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Restriction endonuclease BamHI cleaves bacteriophage Mu DNA within cistrons E and F
Gene, 10 (1980) 79-83
79
© Elsevier/North-Holland Biomedical Press
R e s t r i c t i o n e n d o n u c l e a s e B a m H I cleaves b a c t e r i o p h a g e Mu D N A within c i s t r o n s E and F (Cloning o f Mu DNA; pBR322 vector; plasmid; mapping by marker rescue; recombinant DNA)
Jeffrey A. Engler, Rosalind A. Forgie
* and Martha M. Howe *
Cold Spring ltarbor Laboratory, Cbld Spring lfarbor, NY 11 724, and * Department of Bacteriology, University of Wisconsin, Madison, WI 53706 (U.S.A.)
(Received December 21 st, 1979) (Accepted January 5th, 1980)
SUMMARY We have cleaved phage Mu DNA with restriction endonucleases EcoRI and BamHI and have cloned three specific DNA fragments from the middle of the Mu genome into vector plasmid pBR322. By marker rescue experiments, we have determined that the two BamHl cleavage sites in Mu DNA occur within cistrons E and F.
Mu is a temperate bacteriophage of E. coli K-12 that can insert its DNA into the bacterial chromosome at many sites (for reviews, see Howe and Bade, 1975; Bukhari, 1976). The chromosome of Mu is a linear DNA duplex of approx. 38000 base pairs. Physical maps of Mu DNA have been constructed by restriction endonuclease cleavage analyses (Allet and Bukhari, 1975; Kahmann et al., 1977). A genetic map has been defined by amber mutant marker rescue studies of prophage deletion strains (Abelson et al., 1973) and )~pMu transducing phages (O'Day et al., 1979). Partial correlation of these genetic and physical maps has been obtained by determining the M u DNA segments in ?,pMu transducing phages carrying specific Mu genetic markers (Magazin et al., 1977; J.W. Schumm, D.D. Moore, F.R. Blattner and M.M. Howe, manuscript in preparation) and by defining the genetic markers present in certain Abbreviations: am, umber; amp, ampicillin; pfu, plaqueforming units: tet, tetr:~cycline.
Mu DNA restriction fragments that have been inserted into k (Moore et al., 1977) or plasmids (Zipser et al., 1977; W. Schumann, E.G. Bade, R.A. Forgie and M.M. Howe, manuscript in preparation). In this paper we report the cloning of restriction-endonuclease fragments of Mu DNA generated by the enzymes BamHl and EcoRl and present the results of amber mutant marker rescue studies of these clones. Our findings allow a further correlation of the genetic and physical maps of My by defining the location of the BamHI sites on the genetic map of Mu. Muvir phage were used as the source of Mu DNA for cloning and were grown by infection and purified as described by Bukhari and Ljungquist (1977). Mu DNA was prepared by phenol extraction of phage particles isolated by CsC1 equilibrium density centrifugation. Plasmid pBR322 (Bolivar et al., 1977) was used as the cloning vector and was prepared as described by Clewell (1972). Mu DNA and pBR322 DNA were digested with both l:'coRI and BamHI restriction endonucleases.
80
a)
C
A
b)
B
C
lys
iS
t Immunity end
D
E
H
F G
10
IS
20
,
,
1
,
H,
E¢o
Item
RI
HI
ii
I T J K
Blm
L
M
N POVWR S U
_
k
I
t ECO RI
HI
Y
Variable
end
p J E 107 i
pJE 106
p J E 108
Fig. 1. Mu gene content of pJE plasmids. The genetic (a) (O'Day et al., 1979) and physical (b) (Kahmann et al., 1977) maps of bacteriophage Mu, aligned according to Magazin et al. (1977) and this study. The open boxes represent variable host DNA associated with both ends of the Mu genome. The Mu DNA (including Mu genetic markers) carried by each plasmid are indicated by the bar. Marker-rescue data were obtained from spot and whole-plate marker-rescue tests performed on TCMG plates (Weil et al., 1972), using the method for "Mu-sensitive prophage deletions" described by Howe et al. (1979). A marker-rescue spot test was done for each plasmid with the following Mu anaber mutations (O'Day et al., 1979): A 1093, B1066, C4005, C2005, lysl030, /ys1025, D7330, E7329, E2003,/:'7346, El 006, H7326, H7222, H7315, H7300, H7100, H7007, H1043, F7207, F1065, F7001, F1931, (;1021, I1053, I7349, 17010, T1913, T7327, J1005, K1010, K7332, L1007, L7180, L7156,Mll14, Ml124, Y1027, NI041, P1024, Q1074, V7165, W7334, R1059, S1004, and U1049. In addition, pJE107 was tested by marker-rescue spot test with E mutations 1022, 7112, 7116, 7293, 7367, 1006, 7291 and 7310;pJE106 and pJE108 were tested by marker-rescue spot test with F mutations 1009, 1281, 1527, 1786, 1855, 1902, 7254, 7260, 7285, and 7323;pJE108 was also tested with L mutations 1033, 3010, 7030, 7123, and 7156, andM mutations 1954, 7016, 7061,7174 and 7251. More sensitive whole-plate assays were used to det]ne fragment endpoints, pJE106 was tested with B mutations 1032, 1066, 5177, 5178, 5179, 7096, 7308, and 7342, C1966, F mutations 1527, 1786, 3007, 7254, 7285, 7001,7323, and 1931, and G mutations 1021 and 1042. pJEl07 was tested wit11 the same B and C inutations as pJE 106 and with E mutation 1006, 7112, 7116, 7291, and 7310 and H7055. pJ E 108 was tested with F mutations 1009, 1527, 1786, 1855, 1902, 3007, 7254, 7260, 7285, 7001,7323,and 1931, G mutations 1021 and 1042,and M mutations 1114, 1124, 1954, 7016, 7061,7075, 7174, and 7251.
Fig. 1 shows that each enzyme cuts Mu DNA twice and cuts pBR322 once (Bolivar et at., 1977). The cleaved DNAs were extracted with phenol once and were then precipitated with ethanol. After resuspending the DNAs in double-distilled water, 1/ag of the digested pBR322 DNA and 2/lg of the digested Mu DNA were incubated together with T4 polynucleotide ligase (Miles Laboratories) in a total volume of 100 t-d for 3 h at 15°C, as described by Murray and Murray (1974). A portion of the ligated DNA mixture was used to transfect E. colt strain W3350 strrsu ° (from Dr. W. Dove) by a modification of the method of Mandel and Higa (1970), as described by Glover (1976). Insertion of Mu DNA into pBR322 between the BamHl and the EcoRI cleavage sites inactivates the tetracycline, but not the ampicillin (amp), resistance gene of the plasmid. Transformed colonies were selected on LB ( t r y p t o n e - y e a s t extract-NaCl) plates (Miller, 1972) supplemented with amp (40
/ag/ml). Amp-resistant colonies were further screened for tetracycline (tet) sensitivity on plates supplemented with tet (40 /~g/ml). Nine amp-resistant, tet-sensitive colonies were chosen for further study. The nine transformants were screened in a spot test for their ability to rescue Mu phages containing amber mutations. Several dilutions of Mu ambermutant lysates were spotted onto lawns of each transformant and the plates were incubated overnight. The presence of plaques or cleared areas within a spot was interpreted as marker rescue. As can be seen in Table I, seven of the nine transformants were able to rescue some Mu amber mutations. Five of the transformants allowed rescue of mutations in cistrons C and D, one allowed rescue of mutations in cistrons C, D and F, and another allowed rescue of mutations in only cistrons 1 and K. As expected, none of the transformants was able to allow rescue of mutations in cistrons B or N, which are known to lie outside the
81 TABLE I Spot tests for marker rescue of Mu amber mutants An overnight culture of each transformant was grown in LB broth (Miller, 1972) containing ampicillin (20 t~g/ml). The culture was diluted 1 to 100 in fresh LB + ampicillin and grown to a Klett-Summerson value of 50. A portion (0.l ml) of the culture was mixed with soft agar, plated on an LB plate containing 40 t~g/ml ampicillin and 2.5 mM CaC12, and allowed to cool for 30 minutes. Crude lysates of Mu amber (am) mutants (108-109 pfu per ml, determined by plating on an su host) were diluted in buffer containing 10 mM Tris pH 7.4, 10 mM MgCI2 and 10 mM CaCI 2 and were spotted at three dilutions (10 °, 10 -2, 10 -4) onto the lawns and allowed to dry. The plates were incubated overnight at 37°C. The appearance of plaques or a cleared area in a spot'was taken as a positive (+) result, nd, not determined. Strain
Muvir
Bam1066
783suIII w3350 strrsu° W3350/pJE101 W3350/pJE102 W3350/pJE103 W3350/pJE104 W3350/pJE105 W3350/pJE106 W3350/pJE107 W3350/pJE108 W3350/pJE109
+ + + + + + + + + + +
+ . -
Cam1966
Dam7015
Faro7217
+
+
+ . . . . . . + . .
.
.
.
+ + + + + + -
+ + + + nd + + nd nd
region cloned (Fig. 1). The two remaining transfor-
markers
Iam4037
Kaml010
Nam1034
+
+
+
-
-
+ .
-
.
.
. . . . .
. . . . .
. . . . .
.
.
. .
from
+ .
.
cistrons A-B
or M-U, which were
mants rescued no Mu a m b e r m u t a t i o n s and were not
outside the region cloned. To determine m o r e precisely
characterized further; t h e y m a y have arisen by head
the positions o f the BamHI cleavage sites on the
to head joining o f t w o p B R 3 2 2
genetic map,
D N A molecules,
Plasmid D N A s from six o f the Mu D N A - c o n t a i n i n g transformants
were
isolated
(Clewell,
marker
rescue
spot tests were per-
formed using a large n u m b e r o f phages containing
resulting in amp-resistance and tet-sensitivity. 1972)
and
a m b e r m u t a t i o n s in cistrons B, C, E, H, F, G, L and M. For all m u t a t i o n s located near the ends o f the
digested with restriction endonucleases EcoRI and BamHI. As can be seen in Fig. 2, three types o f
DNA
insertions were f o u n d ; four o f the plasmids were
also performed. The results o f these assays are pre-
identical and c o n t a i n e d Mu D N A f r o m segment I
sented in Figs. 1 and 3. Plasmid p J E 1 0 7 allowed
fragment
carried
by
each plasmid, a m o r e
sensitive marker rescue assay using whole plates was
in Fig. 1 ( e x t e n d i n g f r o m the leftmost EcoRI cut
rescue of all markers tested in cistrons C, lys and
to the l e f t m o s t BamltI cut); one plasmid ( p J E 1 0 6 )
D, but only some in E; this is consistent with the
c o n t a i n e d b o t h segment I and segment II D N A ( e x t e n d i n g f r o m the l e f t m o s t EcoRI cut to the
presence o f D N A segment I. Plasmid p J E 1 0 6 allowed
rightmost BamHI cut) and a n o t h e r ( p J E 1 0 8 ) con-
and H but o n l y some in cistron F; this is consistent
tained segment III D N A ( e x t e n d i n g from the right-
with the presence o f D N A segments I and II. Plasmid
m o s t BamHI cut to the rightmost E c o R I cut).
p J E 1 0 8 did n o t allow rescue o f any o f the markers
One plasmid containing each t y p e o f insertion ( p J E 1 0 6 , p J E 1 0 7 , p J E 1 0 8 ) was further character-
allowed rescue o f the remaining four F markers and
ized b y
all markers tested in cistrons G through L; this is
additional
marker
rescue e x p e r i m e n t s
to
rescue o f all markers tested in cistrons C, lys, D, E,
carried by plasmids p J E 1 0 6 or p J E 1 0 7 but instead
determine the c o n t e n t o f Mu marker alleles. Marker
consistent with the presence o f D N A segment III. If
rescue
phage
the assumption is m a d e that all markers present on a
plasmid
c o n t a i n e d markers f r o m a single contiguous set o f Mu
cloned restriction fragment can be d e t e c t e d in the marker rescue assay, then the results can be used to
cistrons and that
define the l o c a t i o n o f the BamHI restriction sites on
from
spot each
tests using one a m b e r m u t a n t cistron
confirmed none
that
each
o f the plasmids c o n t a i n e d
82
abcdefgh
the genetic map. One site is located between the last E markers that can be rescued from pJE107 and the first E markers that can be rescued from pJE106 but not from pJE107. Similarly the other site is flanked, on the left, by F markers which can be rescued from pJE106 but not from pJEl08 and, on the right, by F markers which can be rescued from pJEl08 but not from pJE106. Fig. 3 presents a detailed genetic map of the region from cistrons E, H, and F (O'Day et al., 1979) and the locations of the BamHI cleavage sites, as determined by the above analysis. The efficient rescue of all F markers from either pJE106 or pJE108 supports the validity of our conclusion from the marker rescue data. In similar experiments W. Schumann, E.G. Bade, R.A. Forgie and M.M. Howe (manuscript in preparation) have isolated plasraids containing either segment I, segment II, or segment 11 (Fig. 1). Marker rescue analysis of those plasraids had confirmed the locations of the BamHI sites determined by this work.
A('K NOWLlq)GEMI~NTS Fig. 2. R e c o m b i n a n t plasmid DNA cleaved with /;coRl and B a m H l restriction endonucleases. Plasmid DNA was isolated according to Clewell (1972). After removal of ellfidium bromide and dialysis against 10 mM Tris pH 7.9, 1 mM H ) T A , 1 ~g of DNA was digested with L'~oRI and B a m t t l in 6 mM Tris • HC1 pH 7.9, 6 mM MgC12, 6 mM i3-mercaptoethanol for 4 h at 37°C. The digested sanrlqes were mixed wifll l,:,ading dye and run on a 1% agarose gel toni:fining l0 ~g/ml ,.~lhidium bronride for 4 h at 4.5 V/cm. The get wa~ ilhn~im~.ted with UV light and photographed on Polaroid type 57 film using a Kodak No. 23 red f'dter. (a) pBR322, (b) p J V l 0 1 , (el p J E l 0 2 , (d) pJE103, (e) p J E l 0 6 , (f) r~JF107, (g) p.t[.'ll)8, (h) Muvir DNA. The u p p e r m o s t band in channel e is a partialdigestion product.
704S 7114 7147 7148
D
Barn H1
Bam H1
1
I
7112 7 2 M 1341 3005 7112 72111 72118 7 1 l J 7311) 7312 72113 7317 7387 7329 7344
E
We thank P.A. Myers and R.J. Roberts for supplying t:coRl and J. Broach for BamH1. D. Kamp provided Muvir phage and valuable advice on cloning techniques. D. Kwoh and D. Erdmann provided the elude lysates of the Mu amber mutants used for the inilial screening of the transformants. We thank L.T. Chow and T.R. Broker for their critical reading of the manuscript. J.M. Scott provided valuable technical assistance. J.E. was supported by NCI Cancer Center CA13106 to the Cold Spring Harbor Lab. and by NIH grant GM23996 to L.T. Chow, and is a recipient of a Fellowship from the Leukemia Society of America. M.M.tt. and R.F. acknowledge support from the
llI? 728l 7170 7324 73211
7300
H
1177 l N l 7 I N lira8 I0N 11141 1044 7007
711$ ~ 714141 7311 731J 7|$4
1~7314 !11~1 1|1| 1033 IIINJ Too I 7333
fill llli 111|6 2i 703J 7207 7217 75'32
1127 lINES IWl 30117 72S4 7280 7383
F
7323
I
G
Fig. 3. I oc~'Jli,~n o f the Ramtll restriction sites on the gt'~ ti~ map of Mu. The m a p of amber m u t a t i o n s is based on the work of O ' D i v et al. (1979t and ~n results ol marker rescue les~s t',erf,~rmed as described in the legend to l:ig. 1.
83 College o f A g r i c u l t u r a l a n d Life Sciences, U n i v e r s i t y o f Wisconsin, M a d i s o n , a n d f r o m N S F grant PCM750 2 4 6 5 , N I H g r a n t A I 1 2 7 3 1 , a n d ACS g r a n t NP-264. MMH is t h e r e c i p i e n t o f N I H R e s e a r c h Career Development Award A100274.
REFERENCES Abelson, J., Boram, W., Bukhari, A.I., Faelen, M., Howe, M., Metlay, M., Taylor, A.L., Toussaint, A., van de Putte, P., Westmaas, G.C. and Wijft'elman, C.A.: Summary of the genetic mapping of prophage Mu. Virology 54 (1973) 90-92. Allet, B. and Bukhari, A.I.: Analysis of bacteriophage Mu and ~.-Mu hybrid DNAs by specific endonucleases. J. Mol. Biol. 92 (1975) 529 540. Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.(., Heyneker, H.L., Boyer, H.W., Crosa, J.H. and Falkow, S.: Construction and characterization of new cloning vehicles. Gene 2 (1977) 9 5 - 1 1 3 . Bukhari, A.I.: Bacteriophage Mu as a transposition element. Annu. Rev. Genet. 10 (1976) 3 8 9 - 4 1 2 . Bukhari, A.I. and Ljungquist, E.: Bacteriophage Mu: Methods for cultivation and use, in Buldaari, A.I., Shapiro, J.A. and Adhya, S.L. (Eds.), DNA Insertion Elements, Plasmids, and Episomes, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1977, pp. 749-756. Clewell, D.B.: Nature of ColE1 plasmid recombination in the presence of chloramphenicol. J. Bacteriol. 110 (1972) 667-676. Glover, D.M.: The construction and cloning of hybrid DNA molecules, in Pain, R.H. and Smith, B.J. (Eds.), New Techniques in Biophysics and Cell Biology, Vol. III, Wiley, London, 1976, pp. 125-145.
Howe, M.M. and Bade, E. : Molecular biology of bacteriophage Mu. Science 190 (1975) 624 632. Howe, M.M., Scllumm, J.W. and Taylor, A.L.: The S and U genes of bacteriophage Mu are located in tile invertible G segment ofMu DNA. Virology 92 (1979) 108 124. Kahmann, R., Kamp, D. and Zipser, D.: Mapping of restriction sites in Mu DNA, in Bukhari, A.l., Shapiro, J.A., and Adhya, S.L. (Eds.), DNA Insertion Elements, Plasmids, and Episomes, Cold Spring ttarbor Laboratory, Cold Spring Harbor, NY, 1977, pp. 335-339. Magazin, M., Howe, M.M. and Allet, B.: Partial correlation of the genetic and physical rnaps of bacteriophage Mu. Virology 77 (1977) 677-688. Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53 (1970) 159-162. Miller, J.H.: Experiments in Molecular Genetics, Cold Spring ttarbor Laboratory, Cold Spring Harbor, 1972, p. 433. Moore, D., Schumm, J.W., ttowe, M. and Blattner, F.R.: Insertion of Mu DNA fragments into phage h in vitro, in Bukhari, A.I., Shapiro, J.A., and Adhya, S.L. (Eds.), DNA Insertion Elements, Plasmids, and Episomes, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1977, pp. 567-574. Murray, N.E. and Murray, K.: Manipulation of restriction targets in phage h to form receptor chromosomes for DNAfragments. Nature251 (1974) 476 481. O'Day, K., Schultz, D., Ericsen, W., Rawluk, L. and Howe, M.M.: Correction and refinement of the genetic map of bacteriophage Mu. Virology 93 (1979) 320- 328. Weil, J., Cunningham, R., Martin III, R., Mitchell, E. and Boiling, B.: Characteristics of ~.p4, a ~ derivative containing 9% excessDNA. Virology 50 (1972) 373 380. Zipser, D., Moses, P., Kahmann, R. and Kamp, D.: The molecular cloning of the immunity gene of phage Mu. Gene 2 (1977) 263-271.
Communicated by W. Szybalski.
79
© Elsevier/North-Holland Biomedical Press
R e s t r i c t i o n e n d o n u c l e a s e B a m H I cleaves b a c t e r i o p h a g e Mu D N A within c i s t r o n s E and F (Cloning o f Mu DNA; pBR322 vector; plasmid; mapping by marker rescue; recombinant DNA)
Jeffrey A. Engler, Rosalind A. Forgie
* and Martha M. Howe *
Cold Spring ltarbor Laboratory, Cbld Spring lfarbor, NY 11 724, and * Department of Bacteriology, University of Wisconsin, Madison, WI 53706 (U.S.A.)
(Received December 21 st, 1979) (Accepted January 5th, 1980)
SUMMARY We have cleaved phage Mu DNA with restriction endonucleases EcoRI and BamHI and have cloned three specific DNA fragments from the middle of the Mu genome into vector plasmid pBR322. By marker rescue experiments, we have determined that the two BamHl cleavage sites in Mu DNA occur within cistrons E and F.
Mu is a temperate bacteriophage of E. coli K-12 that can insert its DNA into the bacterial chromosome at many sites (for reviews, see Howe and Bade, 1975; Bukhari, 1976). The chromosome of Mu is a linear DNA duplex of approx. 38000 base pairs. Physical maps of Mu DNA have been constructed by restriction endonuclease cleavage analyses (Allet and Bukhari, 1975; Kahmann et al., 1977). A genetic map has been defined by amber mutant marker rescue studies of prophage deletion strains (Abelson et al., 1973) and )~pMu transducing phages (O'Day et al., 1979). Partial correlation of these genetic and physical maps has been obtained by determining the M u DNA segments in ?,pMu transducing phages carrying specific Mu genetic markers (Magazin et al., 1977; J.W. Schumm, D.D. Moore, F.R. Blattner and M.M. Howe, manuscript in preparation) and by defining the genetic markers present in certain Abbreviations: am, umber; amp, ampicillin; pfu, plaqueforming units: tet, tetr:~cycline.
Mu DNA restriction fragments that have been inserted into k (Moore et al., 1977) or plasmids (Zipser et al., 1977; W. Schumann, E.G. Bade, R.A. Forgie and M.M. Howe, manuscript in preparation). In this paper we report the cloning of restriction-endonuclease fragments of Mu DNA generated by the enzymes BamHl and EcoRl and present the results of amber mutant marker rescue studies of these clones. Our findings allow a further correlation of the genetic and physical maps of My by defining the location of the BamHI sites on the genetic map of Mu. Muvir phage were used as the source of Mu DNA for cloning and were grown by infection and purified as described by Bukhari and Ljungquist (1977). Mu DNA was prepared by phenol extraction of phage particles isolated by CsC1 equilibrium density centrifugation. Plasmid pBR322 (Bolivar et al., 1977) was used as the cloning vector and was prepared as described by Clewell (1972). Mu DNA and pBR322 DNA were digested with both l:'coRI and BamHI restriction endonucleases.
80
a)
C
A
b)
B
C
lys
iS
t Immunity end
D
E
H
F G
10
IS
20
,
,
1
,
H,
E¢o
Item
RI
HI
ii
I T J K
Blm
L
M
N POVWR S U
_
k
I
t ECO RI
HI
Y
Variable
end
p J E 107 i
pJE 106
p J E 108
Fig. 1. Mu gene content of pJE plasmids. The genetic (a) (O'Day et al., 1979) and physical (b) (Kahmann et al., 1977) maps of bacteriophage Mu, aligned according to Magazin et al. (1977) and this study. The open boxes represent variable host DNA associated with both ends of the Mu genome. The Mu DNA (including Mu genetic markers) carried by each plasmid are indicated by the bar. Marker-rescue data were obtained from spot and whole-plate marker-rescue tests performed on TCMG plates (Weil et al., 1972), using the method for "Mu-sensitive prophage deletions" described by Howe et al. (1979). A marker-rescue spot test was done for each plasmid with the following Mu anaber mutations (O'Day et al., 1979): A 1093, B1066, C4005, C2005, lysl030, /ys1025, D7330, E7329, E2003,/:'7346, El 006, H7326, H7222, H7315, H7300, H7100, H7007, H1043, F7207, F1065, F7001, F1931, (;1021, I1053, I7349, 17010, T1913, T7327, J1005, K1010, K7332, L1007, L7180, L7156,Mll14, Ml124, Y1027, NI041, P1024, Q1074, V7165, W7334, R1059, S1004, and U1049. In addition, pJE107 was tested by marker-rescue spot test with E mutations 1022, 7112, 7116, 7293, 7367, 1006, 7291 and 7310;pJE106 and pJE108 were tested by marker-rescue spot test with F mutations 1009, 1281, 1527, 1786, 1855, 1902, 7254, 7260, 7285, and 7323;pJE108 was also tested with L mutations 1033, 3010, 7030, 7123, and 7156, andM mutations 1954, 7016, 7061,7174 and 7251. More sensitive whole-plate assays were used to det]ne fragment endpoints, pJE106 was tested with B mutations 1032, 1066, 5177, 5178, 5179, 7096, 7308, and 7342, C1966, F mutations 1527, 1786, 3007, 7254, 7285, 7001,7323, and 1931, and G mutations 1021 and 1042. pJEl07 was tested wit11 the same B and C inutations as pJE 106 and with E mutation 1006, 7112, 7116, 7291, and 7310 and H7055. pJ E 108 was tested with F mutations 1009, 1527, 1786, 1855, 1902, 3007, 7254, 7260, 7285, 7001,7323,and 1931, G mutations 1021 and 1042,and M mutations 1114, 1124, 1954, 7016, 7061,7075, 7174, and 7251.
Fig. 1 shows that each enzyme cuts Mu DNA twice and cuts pBR322 once (Bolivar et at., 1977). The cleaved DNAs were extracted with phenol once and were then precipitated with ethanol. After resuspending the DNAs in double-distilled water, 1/ag of the digested pBR322 DNA and 2/lg of the digested Mu DNA were incubated together with T4 polynucleotide ligase (Miles Laboratories) in a total volume of 100 t-d for 3 h at 15°C, as described by Murray and Murray (1974). A portion of the ligated DNA mixture was used to transfect E. colt strain W3350 strrsu ° (from Dr. W. Dove) by a modification of the method of Mandel and Higa (1970), as described by Glover (1976). Insertion of Mu DNA into pBR322 between the BamHl and the EcoRI cleavage sites inactivates the tetracycline, but not the ampicillin (amp), resistance gene of the plasmid. Transformed colonies were selected on LB ( t r y p t o n e - y e a s t extract-NaCl) plates (Miller, 1972) supplemented with amp (40
/ag/ml). Amp-resistant colonies were further screened for tetracycline (tet) sensitivity on plates supplemented with tet (40 /~g/ml). Nine amp-resistant, tet-sensitive colonies were chosen for further study. The nine transformants were screened in a spot test for their ability to rescue Mu phages containing amber mutations. Several dilutions of Mu ambermutant lysates were spotted onto lawns of each transformant and the plates were incubated overnight. The presence of plaques or cleared areas within a spot was interpreted as marker rescue. As can be seen in Table I, seven of the nine transformants were able to rescue some Mu amber mutations. Five of the transformants allowed rescue of mutations in cistrons C and D, one allowed rescue of mutations in cistrons C, D and F, and another allowed rescue of mutations in only cistrons 1 and K. As expected, none of the transformants was able to allow rescue of mutations in cistrons B or N, which are known to lie outside the
81 TABLE I Spot tests for marker rescue of Mu amber mutants An overnight culture of each transformant was grown in LB broth (Miller, 1972) containing ampicillin (20 t~g/ml). The culture was diluted 1 to 100 in fresh LB + ampicillin and grown to a Klett-Summerson value of 50. A portion (0.l ml) of the culture was mixed with soft agar, plated on an LB plate containing 40 t~g/ml ampicillin and 2.5 mM CaC12, and allowed to cool for 30 minutes. Crude lysates of Mu amber (am) mutants (108-109 pfu per ml, determined by plating on an su host) were diluted in buffer containing 10 mM Tris pH 7.4, 10 mM MgCI2 and 10 mM CaCI 2 and were spotted at three dilutions (10 °, 10 -2, 10 -4) onto the lawns and allowed to dry. The plates were incubated overnight at 37°C. The appearance of plaques or a cleared area in a spot'was taken as a positive (+) result, nd, not determined. Strain
Muvir
Bam1066
783suIII w3350 strrsu° W3350/pJE101 W3350/pJE102 W3350/pJE103 W3350/pJE104 W3350/pJE105 W3350/pJE106 W3350/pJE107 W3350/pJE108 W3350/pJE109
+ + + + + + + + + + +
+ . -
Cam1966
Dam7015
Faro7217
+
+
+ . . . . . . + . .
.
.
.
+ + + + + + -
+ + + + nd + + nd nd
region cloned (Fig. 1). The two remaining transfor-
markers
Iam4037
Kaml010
Nam1034
+
+
+
-
-
+ .
-
.
.
. . . . .
. . . . .
. . . . .
.
.
. .
from
+ .
.
cistrons A-B
or M-U, which were
mants rescued no Mu a m b e r m u t a t i o n s and were not
outside the region cloned. To determine m o r e precisely
characterized further; t h e y m a y have arisen by head
the positions o f the BamHI cleavage sites on the
to head joining o f t w o p B R 3 2 2
genetic map,
D N A molecules,
Plasmid D N A s from six o f the Mu D N A - c o n t a i n i n g transformants
were
isolated
(Clewell,
marker
rescue
spot tests were per-
formed using a large n u m b e r o f phages containing
resulting in amp-resistance and tet-sensitivity. 1972)
and
a m b e r m u t a t i o n s in cistrons B, C, E, H, F, G, L and M. For all m u t a t i o n s located near the ends o f the
digested with restriction endonucleases EcoRI and BamHI. As can be seen in Fig. 2, three types o f
DNA
insertions were f o u n d ; four o f the plasmids were
also performed. The results o f these assays are pre-
identical and c o n t a i n e d Mu D N A f r o m segment I
sented in Figs. 1 and 3. Plasmid p J E 1 0 7 allowed
fragment
carried
by
each plasmid, a m o r e
sensitive marker rescue assay using whole plates was
in Fig. 1 ( e x t e n d i n g f r o m the leftmost EcoRI cut
rescue of all markers tested in cistrons C, lys and
to the l e f t m o s t BamltI cut); one plasmid ( p J E 1 0 6 )
D, but only some in E; this is consistent with the
c o n t a i n e d b o t h segment I and segment II D N A ( e x t e n d i n g f r o m the l e f t m o s t EcoRI cut to the
presence o f D N A segment I. Plasmid p J E 1 0 6 allowed
rightmost BamHI cut) and a n o t h e r ( p J E 1 0 8 ) con-
and H but o n l y some in cistron F; this is consistent
tained segment III D N A ( e x t e n d i n g from the right-
with the presence o f D N A segments I and II. Plasmid
m o s t BamHI cut to the rightmost E c o R I cut).
p J E 1 0 8 did n o t allow rescue o f any o f the markers
One plasmid containing each t y p e o f insertion ( p J E 1 0 6 , p J E 1 0 7 , p J E 1 0 8 ) was further character-
allowed rescue o f the remaining four F markers and
ized b y
all markers tested in cistrons G through L; this is
additional
marker
rescue e x p e r i m e n t s
to
rescue o f all markers tested in cistrons C, lys, D, E,
carried by plasmids p J E 1 0 6 or p J E 1 0 7 but instead
determine the c o n t e n t o f Mu marker alleles. Marker
consistent with the presence o f D N A segment III. If
rescue
phage
the assumption is m a d e that all markers present on a
plasmid
c o n t a i n e d markers f r o m a single contiguous set o f Mu
cloned restriction fragment can be d e t e c t e d in the marker rescue assay, then the results can be used to
cistrons and that
define the l o c a t i o n o f the BamHI restriction sites on
from
spot each
tests using one a m b e r m u t a n t cistron
confirmed none
that
each
o f the plasmids c o n t a i n e d
82
abcdefgh
the genetic map. One site is located between the last E markers that can be rescued from pJE107 and the first E markers that can be rescued from pJE106 but not from pJE107. Similarly the other site is flanked, on the left, by F markers which can be rescued from pJE106 but not from pJEl08 and, on the right, by F markers which can be rescued from pJEl08 but not from pJE106. Fig. 3 presents a detailed genetic map of the region from cistrons E, H, and F (O'Day et al., 1979) and the locations of the BamHI cleavage sites, as determined by the above analysis. The efficient rescue of all F markers from either pJE106 or pJE108 supports the validity of our conclusion from the marker rescue data. In similar experiments W. Schumann, E.G. Bade, R.A. Forgie and M.M. Howe (manuscript in preparation) have isolated plasraids containing either segment I, segment II, or segment 11 (Fig. 1). Marker rescue analysis of those plasraids had confirmed the locations of the BamHI sites determined by this work.
A('K NOWLlq)GEMI~NTS Fig. 2. R e c o m b i n a n t plasmid DNA cleaved with /;coRl and B a m H l restriction endonucleases. Plasmid DNA was isolated according to Clewell (1972). After removal of ellfidium bromide and dialysis against 10 mM Tris pH 7.9, 1 mM H ) T A , 1 ~g of DNA was digested with L'~oRI and B a m t t l in 6 mM Tris • HC1 pH 7.9, 6 mM MgC12, 6 mM i3-mercaptoethanol for 4 h at 37°C. The digested sanrlqes were mixed wifll l,:,ading dye and run on a 1% agarose gel toni:fining l0 ~g/ml ,.~lhidium bronride for 4 h at 4.5 V/cm. The get wa~ ilhn~im~.ted with UV light and photographed on Polaroid type 57 film using a Kodak No. 23 red f'dter. (a) pBR322, (b) p J V l 0 1 , (el p J E l 0 2 , (d) pJE103, (e) p J E l 0 6 , (f) r~JF107, (g) p.t[.'ll)8, (h) Muvir DNA. The u p p e r m o s t band in channel e is a partialdigestion product.
704S 7114 7147 7148
D
Barn H1
Bam H1
1
I
7112 7 2 M 1341 3005 7112 72111 72118 7 1 l J 7311) 7312 72113 7317 7387 7329 7344
E
We thank P.A. Myers and R.J. Roberts for supplying t:coRl and J. Broach for BamH1. D. Kamp provided Muvir phage and valuable advice on cloning techniques. D. Kwoh and D. Erdmann provided the elude lysates of the Mu amber mutants used for the inilial screening of the transformants. We thank L.T. Chow and T.R. Broker for their critical reading of the manuscript. J.M. Scott provided valuable technical assistance. J.E. was supported by NCI Cancer Center CA13106 to the Cold Spring Harbor Lab. and by NIH grant GM23996 to L.T. Chow, and is a recipient of a Fellowship from the Leukemia Society of America. M.M.tt. and R.F. acknowledge support from the
llI? 728l 7170 7324 73211
7300
H
1177 l N l 7 I N lira8 I0N 11141 1044 7007
711$ ~ 714141 7311 731J 7|$4
1~7314 !11~1 1|1| 1033 IIINJ Too I 7333
fill llli 111|6 2i 703J 7207 7217 75'32
1127 lINES IWl 30117 72S4 7280 7383
F
7323
I
G
Fig. 3. I oc~'Jli,~n o f the Ramtll restriction sites on the gt'~ ti~ map of Mu. The m a p of amber m u t a t i o n s is based on the work of O ' D i v et al. (1979t and ~n results ol marker rescue les~s t',erf,~rmed as described in the legend to l:ig. 1.
83 College o f A g r i c u l t u r a l a n d Life Sciences, U n i v e r s i t y o f Wisconsin, M a d i s o n , a n d f r o m N S F grant PCM750 2 4 6 5 , N I H g r a n t A I 1 2 7 3 1 , a n d ACS g r a n t NP-264. MMH is t h e r e c i p i e n t o f N I H R e s e a r c h Career Development Award A100274.
REFERENCES Abelson, J., Boram, W., Bukhari, A.I., Faelen, M., Howe, M., Metlay, M., Taylor, A.L., Toussaint, A., van de Putte, P., Westmaas, G.C. and Wijft'elman, C.A.: Summary of the genetic mapping of prophage Mu. Virology 54 (1973) 90-92. Allet, B. and Bukhari, A.I.: Analysis of bacteriophage Mu and ~.-Mu hybrid DNAs by specific endonucleases. J. Mol. Biol. 92 (1975) 529 540. Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.(., Heyneker, H.L., Boyer, H.W., Crosa, J.H. and Falkow, S.: Construction and characterization of new cloning vehicles. Gene 2 (1977) 9 5 - 1 1 3 . Bukhari, A.I.: Bacteriophage Mu as a transposition element. Annu. Rev. Genet. 10 (1976) 3 8 9 - 4 1 2 . Bukhari, A.I. and Ljungquist, E.: Bacteriophage Mu: Methods for cultivation and use, in Buldaari, A.I., Shapiro, J.A. and Adhya, S.L. (Eds.), DNA Insertion Elements, Plasmids, and Episomes, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1977, pp. 749-756. Clewell, D.B.: Nature of ColE1 plasmid recombination in the presence of chloramphenicol. J. Bacteriol. 110 (1972) 667-676. Glover, D.M.: The construction and cloning of hybrid DNA molecules, in Pain, R.H. and Smith, B.J. (Eds.), New Techniques in Biophysics and Cell Biology, Vol. III, Wiley, London, 1976, pp. 125-145.
Howe, M.M. and Bade, E. : Molecular biology of bacteriophage Mu. Science 190 (1975) 624 632. Howe, M.M., Scllumm, J.W. and Taylor, A.L.: The S and U genes of bacteriophage Mu are located in tile invertible G segment ofMu DNA. Virology 92 (1979) 108 124. Kahmann, R., Kamp, D. and Zipser, D.: Mapping of restriction sites in Mu DNA, in Bukhari, A.l., Shapiro, J.A., and Adhya, S.L. (Eds.), DNA Insertion Elements, Plasmids, and Episomes, Cold Spring ttarbor Laboratory, Cold Spring Harbor, NY, 1977, pp. 335-339. Magazin, M., Howe, M.M. and Allet, B.: Partial correlation of the genetic and physical rnaps of bacteriophage Mu. Virology 77 (1977) 677-688. Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53 (1970) 159-162. Miller, J.H.: Experiments in Molecular Genetics, Cold Spring ttarbor Laboratory, Cold Spring Harbor, 1972, p. 433. Moore, D., Schumm, J.W., ttowe, M. and Blattner, F.R.: Insertion of Mu DNA fragments into phage h in vitro, in Bukhari, A.I., Shapiro, J.A., and Adhya, S.L. (Eds.), DNA Insertion Elements, Plasmids, and Episomes, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1977, pp. 567-574. Murray, N.E. and Murray, K.: Manipulation of restriction targets in phage h to form receptor chromosomes for DNAfragments. Nature251 (1974) 476 481. O'Day, K., Schultz, D., Ericsen, W., Rawluk, L. and Howe, M.M.: Correction and refinement of the genetic map of bacteriophage Mu. Virology 93 (1979) 320- 328. Weil, J., Cunningham, R., Martin III, R., Mitchell, E. and Boiling, B.: Characteristics of ~.p4, a ~ derivative containing 9% excessDNA. Virology 50 (1972) 373 380. Zipser, D., Moses, P., Kahmann, R. and Kamp, D.: The molecular cloning of the immunity gene of phage Mu. Gene 2 (1977) 263-271.
Communicated by W. Szybalski.