Download Ability of a Bacterial Chromosome Segment to Invert Is

Copyright 0 1991 by the Genetics Society of America
Ability of a Bacterial Chromosome Segment to Invert Is Dictated by
Included Material Rather Than Flanking Sequence
Michael J. Mahan’.‘ andJohn R. Roth’
Department of Biology, University of Utah, Salt Lake City, Utah 841 12
Manuscript received March6, 1989
Accepted for publication September3, 1991
ABSTRACT
Homologous recombination between sequences present
in inverse order within the same chromosome can result in inversion formation. We have previously shown that inverse order sequences at
some sites (permissive) recombine to generate the expected inversion; no inversions
are found when
the same inverse order sequences flank other (nonpermissive) regionsof the chromosome. In hopes
of defining how permissive and nonpermissive intervalsare determined, we have constructed a strain
that carriesa large chromosomal inversion. Using this inversion mutant
as the parent strain,we have
determined the “permissivity”of a series of chromosomal sites for secondary inversions. For the set
of intervals tested, permissivity seems to be dictated by the nature of the genetic material present
within the chromosomal interval being tested rather than the flanking sequences or orientation of
this material in the chromosome. Almost all permissive intervals include the origin or terminus of
replication. We suggest that the rules for recovery
of inversions reflect mechanistic restrictions on the
occurrence of inversions rather than lethal consequencesof the completed rearrangement.
I
NSPECTION of the chromosomal organization in
Salmonella typhimurium and Escherichia coli reveals
strong conservation of gene order (BACHMAN 1983;
SANDERSON
and ROTH 1988)[for a discussion see
ROTH and SCHMID(1981), RILEY and KRAWIEC
(1987), and KRAWIECand RILEY(1990)]. This is surprising since the two organisms have diverged up to
25% in the nucleic acid sequence of coding regions
(NICHOLSand YANOFSKY1979; CRAWFORD,
NICHOLS
and YANOFSKY1980; CARLOMAGNO
et al. 1988), reducing recombination between them
greater than105fold (MIDDLETON197 1; YANOFSKY,HORNand ROWE
1977). Constraints on the formation of specific types
of genome rearrangements in S. typhimurium and E.
coli have been observed. T h e frequency of spontaneous duplications and deletions is high. Duplications
of chromosomal sites are found at
a frequencyof 1O-*
to 10-5 (ANDERSON
and ROTH 1977, 1981); deletions
of particular sites are found at a frequency of about
to 10” (reviewed in FRANKLIN 197 STARLIN1;
GER 1977). In contrast,rather
few inversion rearrangements have been reported. T h e apparent rarity may be due in part to difficulties in identification
of inversions. However for some sites where homologous sequences were provided, inversions could not
be detected (KONRAD1969,1977; ZIEG and KUSHNER
1978). For other sites, inversions were found but at a
low frequency (ROTHand SCHMID198 1;SCHMIDand
’ To whom correspondence should be sent.
* Present address: Department of Microbiology, Harvard Medical School,
200 Longwood Avenue, Boston, Massachusetts 021 15.
To whom strain and reprint requests should be addressed.
’
Genetics 1 2 9 1021-1032 (December, 1991)
ROTH 1983a,b). T o better understand the process of
chromosome rearrangement, we have developed two
systems for directing the formation of particular rearrangements (MAHANand ROTH 1988; SEGALL,
MAHAN and ROTH 1988). Both systems involve placing
homologous sequences in inverse order at separated
sites in the Salmonella chromosome and selecting for
recombination between them. A
similar system has
been developed for E. coli (FRANCOIS
et al. 1987) and
was used to select the same sorts of rearrangements
(REBOLLO,
FRANCOIS
and LOUARN
1988). All of these
systems are conceptual derivatives of work by KONRAD
(1969, 1977).
In both E. coli and Salmonella, the general results
of these selections are the same. When sequences are
placed at some pairs of chromosomal sites (permissive), the recombinants include the expected inversions; sequences placed at otherpairs of sites (nonpermissive) do notyield inversions. It seems likely that an
understanding of the factors that dictate theseresults
will lead to a better understanding of the structure,
function and possibly evolution of the bacterial chromosome.
Two aspects of our previous work have led us to
conclude that nonpermissive intervals aredueto
mechanistic problems in the recombination process,
rather than to lethal consequences of the final inversion (SEGALL,
MAHANand ROTH 1988). First, we have
successfully used transduction crosses to directthe
inversion of two intervals that are nonpermissive for
inversion by intrachromosomal exchanges. The directed inversions are not lethal; thus we think it un-
M. J. Mahan and J. R. Roth
1022
likely thatthefailure
to detect inversion of these
regions is due to lethality (A. M. SECALL,L. MIE~EL
and J. R. ROTH unpublished results). Second, even
for nonpermissive intervals, both of our two selection
systems detect recombination events between the involved sequences. (MAHANand ROTH 1988,1989;
SECALL
and ROTH1989). Since these alternative sorts
of recombinants (but not inversions) are formed by
sequences at all nonpermissive sites, we conclude that
the sequences involved can interact in some way. We
have proposed that sequences at nonpermissive sites
in the samechromosome cannot interact directly and
therefore cannotform inversions. We suggest that
such sequences can only engage in sister chromosome
exchanges, which can accountfor all the noninversion
recombinants detected.Results of similar experiments
in E. coli have led to slightly different conclusions and
will be discussed later.
In the experiments presented here, we wish to test
the effect of changes in the orientation and chromosomal position of a particular arc of chromosome on
the permissivity of inverting other segments of the
chromosome. To this end, we have constructeda
strain with alarge inversion ( a m to his) involving
almost halfof the bacterial chromosome and have
used this strain as a parent to test the permissivity of
11 chromosomal intervals. The results suggest that
the behavior of recombining sites is a function of the
genetic material located between those sites rather
than orientation, position of endpoints in the chromosome, or sequences flanking the recombining sites.
MATERIALS AND METHODS
Bacterial strains: All strains used in this study (Table 1
are derived from Salmonellatyphimurium strain LT2. All
directed transposition strains were constructed according to
methods described by CHUMLEY
and ROTH(1980), SCHMID
and ROTH(1980). Figure la diagrams the ara-his (minute 2
to minute 42) inversion strain TT11797 described previously (MAHANand ROTH1988). [Unless otherwise specified,
the mutation designations used in thisstudy are as according
to SANDERSON
and ROTH(1988).] This strain is unstable and
can reverse itsinversion by recombination between the
inverse order copies of either his or TnZO material (at ara
and his). By removing these repeated sequences from the
inversion join points (Figure lb), we have stabilizedthe arahis inversion, preventing it from “reflipping” to the normal
chromosomal arrangement. The his and TnZO homologies
were deleted from both inversion join points of TT11797
by methods described by HUGHES and
ROTH(1985).
Removing his and TnZOhomology at his: Mixed lysates of
strains TT8269 (leuAZZ7Y::MudA) and TT7692 (hisD9953::MudA) were used to transduce ara-his inversionbearing strain TT11797 to ampicillin resistance. [MudA
elements (HUGHES and
ROTH1984) referto a conditionally
transposition-defective derivative of the Mud prophages of
CASADABAN and
COHEN(1979).] The Ap’ Leu- His- transductant (TT13904) contains a deletion of genetic material
(leuAZl7Y*MudA*hisDYY53) between the MudA elements
in leuA (minute 3) and hisD (minute 42); the result of
homologous recombination between the twoMudAele-
ments, followed by recombination of this hybrid fragment
with the recipient chromosome (bottom of Figure 1b).
Removing his and TnlO homology at ara: Strain TT13905
is a derivative of TT13904 that contains a cobl-368::TnZOdCam insertion element [(encoding chloramphenicol resistance (ELLIOTand ROTH1988)l. This strain was transduced
to Ara+ with P22 phage grown on TT13903 (ara+,zuc365Y::TnIOd-Cam) (see top of Figure lb). The Ara+ Cm’
Ap’ Leu- His- transductant (TT13906) can arise by recombination between the two TnZOd-Cam elements (one on the
transduced fragment, the other in the recipient chromosome). Such events generate a deletion of genetic material
(zac-3659*TnZOd-Cam*cobZ-368) between the TnZOd-Cam
element in the zac region [adjacent to ara (minute 3)] and
CobI (minute 41). The final recombinant is diagrammed in
Figure IC).The inversion rearrangement of TT13906 was
then transduced intoa
wild type genetic background
(TR6535) according to
a
two-fragment transduction
method developed by SCHMID
and ROTH(1983a). The resultant ara-his inversion strain (TT13909) servesas the
parental strain for testing permissive and nonpermissive
intervals; this strain is isogenicwith the wild-type strain
(TR6535) except for the ara-his inversion, which is stable
due todeleted material near the inversion join points introduced by transduction (Figure IC).The inversion join point
at his (minute
42)
contains
deletion
a (leuAI1 7Y)*MudA*(hisD9953)that fuses the leuA gene (minute
3) to thehisD gene. The inversion join point adjacent to ara
(minute 3) contains a deletion (zac-365Y*TnIOd-Carn*cobl368) that fuses the zac (near ara) region to the CobI operon
(minute 41).
Media: The E medium of VOGEL and BONNER
(1956)
supplemented with 0.2% glucose was used as the defined
minimal medium. Selection for growth on alternative carbon sources was done on NCE medium, described by BERKOWITZ et al. (1968), supplemented with 0.2%of the appropriate carbon source. The complex medium was nutrient
broth (8 g/liter, Difco Laboratories) with added NaCl(5 g/
liter). Solid medium contained Difco agar at 1.5% final
concentration. Auxotrophic requirements were included in
media at final concentrations described by DAVIS, BOTSTEIN
and ROTH(1980). Final concentrations of antibiotics were
as follows: tetracycline hydrochloride (Sigma ChemicalCo.,
16 mg/ml in rich medium, or 10 mg/ml in minimal medium); kanamycin sulfate (Sigma Chemical Co., 50 mg/ml
in rich medium, or 100 mg/ml in minimal medium); chloramphenicol (Sigma ChemicalCo., 20 mg/ml inrich medium,
or 5 mg/ml in minimalmedium); ampicillin (Sigma Chemical
Co., 30 mg/ml in rich medium, or 15 mg/ml in minimal
medium).
Transductional methods: The high frequency generalized transducing mutant of bacteriophage P22 (HT 105/1,
int-201) (SCHMIEGER
1972) was used for all transductional
crosses. Unless otherwise specified, 0.1 ml of an overnight
culture grown in complex medium (ca. 2-4 X lo9 cfu/ml)
was used asa recipient of 0.1 ml transducing phage (ca. 10’lo9 pfu/ml) and plated directly on selective plates. Transductional crosses involving the selection of kanamycin or
chloramphenicol resistance were preincubated overnight on
solidnonselective complex medium, then replica-printed
onto selective medium. Transductant clones were purified
and phage-free isolates were obtained by streaking for single
colonies on green indicator plates (CHANet al. 1972). Phagefree colonies were tested for phage sensitivity by crossstreaking with P22 H5 (a clear plaque mutant of phage
P22).
Construction of TT13913: This strain is used as a donor
in transduction crosses that detect linkage disruption at both
Restrictions on Inversion
1023
TABLE 1
Bacterial strains
Strain"
GenotvDeb
TTll797
TTl3909
TT13913
TT13915
INV768[(ara-651::TnlO)*h~~*(hisC869Z::TnZO-hisOGD646),
zee-P::TnZO, proAB47
DEL859[1NV768((zac-3659)*TnZOd-Cam*(cobZ-368)]
DEL858[(leuA1179)*MudA*(hisD9953)]],
proAB47
DEL859[INV768((zac-3659)*TnlOd-Cam*(cobZ-368)]
DEL860[(leuAlZ79)*MudJ*(hisF9954))1, proAB47
ara-65l::(TnlO-hisOGDC869Z-TnZO),
proAB47,
DEL859[INV768((zac-3659)*TnlOd-Cam*(cobZ-368)]
DEL858[(leuAZZ79)*MudA*(hisD9953))]
pncB165::(TnlO-hisOGDC8691-TnlO),
proAB47,
DEL859[INV768((zac-3659)*TnlOd-Cam*(cobZ-368)]
DEL858[(leuAlZ79)*MudA*(hisD9953))1
pncBZ65::(TnlO-hisOGDC8691-Tn10),
nadAZZ3::MudJ,proAB47,
DEL859[INV768((zac-3659)*TnlOd-Cam*(cobZ-368)]
DEL858[(leuAZZ79)*MudA*(hisD9953))1
pncAl55::(TnlO-hisOGDC869l-TnlO),
proAB47,
DEL859[INV768((zac-3659)*TnZOd-Cam*(cobZ-368)]
DEL858[(leuAZZ79)*MudA*(hisD9953))1
pncA155::(TnlO-hisOGDC8691=TnlO),
nadA213::MudJ,proAB47,
DEL859[INV768((zac-3659)*TnlOd-Cam*(cobZ-368)]
DEL858[(leuAll79)*MudA*(hisD9953)]]
tr~DE-243l::(TnZO-hisOGDC869Z-TnlO),
proAB47,
DEL859[INV768((zac-3659)*TnZOd-Cam*(cobZ-368)]
DEL858[(leuAZZ79)*MudA*(hisD9953)J]
zeg-74::(TnlO-hisOGDC8691-TnlO),
proAB47,
DEL859[INV768((zac-3659)*TnZOd-Cam*(cobZ-368)]
DEL858[(leuAZZ79)*MudA*(hisD9953))1
zfa-3647:(TnlO-hisOGDC869Z-TnlO),
proAB47,
DEL859[INV768((zac-3659)*TnlOd-Cam*(cobl-368)]
DEL858[(leuAZZ79)*MudA*(hisD9953)]]
tyrA555::(TnlO-hisOGDC869Z-TnlO),
proAB47,
DEL859[INV768((zac-3659)*TnZOd-Cam*(cobZ-368)]
DEL858[(leuAZ179)*MudA*(hisD9953))1
cysJZ-Z5Z9::(TnlO-hisOGDC8691-TnZO),
proAB47,
DEL859[INV768((zac3659)*TnlOd-Cam*(cobZ-368)]
DEL858[(leuAZZ79)*MudA*(hisD9953))1
argA1832::(TnlO-hisOGDC8691-TnZO),
proAB47,
DEL859[INV768((zac-3659)*TnlOd-Cam*(cobZ-368)]
DEL858[(leuAZZ79)*MudA*(hisD9953))]
metE866::(TnlO-hisOGDC8691-TnlO),
proAB47,
DEL859[INV768((zac3659)*TnZOd-Cam*(cobZ-368)]
DEL858[(leuAZZ79)*MudA*(hisD9953))I
pyrB692::(TnlO-hisOGDC8691-TnlO),
proAB47,
DEL859[INV768((zac-3659)*TnZOd-Cam*cobl-368)]
DEL858[(leuAZZ79)*MudA*(hisD9953)]]
TT13916
TT13917
TT13918
TT13919
TTl3920
TT13921
TT13922
TT13923
TTl3924
TT13925
TT13926
TTl3927
* All strains are derivatives of S. typhimurium LT2 and were constructed in this laboratory.
Figure l a diagrams the genotype of strain TT11797; Figure I C diagrams the genotype of strains TT13906 and TT13909. Figure 2
diagrams strain TTl3920, which is a representative example of parental ara-his inversion-bearing strain used in the inversion assay, and
strain TT13930, which is a representative example of a secondary inversion recombinant.
the his and non-his endpoints in inversion-bearing recombinants. Mixed lysates of strains TT8269 (leuAII79::MudA)
and TT7693(hisF9954::MudA) were used to transduce the
ara-his inversion strain TTl1797 (Figure la) to ampicillin
resistance. The Ap' Leu- His- transductant strain TTl3910
has acquired a deletion that removes the genetic material
from leuA into hisF (leuA1179)*MudA*(hisF9954);the result of homologous recombination between transduced fragments carrying the twoMudA elements and subsequent
recombination of the hybrid fragment with the chromosome. The MudA element at the joinpoint of this deletion
was converted to an allelic MudJ insertion by homologous
recombination at the ends of Mu, replacing an ampicillin
resistance marker with a kanamycin resistance marker
(TT139 1 1, leuAI 179*MudJ*hisF9954) (CASADABAN
and
COHEN1979; CASTILHO, OLFSON
and CASADABAN
1984; R.
V. SONTIand J. R. ROTH,
unpublished results). This deletion
was introduced
into
ara-his inversion-bearing strain
TT 13906 (see Bacterialstrains in MATERIALS AND METHODS
for discussion). The inversion rearrangement of one Km'
transductant (TT13912) was transduced into a wild type
background (TR6535) by the two-fragment transduction
method of SCHMID
and ROTH (1983a). The resultant arahis inversion-bearing strain TT13913 is identical to
TT13909 (Figure IC) except the large leu-his deletion,
which is associated witha MudJ element, extends from leuA
into hisF (leuA1179*MudJ*hisF9953). Use of this strain is
described below.
Linkage disruption: Linkage disruption was tested at
both join points of an inversion recombinant. (1) Linkage
disruption at the his endpoint. Linkage disruption atthe
his endpoint, inHis+ recombinants derived from parent
strains with an ara-his inversion, was diagnosed as a reduction in ability ofHis+ recombinants to inherit a large his-leu
deletion associated with a Km' determinant (Figure 3); this
deletion/replacement was introduced usingP22
phage
grown on strain TT13913. This donor strain (TT13913)
contains an ara-his inversion; the inversion join point at his
(minute
42/3)
contains a leuA-hisF deletion (leuA1179)*MudJ*(hisF9954) that is associatedwith a MudJ
insertion element (encoding a Km' determinant), constructed according toHUGHESand ROTH (1985). Strain
TT13913 is identical to TTl3909 (Figure IC) except the
large his deletion, which is associated with a MudJ element,
extends from leuA into hisF (leuAI 179)*MudJ*(hisF9954)
(see Construction of TT 139 13 in MATERIALS AND METHODS
for detailed description). When strain TT13913 is used as
a donor in a transduction cross, its kanamycin resistance
determinant can only be inherited by recipients that have
the samejuxtaposition of his and leu sequences as the parent
strain (Figure 3). Strains with a rearrangement that disrupts
this arrangement show a large reduction in ability to inherit
the kanamycin resistance determinant.
A representative example of an inversion-bearing His+
recombinant is diagrammed in Figure 2. Linkage disruption
at the non-his endpoint (at trp, minute 34) was diagnosed
M.
and
J. Mahan
1024
using the same rationale asthat for testing thehis endpoint
(above). That is, since a strain carrying a trp-his inversion
contains normal trp sequence on only one side of the trp
inversionjoin point (andhis material on the other), one will
observe a reduction in the abilityto recover Trp' transductants (when usinga wild type donor).
Nomenclature: Nomenclature is generally as described
et al. (1977),and
in DEMERECet al. (1966),CAMPBELL
CHUMLEY,
MENZELand ROTH (1979). The nomenclature
"z-::TnlO" refers to a TnlO insertion in a "silent" DNA
region; the"z--)' describes the map position of the insertion
(SANDERSON
and ROTH 1988). The nomenclature used for
chromosomal rearrangementsis described in CHUMLEY
and
ROTH (1980),SCHMID and
ROTH (1980) and HUGHES and
ROTH(1 985).
The nomenclature complex
of chromosomal
rearrangements (CRR) in
which
oneborder
consists of
multiple join points
is defined as follows. Commencing with
designation
of
the
outward-most
aspect
of the rearrangement, the terminology proceedsclockwise (from low
minutes to high minutes on the chromosome map) to denote, in order, subsequent aspects of the rearrangement
adjacenttotheoutward
border. In the case where the
outward border is extended by a successive rearrangement,
the CRR of the initial rearrangementis retained. Examples
of strains with successive rearrangements are given below.
INV768[(ara)*TnlO*(hisD)] designates an inversion ofthe
geneticmaterialbetween the TnlO elements in ara and
h i d ; the result of homologous recombination between the
two TnlO elements.
An additional exampleis a derivative of the above strain
that has acquired a deletionremovinghomologyat
the
hisD
inversion
join
point
of
strain
INV768[(ara)*TnIO*(hisD)]; this strain is described as follows: INV768[(ara)*TnlO*DEL860((leuA)*MudA*(hisF)])
designates a deletion of genetic material between MudA
elements in leuA and hisF, removing the TnlO homology at
the inversion join point in h i d ; the result of homologous
recombination between the two
MudA elements. The inversion join point, which previously mapped in the hisD gene
ofstrain INV768[(ara)*TnlO*(hisD)], is nowwithin the
deletion: DEL860[(leuA)*MudA*(hisF)].
A derivative of the above strain that has a deletion of
homology at the ara inversion join point(aswellas
the
leuA-hisD deletion) is designatedDEL859[INV768{(zac)*TnlOdCam*(CobI)]DEL860[(leuA)*MudA*(hisF)]]. This
deletion removes genetic material between the TnlOd-Cam
elements in the zac region (adjacent to ara) and the CobI
operon; the result of homologous recombination between
the twoTnlOd-Camelements. The inversion join point,
which previously mapped in the ara genes, now maps in the
deletion: DEL859[(zac)*TnlOd-Cam*(CobI)].
RESULTS
The assay system: A genetic system has been developed that detects strains containing directed inversions of particular chromosome regions (MAHAN and
ROTH 1988). We have previously used this system to
surveyrecovery of inversions of severalparticular
intervals of the bacterial chromosome(SEGAL,MAHAN
and ROTH 1988; MAHAN, SECALL
and ROTH 1990).
T h e results of these experiments indicated that some
intervals form inversions at high frequency (permissive), whereas other intervals fail to show inversion
(nonpermissive). In this paper, we use a parental strain
J. R. Roth
that contains an ara-his inversion to examine the effects of this parental inversion on the permissivity of
inversion for intervals that are either internal to the
region inverted (subintervals) or external to the parental inversion.
Figure I C is adiagram of strain T T l 3 9 0 9 that
carries a stable ara-his inversion (see Figure Ib and
MATERIALS AND METHODS for construction). T h e inversion join point adjacent toara contains a deletion
that fuses material near theara region (zac, minute 3)
tothe CobIgenes
(normally at minute41) (zac3659*TnlOd-Cam*cobZ-368). T h e inversion join
point athis contains a deletionthat fuses the hisD gene
(minute 42) to the leuA gene (normally at minute 3)
(leuAll79*MudA*hisD9953). T h e hisD-leuA deletion
(leuAll79*MudA*hisD9953) removes the his promoter, thehisG gene and partof the hisD gene. These
deletions remove all shared homology from the two
join point regiowand stabilize the parental (ara-his)
inversion.
Into the trp region of this stable inversion strain
(Figure IC), we introduced a chromosomal fragment
that carries the proximal portion of the his operon
(hisOGDC') flanked by TnlO elements in the same
orientation (C' indicates that only a fragment of the
hisC gene is present, Figure Id). The his material at
trp is in an orientation opposite thatof the normal his
region. This strain is able to grow on histidinol as a
source of histidine (Hal+) due to the functional hisD
gene present at thetrp locus. (The hisD gene product
catalyzes the conversion of the intermediatehistidinol
to histidine.) By selecting for His+ derivatives of this
strain, one can select for recombination between the
inverse order his sequences, generating a complete
his' operon.Thisgeneralprocedure
has been followed to construct a series of strains; each strain has
his sequences placed at a particular site outside the
his
operon in inverse orientationvis a vis the standardhis
region. For each strain,
selection for His+ recombinants demands an exchange between the two separated parts of the his operon. This exchange may or
may not lead to inversion formation. In cases where
inversions are impossible (nonpermissive), His+ recombinants can form by excision of the TnlO-his0GDC'-TnlO material as a circle and integration of
this circle at the his region (MAHANand ROTH 1988,
1989).
Detection of inversions: An homologous reciprocal
exchange event between inverse
order sequences present in the same chromosome results in an inversion
of the intervening material between the sites of exchange. Since the Hol+ His- parent strain TT 13920
contains inverse order his homologies, selection for
His+ recombinants yields a recombinant(such as strain
TT13930 in Figure 2) that contains an inversion of
the arc of chromosomal material lying between the
Restrictions on Inversion
1025
( 1 b)
Removal of shared
homology from
inversion join points
(1 a )
Unstable inversion
(TT11797)
Stable inversion
(Id)
Parent strain
(TT13909)
(TT13920)
(1 C)
Of
hlSD'MudA'leuA
FIGURE .-Construction
1
of a stable inversion-bearing strain. ( l a ) T T l l 7 9 7contains an inversion from ara-his (minute 2 to minute 42) as
described in MAHANand ROTH (1988); homologous recombination between the inverse order his or TnfO homologies present at both
inversion join points (at ara and athis) can reverse the inversion to normal gene order. (1b) Removal of his and TnfOhomologies at ara and
at his. Genetic material was deleted between the leuA (minute 3) and hisD (minute 42) genes (leuAfl79*MudA*hisD9953) of TTI 1797 as
described byHUGHES and ROTH (1985); the result of homologous recombination between two transduced fragments containing MudA
elements (one in leuA, the other in hisD) and the subsequent recombination of the hybrid fragment (dark triangle) with the recipient
chromosome. A derivative of this Ap" Leu- His- transductant strain (TT13905), which contains a TnfOd-Cam element in the cobI genes
[cobl-368::TnfOd-Cam (minute 41)], was used as a recipient of P22 phage grown on TT13903 (zac-3659::TnlOd-Cam);selection for Ara+
requires recombination between the two TnlOd-Cam elements (open triangles), removing the genetic material between the zac region
[adjacent to ara (minute 3)] and thecob1 genes (minute 41). (IC)Strain T T l 3 9 0 9 contains a stable ara-his inversion (constructed as described
in l b , and in MATERIALS AND METHODS).
The ara region contains a deletion (zac-3659*TnfOd-Cam*cobl-368)that fuses material from the
LUC region (minute 3) to the cobI genes (minute 41). The his region contains a deletion ( l e u A l f 79*MudA*hisD9953)that fuses the leuA gene
(minute 3) to the h i d gene (minute 42). (Id) Into the trp region (minute 34) of strain TT13909, we have introduced a chromosomal
fragment that carries the hisOGDC' genes flanked by TnfO elements in the same orientation (C' indicates only a fragment of the hisC gene
is present). The his material at the trp region of strain TT 13920 is in the opposite orientation vis & vis the normal his operon.
his and trp loci. In the rearranged parent strain, this
arc includes minutes 3-34 of the normal chromosome
map (Figure 2). Noninversion recombinant types that
survive this selection have been described in detail
elsewhere (MAHANand ROTH 1988, 1989).
The inversion structure of recombinant TT13930
(bottom of Figure 2) was identified by linkage disruption tests performed on both join points of the inversion. Since the inversion contains normal his material
on only one side of each inversion join point and trp
material on the other, it shouldnotbe possible to
repair either inversion join point with a wild type
transduced fragment. For example, it should be impossible to obtain a Trp+ transductant (using a wild
type donor), since the trp region (thenon-his endpoint)
is disrupted at the inversion join point. Furthermore,
the his inversion join point of TT13930 should not
be
able
to inherit
large
a
his deletion (leuAI I79*MudJ*hisF9954) that is associated with a kanamycin resistance determinant (Figure 3).That is, the
M. J. Mahan and J. R. Roth
1026
Recipient chromosome with
parent gene order
Parent
inversion
strain
(TT13920)
Kmr
+
m
2
orlg
X
a
his+
TT13913
transduced fragment
b
X
b
e
parent
/ /
1,,
Select
b
with
gene order
Kmr
c
d
e
//
Select
HIS+
chromosome
Kmr (HIS’)
transductants
+-T
lnverslon breakpoint in
reclplent
Kmr
Secondary
inversion
recombinant
TT13913
transduced
fragment
b
no shared
(TT13930)
b
2
( homology )
Inversion
chromosome
1
Select
Km
Linkage
disruptlon
No Knf tranductants
FIGURE
2.-Selection for inversions. The trp region (minute 34)
of parent strain TT13920 contains a chromosomal fragment that
carries his material (hisOGDC’) in the opposite orientation vis ii vis
the normal his operon (minute 42). Selection for His’ requires an
exchange event between the shared inverse order hisDC homologies. Such recombination yields strain TT13930: a His+ derivative
that carries an inversion of chromosomal material between the sites
of exchange (trp-his).
his region of parent strain TT13920 (Figure 2, top)
is disrupted at the his inversion join point in recombinant strainTT 13930 (Figure 2, bottom). Transduction of all loci unlinked to either inversion join point
should be normal.A more detailed descriptionof tests
for linkage disruption at the his endpoint and non-his
endpoint is presented in MATERIALS AND METHODS. In
these tests one sees a 6-100-fold (typically a 30-fold)
reduction in transduction frequency, instead of the
expected absence of transductants (MAHAN, SEGALL
and ROTH 1990). Genetic analysis performed on the
few transductants that are found despite the linkage
disruption suggests they are due to reversal of the
inversion; these “backflips” can result from homologous recombination between the inverse order his or
T n l O homologies at both inversion join points of the
His+ derivatives [at trp (minute 34) and his (minute
42) of TT13830, bottom of Figure 2)] (M. J. MAHAN
and J. R. ROTH, unpublished results). Transduction
of loci unlinked to either join point is normal (data
FIGURE3.-Linkage disruption at the his inversion join point in
parent strains with an ara-his inversion. P22 phage grown on
TT13913, which contains a large his deletion associated with a
kanamycin resistance determinant (leuA1 179*MudJ*hisF9954),was
used to transduce His+ recombinants to kanamycin resistance.
Strains that contain parental gene order flanking both sides of the
his region will inherit the deletion with normal transduction ability.
Strains containing an inversion in which the his inversionjoin point
disrupts the his region show a reduction in ability to inherit the
large his deletion. Transduction ability at loci unlinked to either
join point is normal. Strain TT13913 is identical to TT13909
(Figure IC) except the deletion, which is associated with a MudJ
element, extends from leuA into hisF. The arrows denote the two
breakpoints of the inversion.
not shown, also see MATERIALS AND METHODS). The
validity of the linkage disruption test was previously
documented by Hfr mapping of an inversion strain
that was initially characterized by linkage disruption
(MAHANand ROTH 1988). T h e gradient of transfer
of chromosomal markersconfirmed the inversion
structure inferred by linkage disruption.
Permissive and nonpermissive intervals
in parental
strains
containing an uru-his inversion
rearrangement: We have placed the his operon fragment (described above) at 11different locations in
parental strains containingthe stable uru-his inversion;
in each strain the two fragments of the his operon are
in opposite orientation but can recombine to generate
a His+ phenotype. From each of these strains, His+
recombinants were selected on minimal medium. Inversions (listed in Table 1) were identified by linkage
disruption tests performed on both join pointsof the
Restrictions on Inversion
rearrangement (where possible). An interval permissive to inversion is defined asone thatshows a 6- 100fold reduction in transductant frequency at both join
points, but an undiminished transductant frequency
for regions distant from either joinpoint.
For some strains, absence of a selective marker
prevented testing of one endpoint forlinkage disruption.Forexample,
only the his endpoint of strain
TT13916 (PncB) and TT13918(@A) can be tested
for linkage disruption. The non-his endpoint cannot
be tested because the pncA and pncB genes are involved in a salvage pathway of pyridine nucleotides
and there is no positive selection for Pnc+ recombinants in this genetic background. Selection for Pnc+
can beaccomplished if a nadA213::MudJ is introduced
(nadA is involved in the de novo synthesis of pyridine
nucleotides); this allows testing linkage disruption at
the non-his endpoint of the pncB-his (TT139 17) and
pncA-his (TT13919) intervals, but (because of KanR
determinant of the introduced MudJ) prevents the
test for linkage disruption at his. Thus isogenic pairs
of strains (nadA+ and nadA-) were used to permit
detection of disruption at both join points. Further,
the non-his endpoint of TT13915 (ara-his) cannot be
tested because theparentstraincontainsa
proBA
deletion (proAB47). Selection for Ara+ on minimal
medium containing arabinose requiresthe additionof
proline to supplement the auxotrophy. Since proline
can also serve as a carbon source, selection for Ara+
transductants is difficult in this genetic backround.
Finally, the non-his endpoint of TT13921 (metG) and
TT13922 (eut) cannot be tested because the non-his
endpoints map at TnlO insertions near, not in, the
respective genes (Table 1).
Inspection of the results in Table 2 shows that some
intervals show inversion (permissive = P), whereas
other intervals fail to show inversion (nonpermissive
= N). T o permit comparison of the new results with
thoseobtained previously, the materialincluded in
each interval is indicated using the map position (in
minutes) of the intervening material on the wild-type
Salmonella map. A “T”between the minutes at the
end of the segment indicates that the terminus (or
origin) of replication is included in the segment being
tested for inversion. The behavior of this chromosomal segment, permissive (P) or nonpermissive (N)
in the rearranged parent and
in a parentwith a normal
map order is indicated in the two columns at the far
right in Table 2. Designation of wild-type chromosome behavior is based on previously published data
(SEGALL,MAHAN and ROTH 1988). These intervals
are diagrammed in Figure 4. Results obtained previously using this assay in a wild-type parent strain are
presented in Figure 4a; results obtained here in the
rearranged parent strain are diagrammed in Figure
4b.
1027
The entries above the horizontal line in Table 2
describe subintervals that are internal to the region
inverted in the parental strain (ara-his). In the inversion parent, each of these subintervals is located in
reversed orientation and at a slightly different point
in the overall map; material that is clockwise of the
ara locus inwild type is now immediately counterclockwise of the his locus. The ara-trp interval tested
in both strains shows the same inversion behavior as
it did in the wild-type parent. The two intervals that
have not been previously tested do not include the
terminus and proved to be nonpermissive.
Below the horizontal line in Table 2 are presented
results for eight intervals that include material outside
of the parental inversion. The first three intervals (hismetG, his-eut and his-tyrA) are justclockwise of the his
locus. These segments were nonpermissive in the wildtype background and remain so in the parent strain
with the ara-his inversion. The second two intervals
(cysJZ-his and argA-his) includelarger chromosomal
segments clockwise from the his locus and show only
slight differences in behavior that will be discussed
below. The last three intervals (metE-his,pyrB-his and
ara-his) are large regions (nearly half of the chromosome) that include the terminus(ororigin);
these
regions can be viewed as internal (for external) to the
ara-his region, depending on the point of reference.
Two intervals (pyrB-his and ara-his) show the same
permissive character as they did in the wild type
parental strain. The ara-his interval is essentially identical to theparental inversion; inversion of this interval
returns chromosome structure to that of wild type.
T h e third interval (metE-his) changes from nonpermissive to permissive as a consequence of the ara-his
rearrangement.
DISCUSSION
By selecting for recombination between inverse order homologies, we have previously shown that some
intervals of the Salmonella chromosome invertat high
frequency, whereas others fail to show inversion (MAH A N and ROTH 1988;SEGALL, MAHANand ROTH
1988; MAHAN,SEGALL
and ROTH 1990). In the present studies, we test the behavior of 11 intervals in a
rearranged parent strain that alters the chromosomal
position, neighboring sequences and, in some cases,
the orientation of the arcs of chromosome tested for
inversion. T o d o this, we constructed a parent chromosome with a large inversion (ara-his) that reverses
the orientation of a 40-minute segment of chromosome (minutes 3-42). Three of the intervals tested
are internal to this region. Eight other intervals include material outside of this region. With the exceptions noted below, all of these intervals maintained
the same behavior they showed in the wild type parent
strain, suggesting that the permissivity of inversion
M. J. Mahan and J. R. Roth
1028
TABLE 2
Distribution of permissive and nonpermissive intervals in a parent strain with an ara-his inversion
Strain"
Non-his endpoint (position)'
TT13916
TT13917
TT13918
TT13919
TTl3920
pncB (20)
pncB (20)
pncA (27)
pncA (27)
trb (341
TT13921
T T 13922
TT13923
TT13924
TT13925
TT 13926
TTI 3927
TT13915
metG (44)
eut (50)
tyrA (55.4)
C Y ~ J(60)
I
argA (61.2)
metE (84)
PrrB (98)
ara (2.5)
Map units
included in
intervalb
3-20
3-20
3-27
3-27
3-T-34
42-44
42-50
42-55.4
42-60
42-61.2
42-T-84
42-T-98
42-T-2.5
Percent of
His+ clones
with inversions
his endpoint
(no. tested)'
0 (20)
0 (40)
0 (20)
0 (20)
80 (20)
Permissivity
Disruptiond
1 .O
te
n
tc
1 .O
n
0.1 1
0.04
1 .o
1 .O
85 (20)
0 (20)
0 (20)
10 (20Y
2
0
9
"
0.07
0.02
0.10
0.17
0.13
Non-his
endpoint
n te
1.o
nt'
n
1 .O
ntl
0.06
n tC
n te
1.O
0.04
co.01
0.08
0.03
ntc
Inv
parent
Wild type
N
N
N
N
P
ntf
ntJ
tf
P
P
N
N
N'
N'
P
P
P
N
N
N
N
N
P
P
P
90 (20)
80 (20)
90 (20)
a Entries above the horizontal line describe subintervals internal to the region inverted in the parental strain; entries below the horizontal
line describe intervals outside the parental rearrangement. (P) denotes permissive intervals, while (N) denotes nonpermissive intervals.
and ROTH (1988); the his locus is at minute 42. The parent strains contain an inversion of
Map positions are according to SANDERSON
material adjacent to ara (rac region, minute 3) to his. To describe the material within an interval, the ends of that interval are indicated in
map units on the standard wild type genetic map; a "T"is included if the segment includes the terminus (or origin) of replication.
' The frequency ofHis+ recombinants was about for
all intervals tested; a random collection of these clones was tested for linkage
disruption.
Linkage disruption is the reduction in ability of His+ recombinants to be repaired at either inversion join point with P22 phage grown
on TTI 391 3 (see Figure 3 and MATERIALS AND METHODS). For permissive intervals, the numbers, expressed as a fraction, indicate the ratio
of the number of transductants obtained in a His+ clone to the number of transductants obtained in the isogenic parental strain without a
secondary inversion ( i e . , show no linkage disruption). The numbers presented are an average of such ratios determined for five His+
recombinants tested. For intervals judged permissive, the ratio has a value near 1.O, reflecting the fact that all His+ clones recovered show
approximately the same number of transductants (at either inversionjoin point) as the noninversion parental strain ( i e . , no linkage disruption
was detected).
Not tested because the genotype of the parent strain does not allow selection for repair of the endpoint indicated (see RESULTS for a
detailed explanation of each endpoint in question).
/These intervals were not among those tested previously in a wild-type value so no information is available as to their behavior in a strain
without the ara-his inversion.
g The two underlined values are unusual in that they are intermediate between the values found for permissive intervals (80-90%) and
those found for nonpermissive intervals (0%)(MAHAN, SECALLand ROTH 1990). For these two intervals, inversions are found but not at the
high frequency characteristic of fully permissive intervals (see DISCUSSION).
The His+ recombinant strain TT13936, which carries a secondary inversion from argA-his (61-42), shows a marked reduction in growth
rate as judged by colony size on nutrient broth medium.
intervals is dictated by the sequences included and not
by their position in the chromosome or their flanking
regions.
Using the parentalstrain with a stable inversion
rearrangement (40% of the chromosome), we tested
the ability to recover secondary inversions for a series
of chromosomal intervals. The permissivity (or nonpermissivity) of each of the testedintervalsfora
parent strain with the ara-his inversion is presented in
Table 2; the permissivity of the same intervals for a
parentstrain with a wild type chromosome is presented for comparison (data for parent strains
with
wild type gene order were taken from SEGALL,MAHAN and ROTH 1988; MAHAN, SEGALL
and ROTH
1990). Inspection of the distribution of permissive
intervals reveals thatthe ability toinvert is often
correlated with inclusion of the terminus or origin of
replication, regardless of the parental chromosome
arrangement.
One interval internal to the parental inversion 3-
34 (ara-trp) has beentested in both wild-type and
inversion backgrounds. T h e permissivity of this interval is maintainedeventhough,
in therearranged
chromosome, the tested segment is placed in inverse
order with differentflanking sequences [i.e., chromosomal minutes 3 , 4 , 5 , etc. appear near thehis locus
ara(minute 42)]. In the rearranged parent, the entire
trp segment is both inverted and shifted 8 minutes
clockwise. The interval includes the replication terminus and is permissive for inversion regardless of the
parental genome arrangement.
T h e segments 3-20 (ara-pncB)and 3-27 (ara-pncA)
proved nonpermissive in the inversion background
but have not been testedin the wild-type background.
Neither of these two nonpermissive intervals includes
the terminus,thustheir
nonpermissivity obeys the
simplest form of theterminusrule.In
wild-type
strains, we have previously tested the slightly shorter
region 3-17-minute interval (ara-nadA) and found it
permissive (SEGALL,MAHAN and ROTH 1988). This
on
Restrictions
b
FIGURE4.-Distribution of permissive and nonpermissive intervals. Part a diagrams the distribution of permissive (and nonpermissive) intervals in parent strains with wild type gene order. Part
b diagrams the distribution pattern obtained from parent strains
that carry an ata-his inversion. Solid lines (permissive) indicate
chromosomal segments that form inversions at high frequency (6590%of the recombinant types tested) when one selects for recombination between sequences at the his locus and homologous sequences placed in inverse orientation at a distant site; wavy lines
indicate chromosomal segments that are permissive at low frequency (2-10%). Srippled lines (nonpermissive) indicate chromosomal segments that fail to show inversion. The dark arrowheads
in b denote the two inversion breakpoints in parent strains that
contain an ara-his inversion: one at minute 3/41,
the other atminute
4213.
difference in behavior can be interpreted in terms of
the periterminal rule described by REBOLLO,FRANCOIS and LOUARN
(1 988) according to which an interval without aterminuscouldinvert
if it doesnot
disrupt the periterminal regions. Both of the first two
Inversion
1029
inversions have one endpoint within one of the suggested nondivisible zones (NDZ). In contrast, the permissive 3-1 7 minute interval does not disrupt one of
these zones. Thus,the behavior of these intervals
agrees with the model of REBOLLO,FRANCOISand
LOUARN(1988). However,as discussed below, we
prefer to think that endpoints within a NDZ render
inversion mechanistically impossible rather than leading to lethal consequences of the final rearrangement.
If the above intervalsare described in terms of their
endpoints, the his-trp endpoints (42-34), which are
nonpermissive in wild type background, become permissive in the inversion background (compare Figure
4, a and b). Conversely, the his-pncB (42-20) and hispncA (42-27) endpoints are permissive inwild type
and become nonpermissive in the inversion parent.
However, it should be kept in mind that the different
backgrounds cause very different chromosomal material to be located between each set of endpoints. It
seems that the nature of the sequences within each
interval dictates the permissivity of the interval. This
interpretation is reinforced by the behavior of intervals including material outsideof the ara-his region.
The three segments immediately clockwise of the
his operon are not included in the parental ara-his
inversion and that parental inversion does not alter
their nonpermissive character. Thus, despite the fact
that the chromosome has been rearranged and
sequences at one end
of the tested interval are different,
these three regions maintain their nonpermissive character. None of these intervals includes a terminus or
origin region.
In discussing the above inversions, the shortest
chromosomalsegment
between endpoints wasassumed to be undergoing inversion. However, since
the chromosome is circular, the small segment might
be used as a point of reference for inversion of the
rest of the circle. This point becomes troublesome
when discussing larger inversions. For the very large
intervals 42-84, 42-98 and 42-2.5 (last three lines of
Table 2), the smaller arcs of chromosome are 42, 44,
39.5 minutes, respectively, so the endpointsdivide the
chromosomeinto two rather comparabledomains,
making it problematical to discuss these intervals in
terms of alterations of the content of the inverting
segment. The most striking change observed in these
experiments is that shown by the his-metE interval
(42-84); it is converted from nonpermissive (in a wildtype background) to permissive by the parental arahis inversion, which alters the slightly larger arc of
chromosome. Since the metE-his interval includes the
terminus or origin region in both configurations, the
nonpermissivity observed in the wild-type chromosome is an exception to the terminus or origin rule.
The other two large intervals (his-pyrB and his-ara)
are not altered in behavior by the parental ara-his
1030
M. J. Mahan and J. R. Roth
inversion, which slightly modifies the smaller arc of
chromosome;boththeseregions
are permissive in
both the wild type and inversion strains.
The behavior of the cysJZ-his (42-60) and argA-his
(42-61) intervals show at most slight effects of the
parental ara-his inversion. Both intervals were classified as nonpermissive in a wild type background, since
no inversions were recovered (0/20). In therearranged background, both intervalsyielded inversions
but did so at a low frequency (2/20 and 1/40). While
the difference in behavior is not statistically significant, we recovered no inversions from any ofthe
other intervals classified as nonpermissive. Permissive
intervals (for the assay used here) typically gave 80%
inversions among the His+ recombinants. This suggests that the presence of the parental inversion may
have a slight effect on the two intervals clockwise of
the his locus. T o pursue this possibility, we tested 120
more His+ recombinants for the cysJI-his interval arising in the wild type background and found noinversions.
The one (1/40) argA-his inversion observed in the
rearranged parentdisplayed a slow-growth phenotype
as judged by colony size on solid nutrient broth medium. It is possible that poor growth of inversionbearingrecombinantscontributes
totheapparent
nonpermissivity of the argA-his interval.However,
such a slow-growth phenotype is unusual among the
inversion recombinants we have seen in Salmonella.
Of over 40 other intervals tested, only the ara-trp
region (tested in a wild type parent) yielded inversion
recombinants with a reduced growth rate. Thesearatrp inversions were recovered at ahighfrequency
despite their growth impairment.
In any event, the
presence of the ara-his inversion in a parental strain
has at best a small effect on the recovery of inversion
of the adjacent 42-60 and 42-61 minute regions.
It seems likely thatthecorrectexplanationfor
nonpermissive intervals must account for the observations in both Salmonella and E. coli. There is striking agreement regarding the general pattern of permissive and nonpermissive intervals in the two organisms. REBOLLO,FRANCOISand LOUARN(1 988) have
tested several intervals that extend from his toward
the terminus within a periterminal region that does
not include the terminus; they judged these intervals
to be nonpermissive. They have also tested several
intervals that extend fromhis across the terminus and
found them permissive for inversion. They have suggested the existence of critical regions (NDZ) on either
side of the terminusregion; inversions disrupting
these periterminal regions,they suggest, may be lethal
due to difficulties in replicating this material, when it
is presented in the abnormal orientation.We feel our
data may reflect some importance of the terminus
region, but we are less enthusiastic about lethality as
an explanation of nonpermissive intervals.
By a transductional method, we have constructed
inversions of two intervals that are nonpermissive for
inversion by intrachromosomal exchanges. Both constructed inversions appear to grow normally (A. M.
SEGALL, L.MIESELand J. R. ROTH, unpublished results). However, in the data setof REBOLLO,FRANCOIS
and LOUARN( 1 988) and in two of the 45 intervals we
have tested, inversions were recovered that showed
impaired growth. One of these examples is the argAhis region reported here. The other is the ara-trp
region for which slow-growing inversions arose and
were recovered at high frequency (SEGALL,
MAHAN
and ROTH 1988, MAHAN,SEGALL
and ROTH 1990).
While impaired growthor lethality could be suggested
to account for the failure to recover inversions of
some regions, we see normal growth of strains with
constructed inversions of nonpermissive intervals.
These results suggest that a mechanistic barrier is
more likely to represent the general explanation
of
nonpermissive intervals.
The “terminus rule” that invertible segments must
contain a terminus or origin of replication still seems
interesting despite some exceptions. If the two intervals with very infrequent exchanges are classified as
nonpermissive, then the terminus ruleis obeyed by all
of the 1 1 intervals tested here in the altered parental
background. One of the previously reported exceptions to the terminus rule (trp-pyrC) has been reevaluated and now appears to bepermissive in agreement
with the rule (L. MIESEL and J. ROTH, unpublished
results). Some remaining exceptionsmay be explained
by the periterminal rule of REBOLLO,FRANCOISand
LOUARN(1988); e.g., the 3-1’7-minute interval (aranadA) is permissive in the wild type background; this
region does not include the terminus but does not
disrupt
the
periterminal
region.
Regardless of
whether or not some combination of these rules can
be made to predict all inversion behavior, we suggest
that nonpermissivity is not due to lethality of inversions, but rather to a restriction on recombination
between sequences flanking particular chromosome
regions.
We currently entertain two models, both of which
suggest that folding or packaging of the bacterial
chromosomemightcontribute
to limiting recombination between particular sites. (1) If the folding or
positioning of portions of the chromosome restricted
contact between particular sites in the same circular
molecule, inversion formation would be prevented.
T h e noninversion recombinants could arise by sister
chromosome exchanges (SEGALLand ROTH 1989) or
by excision and reintegration of circles in the case of
the recombination system used here (MAHAN and
ROTH 1988). (2)The second possibility is an extension
of the recombination model of MAHAN and ROTH
Restrictions on Inversion
(1989). According to this model, reciprocal exchanges
require repairof double strand breaklgaps generated
in the process of recombination. Excessive degradation of these ends would prevent completion of a
reciprocal exchange within the short inverse repeats
and lead to a failure of inversion formation. Chromosome folding or sequestration of particular chromosomal regions might influence the extent of end
degradation.
Regardless of the specific mechanisms involved, the
distribution of permissive and nonpermissive intervals
and the effects of chromosomealterations such as
those presented here should reflect aspects of chromosome structure that are physiologically relevant.
We have presented data that theability to form inversions is influenced by sequences includedin the tested
segment and is less sensitive to the natureof sequences
that flank that segment. An important aspect may be
possession of the terminus (or origin)of replication.
This work was supported by U.S. Public Health Service grant
GM 27068 from the National Institutes of Health. M. J. M. was
supported by predoctoral training grant T32-GM 07464-1 1 from
the National Institutes of Health. Figure 4a has been published
previously (SEGALL,
MAHANand ROTH, Science 241: 13 14-1 3 17,
1988) and is reprinted here to enhance the clarity of the presentation.
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