Volume 14 Number 24 1986
Nucleic Acids Research
A bacterial protein requirement for the bacteriophage X terminase reaction
Marvin Gold* and Wendy Parris
Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada
Received 20 August 1986; Revised 21 October 1986; Accepted 17 November 1986
ABSTBACT
The bacteriophage lambda terminate enzyme cleaves the cohesive-end
s i t e s of X DNA to y i e l d the protruding 5 ' - t e r n i n i of the nature n o l e c u l e .
In y i t r o . t h i s endonacleolytic event requires a protein factor which has
been i s o l a t e d and p u r i f i e d from extracts of uninfected £ . o o l i . The
terminase host factor (TUF)is a heat stable basic p r o t e i n of M.W.
approximately 2 2 , 0 0 0 . The i n t e g r a t i o n host factor (IHF) p r o t e i n of E. c o l i
can e f f i c i e n t l y s u b s t i t u t e for THF in the terminase r e a c t i o n ; however, THF
can be demonstrated t o be p h y s i c a l l y present in, and i s o l a t e d with f u l l
b i o l o g i c a l a c t i v i t y from e x t r a c t s of c e l l s d e f e c t i v e or d e f i c i e n t in IHF.
INTRODUCTION
The bacteriophage X terminase enzyme p l a y s a c e n t r a l r o l e in X head
morphogenesis and DNA maturation.
Teminase i s a m u l t i f u n c t i o n a l
enzyme
which we have succeeded i n p u r i f y i n g t o a very high l e v e l of homogeneity
(1).
Our f i n d i n g s ,
follows:
as w e l l as those of o t h e r s , can be summarized as
(a) terminase i s composed of the products of the two X genes Nnl
and A ( 2 ) ; (b) terminase can form a binary complex with e i t h e r immature or
mature DNA by binding t o the cos s i t e .
When t h i s complex i s formed with
immature DNA the c o s s i t e s are cut ( 3 ) ; ( c ) a ternary complex can be
formed between terminase, DNA, and proheads; (d) terminase promotes the
packaging of DNA i n t o the proheads and i s i n d i s p e n s i b l e not only for the
packaging of immature DNA, but a l s o of mature DNA;
( e ) terminase can
c l e a v e c i r c u l a r monomers at c o t and package them e f f i c i e n t l y
( 4 ) ; ( f ) the
above r e a c t i o n s , j_n v i t r o , require ATP; (g) terminate i s a DNA-dependent
ATPase s p l i t t i n g ATP i n t o ADP and P i ; and (h) terminase, or at l e a s t gpA.,
i s required at one or more p o i n t s during the l a t e s t a g e s of head formation,
but i s not part of the f i n i s h e d head or phage p a r t i c l e .
F e i s s (5) has
r e c e n t l y reviewed the p o s s i b l e s t r u c t u r e and binding p r o p e r t i e s of the
terminase
subunits.
During p u r i f i c a t i o n of the enzyme, we discovered that some E. c o l i
© IRLPre» Limited, Oxford, England.
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component (s) w«< also required for in vitro terminate a c t i v i t y . Thus,
neither the binary terminate: DNA complex nor the ternary terminate:
DNA:prohead complex could be formed with particular preparations of
terminate unless the react ion mixtures were supplemented with crude extracts
of E. c o l i ( 6 ) . It was not possible to determine whether the same or
different factors were involved in the two complexes. Subsequently, we
found a host factor requirement for in vitro £p_s_-cleavage with partially
purified terminate under conditions where proheads were absent and packaging
considerations irrelevant (3). By using a simple, rapid assay for in vitro
cos-cleavage we have been able to purify to near homogeneity a protein which
snpports this reaction with partially purified terminate preparations. The
purification and properties of this protein which will be referred to as THF
(terminate host factor) are described in this report.
MATERIALS AND METHODS
Terminate was purified by the method of Gold and Becker ( 1 ) . Depending
on the particular preparation, the Biorex 70 (ASIV) and subsequent fraction!
can be uaed in the £p_s.-oleavage reaction to demonstrate the host-factor
requirement.
Phage and pi aim id DMAs were isolated and purified by standard
procedures. The cosmid C25 was constructed by Dr. L. Moran of the
Department of Biochemistry of this University. It i s composed of a 1.85 kb
Balll £p_s.-containing fragment from X Charon 1 inserted into the BamSl site
of plasmid pBR322. A unique Pstl site is located 1.15 kbp from cot. The
cosmid pDJ136 (9.1 kbp) was constructed by D. Hawkins and M. Sumner-Snith of
this department and i s similar to C25 except that cos i s flanked on one side
by a unique Pstl site (1.9 kbp) and on the other by a unique EcoRI site (3.6
kbp).
Phages X 4F106 and 4F103 were gifts of Dr. H. Murialdo.
Terminase reactions (40(il) included: Tris-HCl pfl 8.0, 13mM; MgCl
3mM; EDTA, 0.5mM; 2-nercaptoethanol, 6mM; ATP, lmM; spermidine-HCl,
5mM; KC1, 75nM; and 1 to 2 ug of c_£s_-containing DNA. Incnbation was at
22° for 40 min and the reactions were stopped by the addition of 5 |il of
0.5M EOT A, pH8.0; 5 (il of 10% SDS; and 5ul of a solution of 50% glycerol
- 5% Sarkosyl -0.025% bromophenol blue (all w/v) . The samples were heated
at 65° for 5 min and electrophoresed in 1.4% agarose gels in Tris-acetate pH
7.7 at 25mA for 16 hours.
(7).
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Isoelectric focussing was carried out by the method of O'Farrell ££ tj,
Chromatofocucsing was done with materials obtained from Pharmacia.
Nucleic Acids Research
The varioui proteins tested were commercial preparations.
Purified IHF and
the himA and hip mutant E. coli strains were kind gifts of Dr. H. A. Nash.
RESULTS
(a)
Terminate has a factor requirement
Our i n i t i a l discovery of a host factor requirement for terminate
occured when linear concatamers of X DNA were used as substrate (3).
DNA, labelled with
14
This
C, was extracted from phage-infected c e l l s and the
assay depended on the detection (by autoradiography) of new band on an
agarose gel after complete digestion of the terminase-treated DNA with Eco
Rl.
While terminase by i t s e l f was incapable of promoting this reaction,
crude sonicate of uninfected, non—lysogenic E. coli was effective
in
supplying the required component and this assay first enabled us to
fractionate the bacterial extract and prepare THF (see below).
However,
preparation of pure, radioactive X concatamers is costly and time-consuming
so various other substrates were also tested in an attempt to devise a more
rapid and efficient assay.
The DNA of a phage such as X 4F106 contains an
internal cos site on a fragment located between approximately 46 - 53% of X
(B) .
Agarose gel electrophoresis analysis of reaction mixtures where this
DNA was incubated under various conditions revealed that i t indeed was a
terminase substrate as shown by the appearance of a band whose mobility
corresponded to molecules half the length of X DNA; this conversion was
absolutely dependent on THF.
Similar results were also obtained with X
4F103 DNA which is identical with 4F106 except that the fragment containing
cos has been inserted in the reverse orientation.
These results
confined
that not only could a more simple molecule be used to establish a relatively
convenient terminase assay but also that the requirement for a host factor
was not restricted to reactions with polyneric X DNA containing several cos
sites.
We then asked whether the cleavage of circular molecules containing a
single cos would have similar requirements.
Fig. 1 shows the results of
experiments where the supercoiled DNA of cosmid C25 was used as a substrate.
Lane A shows that our preparations are contaminated with slower moving
material; these additional bands may represent relaxed or multimeric forms
of piism id DNA.
The main band, however, shows a characteristic mobility and
when the DNA i s linearized by digestion with Pstl. a band of 6.1 kb appears
(lane B) .
Incubation of supercoiled C2S DNA with either THF or terminase
alone does not lead to any substantial production of linear DNA (lanes C and
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ABC D E F
Figure 1. Supercoiled DNA of cosmid C25 as a substrate for X t e n i n i i e .
Reaction mixtures vere as described in Materials and Methods; 60 |ig protein
of the Biorex 70 fraction of terminase vere added where indicated and
snpercoiled DNA of cosmid C2S was used. A: DNA alone; B: DNA + P_s_tl (1
unit); C: DNA + THF; D: DNA + terminase; E: DNA + terminase + THF. Lane
F i s a Hind III digest of X DNA as a sixe marker.
D) . When THF and terminase are both present dnring the incubation, nearly
all of the supercoiled DNA is converted into a form which has a mobility
identical to the Pstl linearized molecules (lane E). Similar results are
obtained with aupercoiled DNA of cosmid pDM36 (data not shown).
Cosmid DNA can also serve as substrate for terminase even after i t i s
linearized by digestion with a restriction endonuclease at a unique site
distal from cos. Cosoid pDM36 DNA can be linearized by £JL£1 and if these
linear molecules are subsequently cleaved at cos. 2 fragments of unequal
length (7.2 and 1.9 kb respectively) length should be produced. The
expected results were obtained by the combined action of terminate, THF, and
£ l i l . Similar results are obtained with DNA of cosmid C23 where Pitl
linearized C2S DNA i s converted into 2 fragments (5.0 and 1.1 kb
respectively) by terminase. These experiments were oarried out in the
presence of THF. Fig. 2 represents an experiment using linearized C2S DNA
where the factor requirement is clearly demonatrated. Either THF alone
(lane A) or terminase alone (lanes B, C, F, and 6) produce no cleavage;
however when both are present (lanes D, E, H, and I ) , the 5.0 kb band i s
evident. (The smaller 1.1 kb piece i s not always seen with certain
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ABC D E F G H I
Figure 2. Requirement for host factor with linear DNA a* a substrate for X
terminate. Reaction mixtures were as described in Fig. 1. Co mi id C2S DNA was
f i r s t linearized with £j_tl. A: DNA -I- THF; B and C: DNA + 60 ug and 100 ug of
terminate, respectively; D and E: DNA + THF + 60 ug and 100 ug of terninase
respectively. F, G, H and I are duplicate assays of B, C, D and E,
respectively. J: C25 supercoils; K: Hind III digest of X DNA.
terminate and/or THF preparations as i t appears to be quite susceptible to
nucleate digestion).
Preparations of terminate can vary in their degree of dependence on THF
in the ££s.-cleavage reaction. With some fractions used in these experiments
there was a slight reaction in the absence of factor but significant
stimulation of cutting could always be observed when THF was added,
(b) Purification of THF
In order to elucidate the nature and function of THF, the cosmid assay
outlined above waa used to monitor i t s extensive purification. THF was
prepared from E. coli K12 strain 1100. Frozen c e l l s were disrupted by
sonication and the crude extracts treated with Polynin P to precipitate
factor activity exactly as described for the purification of terminate ( 1 ) .
Polynin pellets obtained from 2 kg of frozen c e l l s vere eluted with 4 l i t r e s
of 0.2H ammonium succinite pH 6.0 and the eluate concentrated by the
addition of solid ammonium snlfate to 80% saturation. After centrifngation,
the pellets were dissolved in 0.02H Trit-HCl pH 8.0. laH EOTA, 7mH
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G100 Fractions
Figure 3 . SDS polyacrylamide gel electrophoresis analysis of TUP. The
Sephadex G-100 column fraction of THF was analysed on a 15* gel. The marker on
the right i t trypsin inhibitor (21,500).
p-aeroaptoethanol and O.OOSBH PMSF (all buffer* uied contained the latter 3
ingredients) and heated in 200 ml aliquot* to 60° for 2 minutea. The
precipitate was removed by centrifugation and the supernatant dialysed
extensively to yield the ammonium sulfate I fraotion (ASI). The latter i t
very (table and can be prepared in good y i e l d . The ASI fraction was loaded
on a 8CB x 36on colunn of DEAE Sephadex A-25 equilibrated with 0.02M Tris
HC1 pH 8 . 0 . The pass-through and wash were collected and precipitated with
80% amnonium sulfate. The pellets were collected and dissolved in 0.02M
Tris-UCl pH 8.0, and dialysed extensively to give the ammonium solfate II
fraction (ASH). A colunn of Cellex-P (Biorad. 6 x 45cm) was equilibrated
in 0.02H Tris-HCl pH 9.0, ASH (385 nl) was loaded and the col nun washed
with 2 l i t e r s of 0.05M potasiium phosphate buffer pH 6.5. containing 0.1M
EC1. The colunn was then developed with a 3 l i t e r linear gradient of KC1
from 0.1 to 2M in the same buffer. 30 fractions of 100ml each were
collected and dialyzed briefly against 0.02M Tris-HCl pH 8.0. Subsequently,
each fraction was precipitated by the addition of solid ammonium sulfate to
80% concentration, and the precipitates collected in 0.0211 Tris-HCl pH 8.0,
and dialyxed against the same buffer. THF a c t i v i t y eluted late in the
gradient. Active fractions were pooled and brought to 80% saturation in
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Rmtatlv Mobility
Figure 4 . Molecular weight determination of XHF. Polyacrylamide gel
electrophoresis was oarried out in a 15% SDS-acrylamide gel containing 8H urea
by the method suggested by BEL. Standard proteins and factor were denatured
and applied. Moleoular weights of the standards were: ovalbumin, 43,000;
chymotrypsinogen, 25,700; soybean trypsin inhibitor, 21,500;
p-lactoglobul in, 18,400; lysoxome, 14,300; aprotinin, 6,200; and insulin,
3,000. The molecular weight of each standard i s plotted against i t s mobility
relative to bromophenol blue. The relative nobility of THF in the same system
is indicated by the arrow.
ammonias sulfate, centrifuged and dialyzed and the resulting fraotion termed
ASIII. ASIII was loaded on a column of Sephadex G100 (8 z 75cm) prepared in
0.02M Tris-HCl pH 8.0, containing 0.1H KC1. A peak of activity was obtained
eluting at a position of approximately 20,000 molecular mass. This peak was
treated as above (ASIV) . A column (0.9 x 12cm) of Pharmacia PBE118 was
poured and equilibrated in 0.025M triethylamine pH 11.0. ASIV was loaded on
the column followed by a 15 ml wash with the above buffer. The column was
developed with 115 ml of Pharmalyte 8-10.5 (Pharmacia) diluted 1:35.
Subsequently fractions were brought to 80% saturation in ammonium sulfate,
centrifuged and resospended, and dialyxed. THF activity eluted at a pH of
about 9 . 3 .
Analysis of the G-100 fractions by SDS-PAGE in a 15% gel is shown in
Figure 3 . It i s evident that the preparation i s nearly homogeneous at this
stage and has a denatured M.f. of 22,000. In another determination using an
SDS-urea gel (Fig. 4) a value of 23,000 was obtained. Gel f i l t r a t i o n on a
column of Sephadex G-100 (Fig. 5) suggests that the native molecular weight
of the protein is approximately 22,600.
(c) Properties of Host Factor
The Sephadex G-100 fraction (ASIV) was analysed by isoelectric
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Figure 5. Molecular weight determination of TflF under native conditions.
Sephadex G—100 chromatography was carried out as described in the text.
Standard proteins were also run: chymotrypsinogen, 25,700; myoglobin,
17,600; and cytochrome C, 12,400. The Kav of THF in the ssme system is
indicated by the arrow.
focussing (Fig. 6 ) . Bovine serun albunin was used as a carrier protein.
There i s one band evident with a molecular weight of 21,900 and an apparent
pi of app. 10.1 indicating that THF is a strongly basio protein. This was
confirmed by its behaviour on DEAE and phoaphocellulose ion-exchange
colunns. THF is also stable to heating at temperatures as high as 80° for
as long as 10 nin and incubation in 1H acetic acid for 2 hrs. at room
temperature has no effect. The purification scheme outlined above has been
carried out with the heating step omitted and similar properties have been
observed. Preliminary characterixation of THF showed that while the protein
can bind to both native and denatured DNA-oellulose, it possesses no
detectable DNase or ATPase a c t i v i t i e s . Incubation of both negatively
twisted and relaxed covalently closed circular DNA molecules with THF in the
presence of Mg++ and ATP has not to date shown any evidence of topoisonerase
a c t i v i t y . Attempts to fractionate THF activity with nuonium sulfate during
early stages of the purification failed because activity was spread almost
equally over all salting-out concentrations. This result suggested that
multiple factors might be present. To test this p o s s i b i l i t y , the
phosphocellulose fraction was analysed by the method of Hager and Burgess
(9) in which SDS-polyacrylamide gels are stained with dilute KC1 solutions,
individual protein bands eluted, and activity recovered after renaturation.
Slices 1 cm in width were cut and eluted; three distinct xones of factor
activity could be detected in regions corresponding to proteins having
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68K
45K
I
25.6K
18.4K
>14.3K
*~ O) * f
CO f *
<D
N
6^K
Figure 6. I i o e l e c t r i c focussing of THF. The ASIV fraction (Sephadex G-100,
tee Fig. 5) of THF was analysed by the i s o e l e c t r i c focussing technique of
O'Farrell e£ ±1 (1977). The pi standards ( l e f t to right) are 4 . 1 , 4 . 9 , 6.4,
8 . 3 , 9.7 and 10.6. Bovine serum albumin (BSA) was nsed as carrier protein.
Molecular weight Barkers are: BSA, 68,000; ovalbumin, 45,000;
chymotrypsinogen, 23,600; myoglobin, 18,400; lysozyne, 14,300; cytochrome
C, 12,300; aprotinin, 6,200. The arrow indicates the position of THF.
molecular weights of app. 22,000, 11,000 and 3,500 respectively. The
identity of the latter two proteins has not yet been determined. Several
other substanoes were also tested for factor activity in the terminase assay
and except for IUF (see below) were a l l found to be inactive (Fig. 7 ) . Also
unable to substitute for THF were E. coli RNA polymerase and chicken
erythrocyte histones. Extraots of M. Intent. B. s n b t i l i s . yeast or CHO
tissue culture c e l l s had no activity (data not shown).
The integration and excision of X DNA into and from the E. coli
chromoscue requires, in addition to the phage int and x l s gene products, an
E. coll protein cofactor, IHF (10, 11, 12). IHF i s a small, heterodimeric,
basic DNA binding protein and we attempted to use i t as a substitute for THF
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A B C D E F G H I J K
Figure 7. Test for sources of t t n i m i o factor activity. Reaction mixtures
were at described in Figure 1 using £s_£l-linearized C2S DNA as substrate. Lane
A: DNA alone; Lane B: DNA + terminaae; lanes C to K: DNA + terminase and
THF (O.Jug); IHF (O.lug); cytochrome C (5 |ig); bovine serum albumin (J
ug); E. c o l i DNA polynerase (1 unit); protanine (5 ug); spennine (5
ug); E. coli single—strand binding protein (1.2 units); and trypsin
inhibitor (S ug), respectively.
in the in vitro terminase resction. As can be seen in Fig. 7, IHF can very
efficiently replace the THF we have purified as described above (cf lanes C
and D). IHF i s composed of 2 subunits: One has a molecular weight of
10,300 and i s the himA gene product and the other has a molecular weight of
9,500 and i s the product of the hip or bimD gene. Although we can
dissociate IHF into i t s subunits under denaturing conditions, we have so far
been unable to observe any decrease in the 22 KD size of IHF under any
conditions. E. c o l l mutations in either of the genes coding for IHF are
defective in X s i t e - s p e c i f i c recombination and also in the synthesis of £ l l
and int protein (13) . Factor preparations from various E. c o l i strains
mutant in IHF were tested in the terminase assay and a l l were found to be
effective in promoting ££s.-cleavage. SDS PAGE analysis of these mutant
preparations revealed the presence of a 22ED polypeptide which possessed
factor activity. The strains tested i n i t i a l l y contained point mutations;
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A B C
Figure 8. Test of an IHF mutant for THF activity. Strains HN 458 (wild type)
and HN 840 (hJsAA82 hipA3) were grown, extracts made, and fractionation carried
out to the ASH ttage as described in the text. Reaction mixtures were as
described in Fig. 7. Lane A: DNA + terminase; Lane B: DNA + terminase + HN
458 ASH (10 ug); Lane C: DNA + terainase + HN840 ASH (10 ug).
we have done similar assays with single and doable deletion mutants and
obtained similar results (Fig. 8 ) . fe therefore conclude that the two
proteins are distinct and we now routinely purify TflF from strain HN840
which contains the double deletion himAA82 hipA3.
DISCUSSION
We have presented evidence that partially purified terminase requires
an E. coli cofactor for .in. vitro cos-cleavage. This is the case with linesr
DNA containing one or more cos sites and also with supercoiled (or relaxed
circular, data not shown) coraids. The cofactor which we have purified to
near homogeneity is a heat-stable basic protein with an apparent molecular
weight of approximately 22,000 under both native and denatured conditions.
As yet, we have been unable to equate i t s identity with any known E. coli
protein. It is possible that THF is one of the several proteins isolated by
acid extraction of E. c o l i nucleoids (14). We have been able to extract THF
by this method and also by that of Kishi sX &1 * 1 5 ' • O o r results suggest
that more than one protein may be able to function in promoting terminase
activity. Several small basic DNA-binding proteins have been isolated from
E. c o l i . like HU (16); H (17); HI (18); and HLP1 (19) and these remain
to be tested. However, our results do show that histone, cytochrome C,
protamine or the polyamine spermine are ineffective. The role for E. coli
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accessory proteins in s i t e - s p e c i f i c interactions is now widely established.
Besides X lysogeny and maturation, bacteriophage Ha exhibits two reactions
where host factors are essential. The _in vitro inversion of the G DNA
segment necessary for host range switching requires the phage-encoded
s i t e - s p e c i f i c reconbination protein Gin and a small (<15,000) DNA-binding
host protein tensed FIS (20). The Mu DNA transposition reactions requires
the phage MuA and MuB proteins as well as HU (21). HU is also a cofactor in
the Hin-mediated s i t e - s p e c i f i c recombination of f l a g e l l i n genes (22). In
this case there i s an additional requirement for a small (12,000) E. coli
protein termed PRE. HU and/or IHF may also be essential for in. vitro TnlO
transposition (23) since snail basic proteins sre involved. The ability of
IHF protein to substitute for our factor in the terminate assay is
strikingly relevant to X DNA function. IHF is necessary for site specific
recombination and for the synthesis of e l l protein in phage X and other
genes as well (24). Mutants in himA show additional phenotypes of a
oleiotropic nature usually involving gene expression (25, 26) and it has
been suggested that IHF or its components f a c i l i t a t e the interaction between
DNA and protein, possibly by some nonspecific melting of the hydrogen-bonded
duplex DNA molecule (27) . Complex DNA-protein structures are formed where
there is local DNA condensation. Binding studies have indicated that IHF
recognizes a consensus sequence in X DNA i . e . T.PyAA.. .PuTTGaT (28) and
Freundlich and Tsui (26) have found at least 65 sequences in £. c o l i ' s and
sone of its phages' promoter regions which are similar. The IHF consensus
sequence does appear several tines in X DNA around oos (29, 30) . However,
l y t i c growth of X is only decreased 2-3 fold in IHF mutants (31) suggesting
that IHF i s not exclusively used by tormina so _in. vivo. The results of jn
vitro experiments (Fig. 8) conf ira this conclusion. While integration and
excision nay have a specific requirement for IHF, X terninase nay be able to
use any of several similar proteins including the one described in this
paper. Phage 21, on the other hand, is unable to grow in c e l l s deficient in
IHF and requires IHF for i a vitro packaging (32). Although 21 and X
sequences around cos are quite similar and both contain IHF consensus
sequences, there are significent differences. Binding studies are currently
in progress to deternine whether THF interacts at any specific s i t e s in
lambda DNA and how this protein and IHF are related. Preliminary results
indicate that while THF exhibits non-specific DNA binding a c t i v i t y it also
can bind specifically to sites near cos.
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ACKNOWLEDGEMENTTS
This work was supported by a grant (MA-7963) from the Medical Research
Council of Canada. We thank Gayle Shinder for her advice in preparing this
manuscript.
•To whom correspondence should be addressed
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