High frequency of triplicated α-globin loci and absence or low frequency of α thalassemia in Polynesian Samoans

Summary Most of the population in certain areas of Melanesia have one -globin gene deletion ( thal₂). It is thought that the high frequencies of thal₂ in this population is due to a selective advantage given by malaria infection to carriers of thal₂. We are interested in neighboring Polynesia which, although adjacent to Melanesia, has always been free of malaria due to the absence of the vector anopheles. We studied 60 Polynesian Samoans and 150 Malaysians by restriction endonuclease gene mapping using Eco RI, Bam HI, and Bgl II and hybridization to ³²P-labeled -globin gene probe. Seven among the 60 (11.7%) Samoans had triplicated -globin loci type 1, while none had thal₂. On digestion with Bgl II the third -globin gene was found in an additional 3.7kb fragment in all seven Samoans with triplicated -globin loci, while digestion with Bam HI produced an abnormal elongated 18.2 kb fragment carrying -globin genes in addition to the normal 14.5 kb fragment. None of the Polynesian Samoans had thal₂ or thal₁. Only two of the Malaysians had triplicated -globin loci.     

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.     

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.     

An H1 histone gene from rainbow trout (Salmo gairdnerii)

Summary Although the major types of vertebrate collagen have a number of structural properties in common, significant DNA sequence homologies have not been detected between different portions of the helical coding domains within the same gene or between different genes. However, under non-stringent hybridization conditions we found considerable cross-homology within and between 1(I) and 2(I) chick cDNAs in the coding regions for helical sequences. Detailed analyses at the DNA sequence level have led us to propose that the gene for chick pro 2(I) collagen arose from a 9-bp primordial sequence. A consensus sequence for the 9-bp repeat was derived: GGTCCTCCT, which codes for a Gly-Pro-Pro triplet. The primordial ancestor of this 9-bp unit, GGTCCTXCT, apparently underwent duplication and divergence. Each resulting 9-bp sequence was triplicated to form a 27-bp domain, and a condensation event produced a 54-bp domain. This genetic unit then underwent multiple rounds of amplification to form the ancestral gene for the full-length helical section of 2(I). A different 9-bp consensus sequence (GGTCCCCCC) seems to have been the basis of the chick pro 1(I) gene.     

Über die Eignung von Naphthyl-α-l-fucosiden zur Untersuchung der α-l-Fucosidasen

Zusammenfassung Verglichen mit 1- und 2-Naphthyl--d-glucosid,--d-galactosid,--d-glucuronid,--d-N-acetylglucosaminid,--d-glucosid,--d-galactosid und--d-mannosid werden 1- und 2-Naphthyl--l-fucosid schneller oder im gleichen Ausmaß von Homogenaten verschiedener Rattenorgane hydrolysiert. Trotzdem fällt der histochemische Nachweis der -l-Fucosidasen methodenunabhängig im Gegensatz zu dem der anderen Glykosidasen überwiegend negativ aus. Ursache dafür ist die massive Hemmung der -l-Fucosidase durch Aldehydfixation und Diazoniumsalze; die Inhibitionsrate liegt bei 90% bzw. zwischen 85 und 98%; die - und -d-Glucosidase, - und -d-Galactosidase, -d-Mannosidase, -d-Glucuronidase sowie -d-N-Acetylglucosaminidase werden durch Aldehydfixation oder Kuppler höchstens zu 70% gehemmt. Daher können 1- und 2-Naphthyl--l-fucosid für die histochemische Darstellung der -l-Fucosidase nicht einschränkungslos empfohlen werden. Kleine Mengen Dimethylformamid hemmen die meisten Glykosidasen nicht.Für biochemische Messungen der -l-Fucosidase eignet sich speziell 1-Naphthyl--l-fucosid und läßt sich an Stelle von p-Nitrophenyl--l-fucosid werwenden. Bei der fluorometrischen Untersuchung der -l-Fucosidase in Rattenorganen mit dem 2-Naphthylderivat ergeben sich bemerkenswerte Aktivitätsunterschiede.

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Suitability of naphthyl--l-fucosides for the investigation of -l-fucosidases

Summary In comparison with 1- and 2-naphthyl -d-glucoside, -d-galactoside, -d-glucuronide, -d-N-acetylglucosaminide, -d-glucoside, -d-galactoside and -d-mannoside 1- and 2-naphthyl -l-fucoside are hydrolyzed more quickly or to the same extent by homogenates prepared from freezedried cryostate sections of various rat organs. Nevertheless, when the fucosides are employed for the histochemical demonstration of -l-fucosidase mostly negative data were obtained independent on the method used, whereas all other naphthyl glycosides deliver positive results. The reasons for these discrepancies are the marked inhibition of -l-fucosidase by aldehyde fixation and diazonium salts. Then, -l-fucosidase activity is suppressed to 90% and between 85 and 98% respectively; the inhibition of - and -d-glucosidase, - and -d-galactosidase, -d-mannosidase, -d-glucuronidase and -d-N-acetylglucosaminidase by the fixative or coupling reagent does not exceed 70%. Therefore 1- and 2-naphthyl -l-fucoside cannot be recommended in general for histochemical purposes. Small amounts of dimethylformamide do not influence the activity of most of the glycosidases investigated.For biochemical measurements, however, especially 1-naphthyl -l-fucoside represents a suitable alternative in a fluorometric procedure instead of p-nitrophenyl -l-fucoside used for the photometric evaluation of -l-fucosidase. With the fluorometric method the enzyme was measured in rat organs, which posses remarkably different activities of -l-fucosidase.

     

Genetic polymorphisms of gene conversion within the duplicated human α-globin loci

Summary Of the 645 Japanese subjects studied, we have identified 10 individuals heterozygous for a chromosome with the triplicated -globin loci. The frequency of the triple -loci was 0.008 in this population, while that of the single -locus, i.e., -thalassemia 2 gene, might be lower than 0.0008. Analysis of haplotypes using particular RsaI site polymorphism in the -globin gene complex strongly suggests that the triple loci may have had multiple origins in this population.     

A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid

Summary Cholic acid (3,7,12-trihydroxy-5-cholanoic acid) was completely and selectively transformed into 12-ketoursodeoxycholic acid (3,7-dihydroxy-12-oxo-5-cholanoic acid) by means of two consecutive enzymatic steps catalyzed, the first, by 7- and 12-hydroxysteroid dehydrogenase and, the second, by 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with -ketoglutarate-glutamate dehydrogenase and glucose-glucose dehydrogenase, respectively.     

Interaction sites on phosphorylase kinase for calmodulin

Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the subunit and a peptide from the subunit present in a region deleted in the isoform were also selected for synthesis. Binding stoichiometry and affinity were determined by following the enhancement in tryptophane fluorescence occurring upon 1:1 complex formation between these peptides and calmodulin. Finally, Ca²⁺ binding to calmodulin in presence of peptides was measured. By this way, the peptides 542–566, 547–571, 660–677 and 597–614 have been found to bind specifically to calmodulin.Together with previously predicted and synthesized calmodulin binding peptides four calmodulin binding regions have been characterized on each the and subunits. It can be concluded that endogenous calmodulin can bind to two calmodulin binding regions in as well as to two regions in and . Exogenous calmodulin can bind to two regions in and in . A binding stoichiometry of 0.8mol of calmodulin/ protomer of phosphorylase kinase has been determined by inhibiting the ubiquitination of calmodulin with phosphorylase kinase. Phosphorylase kinase is half maximally activated by 23nM calmodulin which is in the affinity range of calmodulin binding peptides from to calmodulin. Therefore, binding of exogenous calmodulin to activates the enzyme. A model for switching endogenous calmodulin between , and and modulation of ATP binding to as well as Mg²⁺/ADP binding to by calmodulin is presented.     

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     

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.     

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.     

Site-specific lipophilic modification of interferon-alpha

Interferon- (IFN),⁴ a cytokine with modulatory activities on many cell types, is useful for treating many types of cancer and infectious diseases. This study investigates whether modification of a protein, using IFN as an example, with a lipophilic group can alter its distribution and kinetic properties in the body. Ser163 of IFN2a was mutated to Cys to generate a free sulfhydryl group for site-specific chemical modification. IFN2a(S163C) was conjugated by iodoacetamide derivatives of varying lengths, and the modified IFN2a was purified by gel filtration chromatography. The biological activities of IFN2a(S163C) and lipophilized IFN2a(S163C) were similar to that of IFN2a, as evidenced by their inhibitory effects on the growth of Daudi cells and on the replication of vesicular stomatitis virus in Madin-Darby bovine kidney cells. Lipophilized IFN2a(S163C) bound to human serum albumin and cell membranes more readily than did IFN2a. Future experiments will investigate whether lipophilized IFN2a(S163C) has improved pharmacokinetic properties.     

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.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

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.     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.     

Osteogenesis imperfecta type IV: evidence of abnormal triple helical structure of type I collagen

Summary Skin fibroblasts from a patient with mild osteogenesis imperfecta (OI) type IV synthesize two populations of type I procollagen molecules. One population contains pro1(I) and pro2(I) chains that migrate normally in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a second population contains only slower migrating pro1(I) and pro2(I) chains. The total amount of type I procollagen made by OI cells and the ratio of pro1(I): pro2(I) is normal. When labeled under conditions that inhibit post-translational modification of pro chains, the OI cells produce only single populations of pro1(I) and pro2(I) chains indicating that the apparent increased molecular weight of some OI pro chains is due to excessive post-translational modification rather than peptidyl insertions. Peptide maps indicate that excessive post-translational modification occurs along the entire triple helical segment of some 1(I) and 2(I) chains produced by OI cells. The effect of the mutation is to lower the melting temperature of the molecules containing slow migrating 1(I) and 2(I) chains to 39.5°C (compared to 41.5°C for control), and to delay secretion of the overmodified type I procollagen from OI cells. These data are consistent with a mutation near the carboxyl-terminal end of the triple helical domain which delays triple helical formation and renders all chains available for further post-translational modification amino-terminal to the mutation. Such alterations in triple helical structure, thermal stability, and secretion previously associated only with the lethal OI type II phenotype are thus also seen in the mild OI type IV phenotype.     

Kinetic study of the cytotoxic effect of α-sarcin,a ribosome inactivating protein fromAspergillus giganteus,on tumour cell lines: protein biosynthesis inhibition and cell binding

-Sarcin is a ribosome inactivating protein produced by the mouldAspergillus giganteus. The effect of this protein on eight different tumour cell lines has been studied in the absence of any agent affecting membrane permeability. The protein is cytotoxic for all the tumour cell lines considered. -Sarcin modifies the cell proliferation pattern by inhibiting the protein biosynthesis of the cultured cells. No membrane damage produced by -sarcin has been observed by measuring lactic dehydrogenase leakage. Alteration on the cell mitochondrial activity has not been detected upon treatment with -sarcin. Differences on the extent of the protein binding to the cells have been observed by flow cytometric measurements. The kinetic analysis of the protein biosynthesis inhibition produced by -sarcin reveals an -sarcin concentration-dependent lag phase followed by a first order decrease of the protein synthesis rate. This parameter is dependent on the external -sarcin concentration. A saturable component for the action of -sarcin is also deduced from these experiments. Results are discussed in terms of the protein passage across the cell membrane as the potential rate-limiting step for the action of -sarcin.     

Common expression of a tumor necrosis factor resistance mechanism among gynecological malignancies

Summary The efficacy of tumor necrosis factor (TNF) as an anticancer agent is limited. This limitation might be related to the expression of a protein-synthesis-dependent resistance mechanism that prevents the lysis of tumor cells by TNF. To test this possibility eight randomly selected human cell lines, three derived from ovarian carcinomas and five derived from cervical carcinomas, were tested for their in vitro sensitivity to TNF-mediated lysis. The results of this analysis showed that all eight cell lines are normally resistant to lysis by TNF. However, in the presence of inhibitors of protein synthesis, seven of them showed a significant increase in TNF-mediated lysis. Measurement of protein synthesis showed that there is a linear correlation between the level of inhibition of protein synthesis and the level of TNF-mediated lysis. The fact that seven of eight randomly selected cell lines are resistant to TNF because they express a protein-synthesis-dependent resistance mechanism suggests that this mechanism of resistance may be common among gynecological cancers. The results also suggest that a therapy involving TNF and inhibitors of protein synthesis might be useful for the treatment of gynecological malignancies.     

Partial structure of the human α2(IV) collagen chain and chromosomal localization of the gene (COL4A2)

Summary We have isolated a 2.1-kb cDNA clone from a human placental library encoding part of the 2 chain of collagen IV, a major structural protein of basement membranes. The DNA sequence encodes 446 amino acids in the triplehelical domain plus the 227 amino acids of the carboxy-terminal globular domain. The latter structure is composed of two homologous subdomains and is highly conserved between the 1 and 2 chains. The triple-helical domain contained seven interruptions of the Gly-X-Y repeat and these interruptions were in general larger than their counterparts in the 1 chain. DNA from human rodent hybrid cell lines was analyzed under conditions in which there was no cross-hybridization of the 2(IV) cDNA probe with the gene for the 1(IV) collagen chain. An Eco RI fragment characteristic of the 2 chain had a concordance of 0.97 with chromosome 13. This result was confirmed and extended with in situ localization of the gene at 13q34. Since the 1(IV) gene has previously been localized to 13q34, the two type IV collagen genes reside in the same chromosome region (13q34), possibly in a gene cluster. The presence of the genes for type IV collagen chains on chromosome 13 excludes a primary role for these genes in adult polycystic kidney disease and X-linked forms of hereditary nephritis.