Jan 25, 1989 - ... positive and negative supercoiling are thought to form in DNA molecules whenever free ... Our results favor models in which supercoiling domains are created when ... site, marked with a vertical line at the top of the plasmid.
JOURNAL OF BACTERIOLOGY, Apr. 1989, p. 2181-2187
Vol. 171, No. 4
0021-9193/89/042181-07$02.00/0 Copyright © 1989, American Society for Microbiology
Formation of
Supercoiling
JENNIFER K.
LODGE,'
TONI
Domains
KAZIC,'
Plasmid
in
pBR322
DOUGLAS E. BERG'.2*
AND
Department of Microbiology and Immunology' and Department Of Genetics,2
Washington University Medical School, St. Louis, Received 27
September 1988/Accepted
Missouri 63110
January 1989
25
Twin domains of
positive and negative supercoiling are thought to form in DNA molecules whenever free transcription complex around the DNA helix is impeded. Evidence for these domains has come from findings with Escherichia coil strains that are deficient in DNA I (top mutants) or that have been treated with DNA gyrase inhibitors. Plasmid pBR322 is highly supericoiled in these strains, whereas some of its deletion derivatives are not. The studies of pBR322 derivatives presented here show that high negative supercofiing in top strains requires translation as well as transcription of the first 98 codons of the tet gene and does not require the divergenitly transcribed amp gene. The N-terminal region of the TetA protein is thought to insert into the inner membrane. Our results favor models in which supercoiling domains are created when DNA segments are anichored to a large cellular structure via coupled transcription, translation, and membrane insertion of a nascent protein. rotation of
a
topo'isomerase
negative supercoiling of cellular DNA in a physiologically acceptable range by the opposing actions of DNA gyrase, which introduces negative supercoils, and topoisorherase which relaxes them (for reviews, see references 12 and 39). The importance of proper levels of supercoiling is illustrated by findings that bacterial strains lacking topoisomerase I (top mutants) inevitably accumulate compensatory mutations The
level
of
bound to
Escherichia coli is maintained within
variation in because
are
mutant
strains
with
can
DNA
gyrase
repressor-operator
interactions
come
from
studies with
and its derivatives in cells
pairs (bp) in the tet promoter reduced negative plasmid supercoiling in top cells (26), and a similar deletion also red'uced positive supercoiling in cells treated with gyrase
or
topoisomerase I activities show altered levels of supercoiling. The importance of proper supercoiling could be explained simply by effects on transcription. For example, certain promoters and some strongly affected by
(20, 40).
lacking topoisomerase I or treated with gyrase inhibitors (21, 25, 26, 43). Plasmid pBR322 (3; Fig. 1), which has divergently transcribed amp and tet genes, shows high negative supercoiling in top cells (25) and high positive supercoiling in cells treated with gyrase inhibitors (21). Dele'tions as small as three base
be tolerated, however,
decreased
structure such as the cell membrane or to
plasmid pBR322
in DNA gyrase (10, 27). Some
superhelical density
large
Evidence for this concept has
I,
and that many of these
a
another site in the DNA
inhibitors. Other mutations in the but do not
eliminate
tet
transcription
promoter which reduce from the
tet
promoter
are
supe'rhelical
Pfef
density (9, 31, 41), and the expression of particular genes at an inappropriate level or time could be detrimental. The modulation of specific gene expression by changes in supercoiling is also important in the response to changes in osmolarity (17) or oxygen tension (11). In addition, other vital functions such as replication, recombination, and transposition are also affected by supercoiling (2, 14, 18, 29). Transcription has been postulated to create and maintain domains of supercoiling (20, 40). During transcription, either the RNA polymerase with its associated RNA and proteins must rotate about the DNA or, if this rotation is restrained, the DNA must twist instead. This twisting creates transient twin domains of positive supercoils ahead and negative supercoils behind the transcription complex. Any barrier to the diffusion of positive and negative supercoils along the circular DNA would permit these domains to persist. In wild-type cells, DNA gyrase and DNA topoisomerase I equilibrate these domains, whereas in cells lacking either enzyme the domains are not equilibrated and aberrant supercoiling results. Divergent transcription might be sufficient to block the diffusion of supercoiling domains (20, 43), or barriers might be created when the transcription complex is
of pBR322 (adapted from reference 3). The bp long (24, 38), and numbering begins at the EcoRI site, marked with a vertical line at the top of the plasmid. The two antibiotic resistance genes (amp And tet) are indicated with thick lines, and the known transcripts are shown with arrows. The promoters (with the approximate locations) for these transcripts are ;as follows: P",,,, (4225-4190) and (80-40) for transcription of the amp gene; (5-45) for transcription of the tet gene; P,, (3123-3098) for synthesis of the primer of replication; P4 (2970-2995) FIG.
plasmid
1.
Structure
is 4,363
P,ef
for *
Corresponding
author.
synthesis of the inhibitor of replication; and
P5 (around 2150) 2181
for
synthesis
of RNAs with
P,.rp (23-10-2270)
no
and
known functions.
2182
J. BACTERIOL.
LODGE ET AL.
Analysis of plasmid supercoiling. Fresh overnight cultures of the top strain DM800 carrying the different mutant plasmids were diluted 1/250 into 50 ml of LN broth and grown for 4 h until the optical density at 600 nm was -0.5. The cells were pelleted, suspended in 1 ml of broth, transferred to an Eppendorf tube, and repelleted. They were suspended in 200 ,ul of STET buffer, and plasmid DNA was extracted by the boiling method as described (25) and suspended in 100 ,ul of TE buffer (10 mM Tris [pH 7.6], 1 mM EDTA). A portion (1/10) of each sample was electophoresed in a 1% agarose gel containing 12 ,ug of chloroquine per ml for 12 h at 3 V/cm as described (25).
reduced supercoiling proportionally (23; J. K. Lodge, Ph.D. dissertation, Washington University, St. Louis, Mo., 1988). These results suggest that tet transcription is important for high supercoiling..Large deletions in the tet coding region also reduced supercoiling (26), suggesting that either transcript length or transcription of a particular segment of the tet gene is important for high supercoiling in top cells. However, replacement of the tet gene with the galK gene transcribed from the powerful tac promoter did not affect supercoiling (43); this showed that the tet gene is not absolutely essential for high plasmid supercoiling and supported the divergent transcription model. We report evidence that translation as well as transcription of the 5' end of the tet gene is required and that transcription of the amp gene is not required for high negative supercoiling of pBR322 in top cells. The N terminus of the TetA protein is thought to be used for membrane insertion (15), and our results favor a model in which membrane insertion of the nascent TetA protein, still linked to the plasmid DNA by coupled transcription and translation, blocks the transcription complex from turning about the DNA, thereby generating twin domains of supercoiling.
RESULTS
High supercoiling depends on traniscription of the first part of the tet gene. Chloroquine gel electrophoresis of plasmid DNA from top cells had shown that pBR322 is highly negatively supercoiled (25). In a typical profile (Fig. 2), the bottom band contains the most negatively supercoiled DNA, the intermediate bands contain DNA with fewer supercoils, and the top band contains nicked circular DNA. To assess the role of the tet gene in the high negative supercoiling of pBR322, separate from the roles of transcript length and transcription rate, the coding region of the tet gene was replaced by promoterless kan (ptetkanS, ptetkanP) or lacZ (ptetlacZ) genes. In these substitution derivatives, the transcription levels are assumed to be similar to that of tet and transcript lengths are equal to or greater than those of tet.
MATERIALS AND METHODS Strains and plasmids. The top strain, DM800 (37), is a A(top-cysB)204 acrAB3 gyrB225 derivative of the E. coli K-12 strain W3110 (1) obtained from R. Sternglanz. MC1061 (6) was used for plasmnid constructions. The plasmids used and their constructions are detailed in the figure legends.
B
A QL U3 N] U C\is c
CM
co ) iT
or;
(0
X-
-
-
A
amp
tet
.
-~ I I---rSal
------
pBR322
I
Eo Ban
Pvu
Qi CQ Qc Qc Qc
amp z
or
--'k an
.........
--
ptetkanP Em Ban
or
ptetkanS
Pvu
Z amp 4: --k a n -oI 7;, sY%-'9i -t Ban PM Bain
or
Z
amp
Sal
c .......'Iac
---
PVI
--- -
ptetlacZ Pvu
Eco Ban
or
pkan
_
P
_ amp,
.kan
kan ---
'' -S'ffl; ;".ig ' *-''' '
Eao
Pvu
FIG. 2. Divergent transcription is not sufficient for high supercoiling. (A) Chloroquine gel electrophoresis showing supercoiling of pBR322 and derivatives which have the tet coding region replaced by lacZ or kan. The lowest pBR322 band (marked with an arrow) is the most supercoiled, the intermediate bands are less supercoiled, and the top band (marked with an arrow) is nicked circular DNA. The distribtition of topoisomers shifts upwards with less supercoiling. (B) Schematic diagrams of the pBR322 derivatives, linearized at the PvuII site. ptetkanP contains a promoterless kan gene (a BgiII-SalI fragment from TnS [30]) inserted between the BanI (bp 76) and PvuII (bp 2066) sites; ptetkanS has the same promoterless kan gene inserted between the BanI (bp 76) and Sall (bp 651) sites; ptetlacZ contains a promoterless lacZ gene (from pTK268 [T. Kazic, unpublished data]) inserted between the BanI (bp 76) and PvuII (bp 2066) sites. These three have the same arrangement of Pte,, Pantitet, and Pamp as pBR322. pkan contains the complete TnS kan gene (HindIII-SaIl fragment from TnS) transcribed from its own promoter inserted between EcoRI (bp 4361) and PvuII (bp 2066) of pBR322.
pBR322 SUPERCOILING
VOL. 171, 1989
The entire tet gene was also replaced by the kan gene (pkan) transcribed from its own promoter. None of these plasmids were highly supercoiled (Fig. 2), which indicates that specific transcription of the tet gene is required for high supercoiling at the moderate transcription rate characteristic of the tet promoter. Plasmid pACYC184, which is unrelated to pBR322 except for the same tet gene (7), also exhibited high supercoiling (data not shown). To determine the length of tet transcript needed for high supercoiling, synthetic trpA transcription terminators (8) were inserted at several different points in the tet gene (Fig. 3). This terminator inserted at the EcoRV site (pEv-T; RNA, 140 bases) reduced supercoiling. A terminator at the BamHI site (pBa-T; RNA, 335 bases) or the Sall site (pSa-T; RNA, 610 bases) had a small effect, and a terminator at the NruI (pNr-T; RNA, 900 bases) sites had no effect on supercoiling within the resolution of the one-dimensional gel system used. These results suggest that high supercoiling requires transcription of the first 140 to 335 bases of tet. amp transcription does not affect supercoiling in top cells. Deletion of a segment of pBR322 containing the 5' half of the amp gene and one of the amp promoters (ScaI-EcoRI) did not affect high supercoiling in top cells, although it did reduce positive supercoiling in cells treated with gyrase inhibitors (43). We found that deletion of the entire amp gene and one of the promoters (pDE) or deletion of both amp promoters (pI(39-49)SsAa) had no effect on supercoiling in top cells (Fig. 4). Translation of the N terminus of the TetA protein is required for high supercoiling. Small internal deletions in the tet gene did not affect supercoiling, indicating that high supercoiling does not depend on a complete TetA protein (26). However, the previously tested set of mutations did not include deletions of the 5' end of the coding region. It seemed possible that high supercoiling is an attribute of either the 5' end of the tet RNA or the N terminus of the TetA protein. To test the importance of translation of the TetA protein, we deleted a 107-bp fragment containing the translation start for the TetA protein (pBnEv). Figure 5 shows that this mutation reduced supercoiling substantially. To determine how large a segment of TetA protein must be A C
CMj
CY)H
B
F-F
m mcoC z Q- n- a Q a
made to ensure high supercoiling, a short oligonucleotide containing an amber codon (a translation terminator) was inserted in phase at convenient restriction sites in tet. Terminators inserted at EcoRV (peptide length, 35 amino acids [aa]; pEv-X) and BamHI (99 aa; pBa-X) markedly decreased supercoiling, while terminators at Sall (191 aa; pSa-X) and NruI (300 aa; pNr-X) only weakly affected supercoiling (Fig. 5). These results indicate that translation of a peptide of 99 to 198 aa is required for high supercoiling. An in-frame translational tet-lacZ fusion, preserving only the first 34 codons of tet (pEvlacZ), reduced supercoiling, whereas in-frame fusions preserving the first 98 (pBalacZ) or 190 (pSalacZ) codons of tet did not (Fig. 6). This shows that more than 34 aa of the N terminus of the TetA protein are needed for high supercoiling and that the first 98 aa of TetA are sufficient if fused to a longer peptide. In the reciprocal fusion, a 107-bp fragment of the tet gene including the first 34 codons was replaced by 460 bp of the 5' end of lacZ (ptetlactet). This encodes a fusion protein with the N-terminal 140 aa of ,B-galactosidase and the C-terminal 342 aa of TetA and is transcribed from the tet promoter. Figure 6 shows that this plasmid was not highly supercoiled. Thus, the N-terminal 34 to 98 aa of the TetA protein are necessary for the high supercoiling of pBR322. TetA is an inner membrane protein whose first 98 aa appear to be necessary and sufficient for membrane insertion (15). Expression of membrane protein-lacZ fusions are often deleterious (33). We observed that the colonies made by cells containing pEvlacZ were 1.5- or 2-fold larger than the colonies made by cells containing pBalacZ or pSalacZ, respectively, and that the slow-growing Lac+ strains tended to accumulate more rapidly growing Lac- derivatives. This suggests that fusions containing more than the first 34 aa of TetA are detrimental and is consistent with the idea that the N terminus of the TetA protein is responsible for its membrane insertion. DISCUSSION The experiments presented here show that translation of the N terminus of the tet gene is needed to cause the high tet
45 86 r ATG
ERBIBan I 76
2183
Eco RV Bam H1 375
1 85
Sal 651
Nru 972
FIG. 3. Transcription of the 5' end of tet is important for high supercoiling. (A) Gel electrophoresis showing supercoiling of pBR322 derivatives with trpA transcription terminators (from P-L Biochemicals, Inc.) inserted into the EcoRV (pEv-T), BamHI (pBa-T), Sall (pSa-T), or NruI (pNr-T) sites in the tet gene of pBR322. (B) Schematic diagram of the tet gene including the relevant restriction sites, the RNA start site (bp 45 [5]), and the protein start site (bp 86 [38]). The translation stop site is at bp 1274 (24).
2184
J. BACTERIOL.
LODGE ET AL. tt1
A
B
ci
C,)
or
ON
Dra
Ltwcs m
.............. am p ........
-
- tet
pBR322 Ssp Aat
E&
CD
''--1 o'----
..... .tot
pDE Exo
Dra
or
pJ(39-49) SsAa
tot -e-t
amp
Sp1 rAx Ss Aat
FIG. 4. Deletions in amp do not affect supercoiling. (A) Gel electrophoresis showing supercoiling of pBR322 derivatives with deletions in amp. (B) Schematic diagrams of pBR322 derivatives, linearized at the Pvull site. The three deletion plasmids were made by restriction digestion of pBR322, filling in 5' overhangs with DNA polymerase I and religation. pDE is deleted between the DraI (bp 3232) and EcoRI (bp 4361) sites and lacks Pamp and all of amp. pI(39-49)SsAa is deleted between the SspI (bp 4170) and AatI (bp 4286) sites and lacks Pamp; in addition, the -10 region of Pantitet (bp 43 to 50 of pBR322) has been mutated by inserting the sequence 5' ATTGGAGGTACCC between bp 39 and 49 of pBR322. The arrows indicate the remaining transcripts (for amp, the dashed arrow is from Pan,i,e, and the solid arrow is from
Pamp)
A
C\
C\j
45 86
B
>L
mCm
X X X X
LL,
COI mm cz wo nnnnnn 0 0 0 (
m.
r
te t
ATG
... EoARl Ban 76
Em R 1 85
Barm HI
Sal
o35
65 1
Nru I 972
FIG. 5. Translation of the 5' end of tet is important for high supercoiling. (A) Gel electrophoresis of a translation terminator (5' CTCTAGAG, XbaI linker) inserted into the EcoRV (pEv-X), BamHI (pBa-X), Sall (pSa-X), or NruI (pNr-X) sites in the tet gene of pBR322. (B) Schematic diagram of the tet gene including the relevant restriction sites, the RNA start site (bp 45), the protein start site (bp 86), and the translation stop site (bp 1274).
A
_>~ anchor pBR322 SUPERCOILING
VOL. 171, 1989
CD C\J N N N c0 ('4 cO 0 O 0 O 0 (
c L-
m -
B
:>
I
E R BanI
ui co o _
tet
AM
pBR322
I ECo RV Bam H
Sal
Pvu
0c Ch
pEvlacZ
*
I
- - -
tet ATG
IlacZ
Pa....
VAQ222m
I
PVUIl
ERI Bani fix RV 185
ttet
IacZ
1 ATG
pBalacZ
PvuI
EfiRI BanI EaoRV Bamr HI 375
.... 1 i ATG EoR I Bant fIE?RV BamH tet
ptetlactet
'lacZ
eat
BanI EvRI |uRi
pSalacZ
....
Sai 651
lacZ
Pvul
tet
--
IG
I
&EoRI Ba7I 76
2185
RV BamHI
Ec
PvulI
Sail
1 85
FIG. 6. Translation of the N terminus of the TetA protein is essential for high supercoiling. (A) Gel electrophoresis showing that supercoiling of pBR322 containing tet-lacZ fusions increases with the distance of the fusion from the 5' end of tet. (B) Schematic diagrams of pBR322 derivatives which have tet-lacZ fusions. The same lacZ fragment (16) is fused in frame to tet at EcoRV (pEvlacZ), BamHI (pBalacZ), and Sall (pSalacZ) sites. ptetlactet has a promoterless fragment of lacZ inserted between the BanI and EcoRV sites such that the fusion is in frame and Ptet transcribes the fusion gene. Arrows indicate the start and direction of transcription of the fusion genes.
supercoiling of pBR322 DNA seen in top strains. The most critical evidence comes from the use of protein fusions. With 33 aa of tet fused to lacZ (pEvlacZ), supercoiling was reduced, but with 98 aa of tet fused to lacZ (pBalacZ), supercoiling was not affected. The fusions are transcribed from the tet promoter, translation is initiated at the normal tet start codon, and the length of the transcripts exceeds that of the original tet transcript. This implies that the translation of the N terminus of the TetA protein is crucial for the generation of high supercoiling. The N-terminal 98 aa of the TetA protein contain three hydrophobic regions as judged by two different algorithms (9, 13) and are thought to bind the inner cell membrane (15). When the TetA protein was truncated at 98 aa rather than fused to another protein, supercoiling was reduced, indicating that the total length of the protein is also important. The reduced supercoiling of plasmids in which kan or lacZ genes are substituted for the tet gene is not consistent with the previously favored model (20, 43) in which the normal divergent transcription of the amp and tet genes of pBR322 both creates and maintains twin supercoiling domains. For example, in plasmid ptetkanS, none of the eight known promoters have been altered (Fig. 1 and 3). The only change has been a replacement of 600 bp of the tet coding region with 1,100 bp of the kan coding region, but ptetkanS did not show high supercoiling. We conclude that divergent transcription alone is not sufficient to cause high negative supercoiling of pBR322 in top cells. However, it does not rule out the possibility that divergent transcription is necessary, since the ori transcripts (from Pp and P4 in Fig. 1) are divergently transcribed. We interpret our results in the general framework of the
twin domain model (20, 40). Figure 7 shows our preferred explanation for pBR322. Membrane insertion of the growing TetA protein, still linked to the transcription complex via coupled transcription and translation, firmly anchors the transcription complex to a large cellular structure. This prevents the complex from rotating about the DNA and forces the DNA to twist during transcription, generating supercoils. For persistent domains to arise, a barrier to the diffusion of supercoils would be required elsewhere on the
RNA
-inner membrane
ribosome
main
barrier -^
FIG. 7. A model for the formation of supercoiling domains in pBR322. The anchoring of the nascent TetA protein in the membrane, coupled to the plasmid DNA by transcription and translation of tet, causes the formation of positive supercoils (+) ahead of the transcription complex and negative supercoils (-) behind it (40). For persistent domains to arise, a barrier to the diffusion of supercoils must exist elsewhere on the plasmid, and this barrier might be provided by the replication origin.
2186
LODGE ET AL.
plasmid. This barrier might be provided by the high rate of transcription at the origin of replication (37) or the possible binding of the origin to the membrane (28, 35). If the replication origin is the second barrier, the positively supercoiled domain would stretch between the tet gene and the origin, and it is interesting to note that most DNA gyrase binding sites have been mapped in vivo to that portion of the plasmid (22). Our results on the importance of tet translation are in accord with the deletion data of Pruss and Drlica (26) and most of the data from Wu et al. (43). The latter group of authors had indicated that high supercoiling is seen when tet is replaced by galK, a nonmembrane protein transcribed from the strong tac promoter. This result had indicated that extremely high transcription can elevate supercoiling. Our results emphasize that at the more moderate rates of transcription characteristic of tet and probably of many other genes, the maintenance of different superhelical domains requires a more effective anchor, and this can be provided by anchoring to the inner membrane. It thus seems that each of a variety of events can affect local supercoiling. Wu et al. (43) also had found that deletion of amp or inversion of tet altered supercoiling in cells treated with gyrase inhibitors, although similar mutations did not alter supercoiling in top cells (26, 43; J. K. Lodge and D. E. Berg, unpublished data). These seeming discrepancies may actually reflect subtle differences between the two experimental procedures. In top cells, only the effect of gyrase on the positive domain is monitored, while in cells treated with gyrase inhibitors only the effect of topoisomerase on the negative domain is monitored. Therefore, mutations such as a large deletion in amp or inversions of tet which alter the size, specific sequence, and transcription of the negative domain may actually change the interaction of topoisomerase I with the negative domain rather than affect the formation of the domains. This effect would be seen with gyrase inhibitors but not with top mutations. Alternatively, the seeming discrepancies might reflect the reciprocal interactions of supercoiling and transcription and a dependence on whether negative or positive supercoils are accumulating. Evidence from our studies of transposon Tn5 supports the idea that translation of TetA probably affects supercoiling in top' as well as in top cells. Tn5 transposition is known to require negatively supercoiled target DNA (18), and studies have shown that in top' cells, Tn5 inserts preferentially at a hotspot in the tet promoter of pBR322 (4). This hotspot is not used for transposition to pBX (Lodge and Berg, unpublished data), a plasmid in which tet translation is terminated 345 bp distal to the hotspot and which showed reduced supercoiling in top cells. The E. coli chromosome is divided into -50 large topologically distinct domains, each of which can be relaxed independently of the others (34, 42). Extrapolation from studies of pBR322 suggests that DNA segments of just a few hundred base pairs can differ in superhelical density and that the large chromosomal domains may consist of many smaller transient domains. The resulting heterogeneity has implications for the transcriptional control of genes with supercoiling-sensitive promoters. ACKNOWLEDGMENTS We are grateful to K. Dodson and K. Weston-Hafer for stimulating discussions, to J. Wang and J. Majors for critical reading of the manuscript, and to M. Michener for assistance with the figures. This work was supported by a grant from the Lucille P. Markey Charitable Trust, Public Health Service grant no. GM-37138 from
J. BACTERIOL.
the National Institutes of Health, and grant DMB-8608193 from the National Science Foundation to D.E.B. J.K.L. was supported by a Graduate and Professional Opportunities Program Fellowship, and T.K. was supported by Public Health Service training grant 5T32 Al 07015 from the National Institutes of Health. LITERATURE CITED 1. Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36:525-557. 2. Baker, T. A., K. Sekimizu, B. E. Funnell, and A. Kornberg. 1986. Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome. Cell 45:53-64. 3. Balbas, P., X. Soberon, E. Merino, M. Zurita, H. Lomeli, F. Valle, N. Flores, and F. Bolivar. 1986. Plasmid vector pBR322 and its special-purpose derivatives-a review. Gene 50:3-40. 4. Berg, D. E., M. A. Schmandt, and J. B. Lowe. 1983. Specificity of transposon Tn5 insertion. Genetics 105:813-828. 5. Brosius, J., R. L. Cate, and A. P. Perlmutter. 1982. Precise location of two promoters for the 1-lactamase gene on pBR322. J. Biol. Chem. 257:9205-9210. 6. Casadaban, M., and S. Cohen. 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138:179-207. 7. Chang, A. C. Y., and S. N. Cohen. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P1SA cryptic miniplasmid. J. Bacteriol. 134: 1141-1156. 8. Christie, G. E., P. J. Farnham, and T. Platt. 1981. Synthetic sites for transcription termination and a functional comparison with tryptophan operon termination sites in vitro. Proc. NatI. Acad. Sci. USA 78:4180-4184. 9. Dimri, G. P., and H. K. Das. 1988. Transcriptional regulation of nitrogen-fixation genes by DNA supercoiling. Mol. Gen. Genet. 212:360-363. 10. DiNardo, S., K. A. Voelkel, and R. Sternglanz. 1982. Escherichia coli DNA topoisomerase mutants have compensatory mutations in DNA gyrase genes. Cell 31:43-51. 11. Dorman, C. J., G. C. Barr, N. N. Bhriain, and C. F. Higgins. 1988. DNA supercoiling and the anaerobic and growth phase regulation of tonB gene expression. J. Bacteriol. 170:2816-2826. 12. Drlica, K. 1987. The nucleoid, p. 91-103. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C. 13. Engelman, D. M., T. A. Steitz, and A. Goldman. 1986. Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Annu. Rev. Biophys. Biophys. Chem. 15:321-353. 14. Funnell, B. E., T. A. Baker, and A. Kornberg. 1986. Complete enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome. J. Biol. Chem. 261:5616-5624. 15. Griffith, J. K., T. Kogoma, D. L. Corvo, W. L. Anderson, and A. L. Kazim. 1988. An N-terminal domain of the tetracycline resistance protein increases susceptibility to aminoglycosides and complements potassium uptake defects in Escherichia coli. J. Bacteriol. 170:598-604. 16. Guarente, L., G. Lauer, T. M. Roberts, and M. Ptashne. 1980. Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit 13-globin. Cell 20:543-553. 17. Higgins, C. F., C. J. Dorman, D. A. Stirling, L. Waddell, I. R. Booth, G. May, and E. Bremer. 1988. A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli. Cell 52:569-584. 18. Isberg, R. R., and M. Syvanen. 1982. DNA gyrase is a host factor for transposition of TnS. Cell 50:9-18. 19. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132. 20. Liu, L. F., and J. C. Wang. 1987. Supercoiling of the DNA template during transcription. Proc. Natl. Acad. Sci. USA 84:7024-7027.
pBR322 SUPERCOILING
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