174 RF DNA with the restriction enzyme from Arthrobacter luteus, Alu I, and overlaps some functionally interesting regions of the OX DNA, including the origin of RF DNA replication , the origin of ss DNA synthesis 19, and the terminus of RF DNA replication °. In order to facilitate sequence analysis the fragment A7c was further cleaved into two fragments, one large and one small, by the action of the restriction enzyme from Haemophilus aegyptius, Hae III. The present paper reports the results of sequence analysis of the small fragment.
et al.
MATERIALS AND METHODS PreE2aration of the DNA fragments. *X174 RF DNA was digested by the restriction enzyme from Arthrobacter luteus, Alu I 9. The products were separated 21 . A by continuous gel electrophoresis as described by Lee and Sinsheimer 5% polyacrylamide gel (g = 1 cm, length = 24 cm) was used instead of an agarose gel. The fractions containing the three A7 fragments were pooled and the DNA was precipitated by adding sodium acetate to a concentration of 0.3 M and two volumes of ethanol. The A7 fragments were redigested by the restriction enzyme from Haemophi;lus aegyptius, Hae III, which resulted in two uncleaved A7 fragments, one fragment (A7Z1) of about 180 and one fragment (A7Z2) of 71 base pairs in length 9. This mixture was again subjected to continuous gel electrophoresis on a 5% polyacrylamide gel. The DNA fragments were concentrated by ethanol precipitation and dissolved in 0.01 M Tris/HCl,pH 8.0, 0.001 M EDTA.
RA&221YmErase.
Batches of E. coli DNA dependent RNA polymerase were gifts from mr.H.van Keulen and mr.C.van Kreyl respectively. Both batches of enzyme were ATPase-, DNAase- and RNAase-free. RNA synthesis. Incubation mixtures were essentially the same as described by Blackburn 7: 25 mM Tris/HCl,pH 7.8, 10 mM MgCl2, 5 mM dithiotreitol, 0.5 mM EDTA, 0.8 mM potassium phosphate, three unlabelled ribonucleoside triphosphates (Merck, Darmstadt, Germany) at a concentration of 0.5 mM each and one [a-32 P] ribonucleoside triphosphate (The Radiochemical Centre, Amersham, UK; specific activity 5-10 Ci/mmol, later on 50-100 Ci/mmol) at a concentration of 0.05 mM. The double stranded DNA restriction fragment was denaturated by heating 2828
Nucleic Acids Research 1000C for
3 min and cooling rapidly in ethanol-dry ice. 0.1 pg denaturated DNA was used, and RNA polymerase solution was added to bring the molar template to polymerase ratio to 1: 3. The final volume was 25 pi. The incubation reaction was carried out for 3 h (unless otherwise stated) at 370C and the reaction was stopped by adding 25 p1 0.02 M EDTA, containing 2 mg/ml tRNA (BDH, Poole, UK). This mixture was extracted twice with freshly destilled phenol, saturated with 0.1 M NaCl', 0.01 M Tris/HCl,pH 7.5, 0.001 M EDTA, and eluted over a Sephadex G50 column (in the latter buffer) to remove the low molecular weight materials. The material eluting in the void volume was precipitated with ethanol. After standing for 5 min at -700C (ethanol-dry ice) and 1l h at -200C, the precipitate was spun down, dried in a stream of air, dissolved in 100 pli water and stored at -20 C until use. TCA2_recieitation. The quantity of RNA synthesized during the incubation was determined by trichloroacetic acid precipitation of a 2.5 PI sample of the "stopped mixture" (see above). This was added to 50 -il of a BSA solution (0.25 mg BSA/ml, 0.01 M Tris/HCl,pH 7.5, 0.001 M EDTA). 2.5 ml of an ice cold 10% TCA solution, containing 0.01 M tetra sodium pyrophosphate was added and after 'standing for 15 min at 00C, the precipitate was filtered over a Whatman GF/C 'filter, washed three times with 5 ml of the 10% TCA solution and three times with 5 ml of an ice cold 2% TCA solution containing 0.01 M tetra sodium pyrophosphate. The filter was dried and counted with a Triton based scintillation fluid in a liquid scintillation counter.
at
9[22-R21Y2S[Y12mide
electr2eb2E
10%
polyacrylamide gels (2 mm slab_gel is. thick, 18 cm wide and 40 cm long), in 0.04 M Tris/HCl,pH 8.0, 0.02 sodium acetate, 0.002 M EDTA were prepared as described by Loening 22 with the modification that urea was added to a concentration of 7 M. Samples were dried in a stream of air, dissolved in 20 il formamide, containing sucrose and bromophenol blue as tracking dye. Before layering the gels, the samples were heated at 1000C for 5 min. Electrophoresis was carried out overnight at 7.5 V/cm, until the blue dye had run about 30 cm. Radioactive material was located by atltoradiography. 32P-labelled 5S RNA from Bacillus licheniformis and 32P-labelled-tRNA from yeast were gifts from dr.H.Raue. Ti RNAase and pancreatic RNAase fingererinting. The labelled transcripts were digested with either Ti RNAase (Sankyo, Tokyo, Japan) or RNAase A (Boehringer Mannheim, Germany) for 30 min at 370C at an enzyme to carrier tRNA weight ratio of 1: 10 or 1: 20 respectively. The digestion products were separated by the standard two-dimensional system, described by Brownlee and Sanger 23 , in the modification of Volckaert et al. 24 Cellulose acetate .
2829
Nucleic Acids Research strips were from Schleicher and Schall (Dassel, Germany). The PEI thin layer sheets from Macherey-Nagel & Co. (DUren, Germany) were washed according to the short washing procedure described by Southern and Mitchell 25. The homochromatography was performed at 650 with a 3% homomix. The RNA in this homomix (yeast RNA, sodium salt, BDH, Poole, UK) was hydrolysed for 30 min at pH 12.8 at room temperature. The labelled oligonucleotides were located by autoradiography and eluted as described by Volckaert et al. 24
8a lysis
of the ol i gonucl eot ides. The o ligonucleotides from the Tl RNAase fingerprints were digested with pancreatic RNAase or hydrolysed in alkali. The products were characterised by electrophoresis on DEAE paper at pH 3.5 or on Whatman 540 paper at pH 3.5 respectively, according to Barrell 13. The oligonucleotides from pancreatic RNAase fingerprints were digested with Ti RNAase or hydrolysed in alkali. The products were again characterised by electrophoresis on DEAE paper or Whatman 540 respectively. The products were located by autoradiography. The relative amounts of the products were determined by cutting out the paper and counting the Cerenkov radiation in a liquid scintillation counter. Hybridisations. Hybridisations were carried out as described by Sugimoto et al. 18. A sample of the in vitro synthesised RNA was mixed with 10 hg *X174 viral DNA. Sodiumchloride and sodiumcitrate were added to final concentrations of 0.9 M and 0.09 M respectively. The mixture was heated at 1000C for 5 min and after standing for 4 h at 560C cooled down slowly. Then the mixture was passed over a Sartorius nitrocellulose filter MF14. The filter was washed four times with 0.5 ml of a 0.3 M NaCl and 0.03 M sodiumcitrate solution. The RNA retained on the filter was recovered by heating the filter in 2 ml 0.02 M Tris/HCl,pH 7.5 at 1000C for 5 min and precipitated with ethanol, then it was dried and dissolved in water. The fraction of the RNA that ran through the filter was also precipitated with ethanol, dried and dissolved in water. The hybridising and non hybridising materials were also separated by sucrose gradient centrifugation. After cooling, the hybridisation mixture was layered on a 5-20% linear sucrose gradient. After 16 h of centrifugation in a SW27 1 rotor at 24,000 rpm at 10 C the material hybridised to viral DNA was halfway the gradient. Non hybridising material was present in the top fractions of the gradient. Both fractions were precipitated with ethanol, dried and dissolved in water. Fragent
assay.
Fragment
assays
as
described
by
Weisbeek
et
al.
26were
car-
ried out with the viral DNA of the gene A mutants am 50, to 5, am 8, am 30 and am 86 (the latter as a control). The whole A7Z2 fragment as well as the
2830
Nucleic Acids Research larger part of this fragment after cleavage with the restriction enzyme from Haemophilus influenzae F, Hinf I, were tested.
RESULTS
RNA synthesis
and analysis. The kinetics of RNA synthesis by E. col i RNA polymerase , using the denatured restriction fragment A7Z2, 71 nucleotide pairs in length, as template, is shown in fig. 1. After 3 h 10% of the labelled a-32 P NTP (125 pmol) was incorporated, corresponding to 0.2 ig RNA. This indicates that, on an average, each template molecule has been used twice.
x
z
z 0 0
O
12
0 8
0L
0 6
Z4-
8
04 11
-
18
TI ME (min)
Figure 1 Kinetics of the incorporation of t32P] GMP in RNA under the low salt conditions, using the denatured restriction fragment as template. Analysis on urea gels (fig. 2) of the RNA, synthesised at 10 min, 45 min and 3 h, using 5 S RNA from B. licheniformis (116 nucleotides in length 27) and tRNA from yeast (mean length 77 nucleotides) as references, shows that more or less specific products are formed, resulting in 4 rather distinct bands, corresponding to lengths of about 67, 82, 112 and 132 nucleotides respectively.
!ing2Eprint analysis. T1 RNAase and pancreatic RNAase fingerprints of the four transcripts, labelled separately with each of the four NTP's, are shown in fig. 3 and 4 respectively. The patterns of the intensive spots were very reproducible. The presence of the minor spots differed from batch to batch synthesised RNA (i.e. in the one case more faint spots were found than in the other and their relative intensity was not constant and therefore it was difficult to determine their complete sequences). The faint spots that could be analysed turned out to be incomplete Ti or pancreatic oligonucleotides, or 2831
Nucleic Acids Research 10'
45'
180'
-Zr
R
5S tRNA
start-.,
132
-
112-.---
82--o
9I
67--..
Figure 2 Urea gel analysis of the RNA synthesised at 10 min, 45 min at 3 h incubation, using the denaturated restriction fragment as template.
2'-3' cyclic intermediates. This suggests that they arise from random starts or stops of the RNA polymerase, or incomplete or unspecific digestion during Ti RNAase incubation. lysisof the oli gonucl eot ides. In table I the products formed by digestion with pancreatic RNAase or alkaline hydrolysis of the Ti oligonucleotides and the deduced sequences are listed. When more than one product was formed, the relative amount is given. The oligonucleotide T3, CCG(U), was always underrepresented (but if labelled it contained significantly more label than the minor spots). A possible explanation for this phenomenon is given below. In table 2 the data are given for the pancreatic oligonucleotides, digested with Ti RNAase or hydrolysed in alkali. From these data the sequence of all oligonucleotides except P17 and P18 could be deduced. In oligonucleotide P18 AG(A) and AAG(A) could be interchanged. The right order was determined by twodimensional separation of the products produced by partial spleen phosphodiesterase digestion from the 5' end of P18 obtained from a transcript labelled withra-32P]UTP, in which case only the 3' terminal An
2832
Nucleic Acids Research
I
* .1 .. 4
..
_
W
..
0. ._~~~~~~~~~~%
1
*4 :* t 'a
~~~~~~1 I
e4
11'
0
s
JO
G e
.4
A
3-
F,
.4% 9 I% Oli)
.
.
q
0
U,
Figure 3 Two-dimensional fingerprints of Ti RNAase digest of the four differently labelled transcripts (with [a-32P]GTP:G, with [a-32P]ATP:A, with [a -32P]UTP:U and with [a-32P]CTP:C). Electrophoresis was from left to right and homochromatography was developed from the botton upwards. The numbers refer to those in table 1. nucleotides are labelled. Because of contradictory results in the analyses of oligonucleotide P17, AAGAG(...), which could not be resolved, its complete sequence is not clear. However, the 5' end overlaps the 3' end of oligonucleotide T18 ...AAG(A) (table 1). This phenomenon will be discussed further below. The sequence of oligonucleotide T21, UCUUUUCG(U), in which several sites are possible for the C residue that is flanked by two U residues, was deduced by making the reasonable assumption that it is complementary to T8, AAAAG(A) and P10, GAAAAGAC(A). Similarly, the ambiguities of the 2833
Nucleic Acids Research
34
8X-
4*
,
Nucleic Acids Research
The nucleotide sequence of a DNA fragment, 71 base pairs in length, near the origin of DNA replication of bacteriophage OX174 A. D. M. van Mansfeld, J. M. Vereijken and H. S. Jans z Institute for Molecular Biology and Laboratory for Physiological Chemistry, State University, Utrecht, The Netherlands
Received 23 August 1976 ABSTRACT Part of the nucleotide sequence of a restriction fragment covering the origin of *X174 DNA replication 1 has been determined. The fragment A7c was obtained by digestion of *X174 RF DNA by the restriction enzyme from Arthrobacter luteus, Alu I. It was further cleaved into two fragments, one l-argeand one smal l, by the action of the restriction enzyme from Haemophilus aegyptius, Hae II . The nucleotide sequence of the small fragment has been determined by analysis of the transcription products obtained by the action of Escherichia coli DNA-dependent RNA polymerase on denaturated template under conditions7of low salt. Transcripts longer than the template were found. The whole sequence of 71 nucleotide pairs could be derived from complementary oligonucleotides, obtained after digestion of the transcripts with Ti or pancreatic RNAase. The sequence suggests that at least 4 of the 5 amber mutants 2 that have been mapped on this fragment are identical. On account of this and other evidence a reading frame is proposed.
INTRODUCTION
*X174 is a small single stranded DNA phage (recently reviewed by Denhardt 3). Several investigators have determined nucleotide sequences from different regions of the genome by direct or indirect sequence methods 4,5,6,7. The limited number of possibilities to isolate specific DNA fragments has hampered further sequence analysis considerably. However, thanks to the discovery of the restriction enzymes, the whole double stranded *X174 replicative form (RF) DNA now can be cleaved in specific fragments not longer than 250 base pairs 2,8t9j10,11 which are accessible to the different ways of sequence analysis, or can be used as specific primers in the method for sequence analysis described by Sanger and Coulson 12 Using the standard methods for RNA sequencing 13, the sequence analysis of RNA obtained by asymmetric transcription of native template in vitro has been described by several authors 14,15,16 Air et al. 17 and Sugimoto o Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
2827
Nucleic Acids Research
i8used
denaturated DNA restriction fragments as templates for the transcription. This latter method proves to be very powerful since the overlapping sequences can often be derived from complementary oligonucleotides. By this method part of the sequence of A7c has been determined. This fragment was obtained by digestion of OX174 RF DNA with the restriction enzyme from Arthrobacter luteus, Alu I, and overlaps some functionally interesting regions of the OX DNA, including the origin of RF DNA replication , the origin of ss DNA synthesis 19, and the terminus of RF DNA replication °. In order to facilitate sequence analysis the fragment A7c was further cleaved into two fragments, one large and one small, by the action of the restriction enzyme from Haemophilus aegyptius, Hae III. The present paper reports the results of sequence analysis of the small fragment.
et al.
MATERIALS AND METHODS PreE2aration of the DNA fragments. *X174 RF DNA was digested by the restriction enzyme from Arthrobacter luteus, Alu I 9. The products were separated 21 . A by continuous gel electrophoresis as described by Lee and Sinsheimer 5% polyacrylamide gel (g = 1 cm, length = 24 cm) was used instead of an agarose gel. The fractions containing the three A7 fragments were pooled and the DNA was precipitated by adding sodium acetate to a concentration of 0.3 M and two volumes of ethanol. The A7 fragments were redigested by the restriction enzyme from Haemophi;lus aegyptius, Hae III, which resulted in two uncleaved A7 fragments, one fragment (A7Z1) of about 180 and one fragment (A7Z2) of 71 base pairs in length 9. This mixture was again subjected to continuous gel electrophoresis on a 5% polyacrylamide gel. The DNA fragments were concentrated by ethanol precipitation and dissolved in 0.01 M Tris/HCl,pH 8.0, 0.001 M EDTA.
RA&221YmErase.
Batches of E. coli DNA dependent RNA polymerase were gifts from mr.H.van Keulen and mr.C.van Kreyl respectively. Both batches of enzyme were ATPase-, DNAase- and RNAase-free. RNA synthesis. Incubation mixtures were essentially the same as described by Blackburn 7: 25 mM Tris/HCl,pH 7.8, 10 mM MgCl2, 5 mM dithiotreitol, 0.5 mM EDTA, 0.8 mM potassium phosphate, three unlabelled ribonucleoside triphosphates (Merck, Darmstadt, Germany) at a concentration of 0.5 mM each and one [a-32 P] ribonucleoside triphosphate (The Radiochemical Centre, Amersham, UK; specific activity 5-10 Ci/mmol, later on 50-100 Ci/mmol) at a concentration of 0.05 mM. The double stranded DNA restriction fragment was denaturated by heating 2828
Nucleic Acids Research 1000C for
3 min and cooling rapidly in ethanol-dry ice. 0.1 pg denaturated DNA was used, and RNA polymerase solution was added to bring the molar template to polymerase ratio to 1: 3. The final volume was 25 pi. The incubation reaction was carried out for 3 h (unless otherwise stated) at 370C and the reaction was stopped by adding 25 p1 0.02 M EDTA, containing 2 mg/ml tRNA (BDH, Poole, UK). This mixture was extracted twice with freshly destilled phenol, saturated with 0.1 M NaCl', 0.01 M Tris/HCl,pH 7.5, 0.001 M EDTA, and eluted over a Sephadex G50 column (in the latter buffer) to remove the low molecular weight materials. The material eluting in the void volume was precipitated with ethanol. After standing for 5 min at -700C (ethanol-dry ice) and 1l h at -200C, the precipitate was spun down, dried in a stream of air, dissolved in 100 pli water and stored at -20 C until use. TCA2_recieitation. The quantity of RNA synthesized during the incubation was determined by trichloroacetic acid precipitation of a 2.5 PI sample of the "stopped mixture" (see above). This was added to 50 -il of a BSA solution (0.25 mg BSA/ml, 0.01 M Tris/HCl,pH 7.5, 0.001 M EDTA). 2.5 ml of an ice cold 10% TCA solution, containing 0.01 M tetra sodium pyrophosphate was added and after 'standing for 15 min at 00C, the precipitate was filtered over a Whatman GF/C 'filter, washed three times with 5 ml of the 10% TCA solution and three times with 5 ml of an ice cold 2% TCA solution containing 0.01 M tetra sodium pyrophosphate. The filter was dried and counted with a Triton based scintillation fluid in a liquid scintillation counter.
at
9[22-R21Y2S[Y12mide
electr2eb2E
10%
polyacrylamide gels (2 mm slab_gel is. thick, 18 cm wide and 40 cm long), in 0.04 M Tris/HCl,pH 8.0, 0.02 sodium acetate, 0.002 M EDTA were prepared as described by Loening 22 with the modification that urea was added to a concentration of 7 M. Samples were dried in a stream of air, dissolved in 20 il formamide, containing sucrose and bromophenol blue as tracking dye. Before layering the gels, the samples were heated at 1000C for 5 min. Electrophoresis was carried out overnight at 7.5 V/cm, until the blue dye had run about 30 cm. Radioactive material was located by atltoradiography. 32P-labelled 5S RNA from Bacillus licheniformis and 32P-labelled-tRNA from yeast were gifts from dr.H.Raue. Ti RNAase and pancreatic RNAase fingererinting. The labelled transcripts were digested with either Ti RNAase (Sankyo, Tokyo, Japan) or RNAase A (Boehringer Mannheim, Germany) for 30 min at 370C at an enzyme to carrier tRNA weight ratio of 1: 10 or 1: 20 respectively. The digestion products were separated by the standard two-dimensional system, described by Brownlee and Sanger 23 , in the modification of Volckaert et al. 24 Cellulose acetate .
2829
Nucleic Acids Research strips were from Schleicher and Schall (Dassel, Germany). The PEI thin layer sheets from Macherey-Nagel & Co. (DUren, Germany) were washed according to the short washing procedure described by Southern and Mitchell 25. The homochromatography was performed at 650 with a 3% homomix. The RNA in this homomix (yeast RNA, sodium salt, BDH, Poole, UK) was hydrolysed for 30 min at pH 12.8 at room temperature. The labelled oligonucleotides were located by autoradiography and eluted as described by Volckaert et al. 24
8a lysis
of the ol i gonucl eot ides. The o ligonucleotides from the Tl RNAase fingerprints were digested with pancreatic RNAase or hydrolysed in alkali. The products were characterised by electrophoresis on DEAE paper at pH 3.5 or on Whatman 540 paper at pH 3.5 respectively, according to Barrell 13. The oligonucleotides from pancreatic RNAase fingerprints were digested with Ti RNAase or hydrolysed in alkali. The products were again characterised by electrophoresis on DEAE paper or Whatman 540 respectively. The products were located by autoradiography. The relative amounts of the products were determined by cutting out the paper and counting the Cerenkov radiation in a liquid scintillation counter. Hybridisations. Hybridisations were carried out as described by Sugimoto et al. 18. A sample of the in vitro synthesised RNA was mixed with 10 hg *X174 viral DNA. Sodiumchloride and sodiumcitrate were added to final concentrations of 0.9 M and 0.09 M respectively. The mixture was heated at 1000C for 5 min and after standing for 4 h at 560C cooled down slowly. Then the mixture was passed over a Sartorius nitrocellulose filter MF14. The filter was washed four times with 0.5 ml of a 0.3 M NaCl and 0.03 M sodiumcitrate solution. The RNA retained on the filter was recovered by heating the filter in 2 ml 0.02 M Tris/HCl,pH 7.5 at 1000C for 5 min and precipitated with ethanol, then it was dried and dissolved in water. The fraction of the RNA that ran through the filter was also precipitated with ethanol, dried and dissolved in water. The hybridising and non hybridising materials were also separated by sucrose gradient centrifugation. After cooling, the hybridisation mixture was layered on a 5-20% linear sucrose gradient. After 16 h of centrifugation in a SW27 1 rotor at 24,000 rpm at 10 C the material hybridised to viral DNA was halfway the gradient. Non hybridising material was present in the top fractions of the gradient. Both fractions were precipitated with ethanol, dried and dissolved in water. Fragent
assay.
Fragment
assays
as
described
by
Weisbeek
et
al.
26were
car-
ried out with the viral DNA of the gene A mutants am 50, to 5, am 8, am 30 and am 86 (the latter as a control). The whole A7Z2 fragment as well as the
2830
Nucleic Acids Research larger part of this fragment after cleavage with the restriction enzyme from Haemophilus influenzae F, Hinf I, were tested.
RESULTS
RNA synthesis
and analysis. The kinetics of RNA synthesis by E. col i RNA polymerase , using the denatured restriction fragment A7Z2, 71 nucleotide pairs in length, as template, is shown in fig. 1. After 3 h 10% of the labelled a-32 P NTP (125 pmol) was incorporated, corresponding to 0.2 ig RNA. This indicates that, on an average, each template molecule has been used twice.
x
z
z 0 0
O
12
0 8
0L
0 6
Z4-
8
04 11
-
18
TI ME (min)
Figure 1 Kinetics of the incorporation of t32P] GMP in RNA under the low salt conditions, using the denatured restriction fragment as template. Analysis on urea gels (fig. 2) of the RNA, synthesised at 10 min, 45 min and 3 h, using 5 S RNA from B. licheniformis (116 nucleotides in length 27) and tRNA from yeast (mean length 77 nucleotides) as references, shows that more or less specific products are formed, resulting in 4 rather distinct bands, corresponding to lengths of about 67, 82, 112 and 132 nucleotides respectively.
!ing2Eprint analysis. T1 RNAase and pancreatic RNAase fingerprints of the four transcripts, labelled separately with each of the four NTP's, are shown in fig. 3 and 4 respectively. The patterns of the intensive spots were very reproducible. The presence of the minor spots differed from batch to batch synthesised RNA (i.e. in the one case more faint spots were found than in the other and their relative intensity was not constant and therefore it was difficult to determine their complete sequences). The faint spots that could be analysed turned out to be incomplete Ti or pancreatic oligonucleotides, or 2831
Nucleic Acids Research 10'
45'
180'
-Zr
R
5S tRNA
start-.,
132
-
112-.---
82--o
9I
67--..
Figure 2 Urea gel analysis of the RNA synthesised at 10 min, 45 min at 3 h incubation, using the denaturated restriction fragment as template.
2'-3' cyclic intermediates. This suggests that they arise from random starts or stops of the RNA polymerase, or incomplete or unspecific digestion during Ti RNAase incubation. lysisof the oli gonucl eot ides. In table I the products formed by digestion with pancreatic RNAase or alkaline hydrolysis of the Ti oligonucleotides and the deduced sequences are listed. When more than one product was formed, the relative amount is given. The oligonucleotide T3, CCG(U), was always underrepresented (but if labelled it contained significantly more label than the minor spots). A possible explanation for this phenomenon is given below. In table 2 the data are given for the pancreatic oligonucleotides, digested with Ti RNAase or hydrolysed in alkali. From these data the sequence of all oligonucleotides except P17 and P18 could be deduced. In oligonucleotide P18 AG(A) and AAG(A) could be interchanged. The right order was determined by twodimensional separation of the products produced by partial spleen phosphodiesterase digestion from the 5' end of P18 obtained from a transcript labelled withra-32P]UTP, in which case only the 3' terminal An
2832
Nucleic Acids Research
I
* .1 .. 4
..
_
W
..
0. ._~~~~~~~~~~%
1
*4 :* t 'a
~~~~~~1 I
e4
11'
0
s
JO
G e
.4
A
3-
F,
.4% 9 I% Oli)
.
.
q
0
U,
Figure 3 Two-dimensional fingerprints of Ti RNAase digest of the four differently labelled transcripts (with [a-32P]GTP:G, with [a-32P]ATP:A, with [a -32P]UTP:U and with [a-32P]CTP:C). Electrophoresis was from left to right and homochromatography was developed from the botton upwards. The numbers refer to those in table 1. nucleotides are labelled. Because of contradictory results in the analyses of oligonucleotide P17, AAGAG(...), which could not be resolved, its complete sequence is not clear. However, the 5' end overlaps the 3' end of oligonucleotide T18 ...AAG(A) (table 1). This phenomenon will be discussed further below. The sequence of oligonucleotide T21, UCUUUUCG(U), in which several sites are possible for the C residue that is flanked by two U residues, was deduced by making the reasonable assumption that it is complementary to T8, AAAAG(A) and P10, GAAAAGAC(A). Similarly, the ambiguities of the 2833
Nucleic Acids Research
34
8X-
4*
,
