RESTRICTION TRANSDUCING
ENDONUCLEASE A N A L Y S I S OF T H E BACTERIOPHAGE L A M B D A R I F d18
1MRE BOROSand BI~LASAIN hTstitute q[ Biochemistpj'. Biological Research Center, Hungarian Academy q/Sciences, tt6701 Szeged, P. O. B. 521.. Hungary
(Received 15 July; in revised form 24 August, 1977) Abstract. The Xrifd 18 bacteriophage carries essential parts of the E. coli genome which can not be mapped by conventional methods. The phage DNA was analysed with four restriction endonucleases (endo R. BamHl, Sail, HpaI and EcoR1) and a physical map was constructed.
I.
INTRODUCTION
Several transducing bacteriophages were isolated recently, which carry different ribosomal genes ofE. coli [ 1]. These phages are invaluable in the mapping of these essential genes which are difficult to analyse. One of these phages is the ;krif d 18, which was isolated by Kirschbaum and Konrad [2]. This phage carries bacterial genes from the 88 min region of the E. coli chromosome including genes for two RNA polymerase subunits, five ribosomal proteins, elongation factor Tu and a set of genes for rRNA [3]. The hrifd 18 phage DNA was recently mapped with the restriction endonucleases Hindlll, SmaI and partially with EcoR1 [3]. We constructed a physical map using three other restriction enzymes: Hpal, BamH I, SalI and completed the EcoRI map. This more detailed map may be useful for the analysis and cloning of some important parts (like promoters) of the phage.
II.
M A T E R I A L S AND M E T H O D S
krifdl8 bacteriophage and DNA were purified according to standard methods [4]. [32p]rRNA was isolated from the RNA-ase I defficient strain MRE 600 as described [5]. The restriction endonucleases were purified by standard procedures [6]. Agarose gel electrophoresis was carried out according to Helling et al. [7]. 0.7% agarose gels (Sigma) were used. Hybridization of radioactive RNA to the agarose gels was performed by the method of Southern [8]. The molecular weights are given in million daltons [ Md] and the restriction fragments are designated by their molecular weights.
451 Molecular Biology Reports 3 (1977)451-457.All Rights Reserved. Copyright 9 1977 by Reidel Publishing Company, Dordrecht-Holland.
II1.
RESULTS AND DISCUSSION
The Xrifd 18 phage DNA gives rise to 15, 10 and 5 fragments after cleavage with the restriction enzymes Hpal, Sail and BamHI, respectively. The sizes of these fragments were determined by gel electrophoresis (Figure la) and are listed in Table I. Most of the fragments were analysed by agarose gel electrophoresis but in the case of HpaI the smallest fragments were also analysed by polyacrylamide gel electrophoresis, (not shown). The rRNA genes had been located by Nomura and coworkers [3], therefore the fragments carrying the rRNA genes could be identified by hybridization with radiolabelled rRNA. Figure I b shows the result of such an experiment where the specific radioactivity of 23S rRNA was higher than that of the 16S rRNA. Thus the intensive bands represent sequences complementery to 23S rRNS while the weaker ones are the genes for 16S rRNA. Fragments containing both kinds of sequences are observed as intensive bands. The results of this experiment are also listed in Table 1. TABLE I: enzymes
The molecular weights of the fragments produced by the employed restriction
BamHl
Sail
A B C D E
A B C D E F
w 9.70 7.00** 4.50* 2.90 w 2.25 1.92
A B C D E F
w 5.53 4.97* 4.23** 2.85 2.85 2.71
G H I J
1.72 1.56** 0.80 0.31
G H I J K L M N O
2.20 1.91 1.37 1.15 1.08 1.05 w 0.47 0.26 0.14
w 15.1 4.74** 4.68 w 4.16 4.10
Hpal
stlm
32.78
sum 32.66
sum
32.77 The symbols are as follows: underlining means that the fragment is also present in the wild type lambda DNA, wdesignates the terminal fragments; * fragments hybridizing with 16S r RNA only; ** fragments hybridizing with 23 S rR NA. All molecular weights are given in million daltons (Md). 452
Fig. Ia-b. Separation of fragments ofA.rifd18 DN A and determination of the fragments containing sequences complementary to rRNA. (a) 0.7% agarose gel electrophoresis. The samples loaded to the gelsare: A,/td 18(EcoR 1); B, Xdl8 (Hpal); C, Xdl8 (Sail); D, ~d18 (BamHl); E, hdl 8 (Hindlll): F, Xb2(EcoRI)+ Xb2(uncleaved)(molecular weight references).(b) Hybridization with [32P]rRNA to the gel seen in (a). Slot F does not contain hybridizable material therefore it was omitted from Figure Ib. The terminal fragments could be d e t e r m i n e d easily t a k i n g a d v a n t a g e of the cohesive ends of bacteriophage l a m b d a [9]. If the restricted D N A is heated at 65 ~ for 5 m i n the cohesive ends dissociate giving rise to two new fragments while the b a n d representing the stickedtogether terminal fragments disappears from the electropherogram. The t e r m i n a l fragments determined in this way are indicated with w in Table 1.
453
As the physical map of phage lambda is known with the restriction endonucleases used [ 10, l 1, 12, 13] those fragments could be identified which originated from the lambda part of the ,k rifd 18 phage. These fragments are underlined in Table I. From the data listed in this table the complete BamH 1 map could be deduced. There are only two fragments from the bacterial part of the phage and one of them (the 15. l Md) is terminal. Thus the BamHI map is: 15.1 - 4 . 7 4 - 4 . 1 - 4 . 6 8 - 4 . 1 6 . Knowing that the 16S rRNS gene is right to the 23S r R N A gene o n * r i f d 18 DNA the Sail fragments containing r R N A genes could be ordered as well. The 4.5 Md fragment contains only the 16S r R N A gene so it should be next to the 0.31 Md fragment (which is from the lambda part). As the total r R N A gene set is about 4 Md the only sequence of fragments in this region can be 7.0 - 1.56 -4.5. The most limited information was obtained for the H paI fragments, knowing at this stage only the terminal fragments and that the fragments 4.23 and 4.97 are adjacent in this order as judged by the hybridization experiment. The complete SalI could be deduced from the data of a partial digestion. In order to obtain the partial products, samples were taken from a reaction mixture containing ,Xrif d 18 D N A and SalI enzyme at different intervals of time and subjected to co-electrophoresis (Figure 2a).
Fig. 2a-b. (a) Partial digestion of Jkd18 DNA with Sall. Bandsrepresentingpartial products are marked on the left. (b) Double digestionswith Hpal and EcoRI and with Hpal and Smal. The samplesare: A, hdl8 (Hpal): B. ~d 18(Hpal and EcoRI); C, A.d18(Hpal and SmaI). The arrow shows the position of the missingHpal fragment 1.08 Md.
454
T A B L E II: The analysis of some fragments obtained by partial digestion with Sail restriction enzyme Measured mol. weight op partial product
composition
calculated mol. weight
8.6 7.8 6.4 6.2 5.1 4.9
7.0 0.8 1.56 1.56 2.25 2.9 4.5 1.92 1.72
8.56 7.8 6.37 6.06 5.15 4.82 4.81 3.64 2.52
3.8 2.45
+ 1.56 + 7.0 + 4.5 + 0.31 + 4.5 + 2.9 + 1.92 or + 0.31 + 1.72 + 0.8
All of the bands representing partial products could be considered as the sum of two or three fragments. The data are shown in Table II from which the following sequence of Sail fragments was deduced: 2.25 - 2 . 9 0 - 1.92- 1 . 7 2 - 0 . 8 - 7 . 0 - 1.56-4.50-0.31 -9.70. The analysis of a double digest with SalI and EcoRI confirmed the Sail map and additionally the complete map for EcoRI was obtained as: 4.9- 1.54-0.86- 1.85-0.80- 1.35-5.73-... The underlined sequence of fragments represents new information to the map of Lindahl et al. [3]. The H p a I m a p was largely based on the data obtained from double digestions with
H p a I and EcoRI and with H p a I and S m a I (Figure 2b). One of the 2.85 H p a I fragments was not cleaved by EcoRI, thus it can only be situated within the 4.9 EcoRI fragments. This fragment contains also the 0.47 Md left terminal H p a I fragment so there is room for about 1.6 Md. Since no such fragment could be detected, one of the small H p a I fragments (i.e. 1.05, 1.08 or 1.15 Md) should also be included in this fragment. The next fragment may be either 1.37 or 1.15 (or 1.05) as the 1.54 EcoRI fragment is cleaved. The 5.73 Md EcoRI fragment containes the 4.23 H p a I fragment as was concluded from the hybridization experiment. Here is space for 1.5 Md and since no such fragment was detected, one of the small fragments (1.05, 1.08 or 1.15) can be located here. This fragment must be cleaved with SmaI. F r o m the double digest with H p a I and SmaI the 1.08 Md H p a I fragment is missing so it is the nearest neighbour of fragment 4.23. As EcoRI fragment 1.54 is cleaved by HpaI, the 1.15 or 1.05 H p a I fragment is to be the next to fragment 1.08. This leads to the following sequence: 0.47 - (2.85 and 1.05 or 1.15) - 1.37 - (2.85 and 2.20) - 1.15 or 1.08 - 4.23 -
455
If2.85 - 2.20 were the order of these fragments a new fragment with a molecular weight of about 1.3 - 1.4 Md should be detected after double cleavage with HpaI and EcoRl. Due to the absence of such a fragment the only possible arrangement is 2.20 - 2.85.. The last uncertainties were the order of the fragments of 2.85 Md and 1.05 or 1.15 Md since we could not make a distinction between fragments 1.05 and 1.15 Md. This was solved using partial digests with HpaI (not shown) and the final map was found to be: 0 . 4 7 - 2 . 8 5 - 1.05- 1 . 3 7 - 2 . 2 0 - 2 . 8 5 - 1.15- 1 . 0 8 - 4 . 9 7 - 0 . 2 6 - 1.91 -2.71 0.14-5.53. The Hpal map was confirmed using other double or triple digests. The complete physical map is given in Figure 3. The maps of Lindahl et al. are also listed here. Since many double and triple digests were also analysed and no contradiction was observed with these maps we believe that they are correct. However, minor corrections may be necessary for the exact positions of the sites. This map may be useful for a detailed analysis and the cloning of sequences witkcode for essential functions of E. coli.
I
rpoC rpoB rpl tuf [ bacterial insertion
rRNA 23s1~ ;I ~: ',~
N
int
OP
R ]
t
,,~
E
BomHl
Sol 1 Hpol
EcoR1 Smo 1 Hind Ill
E [ D I vl GIII B 1 Ii C 4 D ILl ll G I E IJIKI C I!i := B N ~ hs~lo~l'.~l~l ~?~ I',!E,, 1 I I
I
,
I
C
l
HJF~o
0 A
I II
1 I
I
Fig. 3. The physicalmap ofJkrifd18bacteriophageDNA.The maps of Hindlll, Smal and partly of EcoRlare redrawn from[3]. Fragmentsare designated bythe letters shownin Table I.
IV.
ACKNOWLEDGEMENTS
The authors wish to thank for the hrif d 18 bacteriophage to Dr J. B. Kirschbaum. We are indebted to Dr P. Venetianer for his critical discussion and to Miss M. Kiss for technical assistance.
V.
REFERENCES
1. Jaskunas, S. R.and Nomura, M., TIBS1, 159(1976). 2. Kirschbaum, J. B. and Konrad, E. B.,J. Bacteriol. 116,517(1973).
456
3. Lindahl, L., Yamamoto, M., Nomura, M., Kirschbaum, J.B., Allet, B., and Rochaix, J. D.,J. Mol. Biol. 109, 23 (1977). 4. Miller, J., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, New York, 1972. 5. Stimegi, J., Udvardy, A., and Venetianer, P., MGG 151,305 (1977). 6. Roberts, R. J., Crit. Rev. Biochem. 3, 123 (1976). 7. Helling, R. B., Goodman, H. M., and Boyer, H. W.,J. Virol. 14, 1235 (1974). 8. Southern, E. M., J. Mol. Biol. 98, 503 (1975). 9. Yarmolinsky, R. M.: in A. D. Hershey (ed.), The Bacteriophage Lambda, Cold Spring Harbor Laboratory, New York, 1971, pp. 97. 10. Thomas, M. and Davis, R. W., J. Mol. Biol. 91,315 (1975). 11. Botchan, P.,J. Mol. Biol. 105, 161 (1976). 12. Haggerty, D. M. and Schleif, R. F.,J. Virol. 18,659,(1976). 13. Nosikov, V. V., Braga, E. A., and Sain, B., Gene, in press.
457
ENDONUCLEASE A N A L Y S I S OF T H E BACTERIOPHAGE L A M B D A R I F d18
1MRE BOROSand BI~LASAIN hTstitute q[ Biochemistpj'. Biological Research Center, Hungarian Academy q/Sciences, tt6701 Szeged, P. O. B. 521.. Hungary
(Received 15 July; in revised form 24 August, 1977) Abstract. The Xrifd 18 bacteriophage carries essential parts of the E. coli genome which can not be mapped by conventional methods. The phage DNA was analysed with four restriction endonucleases (endo R. BamHl, Sail, HpaI and EcoR1) and a physical map was constructed.
I.
INTRODUCTION
Several transducing bacteriophages were isolated recently, which carry different ribosomal genes ofE. coli [ 1]. These phages are invaluable in the mapping of these essential genes which are difficult to analyse. One of these phages is the ;krif d 18, which was isolated by Kirschbaum and Konrad [2]. This phage carries bacterial genes from the 88 min region of the E. coli chromosome including genes for two RNA polymerase subunits, five ribosomal proteins, elongation factor Tu and a set of genes for rRNA [3]. The hrifd 18 phage DNA was recently mapped with the restriction endonucleases Hindlll, SmaI and partially with EcoR1 [3]. We constructed a physical map using three other restriction enzymes: Hpal, BamH I, SalI and completed the EcoRI map. This more detailed map may be useful for the analysis and cloning of some important parts (like promoters) of the phage.
II.
M A T E R I A L S AND M E T H O D S
krifdl8 bacteriophage and DNA were purified according to standard methods [4]. [32p]rRNA was isolated from the RNA-ase I defficient strain MRE 600 as described [5]. The restriction endonucleases were purified by standard procedures [6]. Agarose gel electrophoresis was carried out according to Helling et al. [7]. 0.7% agarose gels (Sigma) were used. Hybridization of radioactive RNA to the agarose gels was performed by the method of Southern [8]. The molecular weights are given in million daltons [ Md] and the restriction fragments are designated by their molecular weights.
451 Molecular Biology Reports 3 (1977)451-457.All Rights Reserved. Copyright 9 1977 by Reidel Publishing Company, Dordrecht-Holland.
II1.
RESULTS AND DISCUSSION
The Xrifd 18 phage DNA gives rise to 15, 10 and 5 fragments after cleavage with the restriction enzymes Hpal, Sail and BamHI, respectively. The sizes of these fragments were determined by gel electrophoresis (Figure la) and are listed in Table I. Most of the fragments were analysed by agarose gel electrophoresis but in the case of HpaI the smallest fragments were also analysed by polyacrylamide gel electrophoresis, (not shown). The rRNA genes had been located by Nomura and coworkers [3], therefore the fragments carrying the rRNA genes could be identified by hybridization with radiolabelled rRNA. Figure I b shows the result of such an experiment where the specific radioactivity of 23S rRNA was higher than that of the 16S rRNA. Thus the intensive bands represent sequences complementery to 23S rRNS while the weaker ones are the genes for 16S rRNA. Fragments containing both kinds of sequences are observed as intensive bands. The results of this experiment are also listed in Table 1. TABLE I: enzymes
The molecular weights of the fragments produced by the employed restriction
BamHl
Sail
A B C D E
A B C D E F
w 9.70 7.00** 4.50* 2.90 w 2.25 1.92
A B C D E F
w 5.53 4.97* 4.23** 2.85 2.85 2.71
G H I J
1.72 1.56** 0.80 0.31
G H I J K L M N O
2.20 1.91 1.37 1.15 1.08 1.05 w 0.47 0.26 0.14
w 15.1 4.74** 4.68 w 4.16 4.10
Hpal
stlm
32.78
sum 32.66
sum
32.77 The symbols are as follows: underlining means that the fragment is also present in the wild type lambda DNA, wdesignates the terminal fragments; * fragments hybridizing with 16S r RNA only; ** fragments hybridizing with 23 S rR NA. All molecular weights are given in million daltons (Md). 452
Fig. Ia-b. Separation of fragments ofA.rifd18 DN A and determination of the fragments containing sequences complementary to rRNA. (a) 0.7% agarose gel electrophoresis. The samples loaded to the gelsare: A,/td 18(EcoR 1); B, Xdl8 (Hpal); C, Xdl8 (Sail); D, ~d18 (BamHl); E, hdl 8 (Hindlll): F, Xb2(EcoRI)+ Xb2(uncleaved)(molecular weight references).(b) Hybridization with [32P]rRNA to the gel seen in (a). Slot F does not contain hybridizable material therefore it was omitted from Figure Ib. The terminal fragments could be d e t e r m i n e d easily t a k i n g a d v a n t a g e of the cohesive ends of bacteriophage l a m b d a [9]. If the restricted D N A is heated at 65 ~ for 5 m i n the cohesive ends dissociate giving rise to two new fragments while the b a n d representing the stickedtogether terminal fragments disappears from the electropherogram. The t e r m i n a l fragments determined in this way are indicated with w in Table 1.
453
As the physical map of phage lambda is known with the restriction endonucleases used [ 10, l 1, 12, 13] those fragments could be identified which originated from the lambda part of the ,k rifd 18 phage. These fragments are underlined in Table I. From the data listed in this table the complete BamH 1 map could be deduced. There are only two fragments from the bacterial part of the phage and one of them (the 15. l Md) is terminal. Thus the BamHI map is: 15.1 - 4 . 7 4 - 4 . 1 - 4 . 6 8 - 4 . 1 6 . Knowing that the 16S rRNS gene is right to the 23S r R N A gene o n * r i f d 18 DNA the Sail fragments containing r R N A genes could be ordered as well. The 4.5 Md fragment contains only the 16S r R N A gene so it should be next to the 0.31 Md fragment (which is from the lambda part). As the total r R N A gene set is about 4 Md the only sequence of fragments in this region can be 7.0 - 1.56 -4.5. The most limited information was obtained for the H paI fragments, knowing at this stage only the terminal fragments and that the fragments 4.23 and 4.97 are adjacent in this order as judged by the hybridization experiment. The complete SalI could be deduced from the data of a partial digestion. In order to obtain the partial products, samples were taken from a reaction mixture containing ,Xrif d 18 D N A and SalI enzyme at different intervals of time and subjected to co-electrophoresis (Figure 2a).
Fig. 2a-b. (a) Partial digestion of Jkd18 DNA with Sall. Bandsrepresentingpartial products are marked on the left. (b) Double digestionswith Hpal and EcoRI and with Hpal and Smal. The samplesare: A, hdl8 (Hpal): B. ~d 18(Hpal and EcoRI); C, A.d18(Hpal and SmaI). The arrow shows the position of the missingHpal fragment 1.08 Md.
454
T A B L E II: The analysis of some fragments obtained by partial digestion with Sail restriction enzyme Measured mol. weight op partial product
composition
calculated mol. weight
8.6 7.8 6.4 6.2 5.1 4.9
7.0 0.8 1.56 1.56 2.25 2.9 4.5 1.92 1.72
8.56 7.8 6.37 6.06 5.15 4.82 4.81 3.64 2.52
3.8 2.45
+ 1.56 + 7.0 + 4.5 + 0.31 + 4.5 + 2.9 + 1.92 or + 0.31 + 1.72 + 0.8
All of the bands representing partial products could be considered as the sum of two or three fragments. The data are shown in Table II from which the following sequence of Sail fragments was deduced: 2.25 - 2 . 9 0 - 1.92- 1 . 7 2 - 0 . 8 - 7 . 0 - 1.56-4.50-0.31 -9.70. The analysis of a double digest with SalI and EcoRI confirmed the Sail map and additionally the complete map for EcoRI was obtained as: 4.9- 1.54-0.86- 1.85-0.80- 1.35-5.73-... The underlined sequence of fragments represents new information to the map of Lindahl et al. [3]. The H p a I m a p was largely based on the data obtained from double digestions with
H p a I and EcoRI and with H p a I and S m a I (Figure 2b). One of the 2.85 H p a I fragments was not cleaved by EcoRI, thus it can only be situated within the 4.9 EcoRI fragments. This fragment contains also the 0.47 Md left terminal H p a I fragment so there is room for about 1.6 Md. Since no such fragment could be detected, one of the small H p a I fragments (i.e. 1.05, 1.08 or 1.15 Md) should also be included in this fragment. The next fragment may be either 1.37 or 1.15 (or 1.05) as the 1.54 EcoRI fragment is cleaved. The 5.73 Md EcoRI fragment containes the 4.23 H p a I fragment as was concluded from the hybridization experiment. Here is space for 1.5 Md and since no such fragment was detected, one of the small fragments (1.05, 1.08 or 1.15) can be located here. This fragment must be cleaved with SmaI. F r o m the double digest with H p a I and SmaI the 1.08 Md H p a I fragment is missing so it is the nearest neighbour of fragment 4.23. As EcoRI fragment 1.54 is cleaved by HpaI, the 1.15 or 1.05 H p a I fragment is to be the next to fragment 1.08. This leads to the following sequence: 0.47 - (2.85 and 1.05 or 1.15) - 1.37 - (2.85 and 2.20) - 1.15 or 1.08 - 4.23 -
455
If2.85 - 2.20 were the order of these fragments a new fragment with a molecular weight of about 1.3 - 1.4 Md should be detected after double cleavage with HpaI and EcoRl. Due to the absence of such a fragment the only possible arrangement is 2.20 - 2.85.. The last uncertainties were the order of the fragments of 2.85 Md and 1.05 or 1.15 Md since we could not make a distinction between fragments 1.05 and 1.15 Md. This was solved using partial digests with HpaI (not shown) and the final map was found to be: 0 . 4 7 - 2 . 8 5 - 1.05- 1 . 3 7 - 2 . 2 0 - 2 . 8 5 - 1.15- 1 . 0 8 - 4 . 9 7 - 0 . 2 6 - 1.91 -2.71 0.14-5.53. The Hpal map was confirmed using other double or triple digests. The complete physical map is given in Figure 3. The maps of Lindahl et al. are also listed here. Since many double and triple digests were also analysed and no contradiction was observed with these maps we believe that they are correct. However, minor corrections may be necessary for the exact positions of the sites. This map may be useful for a detailed analysis and the cloning of sequences witkcode for essential functions of E. coli.
I
rpoC rpoB rpl tuf [ bacterial insertion
rRNA 23s1~ ;I ~: ',~
N
int
OP
R ]
t
,,~
E
BomHl
Sol 1 Hpol
EcoR1 Smo 1 Hind Ill
E [ D I vl GIII B 1 Ii C 4 D ILl ll G I E IJIKI C I!i := B N ~ hs~lo~l'.~l~l ~?~ I',!E,, 1 I I
I
,
I
C
l
HJF~o
0 A
I II
1 I
I
Fig. 3. The physicalmap ofJkrifd18bacteriophageDNA.The maps of Hindlll, Smal and partly of EcoRlare redrawn from[3]. Fragmentsare designated bythe letters shownin Table I.
IV.
ACKNOWLEDGEMENTS
The authors wish to thank for the hrif d 18 bacteriophage to Dr J. B. Kirschbaum. We are indebted to Dr P. Venetianer for his critical discussion and to Miss M. Kiss for technical assistance.
V.
REFERENCES
1. Jaskunas, S. R.and Nomura, M., TIBS1, 159(1976). 2. Kirschbaum, J. B. and Konrad, E. B.,J. Bacteriol. 116,517(1973).
456
3. Lindahl, L., Yamamoto, M., Nomura, M., Kirschbaum, J.B., Allet, B., and Rochaix, J. D.,J. Mol. Biol. 109, 23 (1977). 4. Miller, J., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, New York, 1972. 5. Stimegi, J., Udvardy, A., and Venetianer, P., MGG 151,305 (1977). 6. Roberts, R. J., Crit. Rev. Biochem. 3, 123 (1976). 7. Helling, R. B., Goodman, H. M., and Boyer, H. W.,J. Virol. 14, 1235 (1974). 8. Southern, E. M., J. Mol. Biol. 98, 503 (1975). 9. Yarmolinsky, R. M.: in A. D. Hershey (ed.), The Bacteriophage Lambda, Cold Spring Harbor Laboratory, New York, 1971, pp. 97. 10. Thomas, M. and Davis, R. W., J. Mol. Biol. 91,315 (1975). 11. Botchan, P.,J. Mol. Biol. 105, 161 (1976). 12. Haggerty, D. M. and Schleif, R. F.,J. Virol. 18,659,(1976). 13. Nosikov, V. V., Braga, E. A., and Sain, B., Gene, in press.
457
