Title: The Caenorhabditis elegans Genes sqv-2and sqv-6, Which Are Required for Vulval Morphogenesis, Encode Glycosaminoglycan Galactosyltransferase II and Xylosyltransferase
Abstract: In mutants defective in any of eightCaenorhabditis elegans sqv (squashedvulva) genes, the vulval extracellular space fails to expand during vulval morphogenesis. Strong sqv mutations result in maternal-effect lethality, caused in part by the failure of the progeny of homozygous mutants to initiate cytokinesis and associated with the failure to form an extracellular space between the egg and the eggshell. Recent studies have implicated glycosaminoglycans in these processes. Here we report the cloning and characterization ofsqv-2 and sqv-6. sqv-6 encodes a protein similar to human xylosyltransferases. Transfection of sqv-6 restored xylosyltransferase activity to and rescued the glycosaminoglycan biosynthesis defect of a xylosyltransferase mutant hamster cell line. sqv-2 encodes a protein similar to human galactosyltransferase II. A recombinant SQV-2 fusion protein had galactosyltransferase II activity with substrate specificity similar to that of human galactosyltransferase II. We conclude that C. elegans SQV-6 and SQV-2 likely act in concert with other SQV proteins to catalyze the stepwise formation of the proteoglycan core protein linkage tetrasaccharide GlcAॆ1,3Galॆ1, 3Galॆ1,4Xylॆ-O-(Ser), which is common to the two major types of glycosaminoglycans in vertebrates, chondroitin and heparan sulfate. Our results strongly support a model in which C. elegans vulval morphogenesis and zygotic cytokinesis depend on the expression of glycosaminoglycans. In mutants defective in any of eightCaenorhabditis elegans sqv (squashedvulva) genes, the vulval extracellular space fails to expand during vulval morphogenesis. Strong sqv mutations result in maternal-effect lethality, caused in part by the failure of the progeny of homozygous mutants to initiate cytokinesis and associated with the failure to form an extracellular space between the egg and the eggshell. Recent studies have implicated glycosaminoglycans in these processes. Here we report the cloning and characterization ofsqv-2 and sqv-6. sqv-6 encodes a protein similar to human xylosyltransferases. Transfection of sqv-6 restored xylosyltransferase activity to and rescued the glycosaminoglycan biosynthesis defect of a xylosyltransferase mutant hamster cell line. sqv-2 encodes a protein similar to human galactosyltransferase II. A recombinant SQV-2 fusion protein had galactosyltransferase II activity with substrate specificity similar to that of human galactosyltransferase II. We conclude that C. elegans SQV-6 and SQV-2 likely act in concert with other SQV proteins to catalyze the stepwise formation of the proteoglycan core protein linkage tetrasaccharide GlcAॆ1,3Galॆ1, 3Galॆ1,4Xylॆ-O-(Ser), which is common to the two major types of glycosaminoglycans in vertebrates, chondroitin and heparan sulfate. Our results strongly support a model in which C. elegans vulval morphogenesis and zygotic cytokinesis depend on the expression of glycosaminoglycans. glycosaminoglycan galactose N-acetylgalactosamine glucuronic acid N-acetylglucosamine xylose Ehlers-Danlos syndrome open reading frame Chinese hamster ovary untranslated region cytomegalovirus Glycosaminoglycans (GAGs)1 are important in animal development, and defects in GAGs are responsible for certain human disorders. For example, mutations in the Drosophila melanogastger genes tout-velu (1Bellaiche Y. The I. Perrimon N. Nature. 1998; 394: 85-88Google Scholar) andsulfateless (2Lin X. Perrimon N. Nature. 1999; 400: 281-284Google Scholar), which encode homologs of heparan sulfate co-polymerase and heparan sulfateN-deacetylase/N-sulfotransferase, respectively, cause zygotic lethality and defects in segmentation. Mutations in the mouse tout-velu homolog EXT1 disrupt gastrulation and the generation of mesoderm (3Lin X. Wei G. Shi Z. Dryer L. Esko J.D. Wells D.E. Matzuk M.M. Dev. Biol. 2000; 224: 299-311Google Scholar), while mutations in human EXT1 and EXT2 have been associated with hereditary multiple exostoses (reviewed in Ref.4Zak B.M. Crawford B.E. Esko J.D. Biochim. Biophys. Acta. 2002; 1573: 346-355Google Scholar). Mutations in the human galactosyltransferase I have been associated with a progeroid variant of the connective-tissue disorder Ehlers-Danlos syndrome (EDS) (5Quentin E. Gladen A. Roden L. Kresse H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1342-1346Google Scholar, 6Almeida R. Levery S.B. Mandel U. Kresse H. Schwientek T. Bennett E.P. Clausen H. J. Biol. Chem. 1999; 274: 26165-26171Google Scholar, 7Okajima T. Fukumoto S. Furukawa K. Urano T. J. Biol. Chem. 1999; 274: 28841-28844Google Scholar). EDS is a group of heritable disorders characterized by hyperelasticity of the skin and hypermobile joints. Tout-velu, EXT-1, EXT-2, and Sulfateless affect the biosynthesis of heparan sulfate specifically, while galactosyltransferase I deficiency affects the biosynthesis of both chondroitin and heparan sulfate. The backbones of chondroitin and heparan sulfate consist of repeating disaccharide units: GlcAॆ1,3GalNAcॆ1,4 for chondroitin and GlcAॆ1,4GlcNAcα1,4 for heparan sulfate (reviewed in Ref. 8Esko J.D. Selleck S.B. Annu. Rev. Biochem. 2002; 71: 435-471Google Scholar). Their polymerization occurs on a tetrasaccharide primer (GlcAॆ1,3Galॆ1,3Galॆ1,4Xylॆ-) that is linked to the protein core of a proteoglycan. The addition of these four sugars is catalyzed stepwise in the lumen of the Golgi apparatus and requires three nucleotide sugars, UDP-Xyl, UDP-Gal, and UDP-GlcA, and four glycosyltransferases. Eight sqv (squashed vulva) genes were genetically identified in a screen for Caenorhabditis elegans mutants defective in vulval morphogenesis (9Herman T. Hartwieg E. Horvitz H.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 968-973Google Scholar). Allsqv mutants fail to form a large fluid-filled vulval extracellular space and have a reduced separation of the anterior and posterior halves of the vulva from the early to middle phases of L4 larval development. Strong mutant alleles of all eight sqvgenes also cause maternal-effect lethality. Most progeny of mothers homozygous for a strong sqv mutant allele arrest at the one-cell stage (9Herman T. Hartwieg E. Horvitz H.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 968-973Google Scholar). The nuclei of the arrested progeny divide normally, but the extrusion of the polar bodies and the initiation of cytokinesis are impaired (10Hwang H.-Y. Horvitz H.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14218-14223Google Scholar). These mutant eggs fail to form the normal fluid-filled extracellular space between the membrane of the egg and the eggshell. We have postulated that the sqv genes control the biosynthesis of GAGs that are secreted and become hydrated to form fluid-filled extracellular spaces (10Hwang H.-Y. Horvitz H.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14218-14223Google Scholar, 11Herman T. Horvitz H.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 974-979Google Scholar). The molecular identification of five sqv genes has led to a model implicating the biosynthesis of chondroitin and/or heparan sulfate in C. elegans development. sqv-1,-3, -4, -7, and -8 encode UDP-GlcA decarboxylase (10Hwang H.-Y. Horvitz H.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14218-14223Google Scholar), galactosyltransferase I (12Bulik D.A. Wei G. Toyoda H. Kinoshita-Toyoda A. Waldrip W.R. Esko J.D. Robbins P.W. Selleck S.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10838-10843Google Scholar), UDP-glucose dehydrogenase (13Hwang H.-Y. Horvitz H.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14224-14229Google Scholar), UDP-GlcA/UDP-Gal/UDP-GalNAc transporter (14Berninsone P. Hwang H.Y. Zemtseva I. Horvitz H.R. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3738-3743Google Scholar), and glucuronosyltransferase I (12Bulik D.A. Wei G. Toyoda H. Kinoshita-Toyoda A. Waldrip W.R. Esko J.D. Robbins P.W. Selleck S.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10838-10843Google Scholar), respectively. sqv-3 was used to identify the human galactosyltransferase I, which has been implicated in the progeroid variant of EDS (7Okajima T. Fukumoto S. Furukawa K. Urano T. J. Biol. Chem. 1999; 274: 28841-28844Google Scholar, 15Okajima T. Yoshida K. Kondo T. Furukawa K. J. Biol. Chem. 1999; 274: 22915-22918Google Scholar). In this paper, we show that sqv-6 encodes the xylosyltransferase that adds Xyl to the protein core, thus initiating GAG biosynthesis. sqv-2encodes a galactosyltransferase that adds the second Gal residue to the linkage tetrasaccharide. Strains were cultured as described (16Brenner S. Genetics. 1974; 77: 71-94Google Scholar) and were grown at 20–22 °C unless indicated otherwise. Standard molecular biological techniques were used (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The sequences of all PCR-amplified DNAs used for cloning were confirmed to exclude unintended mutations. Oligonucleotide sequences used for amplification or mutagenesis of DNA are shown in Supplementary Materials and Methods. For germ line rescue, we injected cosmids carrying genomic DNA intosqv-2(n2821) and sqv-6(n2845) unc-60(e677)/unc-34(s138) animals with the dominant roller marker pRF4, as described by Mello et al. (18Mello C.C. Kramer J.M. Stinchcomb D. Ambros V. EMBO J. 1991; 10: 3959-3970Google Scholar). Rol lines were established, and Rol animals and Unc-60 Rol animals were examined for rescue of the sqv-2 and the sqv-6 mutant phenotype, respectively. We injected sqv-2(n2821) hermaphrodites with plasmids containing the sqv-2 open reading frame (ORF) under the control of the C. elegansheat-shock promoters (19Stringham E.G. Dixon D.K. Jones D. Candido E.P. Mol. Biol. Cell. 1992; 3: 221-233Google Scholar) and pRF4 as the coinjection marker. We injected sqv-6(n2845)/nT1(n754) hermaphrodites with plasmids containing the sqv-6 ORF under the control of the C. elegans heat-shock promoters (19Stringham E.G. Dixon D.K. Jones D. Candido E.P. Mol. Biol. Cell. 1992; 3: 221-233Google Scholar) and pRF4. Rol lines were established, and Rol (non-Unc) animals were examined for rescue of thesqv-2 and sqv-6 mutant phenotype following induction of sqv-2 and sqv-6 expression by 30 min of heat-shock treatment at 33 °C. A sequence encoding amino acids 25–330 of SQV-2, thus lacking the presumptive transmembrane domain at the amino terminus, was cloned into pDEST-CMV-protA. This plasmid was designed to express a secreted fusion protein containing protein A and SQV-2 amino acids 25–330. COS7 cells were transiently transfected with pDEST-CMV-protA-sqv-2using LipofectAMINE (Invitrogen) according to the manufacturer's instructions. After 72 h of incubation, the fusion protein was recovered from the cell culture supernatant by affinity chromatography using IgG-agarose (20Wei Z. Swiedler S.J. Ishihara M. Orellana A. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3885-3888Google Scholar). Galactosyltransferase II activity was assayed as described by Bai et al. (21Bai X. Zhou D. Brown J.R. Crawford B.E. Hennet T. Esko J.D. J. Biol. Chem. 2001; 276: 48189-48195Google Scholar). The xylosyltransferase-deficient CHO pgsA-745 cells (22Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Google Scholar) were transfected with sqv-6 ORF, which was cloned into pcDNA3.1. Stable transfectants were selected with 400 ॖg/ml geneticin (Invitrogen). Several drug-resistant colonies were isolated and screened by flow cytometry forsqv-6 expression based on binding of biotinylated FGF-2 as described (23Bai X. Wei G. Sinha A. Esko J.D. J. Biol. Chem. 1999; 274: 13017-13024Google Scholar). Incorporation of 35SO4 into GAG chains of wild-type CHO or pgsA-745 cells with or withoutsqv-6 was assayed essentially as described by Bame and Esko (24Bame K.J. Esko J.D. J. Biol. Chem. 1989; 264: 8059-8065Google Scholar), labeling cells overnight at 30 °C with 50 ॖCi/ml35SO4 (PerkinElmer Life Sciences). Cell extracts of wild-type CHO, pgsA-745, and sqv-6 or empty vector stable transfectants of pgsA-745 were prepared as described previously (22Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Google Scholar). Xylosyltransferase activity was assayed essentially as described (22Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Google Scholar) by incubating 25 ॖg crude cell extract with 50 ॖg of soluble silk acceptor and 6 × 105 cpm UDP- [1-3H]xylose (PerkinElmer Life Sciences, 8.9 Ci/mmol) at 26 °C for 5 h. Product formation was dependent on the addition of silk. The concentration of substrate was saturating. sqv-2 was previously mapped to the left of lin-31 on LGII (25Herman T. Horvitz H.R. Cold Spring Harbor Symp. Quant. Biol. 1997; 62: 353-359Google Scholar). We further mapped sqv-2 to an interval between sup-9and lin-31 (see Supplementary Materials and Methods). We assayed 27 cosmids in this interval for the ability to rescue thesqv-2 mutant phenotype, but none showed rescuing activity (Fig. 1A). We examined the DNA sequence corresponding to the gaps between the cosmids in this interval and found a predicted gene, Y110A2AL.14, that is weakly similar to galactosyltransferases. Because all previously cloned sqv genes are implicated in the biosynthesis of chondroitin and/or heparan sulfate, we suspected that sqv-2also encodes a protein involved in GAG biosynthesis. Specifically, it seemed plausible that Y110A2AL.14 encodes the galactosyltransferase II involved in the formation of the protein core linkage tetrasaccharide and that had not been identified molecularly in any organism at the time. We identified three molecular lesions corresponding to three of the four identified alleles of sqv-2 in the ORF of Y110A2AL.14 (Fig. 1B). The two stronger alleles ofsqv-2, n3037 and n3038, cause a maternal-effect lethal phenotype and are an opal nonsense mutation at arginine 225 and a methionine-to-isoleucine missense mutation of the predicted start codon, respectively. A weak allele, n2826, that results in live progeny is a missense mutation causing a glycine-to-arginine substitution at amino acid position 99. The molecular lesion of the weakest allele, n2840, has not been identified yet. We determined the sequences of two cDNA clones, yk94e4 and yk292g2 (see Supplementary Materials and Methods), that correspond to Y110A2AL.14. The yk292g2 clone contains 990 bases of ORF, 17 bases of 5′-untranslated region (UTR), and 121 bases of 3′-UTR. The 5′ end contains three bases that correspond to the sequence of 5′ SL1trans-spliced leader, which is found at the 5′ end of manyC. elegans transcripts (26Krause M. Hirsh D. Cell. 1987; 49: 753-761Google Scholar). The 3′ end contains a poly(A) sequence. The longest ORF in this cDNA is identical to Y110A2AL.14 and is predicted to encode a protein of 330 amino acids. The yk94e4 clone lacks the 5′ end of Y110A2AL.14. Expression of the longest ORF in yk292g2 under the control of the C. elegans heat-shock promoters (19Stringham E.G. Dixon D.K. Jones D. Candido E.P. Mol. Biol. Cell. 1992; 3: 221-233Google Scholar) rescued the defect in sqv-2 vulval morphogenesis in all five isolated lines. The predicted SQV-2 protein contains a putative transmembrane domain near the amino terminus, suggesting it may be a type II transmembrane protein (Fig. 1B). Most known glycosyltransferases that act in the lumen of the ER and the Golgi apparatus are type II transmembrane proteins. Of 330 amino acids of SQV-2, 93 (287) and 133 (407) are identical to theDrosophila and human homologs, respectively (Fig.1B). Recently, the human homolog of SQV-2 was identified as galactosyltransferase II (21Bai X. Zhou D. Brown J.R. Crawford B.E. Hennet T. Esko J.D. J. Biol. Chem. 2001; 276: 48189-48195Google Scholar). We assayed recombinant protein A-SQV-2 fusion protein expressed in COS7 cells for galactosyltransferase II activity (see 舠Experimental Procedures舡). The SQV-2 fusion protein specifically catalyzed the addition of galactose to a disaccharide acceptor, Galॆ1,4Xylॆ-O-benzyl that had been used to demonstrate the acceptor substrate specificity of the human galactosyltransferase II (21Bai X. Zhou D. Brown J.R. Crawford B.E. Hennet T. Esko J.D. J. Biol. Chem. 2001; 276: 48189-48195Google Scholar) (TableI).Table IThe SQV-2 fusion protein has acceptor specificity consistent with its being galactosyltransferase IIAcceptor substratesEnzyme activitypmol/h/ml mediumMonosaccharides (5 mm) Xylॆ-O-Bn0 Xylॆ-O-naphthol0 Galॆ-O-NM0 GalNAcα-O-Bn0 GlcNAcॆ-O-NM0Disaccharides (5 mm) Galॆ1,4Xylॆ-O-Bn2660 Galॆ1,3GalNAcα-O-NM1 Galॆ1,3Galॆ-O-NM3 Galॆ1,4GlcNAcॆ-O-NM0 Galॆ1,3GlcNAcॆ-O-NM0 GlcNAcॆ1,3Galॆ-O-NM6 Manα1,6Manα-O-C100Galactosyltransferase activity was assayed in vitro using UDP-[3H]galactose together with various acceptor substrates. No substrate controls ranged from 339 to 357 cpm. The range for all substrates shown as “0” activity was 137–619 cpm. The range for the substrate Galβ1,4Xylβ-O-Bn was 141,000–142,000 cpm. Bn, benzyl; NM, naphthalenemethanol; C10, O-decenyl (CH2)8CH=CH2 Open table in a new tab Galactosyltransferase activity was assayed in vitro using UDP-[3H]galactose together with various acceptor substrates. No substrate controls ranged from 339 to 357 cpm. The range for all substrates shown as “0” activity was 137–619 cpm. The range for the substrate Galβ1,4Xylβ-O-Bn was 141,000–142,000 cpm. Bn, benzyl; NM, naphthalenemethanol; C10, O-decenyl (CH2)8CH=CH2 sqv-6 was previously mapped to the left of the polymorphism stP3 on LGV (9Herman T. Hartwieg E. Horvitz H.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 968-973Google Scholar). We further mapped sqv-6 to the left of the cosmid W07B8 and within about 0.2 map units of unc-34 (see Supplementary Materials and Methods). We assayed 11 cosmids to the right of unc-34 for the ability to rescue thesqv-6 mutant phenotype, but none showed rescuing activity (Fig. 2A). We examined the DNA sequences in the gaps in the cosmid coverage near the cosmid W07B8 and unc-34 and found a gene, Y50D4C.d, that is similar to two human xylosyltransferases (27Gotting C. Kuhn J. Zahn R. Brinkmann T. Kleesiek K. J. Mol. Biol. 2000; 304: 517-528Google Scholar). Using DNA from the only allele of sqv-6, n2845, we identified in the ORF of Y50D4C.d an amber nonsense mutation causing the elimination of the last 42 amino acids of the predicted protein product (Fig.2B). We determined the sequence of PCR-amplified cDNA and 5′-rapid amplification of cDNA ends products corresponding to Y50D4C.d (see Supplementary Materials and Methods). We found that this cDNA contains a 5′ SL1 trans-spliced leader, 23 bases of 5′-UTR, and 2418 bases of ORF, including two additional 5′ exons not in Y50D4C.d. The longest ORF in this cDNA including the additional exons is predicted to encode a protein of 806 amino acids. Expression of this ORF under the control of the C. elegans heat-shock promoters (19Stringham E.G. Dixon D.K. Jones D. Candido E.P. Mol. Biol. Cell. 1992; 3: 221-233Google Scholar) prior to the start of vulval morphogenesis rescued thesqv-6 vulval morphogenesis defect in all animals (n = 13) and the maternal-effect lethality of the progeny of sqv-6 homozygotes generated by +/sqv-6heterozygous parents for three of 13 sqv-6 homozygotes studied. Of the 806 amino acids of the SQV-6 protein, 182 (237) and 193 (247) are identical to human xylosyltransferases I and II, respectively (Fig.2B). Both the predicted SQV-6 protein and the human xylosyltransferase II contain a putative transmembrane domain near the amino terminus and are likely to be type II transmembrane proteins. Neither the start codon nor a presumptive transmembrane domain has been defined for human xylosyltransferase I (27Gotting C. Kuhn J. Zahn R. Brinkmann T. Kleesiek K. J. Mol. Biol. 2000; 304: 517-528Google Scholar). We tested the ability of sqv-6 to act as a xylosyltransferase by testing its ability to complement GAG-deficient CHO mutant cells lacking this enzymatic activity (22Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Google Scholar). Mutant pgsA-745 cells were transiently transfected with a plasmid containing sqv-6under the control of a cytomegalovirus (CMV) promoter. These cells showed partial rescue of the defect, as assayed by the restored abilities to incorporate 35SO4 into GAGs (16–277 that of the wild type) and to bind biotinylated FGF-2, which binds cell surface heparan sulfate as assayed by flow cytometry (data not shown). From these transiently transfected cells, we obtained a clonal cell line stably expressing sqv-6. This cell line showed full restoration of FGF-2 binding to heparan sulfate on the cell surface (Fig. 3A). Stable expression of sqv-6 in pgsA-745 cells enhanced the incorporation of 35SO4 into GAGs to ∼507 of wild-type levels, compared with 17 for the untreated mutant or mutant transfected with empty vector (Fig. 3B). The35SO4 incorporation into GAGs was similar in wild-type CHO cells and pgsA-745 cells transfected withsqv-6: 30–407 was released by treatment with chondroitinase ABC and 55–657 by a heparin lyase mixture in both cells, indicating that the composition of chondroitin and heparan sulfate was comparable. Expression of sqv-6 also resulted in restoration of xylosyltransferase activity, as measured by the transfer of xylose from UDP-xylose to a soluble silk acceptor (22Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Google Scholar), whereas pgsA-745 cells transfected with empty vector had virtually no activity (Fig. 3C). Our findings indicate thatsqv-2 and sqv-6 encode galactosyltransferase II and xylosyltransferase, respectively. With the previously identifiedsqv-3 galactosyltransferase I and sqv-8glucuronosyltransferase I, all four C. elegans genes responsible for the biosynthesis of the proteoglycan core protein linkage tetrasaccharide of chondroitin and heparan sulfate have now been defined (Fig. 4). Three previously identified genes, sqv-4 UDP-glucose dehydrogenase,sqv-1 UDP-GlcA decarboxylase and sqv-7UDP-GlcA/UDP-Gal/UDP-GalNAc transporter, act in earlier steps of GAG biosynthesis. All sqv genes identified to date affect the biosynthesis of both chondroitin and heparan sulfate. Based upon these observations, we conclude that in C. elegans early embryonic cytokinesis and epithelial invagination during vulval development depend on the expression of GAGs. We thank Beth Castor for help with DNA sequence determination, Ewa Davison and Ignacio Perez de la Cruz for communicating the locations of the lin-8 andsup-9 loci, respectively, and Mark Alkema and Melissa Harrison for helpful suggestions concerning this manuscript. Download .pdf (.01 MB) Help with pdf files