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Studies on polynucleotides
J. Mel. Sol. (1971) 56, 363-368
Studies on Polynucleotides XCVII. Opposing Orientations and Location of the SU& Gene in the Transducing Coliphages @Ops& and #30&&w&t R. C. MILLER
JR., P. BESMER,
H. G. KHORANA,
Institute for Enzyme Research, The University of Wiswnsin$ M. FIANDTAND W. SZYBALSKI~J McArdb
Memorial Laboratory, The University of Wisconsin Madison, Wise. 53706, U.S.A. (Received 3 August 192’0)
3H- or scP-labeled Escherichia co&i tRNAtYrn was found to hybridize with the r strandof the plaque-forming coliphage #80ppsui& DNA. Since s&&RNA hybridized with the 1 strand of the defective pbage +8Odau&u&, as previously found by Lozeron, Szybalski, Landy, Abelson & Smith (1969), it is concluded that an inversion of the E. coli DNA fragment carrying au& occurred before its incorporation into the @Opsu& genome. Electron micrographs of heteroduplexes between confirmed this conclusion. These the r strands of @Opmr& and +80dsu&eunr micrographs, which showed a short double-stranded DNA segment with a small loop, also indicated that the space between the tandem au& and su;;r genes is less than 50 nucleotides. The length of the E. coli fragment carrying the su& gene m @Op&, corresponded to 5.4% of the length of 480 DNA, and it replaced a segment of the 480 genome bounded by points located 53.8 and 67*9Oh from the left terminus.
1. Introduction Escherichia coli gene su& is the structural gene for a transfer RNA molecule that enables the chain termination codon UAG to be read as tyrosine (Kaplan, Stretton & Brenner, 1965; Weigert, Lanka & Garen, 1965; Goodman, Abelson, Landy, Brenner & Smith, 1968). Russell et al. (1970) h ave described the isolation of the plaqueforming transducing coliphage @Opsu& , which carries one copy of the s& gene. DNA strands of this derivative are useful for hybridization studies with chemically synthesized deoxyribopolynucleotides representing parts of the su& gene. As a prerequisite for these studies it was necessary to determine which of the two DNA strands of @Opsu& serves as a template for @&RNA and where the su& gene is located within the $BOpsu& DNA. Employing the separated DNA strands of the defective transducing coliphage #Odsuf,,-sun, (+SOdsu+ in- ) Lozeron et al. (1969) have shown that au&RNA hybridizes selectively with strands 1 of this phage. In the present work, purified tRNAtY=II, which differs from su&tRNA only in three nucleotide units (Goodman, Abelson, Landy, Zadrazil t Smith, 1970; RajBhandary et al., 1969), has been found to t Paper XCVI in this series is Kleppe, Ohtsuka, Kleppe, Molineux & Khorana, 1971. t Present address: Departments of Biology and Chemistry, Massachusetts Institute of Technology, 8 To
Cambridge, whom the
Mass. reprint
02139, U.S.A. requests should
be addressed. 363
364
R.
C.
ET
MILLER
AL.
hybridize with the r strand of @Opsum. Therefore, these results indicate that an inversion of the suIII gene within the #3Opsu~, genome has occurred relative to $3odsu+&. Electron micrographs of heteroduplexes between the r strands of 98Ops2i, and @Odsu>n- confirm this conclusion. The approximate location of the su& gene within the $8OpsuL, DNA was determined by application of the methods of Westmoreland, Szybalski & Ris (1969) to the study of heteroduplexes of ~#&Opsz&T strands with $80 1 strands.
2. Materials
and Methods
(a) Radioactive laaeliqq of tRNAturl* of 3H-labeled tRNAtYrxl that E. coli tRNAtYPrl contains two thiouridylic acid
(i) Prepar&on It is known residues (RajBhandary et al., 1969). Thiouracil has been shown by Cerutti, Holt & Miller (1968) to be reduced to 2-oxohexahydropyrimidine with sodium borohydride. This property has been used in the present work to prepare 3H-labeled tRNA tyrll (Igo-Kemenes t Zachau, 1969; RajBhandary, 1968). Purified E. coli tRNAtYrn (14 mpmoles) in 1 ml. of 0.2 M-KCl, 20 mM-MgClz and 0.4 Msodium borate, pH 9.8, was treated with [3H]NaBH4 (Amersham-Searle, 100 mc, spec. act. 12 c/m-mole) for 2 hr at room temperature. Unreacted NaBHa was destroyed by addition of 1 ml. of 1 m-potassium acetate, pH 5. After 2 hr at 5°C the tRNAtYrxl was precipitated with 4 ml. of ethanol, and the precipitate was collected by centrifugation. The precipitate then was dialyzed against water. To remove any extraneous 3H radioactivity the preparation of tRNAtyrn was applied to a DEAE-column (5 cm x 1.5 cm) which was washed exhaustively with 0.05 M-NaCl. The tRNAtyPII then was eluted with 1 M-NaCI. The specific activity of the 3H-labeled tRNAtYrn was 480 cts/min/p~mole when measured on Schleicher & Schuell B-6 nitrocellulose filters suspended in toluene-based scintillation fluid. (ii)
Preparation
of
33P-labeled tRNAturlr
Tyrosine tRNA (120 pg) was incubated with O-1 unit (3 pg) of bacterial alkaline phosphatase (electrophoretically purified, BAPF, purchased from Worthington Biochemical Corporation) for 30 min at 70’C. The reaction mixture was incubated further for 5 min at 70°C after addition of 0.15% sodium dodecyl sulfate and 5 x 10V3 M-EDTA and chromatographed on a 90 cm x 1 cm column of O-5 magarose; the column was eluted with 0.1 M -triethylammonium bicarbonate. The recovered tRNAtYPn was found to be free of contaminating alkaline phosphatase ; this was shown by incubating a decanucleotide containing a radioactively labeled 5’-phosphate group with the recovered tRNA preparation under the standard conditions for detection of alkaline phosphatase activity. The tRNAtyrn solution was concentrated and dialyzed against 0.05 M-Tris-HCl buffer (pH 7.4). The dialyzed tRNA preparation was phosphorylated with [Y-~~P@TP using polynucleotide kinase (Richardson, 1965); the incubation mixture (253 ~1.) contained 16 pmoles of Tris-HCl buffer (pH 7*6), 2.5 pmoles of MgCI,, 4.2 pmoles of 2-mercaptoethanol, 0.392 mpmole of tRNA, 1.42 mrmoles of [Y-~~P]ATP (prepared according to Weiss, Live & Richardson, 1968) and 4 units of T4 polynucleotide kinase. The reaction mixture W&B incubated for 60 min at 37°C. The phosphorylated tRNAtYP” was separated from the residual [Y-~~P]ATP on a g50 Sephadex column (30 cm x 1 cm). The specific activity of the labeled tRNAtYrn was approximately 3200 cts/min/rpmole. (b)
Separation
of
the complementary
DNA
strand8
of 48Opsu
& and their hybridization
with
tRNA’Y’” The two intact DNA strands of +3Opsu & Hradecna & Szybalski (1967), as specified by the separation are given in the legend to Fig. The 3H-labeled tRNAtyrn or 33P-labeled hybridize with either of the separated strands
and 480 were separated by the procedure Lozeron et al. (1969). The specific details 1. tRNAtyrn were tested for their ability of #8Opsu& or 480 according to the procedure
of of to
INVERSION
OF
of Lozeron et al. (1969). The conditions in the legend to Table 1.
8uni
GENE
365
for the hybridization
experiments
are described
(c) Electron microscopy microscopy of heteroduplex DNA molecules was carried out aa described by Westmoreland et aZ. (1969), using the Hitachi Hu-11B electron microscope (‘75 kv, x 10,800). All the measurements are projector pole piece III, 60 p objective aperture; expressed aa the percentage of the total length of the 480 DNA molecule, which corresponds The electron
to approximately nucleotide pair
and
42,600 nucleotide for &30 DNA
pairs. (sodium
[Molecular weights salts), respectively].
662
and
28.2
X lo6
for
a
3. Results and Discussion (a) Separation of the DNA strands of +SOpsu& The DNA strands of the phege 48Opsu& were separated in a CsCl density gradient (Fig. 1) according to the procedure of Lozeron et al. (1969). Sedimentation velocity
IO& 3 8 N
-
i2 0.5- _
IO
I 40
20
50
Fraction 110.
Fm. 1. Poly(U,G)-effected fractionation (separation) of the complementary strands of coliphage @psu,+,, DNA by CsCl density-gradient centrifugation. A suspension of &3Op.&, phage (160 pg DNA) was mixed with 300 pg of poly(U,G) in 1.4’7 ml. 10T3 M-EDTA + 6 x 10e4 M-KzHP04 (adjusted to pH 7.8) containing 0.1 y0 Sarkoayl, and heated for 2 mm at 96°C. The samples were cooled in ice, supplemented with 0.30 ml. of 0.60 M-Tris buffer, pH 7.4, and 7.50 ml. of a s&turated solution of CsCl, and adjusted to the density of 1.72 g/cm3. The solution was distributed at 3.0 ml. per Polyallomer tube (Spinco rotor SW39). overlaid with paraffin oil, and oentrifuged for 60 hr at 30,000 rev./min at 5°C. The bar diagram represents the absorbance (260 nm) of four-drop (60 ~1.) fraotions measured in a 2Q-pl. microcuvette (2-mm light path). The shaded areas represent the individual fractions which were combined as the major fractions t and 1. These fractions were self-annealed before being used for hybridization with tRNA.
experiments (Lozeron & Szybalski, 1969) demonstrated that the 1 and r strands of #3Opsu& were integral. Cross-hybridization studies showed that the 1 and r strands of @Opsu& corresponded to the 1 and r strands of $80. (b) Orientation of the sufir gene of 480ps~~~, After separation of the DNA strands of $80 and @Ops&, hybridization experiments were conducted according to Lozeron et al. (1969). As shown in Table 1, the tRNAtvrn hybridizes selectively with the r strand of +Opau:, and not at all with either of the separated strands of 480. This result is just the opposite to that obtained with 480dsu:;; ; i.e. Lozeron et al. (1969) reported that s&&RNA anneals to the 1
R.
366
C.
MILLER TABLE
Hybridization
ET
AL.
1
of tRNA t~TIxwith the separated strands of Wk3uf,, and $80 DNA DNA
Bacteriophage
Strand
; i
480
Hybridized cts/min/ppmole 3H
lOIf lOf6 2 1
tRNAtyrn DNA) 33P
534 29 -
Hybridization between 1 ppmole of separated single-stranded DNA (baaed on a mol. wt of 14x 106 daltone/strand of 480) and 10 ppmoles of labeled tRNAtzPn (for spec. act. see Materials and Methods) was carried out in a 1.0 ml. vol. of 2 X SSC (0.3 na-NaC1; 0.03 &r-sodium citrate) at 66°C for 6 to 8 hr. The samples were diluted to 5 ml. 2 x SSC, slowly filtered through nitrooellulose filters (BB, 26 mm, Sohleicher & Schuell Co.) and washed with 100 ml. 2 x SSC. The filters were exposed to RNase (20 pg/ml. of DNase-free pancreatic RNase; 2 x SSC; 1 hr; 24°C) and washed on both sides with SO-ml. portions of 2 X SSC. They were transferred to scintillation vials, dried, overlaid with toluene-based scintillation fluid, and analyzed for radioaativity in a Pa&ard model 3380 liquid-scintillation counter. Hybridization tests were performed in quadruplicate.
strand of $8OdsU’irr- and that tRNAtyrn competes with su&RNA for binding to this strand. This result leads to the conclusion that an inversion relative to $8Odsu>; has occurred during the formation of +8Ops&. (c) Electron
of heteroduplexes between r strands of $8Opsuf,, and +8Odsu:;
microscopy
The opposing orientations of the su& genome in $8Opsuf,, and 48Odsu&- were confirmed by electron microscopy. If the two phage DNA’s code for the tRNA from the opposite strands, then one would expect the r strands of $8Odsu’& to contain an E. coli DNA fragment with a nucleotide sequence complementary to the E. coli fragment in the r strand of $8Opsz&. A heteroduplex of the r strand of 98Opsu& and the r strand of $80dsu&- should then be non-complementary with the exception of a short duplex segment composed of the complementary base sequences that correspond to the inserted segment of E. coli DNA. (The analogous result also would be expected for a heteroduplex composed of the 1 strands.) This was found to be the case when the heteroduplexes were visualized (Plate I). The electron micrograph shows two single-stranded DNA molecules joined by a short segment of double-stranded DNA corresponding to the complementary E. coli DNA sequences. The length of this duplex segment, based on 20 independent measurements, was 4.8 &0.4% of the $80 DNA length. Since the $8Opsu~, phage used carries only one W& gene, whereas phage $80dsu+& contains two sun, genes in tandem arrangement (Russell et al., 1970; Lozeron et al., 1969), one would expect to see within the duplex structure a short single-stranded loop, the length of which should correspond to the length of the SU& gene (82 or 85 bases) plus the “space” between the two sum genes in $80dsu>r;. Electron micrographs (Plate I) indicate the presence of a very short loop, corresponding to about 0.2 to 0.3% of the 480 length (90 to 130 nucleotides). Thus, the “spacer” between the tandem sum genes appears to be quite short; the sequence of this region might
J'LATE I. Electron micrographs of three heteroduplexes composed of and @Odsu :;IDNA. The DNA segments between points A and B arrows point to small single-stranded loops corresponding to the unpaired ~~~~~~and the space between the two tandem sum genes in QOdsu,,, + ‘- . 10~1) was discernible in 13 out of 20 hetcrodaplexes photographed and
the are
T strands of @Opsu,!,, double-stranded. The segment including gene The small single-stranded examined. [frtcing
p. 366
.._.._...... PLATE strand ponds DNA.
__ .. . .
.. .. . . ..._...
. . ...” . .
..__... - _...._.
.
II. Electron micrograph of a heteroduplex composed of the I strand $80 ad the r of @Opsu&, DNA. In t)he unpaired region, t,hr shark single-stranded segment, (A) comesto thr 480 l>SA and the longer segment (B) t,o the inserted E. coli fragmcsnt in +~O~SU;,~
INVERSION
be revealing, since it probably processing of tRNA molecules.
OF
atin
contains signals for termination
(d) Locatio?t of the a&
387
GENE
and initiation
for
gene of &xm~,,
The location of the E. coli DNA fragment carrying the SU&gene within the #8Op& DNA molecule was determined by electron microscopy of a heteroduplex molecule composed of the 1 strand of $80 and the r strand of #8Opsu&. This heteroduplex molecule (Plate II) contained a region of non-homologous DNA located at a distance of 53.8% from one end of the 480DNA molecule and 425% from the other end. Drs M. Wu and N. Davidson have kindly informed us that they have obtained generally similar results on the position and size of the piece of E. coli DNA in by analogous heteroduplex studies. #3Wu:;; The lengths of the two single strands in the region of non-homology between $80 and ~8Opsu& were 4.1 and 5.7% of the length of the #SO genome. Since the CsCl densities of $80 and $80p& phages were determined to be I.494 and 1.497 g/cm3, (versus h papa = 1508g/cm3), respectively, the shorter strand must correspond to deleted 480 DNA in $8Opsu~,, while the longer strand corresponds to the inserted E. coli DNA containing the su& gene. To determine the left to right orientation of the phage genome, a heteroduplex composed of the r strand of $SOpsu~, and the 1 strand of $SOi” was made. 48Oi’ is a hybrid phage (Signer, 1964; Lozeron BESzybalski, 1969) in which the &q-P segment of X DNA replaces $80 DNA between points 6.50 to 85.5% measured from the left terminus of the 480 genome (Fiandt, Hradecna, Lozeron & Szybalski, 1971.) The +80-h non-homology in the /3+-P segment was used as a marker to determine the orientation of the molecule. Measurements on this heteroduplex showed that the left end of the E. coli fragment carrying the su& gene is located 53.8% of the $80 DNA length from the left terminus of #SOpsu& DNA. This point is very close or identical to the site of $80 attachment (attaa’,,), as determined for 480dsu>$ (Fiandt et al., 1971). In summary then, the E. coli DNA fragment carrying the su& gene is 5.7% of the $80 genome length and replaces a segment of 480 DNA located between 53.8 and 57.9%, measured from the left end of the molecule. Phage 48Opsu& was most probably derived by at least two genetic events, one of which had to shorten the distance between the su& gene and the prophage in the genome of the $80 lysogen (Russell et al., 1970). The present data indicate that the latter event most probably was not simple deletion of the DNA of the lysogenic bacterium between sz& and $80, but more likely involved an inversion of the segment including genes su& and intao. This work was supported by grants from the National Cancer Institute (gram nos. CA05178 and CAO7175), the National Science Foundation (grant nos. GB7484X and GB2096), the Life Insurance Medical Research Fund (grant no. 66-44), and the U. S. Atomic Energy Commission Postdoctoral Fellowship to one of us (R.C.M.) (no. AT( 11 -l)2062). We would like to thank Dr H. A. Lozeron of McArdle Laboratory for the gift of the separated r strands of #30&+& DNA, and for his help with the strand separation of @Opsu& DNA, Dr U. L. RajBhandary for the gift of tRNA=n and his advice conoerning the radioactive labeling of the tRNA, and Dr K. Kleppe for the gift of T4 polynucleotide kineae.
368
R.
C.
MILLER
ET
AL.
REFERENCES P., Holt, J. & Miller, N. (1968). J. Mol. Biol. 84, 505. M., Hradecna, Z., Lozeron, H. A. & Szybalski, W. (1971). In The Bacteriophage Lambda, ed. by A. D. Hershey, Cold Spring Harbor: Cold Spring Harbor Laboratories, in the press. Goodman, H. M., Abelson, 5. N., Landy, A., Brenner, S. & Smith, J. D. (1968). Nature, 217, 1019 Goodman, H. M., Abelson, 5. N., Landy, A., Zadrazil, S. t Smith, J. D. (1970). Europ. J.
Cerutti, Fiandt,
Biochem.
13,
461.
Hradecna, Z. & Szybalski, W. (1967). Virology, 32, 633. Igo-Kemenes, T. & Zachau, H. G. (1969). Europ. J. Biochem. 10, 549. Kaplan, S., Stretton, A. 0. W. & Brenner, S. (1965). J. Mol. Biol. 14, 628. Kleppe, K., Ohtsuka, E., Kleppe, R., Molineux, I. & Khorana, H. G. (1971). J. Mol. BioZ. 56, 341. Lozeron, H. & Szybalski, W. (1969). Virology, 39, 373. Lozeron, H., Szybalski, W., Landy, A., Abelson, J. & Smith, J. D. (1969). J. Mol. BioZ. 39, 239.
RsjBhandery, U. L. (1968). J. BioZ. Chem. 243, RajBhandary, U. L., Chang, S. H., Gross, H. J., (1969). Fed. Proc. 28, 851. Richardson, C. C. (1965). Proc. Nat. Acud. Sci., Russell, R. L., Abelson, J. N., Landy, A., Gefter, J.
MOE.
BioZ.
47,
566. Hareda,
F., Kimura,
F. & Nishimura,
S.
54, 4168. M. L., Brenner, S. & Smith, J. D. (1970).
Wash.
1.
Signer, E. R. (1964). V(viroZogy, 22, 650. Weigert, M., Lanka, E. & Garen, A. (1965). J. Mol. BioZ. 14, 522, Weiss, B., Live, T. R. & Richardson, C. C. (1968). J. BioZ. Chem. 243, 4530. Westmoreland, B. C., Szybalski, W. & Ris, H. (1969). Science, 163, 1343.
Studies on Polynucleotides XCVII. Opposing Orientations and Location of the SU& Gene in the Transducing Coliphages @Ops& and #30&&w&t R. C. MILLER
JR., P. BESMER,
H. G. KHORANA,
Institute for Enzyme Research, The University of Wiswnsin$ M. FIANDTAND W. SZYBALSKI~J McArdb
Memorial Laboratory, The University of Wisconsin Madison, Wise. 53706, U.S.A. (Received 3 August 192’0)
3H- or scP-labeled Escherichia co&i tRNAtYrn was found to hybridize with the r strandof the plaque-forming coliphage #80ppsui& DNA. Since s&&RNA hybridized with the 1 strand of the defective pbage +8Odau&u&, as previously found by Lozeron, Szybalski, Landy, Abelson & Smith (1969), it is concluded that an inversion of the E. coli DNA fragment carrying au& occurred before its incorporation into the @Opsu& genome. Electron micrographs of heteroduplexes between confirmed this conclusion. These the r strands of @Opmr& and +80dsu&eunr micrographs, which showed a short double-stranded DNA segment with a small loop, also indicated that the space between the tandem au& and su;;r genes is less than 50 nucleotides. The length of the E. coli fragment carrying the su& gene m @Op&, corresponded to 5.4% of the length of 480 DNA, and it replaced a segment of the 480 genome bounded by points located 53.8 and 67*9Oh from the left terminus.
1. Introduction Escherichia coli gene su& is the structural gene for a transfer RNA molecule that enables the chain termination codon UAG to be read as tyrosine (Kaplan, Stretton & Brenner, 1965; Weigert, Lanka & Garen, 1965; Goodman, Abelson, Landy, Brenner & Smith, 1968). Russell et al. (1970) h ave described the isolation of the plaqueforming transducing coliphage @Opsu& , which carries one copy of the s& gene. DNA strands of this derivative are useful for hybridization studies with chemically synthesized deoxyribopolynucleotides representing parts of the su& gene. As a prerequisite for these studies it was necessary to determine which of the two DNA strands of @Opsu& serves as a template for @&RNA and where the su& gene is located within the $BOpsu& DNA. Employing the separated DNA strands of the defective transducing coliphage #Odsuf,,-sun, (+SOdsu+ in- ) Lozeron et al. (1969) have shown that au&RNA hybridizes selectively with strands 1 of this phage. In the present work, purified tRNAtY=II, which differs from su&tRNA only in three nucleotide units (Goodman, Abelson, Landy, Zadrazil t Smith, 1970; RajBhandary et al., 1969), has been found to t Paper XCVI in this series is Kleppe, Ohtsuka, Kleppe, Molineux & Khorana, 1971. t Present address: Departments of Biology and Chemistry, Massachusetts Institute of Technology, 8 To
Cambridge, whom the
Mass. reprint
02139, U.S.A. requests should
be addressed. 363
364
R.
C.
ET
MILLER
AL.
hybridize with the r strand of @Opsum. Therefore, these results indicate that an inversion of the suIII gene within the #3Opsu~, genome has occurred relative to $3odsu+&. Electron micrographs of heteroduplexes between the r strands of 98Ops2i, and @Odsu>n- confirm this conclusion. The approximate location of the su& gene within the $8OpsuL, DNA was determined by application of the methods of Westmoreland, Szybalski & Ris (1969) to the study of heteroduplexes of ~#&Opsz&T strands with $80 1 strands.
2. Materials
and Methods
(a) Radioactive laaeliqq of tRNAturl* of 3H-labeled tRNAtYrxl that E. coli tRNAtYPrl contains two thiouridylic acid
(i) Prepar&on It is known residues (RajBhandary et al., 1969). Thiouracil has been shown by Cerutti, Holt & Miller (1968) to be reduced to 2-oxohexahydropyrimidine with sodium borohydride. This property has been used in the present work to prepare 3H-labeled tRNA tyrll (Igo-Kemenes t Zachau, 1969; RajBhandary, 1968). Purified E. coli tRNAtYrn (14 mpmoles) in 1 ml. of 0.2 M-KCl, 20 mM-MgClz and 0.4 Msodium borate, pH 9.8, was treated with [3H]NaBH4 (Amersham-Searle, 100 mc, spec. act. 12 c/m-mole) for 2 hr at room temperature. Unreacted NaBHa was destroyed by addition of 1 ml. of 1 m-potassium acetate, pH 5. After 2 hr at 5°C the tRNAtYrxl was precipitated with 4 ml. of ethanol, and the precipitate was collected by centrifugation. The precipitate then was dialyzed against water. To remove any extraneous 3H radioactivity the preparation of tRNAtyrn was applied to a DEAE-column (5 cm x 1.5 cm) which was washed exhaustively with 0.05 M-NaCl. The tRNAtyPII then was eluted with 1 M-NaCI. The specific activity of the 3H-labeled tRNAtYrn was 480 cts/min/p~mole when measured on Schleicher & Schuell B-6 nitrocellulose filters suspended in toluene-based scintillation fluid. (ii)
Preparation
of
33P-labeled tRNAturlr
Tyrosine tRNA (120 pg) was incubated with O-1 unit (3 pg) of bacterial alkaline phosphatase (electrophoretically purified, BAPF, purchased from Worthington Biochemical Corporation) for 30 min at 70’C. The reaction mixture was incubated further for 5 min at 70°C after addition of 0.15% sodium dodecyl sulfate and 5 x 10V3 M-EDTA and chromatographed on a 90 cm x 1 cm column of O-5 magarose; the column was eluted with 0.1 M -triethylammonium bicarbonate. The recovered tRNAtYPn was found to be free of contaminating alkaline phosphatase ; this was shown by incubating a decanucleotide containing a radioactively labeled 5’-phosphate group with the recovered tRNA preparation under the standard conditions for detection of alkaline phosphatase activity. The tRNAtyrn solution was concentrated and dialyzed against 0.05 M-Tris-HCl buffer (pH 7.4). The dialyzed tRNA preparation was phosphorylated with [Y-~~P@TP using polynucleotide kinase (Richardson, 1965); the incubation mixture (253 ~1.) contained 16 pmoles of Tris-HCl buffer (pH 7*6), 2.5 pmoles of MgCI,, 4.2 pmoles of 2-mercaptoethanol, 0.392 mpmole of tRNA, 1.42 mrmoles of [Y-~~P]ATP (prepared according to Weiss, Live & Richardson, 1968) and 4 units of T4 polynucleotide kinase. The reaction mixture W&B incubated for 60 min at 37°C. The phosphorylated tRNAtYP” was separated from the residual [Y-~~P]ATP on a g50 Sephadex column (30 cm x 1 cm). The specific activity of the labeled tRNAtYrn was approximately 3200 cts/min/rpmole. (b)
Separation
of
the complementary
DNA
strand8
of 48Opsu
& and their hybridization
with
tRNA’Y’” The two intact DNA strands of +3Opsu & Hradecna & Szybalski (1967), as specified by the separation are given in the legend to Fig. The 3H-labeled tRNAtyrn or 33P-labeled hybridize with either of the separated strands
and 480 were separated by the procedure Lozeron et al. (1969). The specific details 1. tRNAtyrn were tested for their ability of #8Opsu& or 480 according to the procedure
of of to
INVERSION
OF
of Lozeron et al. (1969). The conditions in the legend to Table 1.
8uni
GENE
365
for the hybridization
experiments
are described
(c) Electron microscopy microscopy of heteroduplex DNA molecules was carried out aa described by Westmoreland et aZ. (1969), using the Hitachi Hu-11B electron microscope (‘75 kv, x 10,800). All the measurements are projector pole piece III, 60 p objective aperture; expressed aa the percentage of the total length of the 480 DNA molecule, which corresponds The electron
to approximately nucleotide pair
and
42,600 nucleotide for &30 DNA
pairs. (sodium
[Molecular weights salts), respectively].
662
and
28.2
X lo6
for
a
3. Results and Discussion (a) Separation of the DNA strands of +SOpsu& The DNA strands of the phege 48Opsu& were separated in a CsCl density gradient (Fig. 1) according to the procedure of Lozeron et al. (1969). Sedimentation velocity
IO& 3 8 N
-
i2 0.5- _
IO
I 40
20
50
Fraction 110.
Fm. 1. Poly(U,G)-effected fractionation (separation) of the complementary strands of coliphage @psu,+,, DNA by CsCl density-gradient centrifugation. A suspension of &3Op.&, phage (160 pg DNA) was mixed with 300 pg of poly(U,G) in 1.4’7 ml. 10T3 M-EDTA + 6 x 10e4 M-KzHP04 (adjusted to pH 7.8) containing 0.1 y0 Sarkoayl, and heated for 2 mm at 96°C. The samples were cooled in ice, supplemented with 0.30 ml. of 0.60 M-Tris buffer, pH 7.4, and 7.50 ml. of a s&turated solution of CsCl, and adjusted to the density of 1.72 g/cm3. The solution was distributed at 3.0 ml. per Polyallomer tube (Spinco rotor SW39). overlaid with paraffin oil, and oentrifuged for 60 hr at 30,000 rev./min at 5°C. The bar diagram represents the absorbance (260 nm) of four-drop (60 ~1.) fraotions measured in a 2Q-pl. microcuvette (2-mm light path). The shaded areas represent the individual fractions which were combined as the major fractions t and 1. These fractions were self-annealed before being used for hybridization with tRNA.
experiments (Lozeron & Szybalski, 1969) demonstrated that the 1 and r strands of #3Opsu& were integral. Cross-hybridization studies showed that the 1 and r strands of @Opsu& corresponded to the 1 and r strands of $80. (b) Orientation of the sufir gene of 480ps~~~, After separation of the DNA strands of $80 and @Ops&, hybridization experiments were conducted according to Lozeron et al. (1969). As shown in Table 1, the tRNAtvrn hybridizes selectively with the r strand of +Opau:, and not at all with either of the separated strands of 480. This result is just the opposite to that obtained with 480dsu:;; ; i.e. Lozeron et al. (1969) reported that s&&RNA anneals to the 1
R.
366
C.
MILLER TABLE
Hybridization
ET
AL.
1
of tRNA t~TIxwith the separated strands of Wk3uf,, and $80 DNA DNA
Bacteriophage
Strand
; i
480
Hybridized cts/min/ppmole 3H
lOIf lOf6 2 1
tRNAtyrn DNA) 33P
534 29 -
Hybridization between 1 ppmole of separated single-stranded DNA (baaed on a mol. wt of 14x 106 daltone/strand of 480) and 10 ppmoles of labeled tRNAtzPn (for spec. act. see Materials and Methods) was carried out in a 1.0 ml. vol. of 2 X SSC (0.3 na-NaC1; 0.03 &r-sodium citrate) at 66°C for 6 to 8 hr. The samples were diluted to 5 ml. 2 x SSC, slowly filtered through nitrooellulose filters (BB, 26 mm, Sohleicher & Schuell Co.) and washed with 100 ml. 2 x SSC. The filters were exposed to RNase (20 pg/ml. of DNase-free pancreatic RNase; 2 x SSC; 1 hr; 24°C) and washed on both sides with SO-ml. portions of 2 X SSC. They were transferred to scintillation vials, dried, overlaid with toluene-based scintillation fluid, and analyzed for radioaativity in a Pa&ard model 3380 liquid-scintillation counter. Hybridization tests were performed in quadruplicate.
strand of $8OdsU’irr- and that tRNAtyrn competes with su&RNA for binding to this strand. This result leads to the conclusion that an inversion relative to $8Odsu>; has occurred during the formation of +8Ops&. (c) Electron
of heteroduplexes between r strands of $8Opsuf,, and +8Odsu:;
microscopy
The opposing orientations of the su& genome in $8Opsuf,, and 48Odsu&- were confirmed by electron microscopy. If the two phage DNA’s code for the tRNA from the opposite strands, then one would expect the r strands of $8Odsu’& to contain an E. coli DNA fragment with a nucleotide sequence complementary to the E. coli fragment in the r strand of $8Opsz&. A heteroduplex of the r strand of 98Opsu& and the r strand of $80dsu&- should then be non-complementary with the exception of a short duplex segment composed of the complementary base sequences that correspond to the inserted segment of E. coli DNA. (The analogous result also would be expected for a heteroduplex composed of the 1 strands.) This was found to be the case when the heteroduplexes were visualized (Plate I). The electron micrograph shows two single-stranded DNA molecules joined by a short segment of double-stranded DNA corresponding to the complementary E. coli DNA sequences. The length of this duplex segment, based on 20 independent measurements, was 4.8 &0.4% of the $80 DNA length. Since the $8Opsu~, phage used carries only one W& gene, whereas phage $80dsu+& contains two sun, genes in tandem arrangement (Russell et al., 1970; Lozeron et al., 1969), one would expect to see within the duplex structure a short single-stranded loop, the length of which should correspond to the length of the SU& gene (82 or 85 bases) plus the “space” between the two sum genes in $80dsu>r;. Electron micrographs (Plate I) indicate the presence of a very short loop, corresponding to about 0.2 to 0.3% of the 480 length (90 to 130 nucleotides). Thus, the “spacer” between the tandem sum genes appears to be quite short; the sequence of this region might
J'LATE I. Electron micrographs of three heteroduplexes composed of and @Odsu :;IDNA. The DNA segments between points A and B arrows point to small single-stranded loops corresponding to the unpaired ~~~~~~and the space between the two tandem sum genes in QOdsu,,, + ‘- . 10~1) was discernible in 13 out of 20 hetcrodaplexes photographed and
the are
T strands of @Opsu,!,, double-stranded. The segment including gene The small single-stranded examined. [frtcing
p. 366
.._.._...... PLATE strand ponds DNA.
__ .. . .
.. .. . . ..._...
. . ...” . .
..__... - _...._.
.
II. Electron micrograph of a heteroduplex composed of the I strand $80 ad the r of @Opsu&, DNA. In t)he unpaired region, t,hr shark single-stranded segment, (A) comesto thr 480 l>SA and the longer segment (B) t,o the inserted E. coli fragmcsnt in +~O~SU;,~
INVERSION
be revealing, since it probably processing of tRNA molecules.
OF
atin
contains signals for termination
(d) Locatio?t of the a&
387
GENE
and initiation
for
gene of &xm~,,
The location of the E. coli DNA fragment carrying the SU&gene within the #8Op& DNA molecule was determined by electron microscopy of a heteroduplex molecule composed of the 1 strand of $80 and the r strand of #8Opsu&. This heteroduplex molecule (Plate II) contained a region of non-homologous DNA located at a distance of 53.8% from one end of the 480DNA molecule and 425% from the other end. Drs M. Wu and N. Davidson have kindly informed us that they have obtained generally similar results on the position and size of the piece of E. coli DNA in by analogous heteroduplex studies. #3Wu:;; The lengths of the two single strands in the region of non-homology between $80 and ~8Opsu& were 4.1 and 5.7% of the length of the #SO genome. Since the CsCl densities of $80 and $80p& phages were determined to be I.494 and 1.497 g/cm3, (versus h papa = 1508g/cm3), respectively, the shorter strand must correspond to deleted 480 DNA in $8Opsu~,, while the longer strand corresponds to the inserted E. coli DNA containing the su& gene. To determine the left to right orientation of the phage genome, a heteroduplex composed of the r strand of $SOpsu~, and the 1 strand of $SOi” was made. 48Oi’ is a hybrid phage (Signer, 1964; Lozeron BESzybalski, 1969) in which the &q-P segment of X DNA replaces $80 DNA between points 6.50 to 85.5% measured from the left terminus of the 480 genome (Fiandt, Hradecna, Lozeron & Szybalski, 1971.) The +80-h non-homology in the /3+-P segment was used as a marker to determine the orientation of the molecule. Measurements on this heteroduplex showed that the left end of the E. coli fragment carrying the su& gene is located 53.8% of the $80 DNA length from the left terminus of #SOpsu& DNA. This point is very close or identical to the site of $80 attachment (attaa’,,), as determined for 480dsu>$ (Fiandt et al., 1971). In summary then, the E. coli DNA fragment carrying the su& gene is 5.7% of the $80 genome length and replaces a segment of 480 DNA located between 53.8 and 57.9%, measured from the left end of the molecule. Phage 48Opsu& was most probably derived by at least two genetic events, one of which had to shorten the distance between the su& gene and the prophage in the genome of the $80 lysogen (Russell et al., 1970). The present data indicate that the latter event most probably was not simple deletion of the DNA of the lysogenic bacterium between sz& and $80, but more likely involved an inversion of the segment including genes su& and intao. This work was supported by grants from the National Cancer Institute (gram nos. CA05178 and CAO7175), the National Science Foundation (grant nos. GB7484X and GB2096), the Life Insurance Medical Research Fund (grant no. 66-44), and the U. S. Atomic Energy Commission Postdoctoral Fellowship to one of us (R.C.M.) (no. AT( 11 -l)2062). We would like to thank Dr H. A. Lozeron of McArdle Laboratory for the gift of the separated r strands of #30&+& DNA, and for his help with the strand separation of @Opsu& DNA, Dr U. L. RajBhandary for the gift of tRNA=n and his advice conoerning the radioactive labeling of the tRNA, and Dr K. Kleppe for the gift of T4 polynucleotide kineae.
368
R.
C.
MILLER
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AL.
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