DNA binding and transcription activation by chicken interferon regulatory factor-3 (chIRF-3).

Interferon regulatory factors (IRFs) are a family of transcription factors involved in the cellular response to interferons and viral infection. Previously we isolated an IRF from a chicken embryonic liver cDNA library. Using a PCR-based binding site selection assay, we have characterised the binding specificity of chIRF-3. The optimal binding site (OBS) fits within the consensus interferon-stimulated response element (ISRE) but the specificity of chIRF-3 binding allows less variation in nucleotides outside the core IRF-binding sequence. A comparison of IRF-1 and chIRF-3 binding to ISREs in electrophoretic mobility shift assays confirmed that the binding specificity of chIRF-3 was clearly distinguishable from IRF-1. The selection assay also showed that chIRF-3 is capable of binding an inverted repeat of two half OBSs separated by 10-13 nt. ChIRF-3 appears to bind both the OBS and inverted repeat sites as a dimer with the protein-protein interaction requiring a domain between amino acids 117 and 311. In transfection experiments expression of chIRF-3 strongly activated a promoter containing the OBS. The activation domain was mapped to between amino acids 138 and 221 and a domain inhibitory to activation was also mapped to the C-terminal portion of chIRF-3.

Cloning and promoter analysis of the chicken interferon regulatory factor-3 gene.

Interferon regulatory factors (IRFs) are a family of DNA-binding proteins involved in mediating the cellular response to interferons (IFNs) and viral infection. Although extensively studied in mammals, IRFs of other vertebrates have been less well characterized. Previously, we cloned chicken interferon regulatory factor-3 (chIRF-3) mRNA, which is rapidly and transiently induced by double-stranded (ds)RNA. The chIRF-3 mRNA encodes a protein distinct from any known mammalian IRF. Here, we show that chIRF-3 is activated additively by type I and type II IFNs. To delineate the sequence elements required to regulate chIRF-3 expression, we cloned chlRF-3 and 0.48 kb of 5' flanking sequence. Computer analysis of the proximal promoter revealed three putative binding sites for nuclear factor (NF)-kappaB, two overlapping interferon-stimulated response elements (ISREs), and an interferon gamma activating sequence (GAS). The presence of both GAS and ISRE consensus sequences in the chIRF-3 promoter is unique among IRF family members. Both type I and II IFNs, as well as dsRNA and IRF-1, trans-activate the promoter in short-term transfection experiments. Mutational analysis of the promoter demonstrated that the putative NF-kappaB binding sites are needed for stimulation by dsRNA but not by either type I or type II IFN and that both the overlapping ISREs and GAS are required for full induction by type I or type II IFN.

Comparison of the functional characteristics of the nucleotide binding domains of multidrug resistance protein 1.

Multidrug Resistance Protein 1 (MRP1) transports diverse organic anionic conjugates and confers resistance to cytotoxic xenobiotics. The protein contains two nucleotide binding domains (NBDs) with features characteristic of members of the ATP-binding cassette superfamily and exhibits basal ATPase activity that can be stimulated by certain substrates. It is not known whether the two NBDs of MRP1 are functionally equivalent. To investigate this question, we have used a baculovirus dual expression vector encoding both halves of MRP1 to reconstitute an active transporter and have compared the ability of each NBD to be photoaffinity-labeled with 8-azido-[(32)P]ATP and to trap 8-azido-[(32)P]ADP in the presence of orthovanadate. We found that NBD1 was preferentially labeled with 8-azido-[(32)P]ATP, while trapping of 8-azido-[(32)P]ADP occurred predominantly at NBD2. Although trapping at NBD2 was dependent on co-expression of both halves of MRP1, binding of 8-azido-ATP by NBD1 remained detectable when the NH(2)-proximal half of MRP1 was expressed alone and when NBD1 was expressed as a soluble polypeptide. Mutation of the conserved Walker A lysine 684 or creation of an insertion mutation between Walker A and B motifs eliminated binding by NBD1 and all detectable trapping of 8-azido-ADP at NBD2. Both mutations decreased leukotriene C(4) (LTC(4)) transport by approximately 70%. Mutation of the NBD2 Walker A lysine 1333 eliminated trapping of 8-azido-ADP by NBD2 but, in contrast to the mutations in NBD1, essentially eliminated LTC(4) transport activity without affecting labeling of NBD1 with 8-azido-[(32)P]ATP.

Multidrug resistance protein. Identification of regions required for active transport of leukotriene C4.

Multidrug resistance protein (MRP) is a broad specificity, primary active transporter of organic anion conjugates that confers a multidrug resistance phenotype when transfected into drug-sensitive cells. The protein was the first example of a subgroup of the ATP-binding cassette superfamily whose members have three membrane-spanning domains (MSDs) and two nucleotide binding domains. The role(s) of the third MSD of MRP and its related transporters is not known. To begin to address this question, we examined the ability of various MRP fragments, expressed individually and in combination, to transport the MRP substrate, leukotriene C4 (LTC4). We found that elimination of the entire NH2-terminal MSD or just the first putative transmembrane helix, or substitution of the MSD with the comparable region of the functionally and structurally related transporter, the canalicular multispecific organic anion transporter (cMOAT/MRP2), had little effect on protein accumulation in the membrane. However, all three modifications decreased LTC4 transport activity by at least 90%. Transport activity could be reconstituted by co-expression of the NH2-terminal MSD with a fragment corresponding to the remainder of the MRP molecule, but this required both the region encoding the transmembrane helices of the NH2-terminal MSD and the cytoplasmic region linking it to the next MSD. In contrast, a major part of the cytoplasmic region linking the NH2-proximal nucleotide binding domain of the protein to the COOH-proximal MSD was not required for active transport of LTC4.

Closure of tracheoesophageal fistulas with chemotherapy and radiotherapy.

In patients who have esophageal cancer with a tracheoesophageal fistula, chemotherapy and radiotherapy are usually contraindicated because it is thought to enlarge the fistula. The records of 50 patients who had esophageal cancer and received simultaneous chemotherapy and radiotherapy from January 1992 to January 1997 were evaluated in the Medical Oncology Section of the Veterans Administration Medical Center, Washington, D.C. All patients were staged radiographically and endoscopically. Four patients developed a tracheoesophageal fistula while receiving treatment. One patient developed a fistula before treatment and another patient developed a fistula after treatment. Closure of the tracheoesophageal fistulas was achieved in 4 of 5 patients who responded to therapy and in those who developed fistulas before or during therapy. One of the patients whose fistula did not close died during therapy, whereas the other who developed a fistula after therapy underwent stenting. This finding indicates that development of a tracheoesophageal fistula is not a contraindication to chemotherapy and radiotherapy, and patients who are responsive to therapy may have closure of their fistulas.

Analysis of the intron-exon organization of the human multidrug-resistance protein gene (MRP) and alternative splicing of its mRNA.

Overexpression of multidrug-resistance protein (MRP) and P-glycoprotein confers similar but not identical multidrug-resistance phenotypes. However, unlike P-glycoprotein, which comprises two membrane-spanning domains (MSDs) and two nucleotide-binding domains, MRP contains a third NH2-proximal MSD, a feature now identified in several other ATP-binding cassette transmembrane transporters. MRP is located on chromosome 16 at band 13.1 close to the short-arm breakpoint of the pericentric inversion associated with the M4Eo subclass of acute myeloid leukemia. We have defined the intron-exon structure of MRP and characterized a number of splicing variants of MRP mRNA. The gene spans at least 200 kb. It contains 31 exons and a high proportion of class 0 introns, alternative splicing of which results in significant levels of variant transcripts that maintain the original open reading frame of MRP mRNA. Analyses of the conservation of intron-exon organization and protein primary structure suggest that the MRP-related transporters evolved from a common ancestor shared with the cystic fibrosis transmembrane conductance regulator, by fusion with one or more genes encoding polytopic membrane proteins.

Membrane topology of the multidrug resistance protein (MRP). A study of glycosylation-site mutants reveals an extracytosolic NH2 terminus.

Multidrug resistance protein, MRP, is a 190-kDa integral membrane phosphoglycoprotein that belongs to the ATP-binding cassette superfamily of transport proteins and is capable of conferring resistance to multiple chemotherapeutic agents. Previous studies have indicated that MRP consists of two membrane spanning domains (MSD) each followed by a nucleotide binding domain, plus an additional extremely hydrophobic NH2-terminal MSD. Computer-assisted hydropathy analyses and multiple sequence alignments suggest several topological models for MRP. To aid in determining the topology most likely to be correct, we have identified which of the 14 N-glycosylation sequons in this protein are utilized. Limited proteolysis of MRP-enriched membranes and deglycosylation of intact MRP and its tryptic fragments with PNGase F was carried out followed by immunoblotting with antibodies known to react with specific regions of MRP. The results obtained indicated that the sequon at Asn354 in the middle MSD is not utilized and suggested approximate sites of N-glycosylation. Subsequent site-directed mutagenesis studies established that Asn19 and Asn23 in the NH2-terminal MSD and Asn1006 in the COOH-terminal MSD are the only sites in MRP that are modified with N-linked oligosaccharides. N-Glycosylation of Asn19 and Asn23 provides the first direct experimental evidence that MRP has an extracytosolic NH2 terminus. This finding, together with those of previous studies, strongly suggests that the NH2-terminal MSD of MRP contains an odd number of transmembrane helices. These results may have important implications for the further understanding of the interaction of drugs with MRP.

Pharmacological characterization of the murine and human orthologs of multidrug-resistance protein in transfected human embryonic kidney cells.

Overexpression of the human multidrug-resistance protein (MRP) causes a form of multidrug resistance similar to that conferred by P-glycoprotein, although the two proteins are only distantly related. In contrast to P-glycoprotein, human MRP has also been shown to be a primary active transporter of a structurally diverse range of organic anionic conjugates, some of which may be physiological substrates. At present, the mechanism by which MRP transports these compounds and mediates multidrug resistance is not understood. With the objective of developing an animal model for studies on the normal functions of MRP and its ability to confer multidrug resistance in vivo, we recently cloned the murine ortholog of MRP (mrp). To assess the degree of functional conservation between mrp and MRP, we directly compared the drug cross-resistance profiles they confer when transfected into human embryonic kidney cells, as well as their ability to actively transport leukotriene C4, 17beta-Estradiol 17beta-(D-glucuronide), and vincristine; mrp and MRP conferred similar drug resistance profiles, with the exception that only MRP conferred resistance to the anthracyclines tested. Consistent with these findings, accumulation of [3H]vincristine and [3H]VP-16 was decreased, and efflux of [3H]vincristine was increased in both murine and human MRP-transfected cell populations, whereas only human MRP-transfected cells displayed decreased accumulation and increased efflux of [3H]daunorubicin. Membrane vesicles derived from both transfected cell populations transported leukotriene C4 in an ATP-dependent manner with comparable efficiency, although the efficiency of 17beta-estradiol 17beta-(D-glucuronide) transport was somewhat higher with MRP transfectants. ATP-dependent transport of vincristine was also observed with vesicles from mrp and MRP transfectants but only in the presence of glutathione. These studies reveal intrinsic differences between the murine and human MRP orthologs with respect to their ability to confer resistance to a major class of chemotherapeutic drugs.

Reconstitution of ATP-dependent leukotriene C4 transport by Co-expression of both half-molecules of human multidrug resistance protein in insect cells.

Multidrug resistance protein (MRP) confers a multidrug resistance phenotype similar to that associated with overexpression of P-glycoprotein. Unlike P-glycoprotein, MRP has also been shown to be a primary active ATP-dependent transporter of conjugated organic anions. The mechanism(s) by which MRP transports these compounds and increases resistance to natural product drugs is unknown. To facilitate studies on the structure and function of MRP, we have determined whether a baculovirus expression system can be used to produce active protein. Full-length MRP as well as molecules corresponding to either the NH2- or COOH-proximal halves of the protein were expressed individually and in combination in Spodoptera frugiperda Sf21 cells. High levels of intact and half-length proteins were detected in membrane vesicles from infected cells. Although underglycosylated, the full-length protein transported leukotriene C4 (LTC4) with kinetic parameters very similar to those of MRP produced in transfected HeLa cells. Neither half-molecule was able to transport LTC4. However, a functional transporter with characteristics similar to those of intact protein could be reconstituted when both half-molecules were co-expressed. Transport of LTC4 by Sf21 membrane vesicles containing either intact or reconstituted MRP was competitively inhibited by both S-decylglutathione and 17beta-estradiol 17-(beta-D-glucuronide), with Ki values similar to those reported previously for MRP expressed in HeLa cells (Loe, D. W., Almquist, K. C., Deeley, R. G., and Cole, S. P. C. (1996) J. Biol. Chem. 271, 9675-9682; Loe, D. W., Almquist, K. C., Cole, S. P. C., and Deeley, R. G. (1996) J. Biol. Chem. 271, 9683-9689). These studies demonstrate that human MRP produced in insect cells can function as an active transporter of LTC4 and that the NH2- and COOH-proximal halves of the protein can assemble efficiently to form a transporter with functional characteristics similar to those of the intact protein.

Functional expression of the multidrug resistance-associated protein in the yeast Saccharomyces cerevisiae.

The multidrug resistance-associated protein (MRP) is a member of the ATP binding cassette superfamily of transporters which includes the mammalian P-glycoproteins (P-gp) family. In order to facilitate the biochemical and genetic analyses of MRP, we have expressed human MRP in the yeast Saccharomyces cerevisiae and have compared its functional properties to those of the mouse Mdr3 P-gp isoform. Expression of both MRP and Mdr3 in the anthracycline hypersensitive mutant VASY2563 restored cellular resistance to Adriamycin in this mutant. MRP and Mdr3 expression produced pleiotropic effects on drug resistance in this mutant, as corresponding VASY2563 transformants also acquired resistance to the anti-fungal agent FK506 and to the K+/H+ ionophore valinomycin. The appearance of increased cellular resistance to the toxic effect of Adriamycin (ADM) in MRP and Mdr3 transformants was concomitant with a reduced intracellular accumulation of [14C]ADM in spheroplasts prepared from these cells. Moreover, MRP and Mdr3, but not control spheroplasts, could mediate a time-dependent reduction in the overall cell-associated [14C]ADM from preloaded cells, suggesting the presence of an active ADM transport mechanism in MRP and Mdr3 transformants. Finally, human MRP was found to complement the biological activity of the yeast peptide pheromone transporter Ste6 and partially restored mating in a sterile ste6 null mutant. These findings suggest that despite their relatively low level of structural homology, MRP and P-gp share similar functional aspects, since both proteins can mediate transport of chemotherapeutic drugs and the a mating peptide pheromone in yeast.

Expression of multidrug resistance-associated protein (MRP) mRNA in blast cells from acute myeloid leukemia (AML) patients.

A specific and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay was developed for measuring the mRNA of the multidrug resistance-associated protein (MRP). A region corresponding to bp 3897-4471 of MRP cDNA is amplified, which encompasses approximately half of the second nucleotide-binding domain (NBD2). In two multidrug resistant (MDR) sublines of the HL-60 human acute myeloid leukemia (AML) cell line which overexpress MRP but not P-glycoprotein, the assay detects elevated levels of MRP mRNA (4- to 8-fold) relative to the drug-sensitive parental cells (designated HL-60/W). Blast cells from 24 patients with AML were also studied for MRP expression using this RT-PCR method. Expression of MRP was normalized for that of beta-actin in the blast cells, which was also determined by RT-PCR. All of these blast cell samples had MRP expression that was detectable after 35 PCR cycles. Eighteen of these patients samples had levels of expression of MRP mRNA equal to or less than that expressed by HL-60/W cells. In six patient blast cell specimens, the expression of MRP mRNA was up to 1.7-fold higher than that of HL-60/W cells. In 21 specimens, the steady-state intracellular accumulation of daunorubicin (1 microgram/ml, 3h) was also determined. The blast cells with MRP mRNA expression higher than HL-60/W had a lower median accumulation of daunorubicin compared to those whose MRP expression was less than HL-60/W, suggesting a functional defect in drug transport in the cells with higher MRP expression; a similar trend toward lower daunorubicin accumulation was also noted in the one-third of samples that displayed the highest expression of MDR1 mRNA (also determined by RT-PCR). These studies illustrate the range of expression of MRP in AML blast cell specimens. The identification of MRP overexpression in MDR AML cell lines and in some AML patient blast cells with low intracellular daunorubicin accumulation warrants further study of MRP as a component of clinical drug resistance in AML.

cIRF-3, a new member of the interferon regulatory factor (IRF) family that is rapidly and transiently induced by dsRNA.

In mammals, some of the effects of interferon (IFN) on gene transcription are known to be mediated by a family of IFN-inducible DNA-binding proteins, the IFN regulatory factor (IRF) family, which includes both activators and repressors of transcription. Although IFN activities have been described in many vertebrates, little is known about regulation of IFN- or IFN-stimulated genes in species other than human and mouse. Here, we report the cloning of a chicken cDNA, cIRF-3, encoding a protein with a DNA-binding domain similar to that found in the mammalian IRF family of proteins. Similarity between cIRF-3 and the mammalian IRFs is comparable with that between known members of the family. It is most similar to the IRF proteins ICSBP and ISGF3 gamma but is equally divergent from both. Gel mobility shift assays indicate that cIRF-3 is capable of binding a known IFN-stimulated response element that is conserved between the mammalian and chicken Mx genes. Expression of the cIRF-3 gene can be induced to high levels by poly(I).poly(C). Induction is rapid and transient with no requirement for protein synthesis. Co-treatment of cells with cycloheximide results in superinduction of cIRF-3 mRNA. The structural and regulatory characteristics of cIRF-3 indicate that it is the first example of a non-mammalian IRF protein.

Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells.

We have previously identified and characterized a novel member of the ATP-binding cassette superfamily of transport proteins, multidrug resistance protein (MRP), and subsequently demonstrated that its overexpression is sufficient to confer multidrug resistance on previously sensitive cells (Cole et al., Science (Washington DC), 258: 1650-1654, 1992; Grant et al., Cancer Res. 54: 357-361, 1994). In the present study, we have transfected two different eukaryotic expression vectors containing MRP complementary DNA into HeLa cells to study the pharmacological phenotype produced exclusively by overexpression of human MRP. The drug resistance patterns of the two MRP-transfected cell populations were similar. They were characterized by a moderate (5- to 15-fold) level of resistance to doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide, and a low (< or = 3-fold) level of resistance to taxol, vinblastine, and colchicine. The transfectants were not resistant to 9-alkyl anthracyclines, mitoxantrone, or cisplatin. The MRP-transfected cells were also resistant to some heavy metal anions including arsenite, arsenate, and trivalent and pentavalent antimonials but were not resistant to cadmium chloride. Accumulation of radiolabeled vincristine was reduced by 45% in the MRP-transfected cells and could be restored to the levels found in sensitive cells by depletion of ATP. Rates of vincristine efflux did not differ greatly in the sensitive and resistant cells. The cytotoxic effects of vincristine and doxorubicin could be enhanced in a dose-dependent fashion by coadministration of verapamil. Cyclosporin A also increased vincristine toxicity but had less effect on doxorubicin toxicity. The degree of chemosensitization by verapamil and cyclosporin A was similar in MRP-transfected cells and in cells transfected with the vector alone, suggesting that sensitization involved mechanisms independent of MRP expression. Verapamil and cyclosporin A caused a modest increase in vincristine accumulation in the resistant cells but did not restore levels to those of the sensitive cells. Taken together, these data indicate that drug-resistant cell lines generated by transfection with MRP complementary DNA display some but not all of the characteristics of MRP-overexpressing cell lines produced by drug selection in vitro. They further demonstrate that the multidrug resistance phenotype conferred by MRP is similar but not identical to that conferred by P-glycoprotein and includes resistance to arsenical and antimonial oxyanions.

Evaluation of two commercial systems for identification of coagulase-negative staphylococci to species level.

Bloodstream (224) and urine (nine) isolates of coagulase-negative staphylococci (CNS) were evaluated using MicroScan Pos ID and Rapid Pos ID panels. A modification of the conventional method of Kloos and Schleifer served as the reference method. The isolates were selected to include a broad range of CNS species, including 44 S. epidermidis, 50 S. hominis, 39 S. warneri, 33 S. capitis, 21 S. haemolyticus, 12 S. simulans, 11 S. saprophyticus, six S. cohnii, five S. lugdunensis, three S. xylosus, four S. auricularis, two S. schleiferi, two S. intermedius, and one S. sciuri. The Pos ID panel had an overall rate of agreement (correct plus probably correct) with the reference method of 79%, including 95% for S. epidermidis, 95% for S. haemolyticus, 64% for S. hominis, 67% for S. simulans, 79% for S. warneri, and 100% for S. saprophyticus. The Rapid Pos ID panel had an overall rate of agreement (correct plus probably correct) of 76%, including 91% for S. epidermidis, 90% for S. haemolyticus, 64% for S. hominis, 58% for S. simulans, 77% for S. warneri, and 100% for S. saprophyticus. Both systems are acceptable for the identification of the clinically significant species S. haemolyticus, S. saprophyticus, and S. epidermidis, but are less reliable for the infrequently isolated species of CNS.

Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs.

Amplification of the gene encoding multidrug resistance-associated protein (MRP) and overexpression of its cognate mRNA have been detected in multidrug-resistant cell lines derived from several different tumor types. To establish whether or not the increase in MRP is responsible for drug resistance in these cell lines, we have transfected HeLa cells with MRP expression vectors. The transfectants display an increase in resistance to doxorubicin that is proportional to the levels of a M(r) 190,000, integral membrane protein recognized by anti-MRP antibodies. The transfectants are also resistant to vincristine and VP-16 but not to cisplatin. The results demonstrate that MRP overexpression confers a multidrug resistance phenotype similar to that formerly associated exclusively with elevated levels of P-glycoprotein.

Cloning and characterization of chicken YB-1: regulation of expression in the liver.

A cDNA expression library constructed from day 9 embryonic liver was screened with a previously identified protein binding site in the flanking region of the liver-specific, estrogen-dependent avian apoVLDLII gene. Two of the clones isolated were shown to encode the chicken homolog of the Y-box binding protein, YB-1 (dbpb), which we have designated chkYB-1. This protein was originally identified in avian extracts by virtue of its ability to bind to two reverse CCAAT motifs in the Rous sarcoma virus enhancer. Since its identification, additional nucleic acid binding properties have been ascribed to its homologs, or closely related proteins, in other species. We have determined the sequence of chkYB-1, investigated its ability to bind to sites known to be involved in tissue-specific expression in the liver, and examined factors influencing its hepatic expression. These studies have demonstrated that the level of chkYB-1 mRNA in the liver decreases steadily throughout embryogenesis and for several weeks posthatching until adult levels are attained. We present several lines of evidence that YB-1 expression in the liver is positively associated with DNA synthesis or cell proliferation. Its binding characteristics indicate that the protein can interact specifically with a number of binding sites for liver-enriched or specific factors. In addition, although it is not particularly asymmetric in terms of base composition, we find a marked preference in binding to the pyrimidine-rich strand of these sites regardless of the presence or polarity of an intact CCAAT box. The increased levels of expression of YB-1 during proliferation combined with its binding characteristics suggest that it may be involved in the reduced expression of liver-specific genes observed at early stages of development or during liver regeneration.

Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line.

The doxorubicin-selected lung cancer cell line H69AR is resistant to many chemotherapeutic agents. However, like most tumor samples from individuals with this disease, it does not overexpress P-glycoprotein, a transmembrane transport protein that is dependent on adenosine triphosphate (ATP) and is associated with multidrug resistance. Complementary DNA (cDNA) clones corresponding to messenger RNAs (mRNAs) overexpressed in H69AR cells were isolated. One cDNA hybridized to an mRNA of 7.8 to 8.2 kilobases that was 100- to 200-fold more expressed in H69AR cells relative to drug-sensitive parental H69 cells. Overexpression was associated with amplification of the cognate gene located on chromosome 16 at band p13.1. Reversion to drug sensitivity was associated with loss of gene amplification and a marked decrease in mRNA expression. The mRNA encodes a member of the ATP-binding cassette transmembrane transporter superfamily.

Isolation and characterization of cDNA clones encoding polypeptides related to a Dictyostelium discoideum cyclic AMP binding protein.

By screening a cDNA library with a cDNA encoding the Dictyostelium discoideum cAMP-binding protein CABP1, under conditions of reduced stringency, we have isolated clones which code for two closely related molecules. Hybrid selection experiments indicated that these cDNAs encoded polypeptides with molecular masses of 34 (p34) and 31 (p31) kDa, both of which were recognized by anti-CABP1 monoclonal antibodies. Sequence analysis revealed that the clones were identical except for the presence of a 102 nucleotide segment inserted in-frame in the p34 cDNAs, just downstream of the translation initiation codon. DNA blot analysis suggested that p34 and p31 were encoded by the same gene. This hypothesis was strongly supported by the observation that both polypeptides were generated when a single cDNA was expressed under the control of the actin 15 promoter in D. discoideum cells. RNA blot analysis indicated that the cDNAs were complementary to three developmentally regulated transcripts of sizes 1.15 kb, 1.25 kb and 1.4 kb. Comparison of the derived amino acid sequences of p34 and p31 with those of the two subunits of CABP1 indicated that these polypeptides were very closely related, and that the corresponding genes probably arose by duplication followed by sequence divergence. Finally, the carboxy termini of these four polypeptides demonstrated 50% similarity to two polypeptides encoded by a bacterial plasmid which confers resistance to tellurium anions.