©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Expression of a Pituitary Gland Protein Modulating Intestinal Fluid Secretion (*)

(Received for publication, May 5, 1995)

Ewa Johansson (1) Ivar Lönnroth (1)(§) Stefan Lange (2) Ingela Jonson (1) Eva Jennische (3) Christina Lönnroth (4)

From the  (1)Departments of Medical Microbiology and Immunology, (2)Clinical Bacteriology, (3)Anatomy and Cell Biology, and (4)Surgery, Gothenburg University, Guldhedsgatan 10, S-41346 Göteborg, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Antisecretory factor (AF) is a protein known to inhibit intestinal fluid secretion induced by cholera toxin. cDNA clones, expressing immunoreactivity to AF were isolated from a human pituitary gland library and sequenced. The sequence contained 1309 base pairs plus a poly(A) tail; Northern blot analysis of pituitary RNA confirmed this size. One large open reading frame was found to code for 382 amino acids. The protein was expressed in pGEX-1T/Escherichia coli and purified. The recombinant AF was extremely potent, 9 ng (2bullet10 mol), giving a significant antisecretory activity against cholera toxin-induced fluid secretion in rat. Antiserum against recombinant AF was used in immunohistochemical and Western blot analysis. Sections from human pituitary glands manifested specific intracellular staining in cells exclusively located in the anterior part. Both recombinant AF and AF extracted from pituitary gland appeared in SDS-polyacrylamide to have a molecular mass of 60 kDa, although the real value was 41 kDa. The protein sequence manifested homology (29% identity) with one protein, a putative Saccharomyces cerevisiae 30-kDa protein of unknown function.


INTRODUCTION

A large number of regulatory peptides occur both in the gastrointestinal tract and in the central nervous system where they are predominantly confined to the nerves themselves or to endocrine cells (1, 2) . Some of these peptides affect the transport of water and electrolytes across the intestinal mucosa. Thus, vasoactive intestinal polypeptide and substance P stimulate ion secretion from the serosal to the mucosal side of the epithelium(3) . Somatostatin, on the other hand, enhances absorbtion of ion and water in the gut, a property which has been used therapeutically for reducing the stool output during acute diarrhea(4, 5) .

During cholera diarrhea, a pronounced fluid and electrolyte output takes place, due to the action of the potent cholera toxin on the permeability of the intestinal mucosa(4) . As a result, a protein is formed that counteracts the permeability changes by the toxin. This protein, named antisecretory factor (AF), (^1)seems mainly to be formed in the pituitary gland and transported with the blood to the gut(6) . AF appear to have a molecular mass about 60 kDa and an isoelectric point of 5.0; 10 mol of AF is sufficient to abolish intestinal secretion in a rat exposed to cholera toxin, and 10 mol reverses intestinal secretion in pigs exposed to cholera toxin or Escherichia coli heat-labile toxin(7) . The action is not species specific, as AF from man or pig exerts activity in rat. Also, other enterotoxins than cholera toxin, such as those from Campylobacter jejuni, Clostridium difficile, E. coli, and Dinophysis, are inhibited by AF(7) . Moreover, the protein is a potent blocker of chloride ion transport across nerve cell membranes, a property which might be involved in its inhibitory effects on intestinal secretion(8) .

In the present work, we describe the cloning and sequencing of cDNA coding for a protein with the same immunoreactivity as AF. The coding sequence is expressed in E. coli, and the resulting fusion protein is isolated and characterized. Antibodies against the recombinant AF were produced in rabbits and used in immunohistochemical studies.


MATERIALS AND METHODS

Species and Tissues

Human pituitary glands were obtained postmortem from Sahlgrenska Hospital (permission given by the Swedish Health and Wellfare Board. Glands were kept frozen at -70 °C, except those used for histology, which were fixed for 24 h in 4% paraformaldehyde dissolved in phosphate-buffered saline (PBS) (0.15 M NaCl, 0.05 M sodium phosphate, pH 7.2) and thereafter transferred to 7.5% sucrose in PBS. Pituitary glands from pigs, 5-7 months old, obtained from a slaughter house were placed on dry ice during transport and kept frozen at -70 °C until used. Sprague-Dawley rats, 2-3 months old, were obtained for bioassay from B & K Universal AB (Sollentuna, Sweden). Rabbits (New Zealand White) for immunizations were obtained from Lidköping (Kaninfarm, Sweden).

Immunological Screening of a cDNA Library

A 5`-stretch cDNA library from normal human pituitary gland, derived from tissues obtained from a pool of nine caucasians, was purchased from Clontech Laboratories. For screening of the library, phages were plated at 3 10^4 plaque-forming units per 150-mm dish on E. coli Y1090. Rabbit antiserum against porcine AF (6) was absorbed with 0.5 volumes of E. coli Y1090 lysate for 4 h at 23 °C and diluted to a ratio of 1:400; screening was performed according to Young and Davis(9) . Alkaline-phosphatase-conjugated goat anti-rabbit antibodies were used as second antibodies (Jackson). Positive plaques were picked, eluted into phage suspension medium (20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgSO(4), 2% gelatin), replated, and screened until all plaques tested positive.

cDNA Recloning

Phage DNA from AF recombinants was isolated with Wizard Lambda Preps (Promega) and digested with EcoRI. The inserts were purified with Sephaglas BandPrep Kits (Pharmacia Biotech Inc.), recloned into pGEX-1T vector (Pharmacia), and transfected into Epicurian Coli XL1-Blue cells (Stratagene) according to standard techniques(10) .

Amplification of cDNA by PCR

To obtain the missing 5`-end of the cDNA, a PCR-based method called RACE (rapid amplification of cDNA ends) was performed(11) . A modified RACE method (12) that generates 5`-RACE-Ready cDNA with an anchor oligonucleotide ligated to the 3`-ends of the human brain cDNA molecules was purchased from Clontech Laboratories. The 5`-end was amplified from a portion of the 5`-RACE-Ready cDNA in two PCR amplification steps using a 5`-primer complementary to the anchor and two nested gene-specific 3`-PCR primers A and B (A = base 429-411 and B = base 376-359; Fig. 1, top). The amplified DNA fragment was cloned into the pGEX-1T vector by using the EcoRI site built into the anchor and the gene-specific primer. To verify the sequence obtained by the RACE method, double-stranded cDNA from human pituitary gland and brain (Clontech) were amplified with primer pair C/D containing an extra EcoRI cleavage site (Fig. 1, bottom). The primers were designed to allow the entire open reading frame (ORF) to be amplified. The pituitary and brain PCR products of expected size were digested with EcoRI, isolated, and cloned into the plasmid pGEX-1T vector.


Figure 1: Nuclear acid sequence and deduced amino acid sequence of the new human protein. The confirmed amino acid sequence is underlined. Bottom, horizontal map showing cloned cDNA and oligonucleotide primers.



Construction and Purification of Fusion Proteins

The cDNA clones obtained at immunological screening and at PCR amplification of the entire cDNA were ligated to pGEX-1T. This vector allows expression of foreign proteins in E. coli as fusions to the C terminus of the Schistosoma japonicum 26-kDa glutathione S-transferase (GST), which can be affinity purified under nondenaturing conditions(13) . Briefly, overnight cultures of E. coli transformed with recombinant pGEX-1T plasmids were diluted in fresh medium and grown for a further 3 h at 37 °C.

Protein expression was induced by 0.1 mM isopropyl-beta-D-thiogalactopyranoside, and after a further 4 h of growth at 30 °C, the cells were pelleted and resuspended in PBS. Cells were lysed by sonication, treated with 1% Triton X-100, and centrifuged at 12,000 g for 10 min; the supernatant containing the expressed fusion proteins was purified by passing the lysates through glutathione-agarose (Pharmacia). The fusion proteins were either eluted by competition with free glutathione or were cleaved overnight with 10 units of bovine thrombin to remove the AF protein from the GST affinity tail.

Antisera against Recombinant GST-AF Fusion Protein

Antibodies against purified fusion protein GST-AF-2 for use in Western blot and immunohistochemical studies were produced in rabbits. Each rabbit was given 100 µg of antigen in 1 ml of PBS mixed with an equal volume of Freund's complete adjuvant; each immunization was distributed in 8-10 portions injected in the back intracutaneously. Two booster doses with 50 µg of antigen were injected at 3 and 5 weeks, the last one without Freund's complete adjuvant. The rabbits were bled 6 days after last booster, and sera were prepared and stored at -20 °C. The sensitivity of the antiserum was tested with a dot blot assay. GST-AF-2 was applied on an ECL nitrocellulose membrane in 1:5 dilutions, and the antiserum was diluted 1:1000. The membrane was blocked with 1% bovine serum albumin in PBS at 4 °C for 16 h and then incubated for 1.5 h with a 1:800 dilution of rabbit anti-GST-AF or porcine AF antiserum. The blot was developed with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin followed by 5-bromo-4-chloro-3-indolyl phosphate and p-nitro blue tetrazolium (Boehringer Mannheim). The estimated limit for antigen detection was about 1 ng in this test.

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of human and porcine pituitary gland extracts and of pure AF proteins was performed in 10% acrylamide mini-slab gels, essentially as described by Laemmli (14) with the modification that bisacrylamide as a cross-linker was replaced by N,N`-diallyltartardiamide at the corresponding molarity. Pyronin Y (Sigma) was used as a marker of the electrophoretic front. Prestained molecular weight references were purchased from BDH Laboratory Supplies. Proteins were then either stained with Coomassie Brilliant Blue or transferred electrophoretically to 0.45-µm-pore-size ECL nitrocellulose (Amersham) for immunoblotting. The subsequent incubations with bovine serum albumin, conjugated anti-IgG, and alkaline phosphatase substrate were the same as for the dot blot assay described above.

DNA Sequencing and Oligonucleotides

DNA from plasmid pGEX-1T was used as a template for sequencing of the inserts by dideoxy chain termination method (15) with Sequenase version 2.0 (U. S. Biochemical Corp.). Initial forward and reverse primers copying regions of pGEX-1T immediately upstream and downstream of inserted DNA were obtained from Pharmacia. Subsequent primers were synthesized (Scandinavian Gene Synthesis AB) on the basis of sequence information obtained. Three different PCR clones were sequenced to avoid base exchange by Taq polymerase in the 5`-RACE method.

Amino Acid Sequencing

The pure AF proteins were run in 10% macro-slab gel SDS-PAGE(14) , and the proteins were transferred to a Problot membrane (Applied Biosystems) by electroblotting (Bio-Rad). Spots, visualized by Ponceau S staining, were excised from the blot, and the first 20 amino acids of the proteins were sequenced by automated Edman degradation on an automatic sequencer (Applied Biosystems).

DNA and Deduced Protein Sequence Analysis

Nucleotide sequence and the deduced protein sequence data were compiled and analyzed by using MacVector 4.1 (Eastman Chemical Co.). To predict the corresponding amino acid sequence of the cDNA inserts, codon usage of different reading frames was compared according to Fickett (16) and gave one large open reading frame. Interrogation of DNA and protein sequence data was carried out at the National Center for Biotechnology Information using the BLAST network service; in addition, the European Molecular Biology Laboratory BLITZ network service was used for analyzing protein sequences.

Northern Blot Analysis

To obtain RNA, pituitary glands were extracted with guanidinium thiocyanate according to Chomczynski and Sacchi(17) . Polyadenylated RNA was selected by means of a commercial kit (Pharmacia) using columns with oligo(dT)-cellulose. In addition, a pool of human pituitary mRNA from 107 individuals purchased from Clontech was used. 5 µg of each sample of poly(A) RNA was glyoxal-treated and electrophoresed in a 1.2% agarose gel(10) . After capillary alkaline transfer for 3 h in 0.05 M NaOH to Hybond N nylon membranes (Amersham), prehybridization and hybridization were carried out for 24 h each at 42 °C. The hybridization solution contained 50% formamide, 5 SSPE, 10 Denhardt's solution with 250 µg/ml denaturated low-MW DNA, and 50 µg/ml polyadenylic acid. The blots were probed with four different antisense 28-bases oligonucleotides comprising the positions 132-105 (primer E), 297-270 (primer F), 748-721 (primer G), and 833-806 (primer H) of the sequence (Fig. 1); the probes were 3`-end labeled with terminal transferase (Boehringer Mannheim) plus dd-[alphaP]ATP (Amersham) and purified on Nick columns (Pharmacia). Five post-washes in decreasing concentration from 5 SSPE, 0.1% SDS to 0.5 SSPE, 0.1% SDS were made at 42 °C for 30 min each time, with a repeat of the last wash. Filters were exposed to Hyperfilm MP (Amersham) for 7 days.

Immunohistochemistry

The fixed pituitary glands were frozen in liquid nitrogen, and cryo sections, 7 µm thick, were prepared. From each sample, 5-10 sections comprising different parts of the gland were fastened to microscope slides. The sections were blocked in 5% fat-free dried milk and incubated with primary rabbit antiserum (anti-GST-AF-2 fusion protein) diluted 1:4000-1:8000 in a humid chamber overnight at 4 °C. After rinsing in buffer, the specimens were incubated for 1 h at 23 °C with alkaline phosphatase-conjugated swine anti-rabbit immunoglobulins diluted 1:50 (Dako A/S). The immunoreaction was visualized with phosphatase substrates as described elsewhere(19) . Control sections were incubated with immune serum absorbed with an excess of GST-AF-2 protein or with all incubation steps except the primary antibody.

Antisecretory Activity

The antisecretory activity was measured in a previously described rat intestinal loop model(20) . In brief, a jejunal loop was challenged with 3 µg of cholera toxin. Either different doses of purified AF-1 proteins or PBS (control) were injected intraveneously 20-30 s before challenge with cholera toxin. The weight of the accumulated fluid in the intestinal loop (mg/cm) was recorded after 5 h. Each AF preparation was tested in at least six rats. Fisher's protected least significant difference was used for statistical analysis of the data.


RESULTS

Molecular Cloning and Sequence Analysis of cDNA

Polyvalent antisera against AF protein from pig was used for screening cDNA from human pituitary glands. Two clones expressing immunoreactive AF were isolated, rescued from phage , and recloned into the EcoRI site of vector pGEX-1T. Restriction analysis gave insert sizes of 1100 and 900 bp, respectively. DNA sequencing of the two clones revealed homology to be complete except for one substitution (Fig. 1, C replacing T at position 1010). A sequence upstream of the 5`-end of clone 2 was obtained by means of the RACE method. The fragment had a total length of 376 bp (not including the synthetic nucleotide arm at the 5`-end). The total reconstructed cDNA contained 1309 base pairs followed a poly(A) tail, which was preceded by a poly(A) signal (Fig. 1, positions 1289-1295). An ORF of 1146 bp (positions 63-1208) was identified. A data base search of the entire cDNA revealed homology with 12 shorter sequences (100-330 bp) having between 57 and 98% identities. Of these were 10 of human origin, reported to the data base as expressed sequence tags (EST). Two of these sequences, both from fetal lung, manifested 98% identity, whereas the other sequences manifested a high degree of homology in their 5`-end and little or no identity in the 3`-end. The two non-human cDNAcontaining sequences with manifest homology (53 and 56%) both derived from Saccharomyces cerevisiae (bakers' yeast). In one of these sequences, the homology began at the 5`-end of a large open reading frame, which coded for a hypothetical 30-kDa protein (see below).

Sequence and Size of Recombinant AF Proteins

To confirm the coding sequence, we isolated the full-length transcript by using PCR amplification of pituitary and brain cDNA. Using the primer pair C/D, we isolated 1215 bp identical to the sequence of clone 4 (Fig. 1). The open reading frame encoded 382 amino acids with a calculated molecular mass of 41.14 kDa and a calculated pI of 4.9. A data base search revealed that a hypothetical 30-kDa protein from S. cerevisiae manifested 29% identity (score 612 out of 2076 possible) as analyzed with the EMBL Blitz program. This protein was the only one to manifest more than 10% identity.

The AF clones 1, 2, and 3 were ligated into the pGEX 1T plasmid vector so that the ORF was in frame with the GST protein. The constructs were transformed into E. coli, and expression of fusion proteins was induced with isopropyl-beta-D-thiogalactopyranoside. The purified fusion proteins and the thrombin-cleaved AF protein were subjected to SDS-PAGE and Western blotting using antiserum against porcine antisecretory factor (Fig. 2). Coomassie Brilliant Blue staining of the proteins revealed discrete bands for each protein except for the GST-AF-1 protein, which manifested degradation into smaller components. However, in the Western blot analyses, the full-length protein gave a much stronger signal than the degradated products (Fig. 2B). The strong reaction with the antiserum against porcine AF indicated that the recombinant proteins indeed have the same immunoreactivity as AF. The molecular mass of the full-length protein appeared to be about 60 kDa, which is higher than the true molecular mass of 41,139 Da estimated from the amino acid composition. Furthermore, the proteins were also immunoblotted and probed with antiserum raised against GST-AF-2, which bound to the thrombin-cleaved proteins (Fig. 3).


Figure 2: Coomassie Brilliant Blue-stained SDS-polyacrylamide mini-gel (A) and immunoblot probed with antisera against porcine AF (B). Lanes with unprimednumbers contain glutathione-agarose-purified GST-AF fusion proteins AF-1, AF-2, and AF-3, whereas lanes with primednumbers contain the fusion proteins cleaved with thrombin. Molecular weight references (R) are indicated on the left. The GST-AF-1 fusion protein is highly degraded, but the immunoblot analysis shows only the detection of a full-length protein and spontaneous thrombin cleavage product. There is a 26-kDa product in the GST-AF-3 protein, probably the glutathione S-transferase tail that has been independently expressed.




Figure 3: Western blot using antiserum against recombinant protein AF-2. To the left, porcine (P) and three human (H1, H2, H3) pituitary glands were applied; to the right, the three recombinant proteins AF-1, AF-2, and AF-3 (see Fig. 2) were applied; in the center is the molecular weight standard (R).



Protein sequence analysis was performed to further validate the identified ORF. Attempts to obtain the N-terminal protein sequence of clone 1 were unsuccessful, probably because of chemical blocking of the N-terminal end. However, the N-terminal sequences of clone 2 and clone 3 were determined and were shown to perfectly match amino acids 63-75 and 130-140, respectively, of the predicted sequence (Fig. 1).

Expression in Pituitary Gland

Northern blot analyses was performed with a mixture of four oligonucleotide probes hybridizing with different sequences along the cloned cDNA (Fig. 4). The probes hybridized with a single band of about 1400 bp in the separated mRNA from pituitary gland. The strongest signals were obtained with the human material, but the porcine material also cross-reacted.


Figure 4: Autoradiogram of Northern blots of RNA from a human and porcine pituitary gland. 5 µg of purified mRNA was applied in each basin; 3`-end-labeled P oligonucleotide probes were used, and the autoradiogram was developed after 7 days.



Antiserum against recombinant GST-AF-2 reacted with the naturally occurring AF protein of an apparent molecular mass of 60 kDa and with some smaller components, probably enzymatic degradation products (Fig. 3).

The distribution of AF in sections of human pituitary glands was studied with immunohistochemical techniques (Fig. 5). In all specimens investigated, a moderate number of cells in the adenohypophysis were stained; the immunostained material appeared to be located in granules in the cytoplasm; preabsorption of the immune serum with an excess of GST-AF-2 protein abolished the signal. No staining was observed in the posterior part (neurohypophysis).


Figure 5: Cryosections of adenohypophysis stained with antiserum against recombinant protein GST-AF-2. A, sections incubated with immune serum showing scattered cells with varying degrees of positive immunoreactivity (solidarrows). Many cells completely lack staining (openarrows). B, serial sections to A incubated with immune serum preabsorbed with excess of recombinant protein GST-AF-2. There is no specific staining of the cells. C and D, larger magnification of immunopositive cells demonstrating cytoplasmatic staining of the endocrine cells. n, nucleus; c, cytoplasma. Bars in A and B = 100 µm, bars in C and D = 10 µm.



Biological Activity of Fusion Protein

The biological activity of the pure protein of clone 1 produced in E. coli was tested in a rat model. The capacity of the intravenously injected proteins to inhibit cholera toxin-induced fluid secretion in the small intestine is shown in Fig. 6. In control animals injected with buffer only, the cholera toxin caused a pronounced secretion, 412 ± 9 mg of fluid/cm of intestine. The pure protein of clone 1 caused dose-dependent inhibition of the cholera secretion, which was significantly different from the response to the buffer (p < 0.01, n = 6). 9 ng of clone 1 protein is sufficient to reduce the response by 34%, whereas 44 ng (10 mol) and 220 ng reduced it by 46 and 78%, respectively.


Figure 6: Biological activity of recombinant protein AF-1 testing inhibition of cholera toxin-induced fluid secretion. Graded doses of the protein were injected intravenously in rat; 3 µg of cholera toxin was injected into an intestinal loop; after 5 h, the accumulated fluid (mg/cm intestine) in the loop was measured. Each value represents the mean ± S.E. of a group of six animals.




DISCUSSION

The nucleotide sequence of the cloned human pituitary cDNA contained a large open reading frame, which coded for a 382-residue protein. The recombinant protein manifested similar immunoreactivity, localization, migration in SDS-PAGE, isoelectric point, and biological activity as the previously characterized AF(7) . Accordingly, the sequenced protein is in all probability identical with the previously characterized AF.

The sequenced AF resembles no other mammalian protein, but portions of the coding DNA sequence were almost identical to some expressed sequence tags originating from human tissues. The observed lack of homology in the 3`-end of eight of them is probably due to errors arising from automatic sequence determination, and all of them might originate from the AF transcript. The homology of the yeast cDNA is noteworthy since it begins in the start codon of the open reading frame coding for a hypothetical 30-kDa protein(21) . Hopefully, it will be possible to express this protein and compare its biological effect with that of AF.

The terminal parts of the AF protein are basic, the calculated pI of the 100 first and 50 last residues being 9.2 and 12.2, respectively (calculated with the MacVector 4.1 software). The middle part of AF is predominantly acidic, resulting in a pI of the entire protein of 4.9. All of the recombinant proteins migrated abnormally slowly in SDS-PAGE, resulting in an overestimation of their molecular mass. This phenomenon may have been due to the extremely positive charge of the C-terminal combined with a large number of proline residues, which might obstruct the loading with negative SDS molecules. The first 10 residues of the protein appear to be relatively hydrophobic when analyzed according to Kyte-Doolittle (22) and might constitute a signal peptide, which is cleaved out prior to exocytosis of the protein. This interpretation is supported by the Western blot analyses (Fig. 3) in which the recombinant protein appeared to have a slightly higher molecular mass than the protein extracted from pituitary gland. Some of this difference, however, might also be due to the additional five amino acids in the recombinant protein constituting the thrombin cleavage site of the fusion protein.

The biological activity of recombinant AF was similar to naturally occurring AF, and the potency is greater than that of any enterotoxin known to us and greater than that of any intestinal hormone or neuropeptide modifying water and electrolyte transport. Moreover, the level of activity of human AF in rat is surprisingly high, which probably reflects a ubiquitous structure conserved in AF molecules from different species. This hypothesis is supported by the cross-reactivity between human and porcine material obtained in the Western blot and Northern blot analyses. It remains to be seen whether the 30-kDa protein from yeast also show cross-reactivity.

The distribution of immunoreactive material in the pituitary gland demonstrated solely intracellular distribution of AF in secreting cells of the anterior lobe (adenohypophysis). The proteins emanating from this lobe include growth hormone, thyrotropin, corticotropin, prolactin, and luteinizing hormone. The passage of these hormones from intracellular localization to the vascular system is triggered by releasing factors produced by neuroendocrinic cells in the hypothalamus. The secretion of AF into the blood is stress-sensitive (7) , indicating an involvement of hypothalamus and regulation by releasing factors. As protein kinases probably mediate this signal-transmission, it is noteworthy that residues 321-325, 370-373, and 364-367 of the protein (Fig. 1) constitute possible sites for phosphorylation by tyrosine, cyclic AMP-dependent, and calcium-dependent protein kinases, respectively(23- 25). Intestinal challenge with cholera toxin enhances the level of active AF in the pituitary gland(7) . The signal for this induction is probably triggered in the intestinal mucosa by a nerve reflex, which is transmitted to the central nervous system and the pituitary gland via the vagus nerve(7, 18) .


FOOTNOTES

*
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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].

§
To whom correspondence should be addressed. Tel.: 46-31-60-47-25; Fax: 46-31-82-41-22.

(^1)
The abbreviations used are: AF, antisecretory factor; ORF, open reading frame; bp, base pair(s); PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.


REFERENCES

  1. Brown, M. R. (1990) Ann N. Y. Acad. Sci. 579,8-16 [Medline] [Order article via Infotrieve]
  2. Tache, Y., and Wingate, D. (eds) (1991) Brain-Gut Interaction , CRC Press, Boca Raton, FL
  3. Field, M., Rao, M. C., and Chang, E. B. (1989) N. Engl. J. Med. 321,800-806 [Medline] [Order article via Infotrieve]
  4. Field, M., Rao, M. C., and Chang, E. B. (1989) N. Engl. J. Med. 321,879-883 [Medline] [Order article via Infotrieve]
  5. Krejs, G. J. (1987) Am. J. Med. 82,37-48
  6. Lönnroth, I., and Lange, S. (1986) Biochim. Biophys. Acta 883,138-144 [Medline] [Order article via Infotrieve]
  7. Lönnroth, I., Lange, S., and Skadhauge, E. (1988) Comp. Biochem. Physiol. 90A,611-617 [CrossRef]
  8. Lange, S., Lönnroth, I., Palm, A., and Hyden, H. (1987) Pflügers Arch. 410,648-651
  9. Young, R. A., and Davis, R. W. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,1194-1198 [Abstract]
  10. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , pp. 1.74-1.84, 7.40-7.42, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY _
  11. Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 86,8998-9002
  12. Apte, A. N., and Siebert, P. D. (1993) BioTechniques 15,890-893 [Medline] [Order article via Infotrieve]
  13. Smith, D. B., and Johnson, K. S. (1988) Gene (Amst.) 67,31-40 [CrossRef][Medline] [Order article via Infotrieve]
  14. Laemmli, U. K. (1970) Nature 227,680-685 [Medline] [Order article via Infotrieve]
  15. Sanger, F, Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Sci. U. S. A. 74,5463-5467 [Abstract]
  16. Fickett, J. W. (1982) Nucleic Acids Res. 10,5303-5318 [Abstract]
  17. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162,156-159 [CrossRef][Medline] [Order article via Infotrieve]
  18. Jodal, M., Holmgren, S., Lundgren, O., and Sjöqvist, A. (1993) Gastroenterology 105,1286-1293 [Medline] [Order article via Infotrieve]
  19. Jennische, E., and Matejka, G. L. (1992) Acta Physiol. Scand. 146,79-86 [Medline] [Order article via Infotrieve]
  20. Lange, S. (1982) FEMS Microbiol. Lett. 15,239-242 [CrossRef]
  21. Johnston, M., Andrews, S., Brinkman, R., Cooper, J. et al. (1994) Science 265,2077-2082 [Medline] [Order article via Infotrieve]
  22. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157,105-132 [Medline] [Order article via Infotrieve]
  23. House, C., Baldwin, G. S., and Kemp, B. E. (1984) Eur. J. Biochem. 140,363-367 [Abstract]
  24. Taylor, S. S. (1989) J. Biol. Chem. 264,8443-8446 [Free Full Text]
  25. Edelman, A. M., Blumenthal, D. K., and Krebs, E. G. (1987) Annu. Rev. Biochem. 56,567-613 [CrossRef][Medline] [Order article via Infotrieve]

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