©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Topological Determinants of Internal Transmembrane Segments in P-glycoprotein Sequences (*)

(Received for publication, September 6, 1994)

Jian-Ting Zhang (§) Chow Hwee Lee Monika Duthie Victor Ling (¶)

From the Department of Medical Biophysics, University of Toronto, Division of Molecular and Structural Biology, the Ontario Cancer Institute, Toronto M4X 1K9, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

P-glycoprotein (Pgp) is a polytopic membrane protein responsible for multidrug resistance in cancer cells. Previously, we have used a coupled cell-free translation/translocation system to investigate the membrane orientation of Pgp sequences and have made the unexpected observation that predicted transmembrane (TM) segments from both the NH(2)-terminal and COOH-terminal halves inserted in microsomal membranes in two different orientations (Zhang, J.-T., Duthie, M., and Ling, V.(1993) J. Biol. Chem. 268, 15101-15110). How these topological forms of Pgp are regulated is not known. In the present study, we have used site-directed mutagenesis to investigate if the amino acids surrounding the internal TM segments of Pgp may affect their orientation. We discovered that the charged amino acids flanking TM4 are important in determining the membrane orientation of the NH(2)-terminal half molecule of Pgp. This is a novel observation demonstrating the existence of internal topogenic sequences in a mammalian polytopic membrane protein. These findings thus suggest A) that the topological structure of a mammalian polytopic membrane protein does not integrate into the membrane simply by following the lead of the first inserted TM segment but that internal TMs may have independent topogenic information and B) that the TM segments in a multi-spanning membrane protein may be more dynamic than have been previously anticipated, i.e. mutations in the amino acids surrounding internal TMs could drastically change the overall topology of the molecule.


INTRODUCTION

P-glycoprotein (Pgp) (^1)is a polytopic membrane protein responsible for multidrug resistance in cancer cells (1, 2, 3) . Pgp belongs to a superfamily of ATP-binding cassette membrane protein transporters that includes the cystic fibrosis transmembrane conductance regulator, peptide antigen presentation-associated transporters, yeast STE6, and Escherichia coli hemolysin transporter(4, 5, 6) . Over 50 of these ATP-binding cassette transporters are known, and the list of transported substrates is extremely diverse including inorganic ions, amino acids, sugars, peptides, steroids, and hydrophobic anticancer drugs. Substrate specificities of these transporters are likely determined by their transmembrane domains. Hydropathy plot and amino acid sequence analysis of Pgp suggested that it has 12 transmembrane (TM) segments and 2 ATP-binding sites(7, 8, 9) . However, nascent Chinese hamster pgp1 Pgp molecules synthesized in a cell-free translation system in the presence of microsomal membranes (rough microsomes) possess two different topological structures(10, 11) . One has all 12 predicted TMs in the membrane while the other has only 8 in the membrane. Similar observations have been made with the COOH-terminal half of the molecule of human MDR1 Pgp expressed in frog oocytes(12) .

Transmembrane proteins in eukaryotic cells are thought to acquire their final membrane orientations during or immediately after synthesis on the rough endoplasmic reticulum. Two theories have been proposed to explain the orientation of membrane proteins. One is the ``positive-inside rule'' in which it is postulated that membrane proteins orient themselves with the most positively charged end in the cytoplasm(13, 14) . The second is the ``charge-difference rule'' in which membrane proteins orient themselves according to the charge difference flanking the TM segment. In a multi-spanning membrane protein, the charge difference flanking the first (NH(2) terminus) TM segment is thought to play a dominant role in determining the overall membrane topology of the protein. Subsequent TM segments are postulated to simply follow the lead of the first one(15) . In this respect, the Pgp molecule is unusual in that it occurs in more than one topological form. It has been speculated that this feature of Pgp may be associated with its multiple functions(11) . Pgp not only transports anticancer drugs but may also function as an ATP channel (16) and a cell-swelling activated chloride channel(17, 18) . Gill et al.(19) have been able to separate chloride-conducting function from drug-transport function of Pgp by changing cell volume. These two functions may be generated by two different conformations of Pgp.

In this study, we analyze how the two orientations of the hamster pgp1 Pgp observed in an in vitro translation system (10, 11) are regulated. Such in vitro systems have been used for studying in detail the topology of membrane proteins in a wide variety of systems(20) . These topological studies make use of physical and enzymatic properties of rough microsomes (RM) (derived from dog pancreatic endoplasmic reticulum) to detect the orientation and insertion of newly synthesized peptides. We have used site-directed mutagenesis to investigate the effects of charged amino acids on membrane orientation of Pgp sequences. Our results show that the charged amino acids surrounding the TM3 and TM4 determine their orientation in the membrane, independent of the first TM segment, TM1. This type of internal topogenic sequence may dictate the topologies of a wide range of polytopic transport proteins in mammalian cells.


EXPERIMENTAL PROCEDURES

Materials

pGEM-4z plasmid, SP6 and T7 RNA polymerase, RNasin, ribonucleotides, RQ1 DNase, rabbit reticulocyte lysate, and dog pancreatic microsomal membranes were obtained from Promega. [S]Methionine and Amplify were purchased from DuPont NEN and Amersham Corp., respectively. m^7G(5`)ppp(5`)G cap analog was obtained from Pharmacia Biotech Inc. Peptide N-glycosidase F and restriction enzymes were obtained from Boehringer Mannheim. Taq DNA polymerase was obtained from Perkin-Elmer. All other chemicals were obtained from Sigma or Fisher.

Site-directed Mutagenesis

For convenience of analyzing the effect of mutations on the two topologies of Pgp, the truncated construct pGPGP-N4 was used. This construct encodes the NH(2)-terminal four TM segments attached with a COOH-terminal reporter ATP-binding domain (Fig. 1) and has been previously described (11) . The site-directed mutagenesis in pGPGP-N4 construct was performed using two polymerase chain reaction (PCR) steps. In the first PCR step, a universal T7 primer and an oligonucleotide carrying appropriate mutations were used as primers and wild type pGPGP-N4 DNA as templates. The DNA products generated in the first PCR carry the designed mutations. This DNA, together with a universal SP6 primer, were used as primer for the second PCR. The second PCR products were then cloned into a fresh pGEM-4z plasmid, and the mutant clones were identified by restriction mapping and sequencing. A DNA fragment (NcoI-BglII) of 700 base pairs carrying the mutation was then isolated and subcloned into the wild type pGPGP-N4 DNA to replace the wild type NcoI-BglII fragment. The final DNA was sequenced to confirm the mutations and to confirm that no other mutations were generated by the Taq DNA polymerase. The oligonucleotides carrying mutations used for PCR are R207E, 5`-ATTTACTGAAGGCTGG; R207N, 5`-GATTTACTAACGGCTGG; K210D, 5`-AGGCTGGGATCTAACCC; D238K, 5`-CATTTACTAAGAAAGAAC; D238K/E240R, 5`ACTAAGAAACGTCTTCAGGC; K231E, 5`-ATTTGGGCAGAAATATTATC. When the K210D oligonucleotide was used as primer and pGPGP-N4 constructs carrying R207N or R207E mutations were used as templates, double mutants R207K/K210D and R207V/K210D were produced. Double mutations carrying R207N and R207E were not generated as expected due to the addition of an extra oligonucleotide at the 3`-end of the first PCR product by the Taq DNA polymerase.


Figure 1: Coupled in vitro translation with two membrane orientations of a truncated P-glycoprotein sequence. A, schematic linear structure of PGP-N4 molecule. The full-length of PGP-N4 molecule consists of 550 amino acids, 4 TM (TM1-TM4) segments (solidbars), and 4 consensus N-linked glycosylation sites (&cjs1231;&cjs1231;bullet). The arrow indicates the fusion site between the NH(2)-terminal TM domain and the COOH-terminal ATP-binding domain. Amino acids surrounding TM3 and TM4 are shown in single letter codes with charged amino acids marked by (+) or(-). There are no charged amino acid residues within the predicted TM segments. B, two observed membrane orientations of PGP-N4 molecules in RM vesicles. In model I, both NH(2) and COOH termini are on the cytoplasmic side (outside of RM), and all four TM segments (solidbars) are in the membrane. In model II, only three TM segments are in the membrane, and the COOH terminus is in the RM lumen with an oligosaccharide chain (&cjs1231;&cjs1231;bullet) attached. C, in vitro translation and endoglycosidase treatments of PGP-N4 molecules. Two mature full-length products of 62 kDa (representing model II) and 59.5 kDa (representing model I) were translated from the in vitro transcripts in the presence of RM (lane 1). Lanes2-4 show limited endoglycosidase peptide N-glycosidase F (PNGaseF) treatment. The partially deglycosylated peptide is indicated by arrows (lanes 2 and 3), and the fully deglycosylated peptide is 52 kDa (lane 4).



R207E, R207N, R207K/K210D, and R207V/K210D mutations in pGPGP-N3 were constructed from the corresponding pGPGP-N4 mutants. A EcoRI-BalI fragment containing the mutations were released from the pGPGP-N4 DNA and purified. A RsaI-HindIII fragment encoding the ATP-binding reporter was released from the wild type hamster pgp1 cDNA and purified. These two fragments were ligated together and cloned into a new pGEM-4z vector. The final DNA was sequenced to confirm the mutations and correct linkage between the two DNA fragments.

In Vitro Transcription and Translation

About 6 µg of recombinant DNA linearized with HindIII was transcribed in the presence of 5 A units/ml cap analog m^7G(5`)ppp(5`)G as previously described(10) . Removal of DNA templates with RQ1 DNase after transcription and purification of RNA transcripts were carried out according to Zhang and Ling(10) . Cell-free translation using rabbit reticulocyte lysate was performed as before(10) . To enhance the translation efficiency in the absence of RM, 1% Triton X-100 was included in the translation mixture. Proteolysis/membrane protection assay and limited endoglycosidase treatment were performed as described by Zhang et al.(11) . The membrane fraction of translation products was isolated by centrifugation and analyzed using SDS-polyacrylamide gel electrophoresis and fluorography as previously reported(10) .


RESULTS

Previously, two orientations have been observed with the NH(2)-terminal half of the molecules of Chinese hamster pgp1 Pgp translated in vitro(11) . To investigate determinants involved in the generation of the two orientations of Pgp, we have made use of site-directed mutagenesis and the pGPGP-N4 cDNA construct (11) to identify sequences important for determining the topology of the NH(2)-terminal half of Pgp. Fig. 1A shows the linear diagram of the truncated Pgp molecule (named as PGP-N4) encoded by pGPGP-N4. The PGP-N4 protein has been previously shown to be expressed in two different orientations (Fig. 1B, see also (11) ). One orientation, representing about 25% of the molecules (Fig. 1B, model I), has all four TMs in the membrane whereas the other orientation, representing about 75% of the molecules (Fig. 1B, model II), has only three TM segments in the membrane, and the COOH terminus is located in the RM lumen. The model I molecule has three N-linked oligosaccharide chains in the extracellular loop linking TM1 and TM2, while the model II molecule has an extra sugar chain in the COOH-terminal tail (Fig. 1B). Thus, the model I molecule has a faster mobility on SDS-polyacrylamide gel electrophoresis than model II due to one less oligosaccharide chain attached(11) .

Two major protein products (62 and 59.5 kDa) were translated from the pGPGP-N4 RNA transcript in a rabbit reticulocyte lysate supplemented with RM (Fig. 1C, lane 1; see also (11) ). The 62- and 59.5-kDa peptides represent model II and model I molecules, respectively(11) . Limited digestion of translation products with peptide N-glycosidase F generated intermediate deglycosylated products attached with 1, 2, or 3 sugar chains (indicated by arrows in Fig. 1C, lanes 2 and 3). The fully deglycosylated peptide is 52 kDa (Fig. 1C, lane 4). These results demonstrate that the 62-kDa peptide has four oligosaccharide chains while the 59.5-kDa peptide has three. Previously, we have shown that all of the COOH-terminal reporter peptides protected from proteolysis have an oligosaccharide chain(11) . Therefore, the possibility that the 59.5-kDa peptide represents the model II molecules that do not have an oligosaccharide chain in the COOH-terminal tail can be ruled out. The proportion of the 62- and 59.5-kDa peptides serves as a convenient indication for the relative amounts of model I and II molecules.

To determine if the two positively charged amino acids (Arg-207 and Lys-210) between TM3 and TM4 (Fig. 1A) have topogenic information for the membrane orientation of the PGP-N4 molecule, we made mutations of these two amino acids using site-directed mutagenesis and generated PGP-N4-R207E, -R207N, -R207K/K210D, and -R207V/K210D mutant cDNAs (Fig. 2A). Fig. 2B shows the cell-free translation product from wild type (WT) and mutant templates. About 25% of membrane-associated full-length products of WT have model I (59.5 kDa) orientation, and the remaining 75% have model II (62 kDa) orientation (Fig. 2B, lane 1; see also Table 1). The R207E and R207N mutants generated 70% model I and 30% model II molecules (Fig. 2B, lanes 2 and 3). The double mutant R207K/K210D generated 85% model I and 15% model II molecules (Fig. 2B, lane 4). Essentially no model II molecules were generated from the double mutant R207V/K210D (Fig. 2B, lane 5). These observations were confirmed by proteolysis/membrane protection assays of translation products. Upon protease K treatment, the WT, R207N, R207E, and R207K/K210D generated a 42-kDa protease-resistant and glycosylated fragment (COOH-terminal tail located in RM lumen), whereas the R207V/K210D did not (data not shown). Limited peptide N-glycosidase F treatment shows that the major product from R207K/K210D (Fig. 2D, lanes 1-4) and R207V/K210D (Fig. 2D, lanes 5-8) mutants has three oligosaccharide chains (Fig. 1B, model I). These results indicate that both the Arg-207 and Lys-210 between TM3 and TM4 affect the membrane orientation of PGP-N4 molecules. It has been previously suggested that Arg residues have more restrictive effects than Lys residues(21) . This may explain why fewer model II molecules were generated from R207K/K210D than from R207E, although both have similar charge changes. R207E and R207N mutations have similar effects on the membrane topology of PGP-N4, suggesting that the addition of a negative charge does not significantly affect the membrane topology. The origin of the minor band below the major product of R207K/K210D mutant (lane 4) is not known, and it is not consistently observed (see Fig. 2D). Its presence does not affect our interpretation of the experimental data. It should be noted that the mobility difference observed with mutant PGP-N4-R207K/K210D and PGP-N4-R207V/K210D is likely due to charge changes (Fig. 2B, lanes 4 and 5). The effect of charges on the mobility of truncated Pgp peptides has also been previously observed(22) . Sequencing the mutant PGP-N4 cDNA shows no mutation other than the designed ones. When the translation was per-formed in the absence of RM, the mobility of mutant precursors is also different from the WT (Fig. 2C). This shows that the difference in mobility is not due to modification caused by RM-associated enzymes.


Figure 2: Membrane orientation of PGP-N4 molecules with mutations NH(2)-terminal to TM4. A, amino acid sequences flanking TM4 of WT and mutant PGP-N4 molecules. The 7 amino acids at NH(2) terminus and 15 amino acids at COOH terminus of TM4 (hatchedbar) are shown. The dashedlines denote sequence identity of mutants with the WT molecule. The mutations at NH(2) terminus to TM4 are shown at specific positions. Net charge of NH(2)-terminal (N) and COOH-terminal (C) sequences of TM4 and the total net charge (Delta(C-N)) flanking TM4 are shown on the left with the name of each construct. B and C, expression of WT and mutant PGP-N4 molecules. WT and mutant PGP-N4 molecules were translated in the presence (panel B) or absence (panel C) of RM. The membrane fraction (panel B) or total fraction in the absence of RM (panel C) was analyzed by SDS-polyacrylamide gel electrophoresis. I and II in lane 1 of panel B denote molecules with model I and II orientations shown in Fig. 1B. D, limited endoglycosidase treatment of R207K/K210D and R207V/K210D mutant molecules. The membrane-associated translation products were treated with 0 (lanes 1 and 5), 13 (lanes 2 and 6), 17 (lanes 3 and 7), and 60 milliunits (lanes 4 and 8) of peptide N-glycosidase F. Arrows denote the intermediate products.





The fact that a reduction in positive charge in the domain linking TM3 and TM4 generates more model I molecules suggests that these positive charges function as a restriction factor causing TM4 to insert into the membrane with its amino terminus in cytoplasm. This is consistent with previous studies on mammalian type II membrane proteins (23) and asialoglycoprotein receptor H1 (24) where the positive charges at the NH(2) terminus of the TM segment are retained in the cytoplasmic side. In both instances, a membrane protein with a single TM segment was used. The current study is the first to demonstrate that charged amino acids flanking an internal (or subsequent) TM segment of a mammalian polytopic membrane protein are important in determining the membrane topology.

To investigate if the charged amino acids at the COOH terminus of TM4 also affect the membrane orientation of PGP-N4 molecules, we constructed PGP-N4-D238K, -D238K/E240R, and -K231E mutant molecules (Fig. 3A). The translation results of these mutant molecules are shown in Fig. 3B. It can be seen that while the WT molecule has about 75% model II orientation, the D238K and D238K/E240R mutants have 59 and 36% model II, respectively (Fig. 3B, Table 1). This suggests that adding positive charges in the COOH terminus of TM4 generates less model II molecules. The mutation at Lys-231 (mutant K231E) also appears to have increased slightly the proportion of model II molecules. These results, together with that in Fig. 2, indicate that the more positively charged domain tends to be retained in the cytoplasmic side of the membrane.


Figure 3: Membrane orientation of PGP-N4 molecules with mutations COOH-terminal to TM4. A, amino acid sequences flanking TM4 of WT and mutant PGP-N4 molecules. The 7 amino acids at NH(2) terminus and 15 amino acids at COOH terminus to TM4 (hatchedbar) are shown. The dashedlines denote sequence identity of mutants with the WT molecule. The mutations at COOH terminus to TM4 are shown at specific positions. Net charge of NH(2) terminus (N) and COOH terminus (C) sequences of TM4 and the total net charge (Delta(C-N)) flanking TM4 are shown on the left. B, expression of WT and PGP-N4 molecules. I and II in lane 1 denote molecules with model I and II orientations.



The nature and position of the charged amino acids may also play some role in determining membrane orientation. For example, a similar charge change across TM4 appears to generate different effects on the orientation of TM4. As shown in Fig. 2and Fig. 3, the Delta(N-C) (total net charge of NH(2)- and COOH-terminal sequences of TM4) of both R207E and D238K mutants equals zero; however, these two mutations yielded molecules with different ratios between the two orientations (Table 1). Moreover, mutations at the NH(2) terminus appear to affect the topology of PGP-N4 molecules more than mutations at the COOH terminus of TM4. This is probably due to the fact that the charges at the COOH terminus are farther away from TM4 and therefore have less effect than the ones at the NH(2) terminus(25) . Alternatively, the amino acids at the NH(2) terminus of TM4 affect TM3 as well as TM4, while the charged amino acids at the COOH terminus affect only TM4.

To study whether Arg-207 and Lys-210 affect the membrane orientation of TM3, we engineered mutations of Arg-207 and Lys-210 into PGP-N3 molecule, which has only TM1, TM2, and TM3 with an ATP-binding COOH-terminal tail (Fig. 4A). The wild type PGP-N3 is expressed as 40% model I and 60% model II molecules (Fig. 4B; see also Fig. 4C, lane 1, and Table 1). When only one positive charge (Arg-207 or Lys-210) was changed to neutral or negative charge, model I molecules were increased slightly compared with WT (Fig. 4C, lanes 2, 3, and 5; see also Table 1). When both positive charges were changed to neutral and negative charges (R207V/K210D), all of the molecules had the model I orientation. These results suggest that Arg-207 and Lys-210 also affect the membrane orientation of TM3 but to a lesser extent than TM4.


Figure 4: Membrane insertion and orientation of PGP-N3 molecules with mutations COOH-terminal to TM3 A, schematic linear structure of PGP-N3 molecule. The full length of PGP-N3 molecule consists of three predicted transmembrane segments (solidbars) and four consensus N-linked glycosylation sites (&cjs1231;&cjs1231;bullet). The arrow indicates the fusion site between the NH(2)-terminal TM domain and the COOH-terminal ATP-binding domain. Amino acids surrounding the TM3 are shown in single letter code with charged amino acids marked by (+) or(-). B, two models of membrane orientations of PGP-N3 molecules in RM vesicles. The model I molecule has all three TM segments (solidbars) in membrane with NH(2) and COOH termini on the different sides of RM membranes. An extra oligosaccharide chain is attached to the COOH-terminal domain of the model I molecule. Both NH(2) and COOH termini of the model II molecule are in cytoplasmic side (outside of RM) with the TM3 located on the outside of RM. C, expression of WT and mutant PGP-N3 molecules. I and II in lane 1 denote molecules with model I and II orientations. D, amino acid sequences flanking TM3 of WT and mutant PGP-N3 molecules. The 15 amino acids on both sides flanking TM3 (hatchedbar) are shown. The dashedlines denote sequence identity of mutants with the WT. The mutations are shown at specific positions on the COOH terminus of TM3. Net charge of NH(2)-terminal (N) and COOH-terminal (C) sequences of TM3 and the total net charge (Delta(C-N)) flanking TM3 are shown on the left.




DISCUSSION

Using Chinese hamster pgp1 Pgp sequences as a model system, we showed that internal TM segments of a mammalian polytopic membrane protein have topogenic information. We showed that the charged amino acids flanking the TM3 and TM4 of hamster pgp1 Pgp are very important in determining their relative membrane orientation in the in vitro translation system. Deletion of positive charges at the NH(2)-terminal side or addition of positive charges at the COOH-terminal side of the TM4 segment have similar effects on the membrane orientation of TM4. In both cases, more model I molecules were generated. This observation is unique and has not been previously demonstrated for other mammalian polytopic membrane proteins.

Hartmann et al.(15) postulated that TM segments in a polytopic membrane protein simply follow the lead of the first inserted TM segment and insert sequentially into the membrane. This is supported by the observation that the membrane insertion of the subsequent TMs in an artificial polytopic membrane protein does not depend on the signal-recognition particle and the signal recognition particle receptor(26) . Our current study, however, showed that the sidedness of membrane insertion of the TM3 and TM4 segments of Pgp can follow a different pathway. The orientation of TM3 and TM4 in the membrane is dependent on their surrounding charges and does not simply follow the lead of the first TM segment. We hypothesize that the presence of positive charges at the NH(2) terminus of TM4 creates a cytoplasmic ``retention signal'' for the NH(2) terminus of TM4, and thus, model II molecules of PGP-N4 are generated (see Fig. 1). The removal of this positive charge retention signal by site-directed mutagenesis defaults PGP-N4 to a model I probability. Lack of positive charges at the COOH terminus of TM4 may also decrease the potential for TM4 insertion into the membrane in model I orientation. These findings are not in agreement with the prevailing idea that TM segments in a polytopic membrane protein follow the lead of the first inserted TM and insert sequentially. Internal TM segments may have their own topogenic sequences, and mutations of these amino acids may drastically change their insertion and orientation in membrane. This type of internal topogenic sequence may dictate topologies of a wide range of polytopic transport proteins in mammalian cells.

Although it remains to be demonstrated, our hypothesis that more than one topological structure of Pgp is expressed in mammalian cells has functional implications. It is possible that these different topological structures of Pgp are associated with its multiple functions(11, 12) . Pgp not only transports anticancer drugs but also functions as an ATP channel (16) and a cell-swelling activated chloride channel(17, 18, 19) . Gill et al.(19) have been able to separate two conformations of Pgp that are responsible for the chloride channel and drug transport functions. Further studies with site-directed mutagenesis and transfection into mammalian cells to evaluate function should provide insight into the validity of the above hypothesis.


FOOTNOTES

*
This work was supported by the National Cancer Institute of Canada and by the National Institutes of Health (to V. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
A recipient of a postdoctoral fellowship from the National Cancer Institute of Canada. Current address: Dept. of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX, 77555-0641.

To whom correspondence should be addressed. Tel.: 416-924-0671 (ext. 4985); Fax: 416-323-3858.

(^1)
The abbreviations used are: Pgp, P-glycoprotein; TM, transmembrane; RM, rough microsomes; WT, wild type; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank our colleagues in the Ontario Cancer Institute for helpful discussions. We also thank Drs. Luis Reuss, Michael Jennings, and Karl Karnaky, Jr. at UTMB for critical comments on this manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.