Structure of tanacetan,a pectic polysaccharide from tansy Tanacetum vulgare L

Tanacetan TVF was found to have a branched structure with a backbone of linear -1,4-D-galacturonan. The ramified regions consist of linear -1,2-L-rhamno--1,4-D-galacturonan as the core. The side chains appear to attach to the 4-position of the L-rhamnopyranose residues. They are present as single -galactopyranose residues or a branching -1,4-galactopyranan bearing 4,6-substituted -D-galactopyranose residues as branched points. In addition, the ramified regions contain side chains of a branched -1,5-arabinofuranan possessing 2,5- and 3,5-substituted -L-arabinofuranose residues as branching points. Some side chains of rhamnogalacturonan appear to be arabinogalactan which contains branched sugar chains of -1,5-arabinofuranan attached to the linear chains of -1,4-galactopyranan by 1,3- and 1,6-linkages. The residues of -L-arabinofuranose seem to occupy the terminal positions of the arabinogalactan side chains.     

Quantitative analysis of type IV collagen alpha chains in the basement membrane of human urogenital epithelium

Type IV collagen is a major component of the basement membrane (BM), which consists of six genetically distinct (IV) chains. In this study the expression of these six (IV) chains was demonstrated immunohistochemically. In addition, the 2(IV) and 5(IV) chains were analysed quantitatively by confocal laser scanning microscopy in human urogenital epithelial BM. The 1/2(IV) and 5/6(IV) chains were immunoreactive in the epithelial BM, whereas, 3/4(IV) chains were not. The quantitative analysis revealed that the amount of 2(IV) and 5(IV) chains differed in each urogenital epithelial BM. The content of 5(IV) chains in the epithelial BM of the bladder was differentially high, and that of the foreskin was differentially low. It is concluded that the elasticity of epithelial BM of the bladder may be structurally related to the high content of 5/6(IV) chains.     

Long-range physical mapping of the alpha-amylase-1 (alpha-Amy-1) loci on homoeologous group 6 chromosomes of wheat

Summary Long-range physical maps of the small multigene family of the malt -amylase genes (-Amy-1) located on the long arms of wheat chromosomes 6A (the -Amy-A1 locus) and 6B (-Amy-B1) were generated by pulsed-field gel electrophoresis analysis. By using three methylation-sensitive rare-cutter restriction endonucleases, NotI, NruI and MluI, and an -Amy-1 cDNA probe and four gene-specific genomic probes from the -Amy-B1 locus, the size of the -Amy-B1 locus was estimated to be about 700 kb and of the -Amy-B1 locus to be about approximately 4300 kb. These two maps indicate clustering of GC-rich and C-methylation-sensitive restriction enzyme recognition sites. At least five regions reminiscent of CpG islands are apparent in -Amy-B1, and three in -Amy-A1. Correlation between recombination frequency and physical distance within the -Amy-B1 locus suggests that 1 cM approximates to 1 Mb in physical distance.     

A base substitution at IVS-19 3′-end splice junction causes exon 20 skipping in proα2(I) collagen mRNA and produces mild osteogenesis imperfecta

Molecular investigations on a young patient and her family were undertaken to identify the molecular defect responsible for a mild form of osteogenesis imperfecta (OI) with blue sclerae, dentinogenesis imperfecta and joint laxity. Analysis of collagenous proteins from the proband's fibroblasts showed the presence of two populations of 2(I) chains, one normal and one migrating faster on SDS gels, thereby suggesting deletion of amino acid sequences. The faster migrating chains were retained mainly in the cell layer and not found in the extracellular matrix deposited by cultured fibroblasts. Chemical cleavage of mismatch (CCM) analysis on the patient's pro2(I) mRNA:normal cDNA heteroduplexes localized the molecular defect. cDNA sequencing revealed a deletion of exon 20 (54 bp) in about half of the molecules. Genomic DNA sequencing revealed heterozygosity for a G-to-C transversion of the last nucleotide of intron 19, which changed the 3 consensus splicing site. As a consequence pro2(I)mRNA was abnormally spliced from the last codon of exon 19 to the first codon of exon 21. To our knowledge, this is the first acceptor site mutation so far described in an OI patient. Restriction analysis indicated that the mutation was present also in three other affected family members. The full sequence of COL1A2 introns 19 and 20 are reported.     

Phylogenetic Analysis of Vertebrate Fibrillar Collagen Locates the Position of Zebrafish α3(I) and Suggests an Evolutionary Link Between Collagen α Chains and Hox Clusters

Type I collagen in tetrapods is usually a heterotrimeric molecule composed of two 1 and one 2 chains. In some teleosts, a third chain has been identified by chromatography, suggesting that type I collagen should also exist as an 1(I)2(I)3(I) heterotrimer. We prepared, from zebrafish, three distinct cDNAs identified to be those of the collagen 1(I), 2(I), and 3(I) chains. In this study on the evolution of fibrillar collagen chains and their relationships, an exhaustive phylogenetic analysis, using vertebrate fibrillar collagen sequences, showed that each chain constitutes a monophyletic cluster. Results obtained with the newly isolated sequences of the zebrafish showed that the 3(I) chain is phylogenetically close to the 1(I) chain and support the hypothesis that the 3(I) chain arose from a duplication of the 1(I) gene. The duplication might occur during the duplication of the actinopterygian genome, soon after the divergence of actinopterygians and sarcopterygians, a hypothesis supported by the demonstration of a syntenic evolution between a set of fibrillar collagen genes and Hox clusters in mammals. An evolutionary scenario is proposed in which phylogenetic relationships of the chains of fibrillar collagens of vertebrates could be related to Hox cluster history. Present address (Laure Bonnaud): Institut Jacques Monod, Tour 43, CNRS, Universités Paris 6 et 7, Equipe Evolution du Développement des Nématodes, 2 place Jussieu, 75005 Paris, France     

ααααanti-3.7 type II: a new α-globin gene rearrangement suggesting that the α-globin gene duplication could be caused by intrachromosomal recombination

Summary We report here a new human -globin gene rearrangement carrying the two normal, 2 and 1, and two hybrid, 1/2, globin genes in the order 5-2-1/2-1/2-1-3. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the -thalassaemia IVS 1 nt 1 GA mutation caused thalassaemia intermedia. We also report a case of an -globin gene rearrangement in the twin of one of the -globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an -anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the -globin gene rearrangements could be of the intra-chromosomal rather than the interchromosomal type.     

Comparative studies of α-lactalbumin and lysozyme: The proteins of kangaroo (Megaleia rufa and Macropus giganteus) and horse (Equus caballus)

Summary As part of a study of the whey proteins of various mammals, a comparison is made of the -lactalbumins and lysozymes of the kangaroo and horse. In the milk of the red kangaroo (Megaleia rufa) there is only one -lactalbumin and it occurs throughout lactation, but no lysozyme has been detected. There are two -lactalbumins in the milk of the grey kangaroo (Macropus giganteus), one, designated -lactalbumin Zone B, is present throughout lactation; the second, designated -lactalbumin Zone A, is present only in late lactation. One lysozyme is also present. The milk of the horse (Equus caballus) contains one -lactalbumin and at least one lysozyme. Partial amino acid sequences are proposed from sequence determination and from analyses of tryptic peptides compared with the known sequences of other -lactalbumins and lysozymes.     

Regional chromosome mapping of human collagen genes alpha 2(I) and alpha 1(I) (COLIA2 and COLIA1)

Summary For the assignment of the genes for the pro-2(I) (COLIA2) and the pro-1(I) (COLIA1) collagens, cDNA and genomic DNA probes were used in in situ hybridization experiments on human prometaphase chromosomes. An improved staining method is reported for the simultaneous identification of chromosomes and the autoradiographic grains after the hybridization procedures. With this procedure more cells with higher resolution could be used for the assignment of genes by in situ hybridization. Statistical analysis of the grains located on respectively 660 and 302 metaphases using pro-2(I) and pro1(I) DNA probes, confirmed the assignment of these genes to human chromosomes 7 and 17. Analysis of the grain distribution on prometaphase chromosomes showed that the location of the pro2(I) collagen gene is in the region 7q21.3–22.1. The location of the pro-1(I) collagen gene was found to be in band 17q21.31–2005.     

Heterogeneity of the hemoglobin of the Ohrid trout (Salmo L. typicus)

We have analyzed the hemoglobins of five individual trout from the Ohrid Lake (Salmo L. typicus) by electrophoretic methods, by reversed-phase high-performance liquid chromatography, and by limited structural analyses. The two major classes of hemoglobin are type I (35% of total) and type IV (65%). Type IV is the major oxygen-transporting hemoglobin; it consists of three types of chain (in about equal quantities) and three types of chain (one major and two minor types). Several structural differences have been observed between these three (IV) chains and between the three (IV) chains, suggesting a complex genetic system governing the synthesis of these proteins. Moreover, a few amino acid substitutions occur at positions involved in contacts between chains, which suggests that differences in oxygen affinity may exist between these various type IV hemoglobins. Type I hemoglobin is less complex because it contains one type of chain and two chains; the latter two differ in numerous positions, suggesting duplications of the (I)-globin gene. The and chains of type I hemoglobin differ considerably from the and chains of type IV hemoglobin, indicating the existence of (I)- and (I)-globin genes separate from the (IV)- and (IV)- globin genes.This study was supported in part by the Yugoslav-American Joint Funds, pp 812 (to G.D.E.), and by United States Public Health Service Research Grant HLB-05168 (to T.H.J.H.).     

Two forms of α-glucosidase from sugar-beet seeds

Two forms of -glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from sugarbeet (Beta vulgaris L.) seeds by a procedure including fractionation with ammonium sulfate and ethanol, carboxymethyl-cellulose column chromatography, and preparative disc gel electrophoresis. The two enzymes were homogeneous by polyacrylamide disc gel electrophoresis. Their molecular weights were 98,000 (I) and 60,000 (II). -Glucosidase I readily hydrolyzed maltose, isomaltose, kojibiose, maltotriose, panose, amylose, soluble starch, amylopectin and glycogen. -Glucosidase II also hydrolyzed maltose, kojibiose and maltotriose but hydrolyzed the other substrates only very weakly or not at all. -Glucosidase I hydrolyzed soluble starch at a faster rate than maltose. It produced isomaltose and panose as the main -glucosyltransfer products from maltose, whereas maltotriose was the main product of -glucosidase II. -Glucosidase I hydrolyzed amylose liberating -glucose. The neutral-sugar content was calculated to be 2.7% for -glucosidase I and 8.8% for -glucosidase II. The main neutral sugar was mannose in -glucosidase I, and glucose in -glucosidase II.     

The sialyl-α2,6-lactosaminyl-structure: Biosynthesis and functional role

Sialylation represents one of the most frequently occurring terminations of the oligosaccharide chains of glycoproteins and glycolipids. Sialic acid is commonly found ,3- or ,6-linked to galactose (Gal), ,6-linked to N-acetylgalactosamine (GalNAc) or ,8-linked to another sialic acid. The biosynthesis of the various linkages is mediated by the different members of the sialyltransferase family. The addition of sialic acid in ,6-linkage to the galactose residue of lactosamine (type 2 chains) is catalyzed by -galactoside ,6-sialyltransferase (ST6Gal.I). Although expressed by a single gene, this enzyme shows a complex pattern of regulation which allows its tissue- and stage-specific modulation. The cognate oligosaccharide structure, NeuAc,6Gal1,4GIcNAc, is widely distributed among tissues and is involved in biological processes such as the regulation of the immune response and the progression of colon cancer. This review summarizes the current knowledge on the biochemistry of ST6Gal.I and on the functional role of the sialyl-,6-lactosaminyl structure.     

α-Thalassemia in Saudi Arabia: deletion pattern

Summary -Thalassemia exists at a high prevalence in several regions of Saudi Arabia. The restriction endonucleases Bam HI and BglII were used to investigate the molecular basis of deletion type of -thalassemia in 226 subjects from the eastern and 61 subjects from the northwestern regions of the country. The arrangements-/ and-/- were common. BglII digestion revealed the existence of rightward deletion in a majority of the cases. Leftward deletions, both homozygous and heterozygous, were also identified. Triple -gene arrangements -/ and -/- were observed at a low frequency in both regions.     

Detection of a new hybrid α2 globin gene among American Blacks

Summary We have examined the globin gene complex for 49 individuals with -thalassemia-2 (–^3.7). Crossovers resulting in -thalassemia-2 (type I) were observed in all 57 chromosomes with the –^3.7 defect. Except for one -thalassemia-2 chromosome, all were linked to the absence of an Rsa I restriction site located 0.7 kb 5 to the 2-globin gene; this polymorphic site was observed for 10 of 38 non--thalassemia chromosomes from Black Americans. In four Black families with a heterozygous -thalassemia-2 [–^3.7 (I)], an Apa I restriction site has been identified in the IVS-2 of the 2 gene of the normal chromosome (labeled the ^*2 gene). The ^*2 gene of one Black subject was cloned and a segment located 5 to the Cap site as well as the IVS-2, exon 3, and a 3 segment were sequenced. The data show that the ^*2 gene is an 2 gene except for a segment between nucleotides (nts) 580–81 and nt 509 (Cap site=nt 1), and perhaps as far upstream as nt-634, which has an 1 sequence. This ^*2 hybrid gene probably originated through a double crossover; the structural identity of its IVS-2 with that of the 1 gene adequately explains the presence of the Apa I restriction site.     

Somatic cell mapping of the bovine interferon-α receptor

The bovine interferon- receptor (BoIFN-R) mediates the activity of bovine IFN-s and IFN-. In addition, human IFN-s have uniformly high biological activity on bovine cells. A ³²P-labeled derivative of human recombinant IFN-A (HuIFN-A-P1) binds well and can form a characteristic 130-kDa complex on bovine cells, but not on hamster cells. We have, therefore, analyzed the binding and covalent crosslinking of [³²P]HuIFN-A-P1 to a panel of bovine-hamster somatic cell hybrids. Binding to several bovine-hamster hybrid cell lines was strong (about 30–50% of that seen with bovine MDBK cells) and specific. The binding correlated uniquely with bovine syntenic group U10. In several of the hybrid lines, the ability of human IFN-B to enhance the expression of endogenous MHC class I molecules correlated with the binding results. We thus conclude that the bovine IFN-R structural gene (locus designation IFNAR) localizes to syntenic group U10. This group includes a number of other genes whose homologs map to human Chromosome (Chr) 21.A summary of this work was presented at the annual meeting of the International Society for Interferon Research (November 1991, Nice, France) and appeared as an abstract for that meeting (Langer et al., J Interferon Res 11 (Suppl): S203, 1991).     

SSCP detection of a Gly565Val substitution in the proα(I) collagen chain resulting in osteogenesis imperfecta type II

A patient with perinatal lethal osteogenesis imperfecta (OI) type II has been studied in order to identify the causative mutation. By analysis of the type I collagen produced by cultured fibroblasts from the patient, the defect was mapped to 1 cyanogen bromide peptide 7, a region corresponding to 271 amino acid residues of either the 1(I) or 2(I) collagen chains. Polymerase chain reaction (PCR) amplification of the corresponding region of the 1(I) mRNA followed by single-strand conformation polymorphism analysis of restriction enzyme digestions of the PCR products allowed further mapping of the mutation to a small region of COL1A1. A heterozygous transversion of G to T within the last glycine codon of exon 32 was identified by DNA sequence analysis. This resulted in the substitution of glycine-565 by a valine residue, disrupting the repeating Gly-Xaa-Yaa sequence that is obligatory for correct formation of the collagen molecule. The mutation was shown to have occurred de novo and is thought to result in the OI phenotype.     

Physicochemical characterization of alpha-crystallins from bovine lenses: hydrodynamic and conformational properties

A detailed investigation of hydrodynamic and conformational behavior has been made of the HM-crystallin and -crystallins of bovine lens. Results from this study indicated that HM (high-molecular-weight -crystallin) and (low-molecular-weight -crystallin) possess considerable size and charge heterogeneities in their native structures and subunit polypeptides, respectively. Sedimentation velocity showed a heterogeneous polydisperse system of HM with an average sedimentation coefficient of about 50 S and a more homogeneous system of -crystallin of 20 S. Viscosity and circular dichroism studies pointed to a compact and globular shape of dominant -sheet conformation for -crystallin, yet a highly asymmetrical and aggregated form for HM. The conformational stability of -crystallin was investigated in the presence of various denaturants. The evidence presented shows that hydrogen bonding is the main force in maintaining the quaternary structure of compact native -crystallin. Conformational flexibility of -crystallin demonstrated in the equilibrium unfolding study indicated a multistep transition that made the extraction of thermodynamic data from the heat denaturation study difficult. Temperature perturbation on -crystallin suggested the possible involvement of hydrophobic interaction in the aggregation process, leading to the formation of HM from -crystallin. The comparison of conformational properties between HM and -crystallin strongly indicated that HM is a denatured form of -crystallin.     

Primary structure of alpha-globin chains from river buffalo (Bubalus bubalis L.) hemoglobins

Primary structure analysis of the four river buffalo -globin chains showed that haplotypes A and B differ from each other by a substitution at codon 64 that may encode Ala or Asn. The A haplotype encodes two -globin chains, ^I1 and ^II3, which differ at positions 129 and 131: ^I1 has 64 Ala, 129 Phe, 131 Asn; ^II3 has 64 Ala, 129 Leu, 131 Ser. The B haplotype encodes two -globin chains, ^I2 and ^II4, which differ at positions 10 and 11: ^I2 has 10 Ile, 11 Gln, 64 Asn; ^II4 has 10 Val, 11 Lys, 64 Asn. Apart from the Ala/Asn polymorphism at position 64, amino acid substitutions in allelic and nonallelic -globin chains seem to have arisen by single point mutations. Detection of electrophoretically silent mutations due to neutral amino acid substitutions and their influence on the isoelectric point are discussed. Furthermore, primary structures of river buffalo -globin chains are compared to other species of the Bovidae family to suggest evolutionary events that have characterized the amino acid substitutions of river buffalo hemoglobin.     

The complete primary structure of the marine carnivora, galapagoes fur seal (Arctocephalus galapagoensis, Otariidae) hemoglobins

The complete primary structure of the two hemoglobin components of the fur seal (Arctocephalus galapagoensis) is presented. The two components (HbI and HbII) occur in nearly equal amounts and have identical -chains; whereas the two -chains (I/II) differ by six exchanges Ile/Val, Met/Thr, Ser/Ala, Pro/His, Lys/Gly, and Thr/Ala at positions 10, 34, 35, 50, 78, and 131, respectively. The components were isolated by DEAE-Sephacel chromatography and were separated into the globin chains by RP-HPLC on a column of Nucleocil-C4. The sequences have been determined by Edman degradation in liquid- and gas-phase sequencer, using the native chains and tryptic peptides. The sequences compared with those of other Carnivora species and an adult human globin chains. An identical -chain is found in fur seal and walrus, whereas larger differences were found between I and II compared to -chains.Deceased on May 27, 1989.     

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin

Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.