Genetic polymorphism of complement component C8

Summary Extensive genetic polymorphism of complement component C8 was demonstrated by isoelectric focusing of serum or plasma samples followed by immunoblotting procedures. Using these methods, we could detect both - (C81) and (C82) chain polymorphisms in the same gel. Two-dimensional (2D) electrophoresis of C8 immunoprecipitates was used to obtain further information of the C8 patterns. Evidence was obtained that the C81 polymorphism resides in the structural gene of the C8 chain. Both C8 systems show autosomal, chiefly codominant inheritance, and the distribution of phenotypes agrees with the Hardy-Weinberg equilibrium. Our findings suggest at least five different alleles in the C81 system; the gene frequencies of the two most common ones, C81 ^*A and C81 ^*B being 0.59 and 0.39, respectively. In C82 we found evidence for at least three codominant alleles, the gene frequencies for the two most common ones, C82 ^*B and C82 ^*A being 0.94 and 0.05, respectively. In addition, family studies disclosed the existence of a null allele, C82 ^* Q0.     

Genetics of human C4 polymorphism: Detection and segregation of rare and duplicated haplotypes

Applying a combined technology for the detection of allotypec variation of the fourth component of human complement (C4), including immunofixation with anti-C4 and C4-dependent lysis after agarose electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of C4 to separate the C4A and B -chains, and the determination of Rodgers (Rg) and Chido (Ch) determinants of C4 in serum and at the blotted C4 -chains, we detected rare human C4 allotypes and studied the genetic linkage. Partial inhibitors (p. i.) of anti-Rg and anti-Ch sera were found; the C4A51 allotype characterized as Rg p. i. and the C4A1 and C4B51 allotypes as Ch p. i. were genetically inherited. The C4A1 allotype has a unique Rg^- Ch⁺ C4A -chain. Duplicated C4A loci, A ^*3, A ^*2, and A ^*5, A ^*2 were both associated with a C4BQO and the HLA haplotype A3-Cw4-Bw35-DR1. These additions to the already known extensive C4 polymorphism may help to sort out their significance for the biological functions of human C4.Abbreviations used in this paper BF Factor B polymorphism of the alternative pathway of complement activation - C2 second component of complement - C4 fourth component of complement - C4D C4-deficient (C4^*QO/QO) - Ch Chido determinant on C4B^* products - EDTA ethylendiaminetetraacetic acid - GLO I glyoxalase I - HLA human leucocyte antigens, A, B, C and DR (D =related) loci - PAGE polyacrylamide gel electrophoresis - PGM3 phosphoglucomutase, third locus - p. i. partial inhibitor = serological inhibition of some, but not all anti-Ch and anti-Rg sera at selected dilutions - SDS sodium dodecyl sulphate; 94k/96k, 94 000 and 96 000 dalton molecular weight Presented in part at the 1V International Workshop on the Genetics of Complement, July 13–15, 1982, Boston, MA, and the Xth International Complement Workshop, May 25–27,1983 in Mainz, Federal Republic of Germany.     

The study of a French family with two duplicated C4A haplotypes

Summary The finding of two duplicated C4A haplotypes in a normal French family led to a detailed study of their C4 polymorphism. The father had an extremely rare A^*6A^*11, B^* QO haplotype inherited by all of his children and the mother had the more common A^*3A^*2, B^*QO haplotype. Two HLA identical daughters only have four C4A alleles. The father's A11 allotype expresses Ch: 1 (Chido) rather than Rg:1 (Rodgers) and represents a new Ch phenotype Ch: 1,-2,-3,-4,-5,-6. In order to clarify the genetic background in this unusual family, DNA studies of restriction fragment length polymorphisms (RFLPs) were undertake. The father's rare haplotype, which expresses two C4A allotypes, results from a long and a short C4 gene normally associated with the A^*6, B^*1 that also exhibits the BglII RFLP. As it travels in an extended MHC haplotype HLA A2, B57 (17), C2^*C, BF^*S, DR7 that is most frequently associated with A^*6, B^*1, we postulate that the short C4B has been converted in the chain region to a C4A gene which produces a C4A protein. This report of a short C4A gene is the first example in the complex polymorphism of C4.     

Three new rare variants of α1-Antitrypsin

Summary Three new rare genetic variants of the serum protein ₁-antitrypsin (₁-protease inhibitor) have been identified in a Caucasian population. The new alleles in the PI system are PI ^*EFRA, PT^*PCAS, and PI ^*XALB. When compared with the normal type M by isoelectric focusing in polyacrylamide, Efranklin (EFRA) is anodal, and Pcastoria (PCAS) and Xalban (XALB) are cathodal. These variants have been compared with previously described variants by isoelectric focusing and by electrophoresis in agarose and acid starch gels. All three variant alleles appear to be associated with normal amounts of ₁-antitrypsin, assayed both by functional and immunological methods.This work was supported by a grant from the Medical Research Council of Canada     

The molecular basis of HbH disease in Taiwan

Summary We have determined the molecular characteristics of -thalassemia in 12 HbH subjects from Taiwan by restriction endonuclease mapping with -and -specific probes. We have found four types of defects in the -thalassemia-2 genetic determinant: ^3.7 type I; ^4.2; ^CS; and ^T. All HbH subjects carried the ——^SEA genotype in the -thalassemia-1 determinant. At least two different subtypes of ——^SEA genotype were observed in this study.     

Molecular basis for erythrocyte Le(a+b+) and salivary ABH partial-secretor phenotypes: expression of a FUT2 secretor allele with an A→T mutation at nucleotide 385 correlates with reducedα(1,2) fucosyltransferase activity

TheSe ^wA385T mutation of the FUT2 gene was found to correlate with both the erthrocyte Le(a+b+) and/or salivary ABH partial-secretor phenotypes of Polynesians. Constructs with FUT1 and FUT2 wild type genes, and the FUT2Se ^wA385T,se ^G428A andse ^C571T mutated alleles, were cloned into pcDNAI, and expressed in COS-7 cells. COS-7 cells transfected with theSe ^wA385T allele had weak, but detectable, (1,2)fucosyltransferase activity, with an acceptor substrate pattern similar to the wild type FUT2 gene. Comparative kinetic studies from cell extracts with mutatedSe ^wA385T and wild type FUT2 alleles gave similarK _m values, but less enzyme activity was present in cells transfected withSe ^wA385T (V _max 230 pmol h^–1 mg^–1), as compared to those transfected with FUT2 (V _max 1030 pmol h^–1 mg^–1), suggesting that the mutated enzyme is more unstable. These results confirm that the molecular basis for the erythrocyte Le(a+b+) and the associated ABH salivary partial-secretor phenotype, is an amino acid change of Ile 129Phe in the secretor (1,2)fucosyltransferase.Abbreviations (1,3/1,4)fucosyltransferase GDP-L-fucose:-D-N-acetylglucosaminide 3/4--L-fucosyltransferase - (1,2)fucosyltransferase GDP-L-fucose: -D-galactoside-2--L-fucosyltransferase - bp base pairs - FUT1 H gene; FUT2,Se gene - FUT3 Lewis gene or Fuc-TIll gene - FUT4 Fuc-TIV gene - FUT5 Fuc-TV gene - FUT6 Fuc-TVI gene - MAb monoclonal antibody - PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - se ^G428A FUT2 nonsecretor GA mutation at nucleotide 428 - se ^C571T FUT2 nonsecretor CT mutation at nucleotide 571 - Se ^wA385T FUT2 secretor weak AT mutation at nucleotide 385 - SSP sequence specific primer     

A Gln-281 to Arg substitution in α-l-fucosidase is responsible for a common polymorphism detected by isoelectric focusing

A common polymorphism of human -l-fucosidase consists of three phenotypes (Fu 1, Fu 2, and Fu 2-1) assigned by isoelectric focusing. The phenotypes are determined by two codominant alleles (Fu¹ and Fu²). Isozymes with the Fu 2 phenotype have more basic pI than Fu 1, while Fu 2-1 is a mixture of Fu 2 and Fu 1. Recently, a missense mutation (A⁸⁶⁰G) in the -l-fucosidase gene was described that did not affect -l-fucosidase activity. The mutation causes the substitution of Arg (pKa^Guan=12.5) for Gln-281, which has no ionizable side chain. Isoelectric focusing profiles of extracts of COS-1 cells transfected with wild-type or mutant -l-fucosidase cDNAs had phenotypes of Fu 1 and Fu 2, respectively. Next, 20 human lymphoid cell lines were examined for the occurrence of the A⁸⁶⁰G mutation and expression of the Fu 1, Fu 2, and Fu 2-1 phenotypes. Eight lines with Fu 2 were homozygous for the A⁸⁶⁰G mutation; six lines with Fu 1 were homozygous for the normal nucleotide (A⁸⁶⁰); and six lines with Fu 2-1 were heterozygous. Thus, the A⁸⁶⁰G mutation is the molecular basis for the protein phenotypes and the Fu¹ and Fu² alleles. The normal nucleotide (A⁸⁶⁰) is responsible for Fu¹ and the A⁸⁶⁰G mutation for Fu².     

A novel wheat alpha-amylase gene (alpha-Amy3)

Summary A genomic clone of a wheat -amylase gene (Amy3/33) was identified, on the basis of hybridisation properties, as different from -Amy1 and -Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of -amylase from the -Amy1 and -Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca⁺⁺ binding region. In addition, the introns are at positions equivalent to the position of introns in the -Amy1 and -Amy2 genes. However, the sequence was less similar to -Amy1 and -Amy2 than these are to each other. Southern blot analysis showed that the Amy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the -Amy1 or -Amy2 genes. A further difference from the -Amy1 and -Amy2 genes was the pattern of expression. Amy3/33 was expressed only in immature grains and, unlike the -Amy1 and -Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of -amylase gene, not described before, which shares a common evolutionary ancestor with the -Amy1 and -Amy2 genes.     

Amino acid sequence of Kalinowaski's Tinamou (Nothoprocta kalinowskii) hemoglobin and the rate of evolution of bird alphaD-globin

Two hemoglobin components are recognized in erythrocytes of the adult Tinamou. We determined the amino acid sequences of Tinamou ^D-, ^A-, and -globins from intact globin chains and several chemically cleaved fragments. A remarkable feature of Tinamou hemoglobin was a deletion in the ^D-globin chain. This has not been reported in the literature, except in pigeon embryonic ^D-globin. The amino acid sequences of Tinamou globin were highly similar to those of Ostrich and Rhea hemoglobin. Comparison between Tinamou, Ostrich, and Rhea that suggested the evolution speed of globin, ^D = ^A > , was related with the early appearance birds. The important residues in Tinamou hemoglobin as the heme contact and oxygen binding regions were highly conserved in other species.     

Efficient and precise measurement of H(alpha)-C(alpha), C(alpha)-C', C(alpha)-C(beta) and H(N)-N residual dipolar couplings from 2D H(N)-N correlation spectra

A suite of experiments are presented for the measurement of H–C, C–C, C–C and H^N–N couplings from uniformly ¹⁵N, ¹³C labeled proteins. Couplings are obtained from a series of intensity modulated two-dimensional H^N–N spectra equivalent to the common ¹H–¹⁵N–HSQC spectra, alleviating many overlap and assignment issues associated with other techniques. To illustrate the efficiency of this method, H–C, C–C, and H^N–N isotropic scalar couplings were determined for ubiquitin from data collected in less than 4.5 h, C–C data collection required 10 h. The resulting couplings were measured with an average error of ±0.06, ±0.05, ±0.04 and ±0.10 Hz, respectively. This study also shows H–C and C–C couplings, valuable because they provide orientation of bond vectors outside the peptide plane, can be measured in a uniform and precise way. Superior accuracy and precision to existing 3D measurements for C–C couplings and increased precision compared to IPAP measurements for H^N–N couplings are demonstrated. Minor modifications allow for acquisition of modulated H^N–C 2D spectra, which can yield additional well resolved peaks and significantly increase the number of measured RDCs for proteins with crowded ¹H–¹⁵N resonances.     

Hemoglobin M Iwate is caused by a C→T transition in codon 87 of the human α1-globin gene

Summary DNA restriction, molecular cloning, and sequencing methods have been used to characterize the mutation leading to the methemoglobinemia HbM Iwate. It could be demonstrated that the HbM Iwate defect is caused by a point mutation involving a transition from C to T in the first position of codon 87 of the ₁-globin gene. Furthermore, the HbM Iwate mutation can directly be identified upon RsaI digestion. This direct detection of the mutation on the gene level is of significant advantage for differential diagnostic purposes.     

Population and formal genetics of the human C81(α-γ) polymorphism

Summary One hundred and ninety-six unrelated healthy individuals and 30 families with 75 offspring have been studied for the C81(-) polymorphism. The following allele frequencies were calculated: C81^*A=0.5536; C81^*B=0.4286; C81^*A1=0.0178. Observed and expected phenotype frequencies were in a good agreement according to the Hardy Weinberg law. No exceptions from the mode of inheritance were found. In family W the segregation of the rare allele C81^*A1 could be followed. Comparing the results of this study with previous data from Boston and Oslo, a combined technology including C8-dependent lysis and C8 structural variation is suggested for future investigations.     

Frequency and molecular types of deletional α-thalassemia in Egypt

Summary The frequency of deletional -thalassemia in the Egyptian population was estimated at 0.08 by DNA analysis of a newborn random sample. No ⁰ determinants were found. The most frequent ⁺ determinant was the –^3.7 type I in association with the medium allele at inter-zeta HVR. The –^4.2 and ^{anti 3.7} arrangements were found at very low frequencies.     

Three unrelated individuals with perinatally lethal osteogenesis imperfecta resulting from identical Gly502Ser substitutions in the α2-chain of type I collagen

In general, osteogenesis imperfecta is caused by heterozygous mutations in either of the genes encoding the 1 or 2 chains of type I collagen (COL1A1 and COL1A2, respectively). Usually, these mutations are unique to the affected individual or individuals within a family. In this study, single-strand conformation polymorphism mapping analysis has been coupled with sequence analysis to identify a single base mutation in the 2(I) gene of type I collagen; this mutation is identical in three unrelated individuals with perinatal lethal osteogenesis imperfecta. The heterozygous G to A transition at a CpG dinucleotide results in a Gly502Ser substitution in the 2 chain of type I collagen.     

Regulation of NaK-ATPase by platelet-derived growth factors in cultured rat thoracic aortic smooth muscle cells

Regulation of (Na⁺ + K⁺)-adenosine triphosphatase (NaK-ATPase) by platelet-derived growth factor (PDGF) in cultured rat thoracic aortic smooth muscle cells (SMC) was examined. PDGF-BB enhances SMC proliferation and NaK-ATPase activity. Ouabain, an inhibitor of NaK-ATPase activity, prevents PDGF-BB-induced SMC proliferation. As shown by Western blot and immunochemiluminescence analysis, PDGF-BB also enhances ₁, truncated ₁, and ₁ NaK-ATPase subunit levels. PDGF-AA and PDGF-AB show no effect on ₁ and truncated ₁ levels in slot blot analysis. Induction of NaK-ATPase subunit levels by PDGF-BB could be one of the initial events in vascular SMC proliferation.     

Cobra (<Emphasis Type="Italic">Naja</Emphasis> spp. ) Nicotinic Acetylcholine Receptor Exhibits Resistance to Erabu Sea Snake (<Emphasis Type="Italic">Laticauda semifasciata</Emphasis>) Short-Chain α-Neurotoxin

Snake -neutotoxins of Elapidae venoms are grouped into two structural classes, short-chain and long-chain -neutotoxins. While these two classes share many chemical and biological characteristics, there are also distinct dissimilarities between them, including their binding site on the nicotinic acetylcholine receptor (nAChR), specificity among species of Chordata, and the associated pharmacological effects. In the present study we test the hypothesis that structural motifs that evolved to confer natural resistance against conspecific long-chain -neurotoxins in Elapidae snakes also interfere with the biological action of short-chain -neurotoxins. We expressed functional nAChRs that contains segments or single residues of the Elapidae nAChR ligand binding domain and tested the effect of short-chain -neurotoxin erabutoxin-a (ETX-a) from the Erabu sea snake Laticauda semifasciata on the acetylcholine-induced currents as measured by two-microelectrode voltage clamp. Our results show that the Elapidae nAChR subunit segment T¹⁵⁴–L²⁰⁸ ligand binding domain has an inhibitory effect on the pharmacological action of ETX-a. This effect is primarily attributed to the presence of glycosylation at position N¹⁸⁹. If the glycosylation is removed from the T¹⁵⁴–L²⁰⁸ segment, the nAChR will be inhibited, however, to a lesser extent than seen in the mouse. This effect correlates with the variations in -neurotoxin sensitivity of different species and, importantly, reflects the evolutionary conservation of the binding site on the nAChR polypeptide backbone per se. Phylogenetic analysis of -neurotoxin resistance suggests that -neurotoxin-resistant nAChR evolved first, which permitted the evolution of snake venom -neurotoxins. A model describing -neurotoxin resistance in Elapidae snakes is presented. Present address: Schering-Plough Research Institute, CNS-CV Research, K-15 C205/2600, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA     

Complement component C3: molecular basis of the C3*S025 variant and evidence for molecular heterogeneity of other variants

Complement component 3 (C3) is the central molecule of the complement system. It displays a number of polymorphic variants with, as yet, unclear functional consequences. We have investigated a number of rare C3 variants by PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) analysis and could identify the molecular basis of a C3^*S025 variant. The decreased electrophoretic mobility of this protein is caused by the exchange of a neutral serine residue to an arginine residue (positively charged). This exchange is unlikely to have functional consequences as it maps to the C-terminus of the -chain. C3 variants appear to have originated from various independent mutations as we could not detect this mutation in different allotypes.     

Localisation régionale des gènes humains LDHB,TPI, ENO2, PepB,PGK, αGALA,G6PD et HGPRT par l'hybridation cellulaire interspécifique

Summary Twenty-two independent man-mouse (A9, HGPRT^-) and manhamster (CH, HGPRT)-hybrids using male human with balanced reciprocal translocation XY t(X;12)(q2,3;q1,2) were analysed for human genes localized on chromosome 12 (LDH_B, PepB, ENO₂, TPI), on chromosome X (PGK, GAL_A, G6PD) and for human chromosomes in relation with the balanced reciprocal translocation (chromosome 12, 12q^-, Xq⁺). The human chromosome 12q^- was not analysed because of its morphology is similar to some rodent chromosomes.The following results were obtained:1.Man-mouse hybrids (12 independent hybrids):The chromosome 12 is absent. A positive correlation is observed between Xq⁺, PGK, and PepB. Four hybrids are Xq⁺+PGK+PepB+ and eight hybrids are Xq⁺-PGK-PepB-This result indicate that the genes for human PGK, PepB are on the chromosome Xq+. 2.Man-hamster hybrids (10 independent hybrids):A positive correlation is observed between Xq⁺, PGK, GAL_A. Two hybrids are Xq⁺+PGK+GAL_A+ and eight hybrids are Xq⁺-PGK-GAL_A-.A positive correlation is observed between Xq⁺, PGK, GAL_A, PepB with the seven hybrids missing the normal human chromosome 12. One hybrid is Xq⁺+PGK+GAL_A+PepB+ and six hybrids are Xq⁺-PGK-GAL_A-PepB-. These results indicate that the genes for human PGK, GAL_A, PepB are on the chromosome Xq⁺. 3.Man-mouse and man-hamster hybrids:The highest percentage of presence in the hybrids is observed for the following markers: G6PD (100%), LDH_B, TPI, ENO₂ (90%). This is explained by the fact that these hybrids selected in HAT medium, had to retain a segment of X (Xq⁺ or 12q^-) bearing the human HGPRT gene. The different results indicated that the segment of X retained in these hybrids must be on the 12q^- and not on the Xq⁺, for Xq⁺ is rarely present in man rodent hybrids.The different results implicate finally the following localisations:LDH_B, TPI, ENO₂ on 12 q12 12pter,PepB on 12 q12 12qter;PGK, GAL_A on X q23 Xpter;HGPRT, G6PD on X q23 Xqter.     

C-Terminal exchange between Gα o and Gα i3 proteins differently modulates α2A-adrenoceptor activation

Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The _2A-adrenoceptor (_2AAR), which yielded an efficacious and weak [³⁵S]GTPS binding response by respectively G _o and G _i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G _o and G _i3 proteins, and reciprocally, were permuted. Activation of the chimeric G _{o / i3} protein was highly efficient whereas the G _{i3 / o} protein yielded a weak stimulation. These [³⁵S]GTPS binding responses were not different from their parental wild-type G _o and G _i3 proteins. Similar results were obtained with an _2AAR carrying a facilitating Thr³⁷³Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G _o protein amino acids do not constitute a major _2AAR interaction domain for G protein activation.     

Different ζ globin gene deletions among Black Americans

Summary Four types of chromosomes with a deletion between the human embryonic and globin genes were identified among 2.8% of 321 Black Americans from Georgia. Two deletions of approximately 11 kb which differed by about 300 bp occurred on chromosomes with or without a polymorphic Xba I site 5 to the globin gene [(X⁺) or (X^-)]. The deletions are identifiable in Xba I digests of genomic DNA using an or a globin gene probe which yield fragments of 23 kb from (X⁺)–^* chromosomes or 27 kb from (X^–)–^* chromosomes. Digestion with other enzymes and probing with both and probes gave fragments typical of the two globin gene deletions previously identified in Polynesians. Among Black Americans, these globin gene deletions have been found in combination with globin gene deletions in trans but not in cis. Homozygotes have not been found. Hematologic data on carriers of the globin gene deletions in association with Hb AS, SS, and SC suggest that these deletions have no effect on the function of the adult globin genes.