From the
Cold Spring Harbor Laboratory and
¶Howard Hughes Medical Institute, Cold Spring
Harbor, New York 11724
Received for publication, March 24, 2003 , and in revised form, May 14, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The known POZ-domain protein DNA binding sites display considerable sequence heterogeneity. Two POZ-domain zinc finger proteins whose DNA binding properties have been characterized in some detail, i.e. the GAGA factor and Kaiso, bind DNA with surprising flexibility (68). The GAGA factor can bind variable numbers of sites with flexible spacing, and Kaiso recognizes distinct binding sites of different sequences. It is not known whether this flexibility is a frequent feature of POZ-domain proteins, but at least in these cases it is consistent with these proteins playing a role in the regulation of a variety of genes.
We have previously described FBI-1 (factor binding to
IST) (9), a human
protein that binds specifically to an unusual human immunodeficiency virus,
type 1 (HIV-1)1
promoter element, the inducer of short transcripts (IST)
(10,
11). Molecular cloning
revealed FBI-1 to be a 61.5-kDa Krüppel-type zinc finger protein with a
POZ domain at the N terminus and four C2-H2 zinc fingers
at the C terminus (12). FBI-1
can self-associate via both the POZ and zinc finger domains
(12). In addition, it
associates via its POZ domain with activation competent, but not with
activation-deficient, HIV-1 Tat protein
(13). Indeed, overexpressed
FBI-1 stimulates Tat trans-activation in transient transfection assays and
partially co-localizes with Tat and the cellular Tat co-factor P-TEFb in the
splicing factor-rich nuclear speckles
(13). Consistent with playing
a role in cellular transcription regulation, a less soluble fraction of FBI-1
also localizes to an unusual subnuclear domain where it appears to associate
with active chromatin (13).
Indeed, FBI-1 has been shown to repress transcription of some extracellular
matrix genes (14) (these
authors refer to FBI-1 as hcKrox-) and of the ADH5/FDH
promoter (15). In the latter
case, the proposed mechanism of repression is interference by FBI-1 with the
binding of SP-1 to a GC-box adjacent to an FBI-1 binding site. Most recently,
FBI-1 was identified as one of several genes overexpressed in precursor
B-leukemia cells protected from apoptosis by integrin stimulation
(16).
Two mammalian homologues of FBI-1, whose DNA binding sites are unknown, have been isolated. The mouse homologue, leukemia/lymphoma-related factor, is a developmentally highly regulated nuclear protein that associates with the POZ-domain protein BCL-6 (17). The rat homologue, osteoclast-derived zinc finger protein, has been shown to play a role in osteoclast differentiation (18). Together, these observations suggest that FBI-1 is a transcription factor involved in diverse aspects of development and differentiation.
Considering the paucity of information on DNA binding of POZ-domain proteins, we have used the binding site of FBI-1 in the HIV-1 promoter, as well as other viral and cellular FBI-1 binding sites of unknown function, to characterize the DNA binding properties of a POZ-domain protein in detail. We find that high affinity FBI-1 binding sites consist of either a single guanine-rich site or two half-sites each with the consensus sequence G(A/G)GGG(T/C)(C/T)(T/C)(C/T), whose relative orientation and spacing vary greatly. Taken together, our results demonstrate that FBI-1 binds DNA with remarkable flexibility. Consistent with it playing a role in diverse biological processes, FBI-1 thus has the potential to act at a variety of cellular sequences.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
pHIV-1/RpUC119 was constructed by ligating the
PvuII/BamHI fragment from pHIV-1/R containing HIV-1
sequences 19 to +82 into pUC119 cleaved with HincII and
BamHI. 3'pIST/pUC was constructed by ligating the
BglII/BamHI fragment from pIST, containing HIV-1 sequences
+21 to +82, into pUC119 cleaved with BamHI. 5'IST/pUC was
generated by ligating the PvuII/BglII fragment from
pHIV-1/R, comprising HIV-1 sequences 19 to +24, into pUC119 cleaved
with BamHI and HincII. These constructs allowed for the
amplification of probe fragments containing the respective HIV-1 sequences
with universal primers hybridizing on either side of the pUC119 polylinker.
52/R was constructed by oligonucleotide-mediated site-directed
mutagenesis (19).
The deletion mutants D1 through D8 were constructed by ligating double-stranded oligonucleotides carrying the appropriate deletions into the vector pHIV-1/R cleaved with XhoI and AflII. The latter, as well as plasmids pIST and pIST/Bu-, are described in Ref. 11. WT[ABC]WT is identical to WT[WT]WT described in Ref. 11, except that the insert between positions +24 and +40 corresponds to the +1 to +59 fragment from msABC (3).
Electrophoretic Mobility Shift AssaysElectrophoretic mobility shift assays (EMSA) were performed with end-labeled DNA probes generated by PCR amplification as described previously (9). The use of a common radiolabeled PCR primer assured equal specific activity of all probes. The sources of FBI-1 were either heparin-Sepharose or hydroxy apatite column fractions from HeLa cell nuclear extract (9). In Fig. 4C, 2 µl of heparin-Sepharose column fraction (20-fold purified in FBI-1 binding activity as compared with the nuclear extract, containing 3.5 µg/µl total protein and 5 ng/µl FBI-1 protein (9, 20)) were used per binding reaction. In all other experiments, 0.5 to 1 µl of hydroxy apatite column fraction (54-fold purified in FBI-1 binding activity, containing 3.9 µg/µl total protein and 15 ng/µl FBI-1 protein (9, 20)) was used per binding reaction. FBI-1 contained in nuclear extract or the column fractions of different degrees of purification binds to DNA in the same sequence-specific manner (9).
|
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Each HIV-1 Half-site Is Capable of Binding FBI-1To define further the role of each HIV-1 half-site in FBI-1 binding, we generated probes containing HIV-1 sequences from 18 to +24 and thus including the specific sequences constituting the 5' half-site or HIV-1 sequences from +21 to +82 (and thus including the 3' half-site) and tested them in an EMSA with partially purified FBI-1 [hydroxy apatite column fraction (see Ref. 9); this partially purified FBI-1 forms the same complex in an EMSA as recombinant FBI-1 expressed in Escherichia coli (see Ref. 12).2 The results are shown in Fig. 2A. As expected, the FBI-1·DNA complex formed efficiently on the wild-type probe (Fig. 2A, lane 1, arrow). Signals of decreasing intensities were observed with probes containing the 5' (lane 2) and 3' (lane 3) half-sites, respectively, whereas only a background signal was obtained with a probe containing just the polylinker (lane 4). The signals observed were of the sizes expected for FBI-1·DNA complexes considering the different lengths of the probes. In Fig. 2B, the same probes were used as unlabeled competitors against the labeled wild-type probe. Consistent with the results in Fig. 2A, the complete FBI-1 binding site was an efficient competitor, the 3' half-site was poor, and the 5' half-site was intermediate (compare lanes 24 with lane 1). However, even the 3' half-site was a better competitor than the full FBI-1 binding site debilitated by point mutations (msABC, lane 5; see Fig. 1B for the sequence of this mutant) or a DNA fragment carrying just the polylinker (lane 6).
We then used each half-site as unlabeled competitor against the other half-site and against itself. As expected, each unlabeled half-site competed efficiently against itself (Fig. 2C, lanes 25 and 1720). In addition, each half-site competed against the other half-site for formation of the complex, and the 5' half-site competed more efficiently than the 3' half-site against both a labeled 5' half-site (compare lanes 25 with lanes 710) and a labeled 3' half-site (compare lanes 1215 with lanes 1720). This was similar to the pattern observed above with the labeled wild-type probe, where the 5' half-site was also a better competitor than the 3' half-site (Fig. 2B).
The binding of FBI-1 to an isolated 3' half-site was unexpected, because we observed previously (9) that FBI-1 did not bind detectably to a mutant probe (msABC(5')) carrying an intact 3' FBI-1 half-site and a mutated 5' half-site. This discrepancy probably results from the higher concentration of nonspecific competitor nucleic acids used in our previous experiments. In addition, it is possible that the mutated 5' half-site, which is not present in the probes used here (Fig. 2), in some way interfered with FBI-1 binding to the 3' half-site. At any rate, the present findings indicate that complexes with similar binding specificities form on each half-site and on the complete FBI-1 binding site. Together with our previous observation that on probes containing the entire FBI-1 binding site, point mutations in either FBI-1 half-site weaken the FBI-1·DNA complex but do not change its mobility (9), the results suggest that each individual half-site can bind FBI-1 but with lower affinity than the complete wild-type binding site.
The Half-sites Contain an Imperfect PalindromeAs indicated
by the arrows in Fig.
1A, the sequences most important for FBI-1 binding in
each half-site constitute a pair of imperfect inverted repeats, which
corresponds to the sequences encoding the base of the TAR stem-and-loop
structure. This suggests that each half-site may be recognized similarly by
FBI-1. If this is the case, making the 5' half-site more similar to the
weaker 3' half-site should decrease binding of FBI-1, whereas making the
3' half-site more similar to the stronger 5' half-site should
increase binding of FBI-1. To test this hypothesis, we introduced the
mutations shown in Fig.
3B and tested the resulting probes in the EMSA shown in
Fig. 3A. Deleting the
C-G base pair at position +5 and thus making the 5' repeat more similar
to the 3' repeat reduced FBI-1 binding (mutant
5'2; compare lanes 3 and 2).
Strikingly, however, inserting a G-C base pair after position +55 and thus
making the 3' repeat more similar to the 5' repeat enhanced FBI-1
binding (mutant pISTBu, compare lanes 5 and
4). Taken together, these results suggest that FBI-1 recognizes
sequences in each half-sites with the same specificity and that the 3'
repeat is "imperfect" with respect to the 5' repeat.
|
FBI-1 Binds to DNA in a Flexible MannerThe segments of the FBI-1 half-sites most critical for binding are located in transcribed DNA that encodes the base of the TAR RNA stem-and-loop structure. It is therefore possible that the structure of TAR, e.g. the binding sites for Tat and its co-factors, imposes a suboptimal spacing of the FBI-1 half-sites. To test this hypothesis, we altered the spacing between the half-sites by insertions or deletions and determined the relative affinities of the resulting probes for FBI-1 by EMSA. Fig. 4A shows the 5' and 3' half-sites (shaded sequences) as determined previously by clustered point mutations, as well as the locations of the clustered point mutations (brackets above sequences) that delineated the internal borders of the 5' and 3' half-sites (constructs ms153 and ms23/fp in Ref. 9). The spacing between the two half-sites in the various constructs, calculated between the guanine at position +1, near the 5' end of the 5' repeat, and the corresponding guanine on the lower strand at position +59, near the 5' end (on the lower strand) of the 3' inverted repeat, is indicated above the lanes in Fig. 4B, as well as the rotation angle between these same guanine residues.
As shown in Fig.
4B, increasing the spacing between the half-sites by 44
bp reduced the binding efficiency by 50% (construct
WT[ABC]WT; compare lanes 2 and 3). In
contrast, progressive deletions between the half-sites initially either
enhanced binding in a minor way (mutants pIST, D1, and D5;
compare lane 3 with lanes 4, 5, and 9) or had no
effect (mutants D3, D4, and D6; compare lane 3 with
lanes 7, 8, and 10). Thus, D6, which lacked the sequences
from +8 to +52, bound FBI-1 with wild-type efficiency. In contrast, the next
deletion, D7, which lacked sequences from +6 to +55, abolished detectable
binding (compare lanes 3, 10, and 11), either because it
removed sequences required for binding, or because it gave rise to a
suboptimal spacing between the half-sites. The D6 mutant thus localizes the
maximal extent of the "inward" borders of the 5' and
3' half-sites more precisely: +7 for the 3' border of the 5'
half-site, and +53 for the 5' border of the 3' half-site. As
described below (see Fig.
5A and Fig.
6A), methylation of the Gly residue at position +7
interfered with binding of FBI-1, thus localizing the 3' border of the
5' half-site at +7. Moreover, our previous analysis of clustered point
mutations mapped the 5' border of the 3' half-site between +49 and
+56 (see bracket on top in
Fig. 4A). Thus, we can
now localize the 5' border of the 3' half-site between +53 and
+56. These new maximal inward borders of the 5' and 3' half-sites
are indicated below the sequences in
Fig. 4A.
|
As shown in Fig. 4B, the deletions rotated the half-sites with respect to each other to different extents (labeled in degrees above the lanes), but there was no correlation between the rotational displacement and formation of the FBI-1·DNA complex. Taken together, the above results indicate that (i) any distance between the half-sites from the natural spacing of 58 bp in HIV-1 to near juxtaposition is compatible with efficient binding of FBI-1, and (ii) there is no requirement for a particular rotational alignment of the half-sites on the DNA surface.
We then tested whether the flexible binding of FBI-1 to DNA would extend to
variations in the orientation of the half-sites with respect to each other. As
outlined above, FBI-1 recognizes an imperfect inverted DNA sequence repeat
downstream of the HIV-1 transcription start site. In mutant DD/pUC, sequences
containing the 3' half-site are flipped such that the 5' and
3' half-sites now form a direct repeat. In mutant UD/pUC, both
half-sites are flipped, resulting in an everted repeat. In DD30/pUC and
UD
25/pUC, the spacing between the flipped half-sites is reduced by 30
and 25 bp, respectively, by deletion of non-binding sequences between the
half-sites (see Fig.
1B, and see diagram above
Fig. 4C). As shown in
Fig. 4C, the
FBI-1·DNA complex formed efficiently on all probes but was slightly
enhanced on probes containing direct repeats and slightly reduced on probes
containing everted repeats (probes UD, lane 4 and
UD
25, lane 5) as compared with the wild-type HIV-1
sequences (lane 1). Quantification with a PhosphorImager of the
shifted complexes in this and two replicate experiments revealed the following
signals with respect to the wild-type probe: DD, 108123%; DD
30,
115122%; UD, 8391%; UD
25, 7380%. Thus, although
small differences in FBI-1 binding efficiency were observed, no particular
orientation of the half-sites was required, suggesting that FBI-1 can adopt a
variety of conformations on the DNA.
FBI-1 Contacts Guanine Residues in Each Half-siteTo identify nucleotides in close contact with FBI-1, we used a methylation interference assay. Probes carrying HIV-1 sequences from 45 to +82 were modified with dimethyl sulfate, which methylates guanine residues at the N7 position in the major groove of the DNA, and were then used in an EMSA. Free DNA and DNA complexed with FBI-1 were then cleaved with piperidine and analyzed on a sequencing gel. The results are shown in Fig. 5. On the upper strand, methylation of three guanine residues at positions +1, +2, and +3 interfered significantly with formation of the FBI-1·DNA complex, with minor interference observed at position 10 (compare lanes 1 and 2; the wedges mark residues where interference was observed, and the size of the wedges correlates with the degree of interference). On the lower strand, methylation of two guanine residues at +5 and +7, as well as three guanine residues at +57, +58, and +59, interfered the most with FBI-1 binding, with less significant interference also observed at positions +61, +71, and +73 (compare lanes 3 and 4). Together with the deletion analysis described above (Fig. 4B), the observation that methylation of guanine +7 interferes strongly with binding suggests that the 3' border of the 5' half-site is at this position (see Fig. 4A).
In summary, the methylation interference assay identified two clusters of guanine residues whose chemical modification within the major groove of the DNA interfered severely with FBI-1 binding: one located between +1 and +7, and one located between +57 and +59 (see bottom panel of Fig. 6A). These two clusters reside within the two regions in the HIV-1 promoter that were identified previously (9) by EMSA as contributing most to FBI-1 binding (see also Fig. 1A).
Establishment of an FBI-1 Consensus Binding SiteWe have shown previously (9) that FBI-1 binds to a variety of cellular promoters and the Ad2 MLP. These promoters do not, however, contain any obvious sequence similarities to the HIV-1 IST, and the exact locations of the FBI-1 binding sites are unknown. We therefore used the dimethyl sulfate methylation interference assay to identify FBI-1 binding sites in several cellular genes and the Ad2 MLP and then aligned the identified half-sites to derive a consensus sequence. The results for the Ad2 MLP, c-fos intron 1, and c-myc P1 promoter are shown in Fig. 5, B, C, and D, respectively. All the results are summarized schematically in Fig. 6A, where the binding sites are depicted from top to bottom with decreasing affinity for FBI-1.
In the Ad2 MLP, methylation of three clusters of guanine residues flanking the TATA box interfered severely with binding, revealing a curious guanine-rich binding site, where the 5' cluster and the most 3' cluster are followed by a cytosine and resemble a pair of direct repeats (Fig. 5B). In the first intron of the c-fos gene, the FBI-1 binding site consisted of two direct repeats (Fig. 5C). In the c-myc P1 promoter, we identified an FBI-1 binding site consisting of two inverted repeats reminiscent of the site in the HIV-1 promoter, except that the distance between the half-sites was significantly shorter (Fig. 5D). Moreover, in the c-myc P2 promoter, we found a binding site consisting of a single half-site, and in the first exon of the c-fos gene, we found a site consisting of two abutting everted repeats (data not shown but summarized in Fig. 6A). The number of guanines whose methylation interfered with binding varied from 5 (c-myc P2) to 17 (MLP), and the distance between the half-sites (calculated between the first of the three strictly conserved guanines identified in the consensus sequences in Fig. 6B) varied between 7 (c-fos, exon 1) and 51 bp (c-fos, intron 1).
This analysis revealed no correlation between the affinity for FBI-1 and the distance between the half-sites or the orientation of the half-sites with respect to each other. Moreover, as exemplified by the c-myc P2 promoter binding site, a single site was sufficient for binding. These results agree well with the above observations that each HIV-1 half-site can bind FBI-1 on its own (see Fig. 2) and that the relative orientation and spacing of the HIV-1 half-sites can be varied (see Fig. 4, B and C). Thus, even though the functional significance of FBI-1 binding to these sites is unknown, these findings provide further evidence that FBI-1 binds DNA with remarkable flexibility.
From the half-sites identified in HIV-1 and the cellular promoters, the single site found in the c-myc P2 promoter, and the 5' and 3' Ad2 MLP sites, we then derived the consensus FBI-1 half-site shown in Fig. 6B. It consists of two well but not absolutely conserved guanines followed by a core of three strictly conserved guanines and four well conserved pyrimidines, with a preference for thymine at the first and cytosine at the second position after the three guanines. This consensus sequence resembles the FBI-1 binding site in the ADH5/FDH promoter (15), which is depicted on the bottom of Fig. 6B. FBI-1 binds to this site in vivo (15), suggesting that the consensus sequence reflects the properties of natural FBI-1 binding sites. The consensus sequence is also reminiscent of the guanine-rich binding sites of some other Krüppel-type zinc finger and POZ-domain proteins (see "Discussion").
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
What is the stoichiometry of FBI-1 on DNA? Probes carrying mutations in one half-site form FBI-1·DNA complexes of the same electrophoretic mobilities as wild-type probes of the same length (9). Moreover, the FBI-1·DNA complexes that form on probes carrying a single binding site migrate only slightly faster than those formed on full-length probes carrying two half-sites (see Fig. 2A), consistent with these complexes differing only by the size of the probe. This suggests that FBI-1 binds to single sites with the same stoichiometry as to duplicated sites. This could be a monomer, with similar or identical DNA binding domains recognizing each half-site, or a homodimer (or multiple thereof), with each FBI-1 molecule contacting one half-site. Using glycerol gradient equilibrium centrifugation, we have previously estimated the molecular mass of native FBI-1 to be 140 kDa (20), which is close to the predicted molecular mass of a homodimer, 123 kDa. This result and the observations that (i) FBI-1 does not contain a duplicated DNA binding domain but rather four different zinc fingers (12), (ii) recombinant FBI-1 can self-associate in solution via both the POZ domain and the zinc fingers (12), and (iii) the crystal structure of the POZ domain of the related POZ-domain zinc finger transcription factor PLZF reveals a tightly intertwined dimer with an extensive dimerization surface (22) all strongly suggest that, prior to binding to DNA, FBI-1 exists as a preformed dimer in which each FBI-1 molecule contributes one DNA binding domain. Because mutation of the first or second (but not the third or fourth) zinc fingers severely reduces FBI-1 binding (12), this binding domain includes, minimally, the first two FBI-1 zinc fingers. The ability of FBI-1 to bind to very differently spaced and oriented half-sites suggests that the zinc finger DNA binding domains are separated from the dimerized POZ domains by flexible linkers. It also suggests that self-association of the FBI-1 zinc finger domains on DNA may occur only on DNA binding sites with appropriately spaced and oriented half-sites.
Our results reveal a remarkable coincidence between the HIV-1 FBI-1 binding site and the IST promoter element. Consistent with this, the affinities of various IST mutants for FBI-1 and their abilities to support the synthesis of short transcripts correlated extensively in previous experiments (9, 23). Nevertheless, transient transfection and cell-free transcription assays have not revealed a specific effect of FBI-1 on IST function.3 Rather, overexpressed FBI-1 stimulates Tat trans-activation in transient transfection assays (13). Interestingly, mutations that debilitate the IST and abrogate FBI-1 binding magnify this effect, indicating that endogenous FBI-1 binds to the IST and may actually function as a repressor of HIV-1 transcription.4 Conceivably, this latter effect may relate to a role of FBI-1 in the synthesis of the short transcripts.
Flexible Binding of FBI-1 to DNAThe known DNA binding sites of other POZ-domain proteins display considerable sequence heterogeneity. Some of them, however, resemble the FBI-1 binding sites. For instance, Egr-1 and MAZR recognize single guanine-rich sites (24, 25), and the c-Krox protein binds alternatively to a single guanine-rich site (26) or to tandem repeats of the sequence 5'-GGAGGG-3', separated by nine base pairs (18). Consistent with these similarities, the rat homologue of FBI-1, osteoclast-derived zinc finger protein, binds to both the c-Krox tandem site and the Egr-1 single site, albeit with weaker affinity than their cognate proteins (18). Moreover, at least two other POZ-domain zinc finger proteins interact with DNA in a flexible manner; the Kaiso protein recognizes the specific consensus sequence TCCTGCNA, as well as methyl-CpG dinucleotides (8), and the GAGA factor binds cooperatively to repeats of the sequence motif GAGA that can vary in number and spacing (6, 7).
Our analysis of FBI-1 binding to various permutations of its HIV-1 binding
site and to other cellular and viral binding sites reveals that FBI-1 binds
efficiently to a variety of sequences with different spacings and
orientations. This flexibility is reminiscent of the versatile interactions of
other proteins with DNA. For example, the yeast homeodomain protein 2
binds as a dimer to variously spaced half-sites forming inverted, direct, or
everted repeats, because the relative orientations of the two homeodomains in
the dimer are unconstrained
(27). The POU domain proteins
constitute another example of highly flexible binding to DNA. The POU domain
consists of two DNA binding domains, the POU-specific (POUS) domain
and the POU homeo (POUH) domain, joined together by a flexible
linker. Oct-1, a broadly expressed transcription factor, is capable of binding
to a wide variety of sequences
(21). Such flexibility derives
in part from the ability of the POUS and POUH domains to
contact DNA in different orientations and different positions relative to one
another (28,
29). In the above cases, DNA
binding flexibility is achieved in part through the presence of two DNA
binding domains, which can be carried either on two polypeptides or on a
single polypeptide, whose orientation and relative position on the DNA are
unconstrained. In the case of FBI-1, it probably results from the interaction
between two monomers via their POZ domains and, perhaps on some binding sites,
zinc finger domains. This flexibility may allow FBI-1 to recognize a variety
of cellular binding sites. The nature of the binding site (e.g.
single versus bipartite) and the orientation of the half-sites with
respect to each other may allow an additional level of transcriptional
regulation, for instance by exposing different surfaces of FBI-1 to the
transcriptional machinery or by allowing it to engage in different
protein-protein interactions.
![]() |
FOOTNOTES |
---|
Present address: Div. of Rheumatology, Children's Hospital of Philadelphia,
3516 Civic Center Blvd./1102 ARC, Philadelphia, PA 19104.
|| To whom correspondence should be addressed. Tel.: 215-590-7180; Fax: 215-590-1258; E-mail: pessler{at}email.chop.edu.
1 The abbreviations used are: HIV-1, human immunodeficiency virus, type 1;
EMSA, electrophoretic mobility shift assay; Ad2, adenovirus 2; MLP, major late
promoter; IST, inducer of short transcripts; p, plasmid; WT, wild-type.
2 D. J. Morrison and N. Hernandez, unpublished data.
3 F. Pessler and P. S. Pendergrast, unpublished results.
4 P. S. Pendergrast and N. Hernandez, unpublished data.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|