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Isolation and Genetic Localization of Three φX174 Promoter Regions
C. Chen et al .
Editor’s Note
Four years before the φ X174 bacteriophage became the first DNA-based organism to have its genome completely sequenced, Cheng-Yien Chen and colleagues had used restriction enzymes (which cut DNA at sequence-specific locations) to produce a genetic map of this model molecular biology system. Importantly, they managed to isolate and localize three promoter regions (DNA segments that facilitate the transcription of a particular gene) by clever use of a “ protective ” RNA polymerase protein that let them digest away the exposed, unwanted nucleic acid. Three decades later the φ X174 bacteriophage was to court attention again, when researchers reported that they had synthetically assembled its genome from scratch. 中文
SPECIFIC sequences of nucleic acids such as ribosome binding sites 1 (W. Gilbert, cited in ref. 2) and portions of the promoter region 3,4 have been isolated by protecting those sequences with the relevant protein or organelle and digesting away the exposed nucleic acid. We have isolated specific sequences from the promoter regions in bacteriophage φ X174 replicative form (RF) DNA using RNA polymerase as the protecting protein. We wish to describe that isolation and the procedures used to localize these RNA-polymerase-protected sequences within the φ X174 genome. 中文
We have previously described the use of restriction enzymes to cleave φ X174 DNA into specific fragments 5,6 and the genetic assay used to order these fragments with respect to the φ X174 recombination map 7 . The φ X genome is separated into 11-14 specific pieces using either of the Haemophilus restriction enzymes, endonuclease R 5,8 or endonuclease Z 6 . Our approach has been to determine which restriction fragments contain sequences protected by RNA polymerase. As most of the restriction fragments have been ordered with respect to the φ X174 genetic map (manuscript in preparation), this localizes the protected sequences within the map as well. 中文
Two procedures were used to identify DNA fragments bearing sequences protected by RNA polymerase. In the first, Escherichia coli RNA polymerase was bound to 3 H-labelled φ X174 RFI (covalently closed circular DNA). After extensive digestion with pancreatic DNase, the fraction of the DNA protected by RNA polymerase was isolated. The protected 3 H-DNA was then hybridized to purified 32 P-labelled restriction fragments of φ X174 RF immobilized on nitrocellulose filters. Retention of 3 H counts indicated the presence of homologous sequences within that particular specific restriction fragment. 中文
About 1.9% of φ X174 RFI is protected from DNase digestion by RNA polymerase. The DNA-RNA polymerase complex formed is quite stable because an excess of cold RF during digestion does not change the fraction of labelled DNA protected. The sites which can be protected by RNA polymerase are saturated, as increasing the ratio of RNA polymerase to DNA beyond 4 μg polymerase to 0.02 μg DNA (our standard conditions) does not increase the amount of DNA protected. 中文
Assuming the average chain length of a protected site is thirty-five base pairs long 3 , the resistant counts represent approximately three to four such sites per genome. The protected 3 H-labelled DNA sequences were eluted from a nitrocellulose filter with 0.2% SDS and hybridized to a set of filters loaded with purified restriction fragments of φ X174 RF produced by endonuclease R and to a separate set of filters containing the fragments produced by endonuclease Z. The RNA polymerase protected DNA hybridized to filters bearing endonuclease R fragments r2, r4 and r6 and to endonuclease Z fragments z1,z2 and z3 (Table 1). 中文
Table 1. Protection of DNA
* A blank filter background of 0.39% has been subtracted.
† A blank filter background of 0.35% has been subtracted.
Hybridization of RNA-polymerase-protected 3 H-labelled φ X174 RF DNA to specific 32 P-labelled, endonuclease R and Z fragments. The preparation of 32 P-RFI, endonuclease R and Z digestions, electrophoresis and autoradiography have been described previously 5,6 . The 32 P endo Z and endo R fragments were eluted from the bands of dry gel in 2×SSC at 65℃ for 24 h (N. Axelrod, personal communication). Equimolar amounts of the 32 P restriction fragments were immobilized on the membrane filters (25 mm) (Schleicher and Schuell, B6). Each fragment was made up to 20 ml at a final concentration of 0.1×SSC. After heating at 100℃ for 5 min and quickly quenching in the ice bath, each fragment was immobilized on a B6 filter according to the method of Raskas and Green 18 . The 3 H-RFI-DNA-RNA polymerase complexes were produced as follows. The reaction mixture contained 0.02 μg of 3 H-RFI-DNA (about 75,000 c.p.m.) (prepared as in ref. 6), 20 μg of RNA polymerase and 0.1 mM each of ATP and GTP in 0.5 ml of buffer A (8 mM MgCl 2 , 50 mM KCl, 20 mM Tris, p H 7.9, 0.1 mM dithiothreitol). The RNA polymerase had been purified through a glycerol gradient centrifugation as in the method of Burgess 16 . Acrylamide gels had verified that sigma factor was present in our preparation. After 5 min at 37℃, 10 μg of unlabelled RFI-DNA, followed by 200 μg of pancreatic DNase, was added and the digestion was continued for 40 min. The mixture was chilled, diluted with cold buffer A and the complexes were collected on a nitrocellulose filter (“Millipore”, HA). The protected sequences were freed from the filter by treatment with 0.2% SDS. An aliquot (430 c.p.m.) of the protected sequences was heated at 100℃ for 5 min and quickly quenched in ice and hybridized to each filter containing 32 P endo Z and endo R fragments. The filters were then counted with a Packard Tri-Carb scintillation counter in a toluene-based scintillation fluid. The percentage of the 3 H bound was calculated as the fraction of input counts bound minus the percentage of counts bound to a blank filter. The counts were determined from 100 min counts and were reproducible in four different experiments.
The second procedure used to identify DNA fragments bearing RNA-polymerase-protected sequences was to bind RNA polymerase to 32 P-labelled restriction fragments of φ X174 RF and then to pass the reaction mixture through a nitrocellulose filter which should retain only the RNA polymerase- 32 P-labelled DNA fragment complex 9 . The filtrate was then electrophoresed in 3% polyacrylamide gels to determine which DNA fragments were missing. The gel was then fractionated and counted to obtain quantitative data on the recovery of each restriction fragment. The recoveries of these fragments compared with fragments which were not reacted with RNA polymerase showed that fragments r2, r4 and r6 were retained by the filter (Fig. 1). A similar experiment with restriction fragments produced by endonuclease Z showed that fragments z1, z2 and z3 were retained by filtration. 中文
Fig. 1. Quantitative determination of fragment recovery with and without RNA polymerase binding before nitrocellulose filtration. A sample of 200 μl of 32 P- φ X RF DNA (2.6×10 5 c.p.m., 0.08 μg) was digested with endonuclease R 5 . RNA polymerase (32 μg) was then mixed with 100 μl of the endo R limit digest of the RF to give a final volume of 200 μl in buffer A (8 mM MgCl 2 , 50 mM KCl, 20 mM Tris, p H 7.9, 0.1 mM dithiothreitol) and 0.1 mM each of ATP and GTP. After 5 min at 37℃ the reaction mixture was passed through a nitrocellulose filter (“Millipore”, HA). The filtrate containing approximately 150 μl was subjected to electrophoresis 5 . The remaining 100 μl of the endo R limit digest of RF was treated as above except that RNA polymerase was omitted. After electrophoresis the gels were dried on a filter paper, cut into 1 mm segments and counted with a scintillation counter. The tracking dye migrated 142 mm. The integrated counts in the various bands were plotted (log scale) against mobilities (band position in mm). Band R6 has been shown to contain three unresolved fragments of about the same size 5 and so has three times (n=3) the number of counts as those bands containing a single fragment (n=1). Band R7 as those bands containing a single fragment (n=1). Band R7 contains two unresolved fragments (n=2). ●, No RNA-P; ○, plus RNA-P.
The data from these experiments are consistent with the restriction fragment map for φ X174. That is, each r fragment containing sequences protected by RNA polymerase overlaps with a z fragment also shown to contain such a sequence (Fig. 2). 中文
Fig. 2. Locations on the genetic map of φX174 of restriction fragments containing sequences protected by RNA polymerase. The order of eight φX174 genes ( A through H ) has been determined by genetic recombination 15 . The gene sizes shown on this map were calculated from published estimates of the molecular weights of the corresponding gene products 19–21 . (The assignments of sizes for genes A , B , C and E are uncertain; however, the exact sizes of these genes do not affect the general picture presented here.) As the estimated gene sizes add up to 90% of the total genome size (5,500 nucleotide pairs), the remaining 10% has been arbitrarily distributed as intergenic spacers. Fragments z1, z2 and z3 have been mapped by the genetic assay for DNA fragments 5-7,22 and contain the indicated genetic sites. Fragment r2 is homologous to z1 by hybridization, but does not contain any of the genetic sites shown. A different endonuclease R fragment (r1) contains the site F ts 41D and extends counterclockwise beyond the end of z1. Therefore, r2 must lie between the sites F ts 41D and G am 9 as shown. Fragment r4 is homologous to z2 by hybridization. Also, r4 is adjacent to a fragment (r3) containing the site am 33 in gene A . So r4 must be located in the region indicated. (We know that r4 and r3 are adjacent because electrophoresis of an endonuclease R digest of phage S13 RF gives the normal φX174 pattern of fragments except that r4, r3 and r5 (gene B ) are missing and are replaced by a single large fragment.) Band R6 has been shown to contain three fragments 5 (named r6.1, r6.2 and r6.3) which have been partially resolved on prolonged electrophoresis. Fragments r6.1 and r6.2 contain genetic sites within cistrons G and H; therefore, fragment r6.3 (containing the indicated sites in cistron D) must be the component of band R6 which shares a polymerase protected sequence with fragment z3.
The RNA polymerase DNA fragment complexes were also visualized in the electron microscope. RNA polymerase was reacted with purified restriction fragments r2 and z1 and stained by the Kleinschmidt procedure for microscopy. When r2 was mixed with RNA polymerase the structures shown in Fig. 3 a , b , and c were seen. Polymerase was seen bound only at the end of r2, although not every r2 fragment had an enzyme molecule associated with it. On the other hand, when RNA polymerase was mixed with fragment z1 all the binding seen was internal, about one-third of the way from the end (Fig. 3 d , e and f ). These fragments have not been placed precisely enough with respect to the recombination map to be sure which end of r2 contains the sequences protected by RNA polymerase. 中文
Fig. 3. Electron microscopic pictures of RNA polymerase-restriction fragment mixtures. RNA polymerase was bound to the fragments r2 ( a , b , c ) and z1 ( d , e , f ) as described in Fig.1. The RNA polymerase-DNA fragments were prepared for microscopy according to Kleinschmidt’s procedure, as modified by Davis and Davidson 17 . Grids containing DNA-RNA polymerase complexes were stained with uranyl acetate and then photographed at a magnification of 20,000 with an AEI model EM6B electron microscope. The bar represents 0.2 μm.
It is clear that RNA polymerase when reacted with DNA in this way protects specific sequences from nuclease digestion. It seems likely to us that these sequences contain the start sites 10 within the promoter regions. RNA polymerase in the presence of ATP and GTP presumably becomes associated with the DNA at entry sites 10 and then drifts to the start site where it is prevented from synthesizing mRNA by the absence of all four triphosphates. It has been shown that the sequences from bacteriophage fd protected in a similar fashion by RNA polymerase contain sequences which specify the initial nucleotides in the in vivo messages 3 . Rüger 11 showed that similar sites from T4 phage DNA still serve as a template for RNA polymerase. There is no clear evidence, however, that every sequence protected in this fashion by RNA polymerase contains a start site. 中文
Previous studies of the in vivo and in vitro transcription products of φ X174 12,13 and fd 14 have suggested the existence of multiple sites for message initiation and termination. These sites, however, were not ordered with respect to the genetic map. Studies on φ X174 in vivo translation 15 led to the conclusion that synthesis was initiated with the cistron A and D products. We have now mapped three φ X174 sequences which are protected by RNA polymerase. These sequences lie within restriction fragments which span the beginnings of cistrons A, D and G (Fig. 2). However, our data do not prove that these protected sequences are in fact located at the beginnings of the cistrons. 中文
It is interesting that sequences can be isolated from E . coli DNA by protection with E . coli RNA polymerase which will hybridize to these same restriction fragments from φ X174 RF DNA (Table 2) which we have shown to contain the φ X174 sequences protected by RNA polymerase. Apparently E . coli DNA contains some promoter regions which have start sites similar to those found in φ X174 RF DNA. 中文
Table 2. Hybridization of 3 H-labelled E. coli DNA Sequences Protected by RNA Polymerase to Specific 32 P-labelled Endonuclease R and Z Fragments
* A blank filter background of 0.21% has been subtracted.
† A blank filter background of 0.18% has been subtracted. See legend to Table 1 for the procedures used.
The hybridization data in the two tables indicate a different degree of hybridization with the various restriction fragments. These are reproducible differences. This suggests that the φ X174 promoter regions are not necessarily identical. It may be possible to use these techniques to divide promoter regions into several classes. The overlap between r6.3 and z3 containing an RNA-polymerase-protected sequence is less than 100 nucleotide pairs long. This overlap fragment has been isolated from φ 174 RF cleaved simultaneously by endonucleases R and Z and has been shown to contain a genetic site (D am H81) near the beginning of cistron D (J. H. Middleton, unpublished results). 中文
We thank June H. Middleton for stocks of endonulease R and endonuclease Z. This work was supported by US Public Health Service grants from the National Institute of Allergy and Infectious Diseases. 中文
( Nature New Biology , 243 , 233–236; 1973)
Cheng-Yien Chen, Clyde A. Hutchison, III and Marshall Hall Edgell
Department of Bacteriology and Immunology and Curriculum in Genetics, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514
Received December 14, 1972; revised March 7, 1973.
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