was placed in a 5% CO2 incubator at 37°C. After 3.5 h, cells were fixed with 0.2% ..... by The William H. Welch Medical Library of The Johns Hopkins. University ...
Journal of Cell Science 106, 401-409 (1993) Printed in Great Britain © The Company of Biologists Limited 1993
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Cell binding specificity of mouse R-cadherin and chromosomal mapping of the gene Hiroaki Matsunami1, Seiji Miyatani1, Takayoshi Inoue1, Neal G. Copeland2, Debra J. Gilbert2, Nancy A. Jenkins2 and Masatoshi Takeichi1,* 1Department 2Mammalian
of Biophysics, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-01, Japan Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA *Author for correspondence
SUMMARY R-cadherin was originally identified as a chicken cadherin expressed by the retina. Here, we describe the identification of a mouse homologue of R-cadherin. We isolated mouse cDNAs encoding a cadherin with 94% identity in amino acid sequence to the chicken R-cadherin, and defined this molecule as mouse R-cadherin. L cells transfected with the mouse R-cadherin cDNA acquired a cadherin-mediated cell-cell adhesiveness as found for other cadherins. To examine the binding specificity of mouse R-cadherin, L cells expressing this cadherin (mRL) were mixed with L cells expressing chicken R-cadherin (cRL), mouse N-cadherin (mNL), mouse E-cadherin (mEL) and mouse P-cadherin (mPL). While mRL cells randomly intermixed with cRL cells,
INTRODUCTION Cadherins are a family of homophilic cell-cell adhesion molecules that are crucial not only for physical cell-cell associations but also for selective cell-cell adhesion (Takeichi, 1991). This family contains more than 15 members, and some of the members have been well characterized for their cell-cell binding specificities (Nose et al., 1988; Friendlander et al., 1989; Miyatani et al., 1989; Inuzuka et al., 1991a). These include E-, P-, N- and R-cadherin. When L cells are transfected with cDNAs of these cadherins and mixed with each other, they form separate aggregates, indicating that each cadherin type has a homophilic binding specificity. There was, however, an exception, in that cells expressing N- and R-cadherin, both derived from the chicken, formed chimeric aggregates although they tended to segregate within the aggregates (Inuzuka et al., 1991a). This phenomenon was explained by relatively high sequence similarity between N- and R-cadherin (74%) as compared to that between others (43 to 58%). Selective cell adhesion based on these cadherin specificities was implicated in a variety of morphogenetic phenomena (Takeichi, 1988, 1991). In the cell mixing experiments mentioned above, cell
those cells aggregated separately from mEL or mPL cells. Mixing of mRL with mNL cells gave an intermediate result; that is, they formed both separate and chimeric aggregates, suggesting that R- and N-cadherin can interact with each other although each has a preference to bind to its own type. Similar properties were previously found for chicken R-cadherin. Thus, the cell binding specificity of R-cadherin is entirely conserved between the two species, suggesting a conserved role for this protein in morphogenesis. We also located the mouse R-cadherin gene to chromosome 2. Key words: cadherins, cell adhesion, cell sorting, chromosomal mapping
combinations were made beyond species, as E- and P-cadherin cDNAs were isolated from the mouse and R-cadherin was identified in the chicken, although N-cadherin cDNAs were cloned from both species. The binding specificity of these cadherins, therefore, eventually has to be determined using the same species, to exclude the possibility of speciesspecific differences in their binding specificities. In the present study, we identified a mouse R-cadherin homologue. The mouse R-cadherin had 94% identity to the chicken R-cadherin and its cell binding specificity was conserved between the species. We also found that the mouse R-cadherin gene was located on chromosome 2, although the mouse N-cadherin gene was mapped to chromosome 18.
MATERIALS AND METHODS Isolation of mouse R-cadherin cDNAs cDNA fragments of mouse R-cadherin were initially isolated as polymerase chain reaction (PCR) products as follows. Total RNAs were prepared from 14-day fetal mouse brains by a modification of the method of Chirgwin et al. (1979). cDNAs were synthesized from these RNAs with the oligo(dT) primer by reverse transcrip-
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tase. PCR was then carried out by a method similar to that of Suzuki et al. (1991). The primers used for PCR were selected from two highly conserved amino acid sequences located in the cytoplasmic region of the known cadherins. The nucleotide sequences of the two primers were identical to those used by Suzuki et al. (1991). EcoRI linkers were added to the 5′ ends of the primers for subsequent subcloning. PCR conditions were chosen as follows. Denaturation was performed at 95°C for 1.5 min, annealing at 45°C for 2 min, and polymerization at 75°C for 3 min. The reaction was initiated by adding two units of Taq DNA polymerase (Toyobo, Japan), after which 35 reaction cycles were carried out. After PCR, the reaction products were digested with EcoRI, separated by 2% low-melting agarose gel electrophoresis. Only a single band of ~160 nt was found in this electrophoresis. The DNA band was purified from the gel by the phenol extraction method (Maniatis et al., 1982). The purified DNAs were subcloned into pUC18 vector, and 200 clones were isolated and sequenced. Among them, 11 clones encoded R-cadherin, and one of these PCR fragments, radiolabelled by the random primer labeling kit (Takara, Japan), was used for screening a λgt10 cDNA library of 14-day fetal mouse brain poly(A)+ RNA (Miyatani et al., 1989). A 2.1 kb Rcadherin cDNA fragment (λMR1) was thus obtained. For the second screening of a λgt10 cDNA library of newborn mouse eye poly(A)+ RNA, the 0.7 kb EcoRI-BglII fragment of λMR1 was used, and λMR4 was isolated. Sequencing of double-stranded DNA was carried out using the Sequenase Kit (U.S. Biochemical, USA).
RNA blot analysis A 5 µg sample of poly(A)+ RNA isolated from newborn mouse brains was resolved in a 1% agarose-formaldehyde gel. RNA was transferred to nitrocellulose filters and hybridized at 42°C in a buffer containing 30% formamide with a probe of the 32P-labeled λMR1 insert prepared with the Random Primer DNA Labeling Kit (Takara, Japan). After hybridization, the filters were washed at 65°C in 0.1× SSC and 0.1% SDS.
cDNA transfection The expression vector of mouse R-cadherin cDNA, pMiwMR, was constructed as follows. λMR4 was inserted into pBluescript SK(+) (Stratagene, USA) to yield pBSMR4. With the HindIII-SmaI fragment of this pBSMR4, the HindIII-HpaI fragment encoding the CAT gene of the expression vector pMiwCAT (Kato et al., 1990) was replaced to make pMiwMR. L cells, which express no cadherins (Nagafuchi et al., 1987), were cotransfected with pMiwMR and the neomycin-resistant gene pBATneo (Nagafuchi and Takeichi, 1988) by the calcium phosphate precipitation method, as described previously (Nagafuchi et al., 1987). After selection with G418, cells expressing Rcadherin proteins were cloned. One line of these cells, mRL12, was used for the following experiments.
Cells and cultures mRL12 and L cell lines expressing mouse E-cadherin (ELβ1) (Nose et al., 1988), mouse P-cadherin (PLβ2) (Nose et al., 1988), mouse N-cadherin (mNLβ1) (Miyatani et al., 1989), chicken Ncadherin (cNLm1) (Fujimori et al., 1990) and chicken R-cadherin (RLβ3) (Inuzuka et al., 1991a) were used for mixed cell aggregation assays. Cells were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium supplemented with 10% fetal calf serum (DH10).
Mixed cell aggregation To label cells fluorescently, those in monolayer cultures were incubated with 15 µg/ml DiO (Molecular Probes, USA) in DH10 for 12 h. The cells were then dispersed by treatment with a mixture of 0.05% trypsin (Difco 1:250, USA) and 1 mM EDTA for 5 min at 37°C. (Cadherins were temporarily removed from the cell surface by this treatment, but they were restored during the following incubation.) These cells were mixed with unlabeled cells, which were prepared by the same trypsin treatment, in which the cell numbers were adjusted to 2×105/ml. A 2 ml sample of this cell suspension was placed in a 3.5 cm dish with DH10 and incubated
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Fig. 1. Mouse R-cadherin cDNA clones and the translation product. (A) cDNAs isolated and the predicted molecular organization of mouse R-cadherin. E, EcoRI; B, BamHI; Bg, BglII. PRE, precursor region; EC, extracellular domain of the mature form; TM, transmembrane domain: CP, cytoplasmic domain. (B) Northern blot analysis of R-cadherin mRNA. A 8.2 kb transcript is detected (arrow). (C) Western blot analysis of a lysate of L cells transfected with mouse Rcadherin cDNA. An antiserum raised against a fusion protein of R-cadherin was used for detecting the antigens. Arrow indicates the major 126 kDa band.
Mouse R-cadherin on a gyratory shaker rotating at 80 rpm (Marysol KS6320) that was placed in a 5% CO2 incubator at 37°C. After 3.5 h, cells were fixed with 0.2% glutaraldehyde for 5 min. The cell aggregates formed were placed on glass slides and observed by fluorescence microscopy (Zeiss, Germany). A hundred aggregates, each composed of 5 to 20 cells, were selected randomly, and the numbers of aggregates consisting of only labeled or non-labeled cells, and of aggregates containing both cell populations, were counted.
Antibodies to mouse R-cadherin Antibodies to mouse R-cadherin were prepared by generating a fusion protein as the antigen. The BamHI fragment of mouse R-
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cadherin cDNA, encoding 111 amino acids from position 480 to 590, was inserted into the BamHI site of pGEX-3X vector (Pharmacia, Sweden). Expression of the fusion protein was induced by adding isopropyl-β-D-thiogalactopyranoside to Escherichia coli cultures. Bacterial extracts were prepared by sonicating the induced bacteria in phosphate-buffered saline, and the proteins in the extracts were separated by electrophoresis in 12.5% polyacrylamide gels containing 0.1% SDS. After brief staining with Coomassie blue, the fusion proteins were electroeluted from the gels, precipitated with acetone, and dialyzed against Trisbuffered saline supplemented with 1 mM CaCl 2 (TBS-Ca). The purified fusion proteins were emulsified with complete Freund’s
Fig. 2. Nucleotide and deduced amino acid sequences. The nucleotide sequence is numbered starting with the first base in λMR4.
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Fig. 3. Sequence similarity of mouse R-cadherin to chicken R-cadherin and mouse N-cadherin. The predicted amino acid sequences of chicken R-cadherin (cR), mouse R-cadherin (mR), and mouse N-cadherin (mN) are compared. In the stippled regions, amino acid residues are identical between mR and either cR or mN. SIG, signal peptide; PRE, precursor region; EC1-5, extracellular domain; TM, transmembrane domain; CP, cytoplasmic domain. Asterisks, stop codons. adjuvant, and injected into rabbits. The animals were boosted three times with the antigens mixed with incomplete Freund’s adjuvant, and antisera were collected 7-10 days after the last boosting. Antibodies were affinity-purified to the mouse R-cadherin fusion protein as described previously (Inuzuka et al., 1991a).
Immunoblot analysis Proteins of mRL12 cells were separated by SDS-PAGE (7.5% polyacrylamide) and transferred to nitrocellulose filters. After incubating the blots with anti-mouse R-cadherin antibodies and then with HRP-linked anti-rabbit Ig, signals were detected by the ECL Western blotting system (Amersham, UK).
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Immunocytochemistry mRL12 cells were fixed with Carnoy’s solution (75% ethanol and 25% acetic acid). The fixed cells were rinsed with TBS-Ca and incubated with 5% skim milk in TBS-Ca. Then, the samples were treated with anti-mouse R-cadherin antibodies, biotinylated antirabbit Ig and Texas Red dye-coupled streptavidin (Amersham, UK). After washing with TBS-Ca, the samples were mounted in 90% glycerol containing 1 mg/ml paraphenylenediamine. These samples were examined using a Zeiss fluorescence microscope.
Interspecific backcross mapping Interspecific backcross progeny were generated by mating (C57BL/6J × Mus spretus)F1 females and C57BL/6J males as described (Copeland and Jenkins, 1991). A total of 205 N2 progeny were obtained; a random subset of these N2 mice was used to map the R-cadherin gene (Rcad) locus (see text for details). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer and hybridization were performed essentially as described (Jenkins et al., 1982). All blots were prepared with Zetabind nylon membrane (AMF-Cuno). The probe, a 2.1 kb EcoRI fragment of mouse R-cadherin cDNA, was labeled with [α-32P]dCTP using a nick-translation labeling kit (Boehringer Mannheim, Germany); washing was done to a final stringency of 0.4× SSCP, 0.1% SDS, 65°C. Fragments of 5.3, 4.2, 1.7 kb were detected in KpnI-digested C57BL/6J DNA and fragments of 5.3, 2.4, 1.8 and 1.5 kb were detected in KpnI-digested M. spretus DNA. The presence or absence of the 2.4, 1.8 and 1.5 kb M. spretus-specific KpnI fragments, which cosegregated, was followed in backcross mice. A description of the probes and restriction fragment length polymorphisms (RFLPs) for the loci linked to Rcad including Rous sarcoma virus proto-oncogene (Src), adenosine deaminase (Ada) and guanine nucleotide binding protein, alpha stimulating (Gnas) has been reported previously (Siracusa et al., 1989; Wilkie et al., 1992). Recombination distances were calculated as described (Green, 1981) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.
RESULTS AND DISCUSSION Isolation of mouse R-cadherin cDNA Among the PCR products obtained as described in Materials and Methods, one DNA fragment encoding a polypeptide with 100% amino acid sequence identity to the chicken R-cadherin was chosen and used to screen a fetal mouse brain cDNA library (Miyatani et al., 1989). One positive clone, λMR1, was isolated and sequenced. λMR1 showed extensive similarity to the chicken R-cadherin cDNA, but it was only 2.1 kb and lacked the 5′ end of the open reading frame. We, therefore, further screened a cDNA library of newborn mouse eye poly(A)+ RNA using λMR1, and isolated a longer clone, λMR4. Restriction maps and partial sequences of these two clones were identical, indicating that they were overlapping clones (Fig. 1A). RNA blot analysis using λMR1 detected a 8.2 kb band from newborn mouse brain RNA (Fig. 1B). The complete nucleotide sequence of the insert of λMR4 was determined (Fig. 2). The longest open reading frame started with a methionine codon at nucleotide 55 and ended with a TAA stop codon at nucleotide 2,794. The putative protein encoded by this cDNA clone had 913 amino acids,
Fig. 4. Mouse R-cadherin transfectants. (A) Phase-contrast photomicrograph of untransfected L cells. (B) Phase-contrast photomicrograph of mRL12 cells, which adhere to each other. (C) Immunofluorescence detection of R-cadherin in the same field as in (B). Bar, 50 µm.
consisting of a signal peptide, a precursor peptide and a mature protein region, which was divided by a single transmembrane region. The overall identity between the deduced amino acid sequence of this protein and that of chicken Rcadherin.(Inuzuka et al., 1991a) was 94%, and the identity in the extracellular and cytoplasmic regions was 93% and 96%, respectively. On the basis of this high sequence similarity, we concluded that the encoded molecule was a mouse homologue of R-cadherin. The overall amino acid sequence identity between R-cadherin and N-cadherin of the mouse was 72%; the identities at the extracellular and cytoplasmic domains were 69% and 82%, respectively. Amino acid residues identical between these two cadherins are shown in Fig. 3. Compared to the
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Fig. 5. Mixed aggregation of L cells expressing different cadherins. mRL12 cells expressing mouse R-cadherin were labeled with DiO, mixed with unlabeled L cells transfected with different cadherins, and incubated for 3.5 h. (A) mRL12 cells + ELβ1 cells expressing mouse E-cadherin. (B) mRL12 cells + PLβ2 cells expressing mouse P-cadherin. (C) mRL12 cells + mNLβ1 cells expressing mouse Ncadherin. (D) mRL12 cells + cNLm1 cells expressing chicken N-cadherin. (E) mRL12 cells + unlabeled mRL12 cells. (F) mRL12 cells + RLβ3 cells expressing chicken R-cadherin. Note segregation in (A) and (B), partial segregation in (C) and (D), and random intermixing in (E) and (F) of DiO-labeled and unlabeled cells. Bar, 100 µm.
relatively high similarity to N-cadherin, the mouse R-cadherin was less similar to mouse E- and P-cadherins: 49% identical to E-cadherin and 47% identical to P-cadherin. Establishment of L cell lines expressing mouse Rcadherin L cells without endogenous cadherin activity were trans-
fected with mouse R-cadherin cDNA using pMiwMR. The morphology of the transfected cell lines was altered as a result of the cDNA transfection, as observed for other cadherin cDNA transfectants. While the parent L cells do not form tight intercellular associations, the mouse R-cadherin transfectants were closely connected to each other, forming cell clusters (Fig. 4A, B). Immunoblotting analysis using
Mouse R-cadherin antibodies to R-cadherin detected the major 126 kDa protein (Fig. 1C). Also, these antibodies stained cell-cell boundaries in the transfected cell lines (Fig. 4C). Untransfected cells were completely negative by this staining. Binding specificity of mouse R-cadherin In order to determine the cell binding specificity of mouse R-cadherin, one of the mouse R-cadherin-transfected lines, mRL12, was mixed with L cell lines expressing mouse Ecadherin (ELβ1), mouse P-cadherin (PLβ2), mouse N-cadherin (mNLβ1), chicken N-cadherin (cNLm1) and chicken R-cadherin (RLβ3). These cells showed similar aggregation rates and similar levels of cadherin. To distinguish between two cell lines that were mixed together, mRL12 cells were fluorescently pre-labeled with DiO. In these mixtures, mRL12 cells aggregated separately from ELβ1 and PLβ2 cells, indicating that R-cadherin did not interact with E- and P-cadherin (Fig. 5A, B). When mRL12 cells were mixed with mNLβ1 or cNLm1 cells, both separate and chimeric aggregates were formed (Fig. 5C, D). This result suggests that R- and N-cadherin can bind to each other, but their homophilic binding affinities are higher than the heterophilic binding affinity. When mRL12 cells were mixed with RLβ3 cells, the two cell lines randomly intermixed forming chimeric aggregates, indicating that the mouse and chicken R-cadherin shared the same binding specificity (Fig. 5F). These results were confirmed by quantitative measurements (Fig. 6). R- and N-cadherin are differentially expressed in the neural tube and also in other parts of the nervous system (Inuzuka et al., 1991a, b). In chicken central nervous system (CNS), the localization of these two molecules has been studied in detail (Redies et al., 1992, 1993). Results of these studies demonstrated that these molecules were expressed by different cell groups or by distinct nuclei in the brain stem. Interestingly, cell groups expressing R- and N-cad-
Fig. 6. Ratio of chimeric aggregates to total aggregates formed in mixtures of mRL12 cells expressing mouse R-cadherin, and L cells expressing mouse E-cadherin (mE), mouse P-cadherin (mP), mouse N-cadherin (mN), chicken N-cadherin (cN), mouse Rcadherin (mR) or chicken R-cadherin (cR). Cells were cultured as described for Fig. 6, and % chimeric aggregates were measured as described in Materials and Methods. Each value represents the mean of duplicate cultures ± s.e.
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herin are often located in overlapping areas. In certain cases, axons express N-cadherin, and glial cells, which sup port the migration of these axons, express R-cadherin; for example, optic axons express N-cadherin and glial cells in the optic stalk express R-cadherin (Redies and Takeichi, 1993). Our recent observations indicate that, also in the mouse, R-cadherin is expressed by restricted cell groups, in particular zones of the neural tube (H. Matsunami and M. Takeichi, unpublished), and N-cadherin is distributed in wider areas (C. Redies and M. Takeichi, unpublished). Such an arrangement of R- and N-cadherin-expressing cells could at least in part be controlled by the binding specificity of each adhesion molecule. Cells expressing R-cadherin should be able to associate with cells expressing N-cadherin, but each cell group may maintain homotypic clusters via its own specificity. This may be a mechanism by which heterogeneous cell types could segregate to form a continuous layer. Chromosomal localization of the R-cadherin gene The mouse chromosomal location of Rcad was determined by interspecific backcross analysis using progeny derived from matings of [(C57BL/6J × Mus spretus)F1 × C57BL/6J] mice. This interspecific backcross mapping panel has been typed for over 1200 loci that were well distributed among all the autosomes as well as the X chromosome (Copeland and Jenkins, 1991). C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative RFLPs using a mouse cDNA Rcad probe. The 2.4, 1.8 and 1.5 kb M. spretus KpnI RFLPs (see Materials and Methods) were used to follow the segregation of the Rcad locus in backcross mice. The mapping results indicate that Rcad is located in the distal region of mouse chromosome 2 linked to Src, Ada and Gnas (Fig. 7). Although 111 mice were analyzed for every marker and are shown in the segregation analysis (Fig. 7), up to 153 mice were typed for some pairs of loci. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are: centromere - Src - 4/150 Ada - 11/139 - Gnas - 4/153 - Rcad. The recombination frequencies (expressed as genetic distances in centiMorgans (cM) + the standard error) are - Src - 2.7 ± 1.3 - Ada - 7.9 ± 2.3 Gnas - 2.6 ± 1.3 - Rcad. We compared our interspecific map of chromosome 2 with a composite mouse linkage map that reports the map location of many uncloned mouse mutations (compiled by M.T. Davisson, T.H. Roderick, A.L. Hillyard, and D.P. Doolittle, and provided from GBASE, a computerized database maintained at The Jackson Laboratory, Bar Harbor, ME). Rcad mapped in a region of the composite map that lacks mouse mutations with a phenotype that might be expected, from alteration in this locus (data not shown). The distal region of mouse chromosome 2 shares a region of homology with human chromosome 20q (summarized in Fig. 7). In particular, Gnas has been placed on human 20q 13.2 - ql3.3. The tight linkage between Gnas and Rcad in mouse suggests that Rcad will reside on 20q, as well. The placement of Rcad on mouse chromosome 2 demon-
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H. Matsunami and others National Cancer Institute, DHHS, under contract NOl-CO-74101 with ABL. The GenBank accession number for the mouse R-cadherin cDNA sequence is D14888.
REFERENCES
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Fig. 7. Rcad maps in the distal region of mouse chromosome 2. Rcad was placed on mouse chromosome 2 by interspecific backcross analysis. The segregation patterns of Rcad and flanking genes in 111 backcross animals that were typed for all loci are shown at the top of the figure. For individual pairs of loci, more than 111 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J × M. spretus) F1 parent. The filled boxes represent the presence of a C57BL/6J allele and open boxes represent the presence of a M. spretus allele. The numbers of offspring inheriting each type of chromosome are listed at the bottom of each column. A partial chromosome 2 linkage map showing the location of Rcad in relation to linked genes is shown at the bottom of the figure. Recombination distances between loci in centiMorgans are shown to the left of the chromosome and the positions of loci in human chromosomes, where known, are shown to the right. References for the map positions of most human loci can be obtained from GDB (Genome Data Base), a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).
strated that it was unlinked to other cadherin structural genes thus far identified. We have shown previously that Pcad and Ecad are tightly linked on mouse chromosome 8 (Hatta et al., 1991) and Ncad resides in the proximal region of mouse chromosome 18 (Miyatani et al., 1992). The genes of L-CAM and K-CAM, chicken cadherins, are also arranged in tandem (Sorkin et al., 1991). N- and R-cadherin are exceptionally similar to each other in amino acid sequence compared to other cadherins, but this similarity is not correlated with their gene localization. This finding suggests a complex history in the evolution of this molecular family. We thank M. Hatta, I. Gamou, T. Uemura, K. Shimamura for technical advice. We also thank B. Cho for excellent technical assistance. This research was supported by research grants from the Ministry of Education, Science and Culture of Japan, a grant from the Human Frontier Science Program, and, in part, by the
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Korenberg, J. R., Trask, B. J., de Jong, P., Reed, R. R., Simon, M. I., Jenkins, N. A. and Copeland, N. G. (1992). Evolution of the mammalian G protein α subunit multigene family. Nature Genet. 1, 8591. (Received 14 April 1993 - Accepted 28 May 1993)
