C4 urernic variant: An acquired C4 allotype

The major histocompatibility complex (MHC)-linked complotype region includes alleles for B, C2, and C4 loci. These loci are closely linked to each other and to HLA-DR on chromosome 6. The duplicated C4 loci,, C4A and B, are especially polymorphic. In seven patients with renal insufficiency, we observed a C4 variant with electrophoretic mobility between C4B2 and C4B3. Four of these patients were detected during a study of MHC markers in mernbranoproliferative glomerulonephritis. Complete complotype and HLA data from families of five of the seven patients demonstrated that the variant was not inherited. The pattern was revealed by immunofixation electrophoresis and also by C4-specific hemolytic overlay. In serial plasma specimens taken from one patient, the C4 variant appeared only after the patient became uremic. However, the variant could not be produced in normal plasma after incubation with C4-depleted uremic plasma. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions of immunoprecipitated C4 from these patients showed C4A and C4B chains of normal molecular mass; incompletely processed forms of C4 were not observed. We believe that this variant is probably acquired in the presence of uremia and may represent the C4B2.9 allele found by Wank and co-workers in many patients with glomerulonephritis. Family studies are mandatory to distinguish genetic variants from acquired alterations in the C4 phenotype.     

Multiple sequences related to a constant-region kappa light chain gene in the rabbit genome

Four allelic genes control kappa light chain allotypes in the rabbit. Amino acid sequence studies have revealed an extensive divergence (22%--33%) in the alternative forms of the kappa constant region (b4, b5, b6 and b9). Furthermore, independent studies have shown that a rabbit could express a wrong allotype. To assess the hypothesis that b allotypes are encoded by duplicated genes with a polymorphic control mechanism, we have analyzed the DNAs of four different homozygous rabbits using the Southern blot hybridization technique, with a cloned b4 C kappa probe. DNAs of individual b4, b5, b6 and b9 rabbits were cleaved with Eco RI, Kpn I and Pst I restriction endonucleases. Comparative analysis of restriction patterns shows that all four rabbit DNAs contain multiple DNA fragments hybridizing with the probe under low- and high-stringency washing conditions. In addition, each restriction pattern is distinct, suggesting that the C kappa genes are organized differently in animals expressing different allotypes.     

Structural basis of the polymorphism of human complement components C4A and C4B: gene size, reactivity and antigenicity.

The human complement components C4A and C4B are highly homologous proteins, but they show markedly different, class-specific, chemical reactivities. They also differ serologically in that C4A generally expresses the Rodgers (Rg) blood group antigens while C4B generally expresses the Chido (Ch) blood group antigens. C4A 1 and C4B 5 are exceptional variants which possess their class-specific chemical reactivities, but express essentially the reversed antigenicities. The genes encoding the typical Rg-positive C4A 3a and Ch-positive C4B 3 allotypes and the interesting variants C4A 1 and C4B 5 have been cloned. Characterization of the cloned DNA has revealed that the genes encoding the A 3a, A 1 and B 3 allotypes are 22 kb long, but that encoding B 5 is only 16 kb long. Comparison of derived amino acid sequences of the polymorphic C4d fragment has shown that C4A and C4B can be defined by only four isotypic amino acid differences at position 1101-1106. Over this region C4A has the sequence PCPVLD while C4B has the sequence LSPVIH, and this presumably is the cause of their different chemical reactivities. Moreover, the probable locations of the two Rg and the six Ch antigenic determinants have been deduced. Our structural data on the C4A and C4B polymorphism pattern suggests a gene conversion-like mechanism is operating in mixing the generally discrete serological phenotypes between C4A and C4B.     

Human C4 polymorphism: Pedigree analysis of qualitative,quantiative, and functional parameters as a basis for phenotype interpretations

Summary Ten families with 82 members were investigated for C4A- and B polymorphism in a blind trial. Phenotyping was done on neuraminidase treated sera by immunofixation and simulataneously by hemolytic overlay electrophoresis. In addition Rg, Ch, BF, C2, HLA-A, B, C, DR, and GLO were determined. After decoding the samples the reliability of blind typing was found to be 84.4% according to segregation patters. Inconsistencies occurred mostly when A 4, A 2, or A 92 were present. The detection of silent A*Q0 and B*Q0 alleles was more critical than that of difficult allotypes. The quantitation of the C4A/B ratio by densitometry of stained gels or by conventional immunochemical measurements of serum C4 level could not substantially improve the identification of A*Q0 or B*Q0. C4 dependent activity in radial diffusion hemolysis showed satisfactory correspondence with the number of expressed C4B alleles. At least three haplotypes with two C4A genes (duplicated A genes) were observed as ascertained from offspring analysis in accordance with the MHC segregation pattern. Individuals with the duplicated C4A gene (C4A*3. A*2. in the absence of any other expressed A allele or together with C4A*92) showed only partial inhibition of Rodgers antisera. Partial inhibition of Chido antisera was seen in individuals with C4B 2 (in the absence of other B allotypes). The findings support the hypothesis of at least two structural C4 loci. The also demonstrate the inconsistency of quantitative data in the recognition of silent alleles.     

Rodgers (Rg) and Chido (Ch) determinants on human C4: characterization of two C4 B5 subtypes, one of which contains Rg and Ch determinants

The genetically determined polymorphism of the fourth component of human complement was further extended with the aid of a panel of human allo-anti-C4 sera, anti-Rodgers and anti-Chido. These antisera were found previously to react with the alpha-chains of the C4 molecules controlled by the C4A and C4B loci, respectively. We analyzed a number of new and rare C4 allotypes, and found that they generally followed the expected pattern. Some interesting exceptions, however, were found. The alpha-chain of the allotype C4A1 was found to react with anti-Chido, unlike all other C4A allotypes. Also the C4B5 allotype could be subdivided into two subtypes on the basis of their reaction with anti-Rodgers. They were tentatively named B5Rg+ and B5Rg-. Moreover, the B5Rg+ subtype reacted not only with anti-Rodgers but also with some anti-Chido sera, indicating for the first time that Chido and Rodgers determinants are present on the same allotype.     

Comparison of one-dimensional IEF patterns for serologically detectable HLA-A and B allotypes

A new mouse monoclonal antibody (MoAb) 4E, which detects an epitope shared by HLA-B locus antigens, together with the MoAb W6/32, detecting a common HLA, B, C, determinant, and the MoAb 4B, detecting HLA-A2 and A28, were used to isolate HLA-A and -B antigens in sequential immunoprecipitation. The HLA antigens obtained from metabolically labeled cell extracts of B-lymphoblastoid cell lines or from phytohemagglutinin (PHA) activated peripheral blood lymphocytes were compared by one-dimensional isoelectric focusing (1D-IEF). The IEF banding patterns obtained with native HLA antigens segregated in a family with HLA. Neuraminidase treatment of isolated antigens reduced the number of bands to one or two, simplifying the analysis of characteristic patterns. Thus, we have cataloged IEF banding patterns for the majority of the serologically recognized HLA-A and -B allotypes obtained from 57 unrelated American Caucasians. While no HLA-A locus or HLA-B locus specific banding patterns were observed, the HLA-A antigens had, in general, slightly higher pl values than the HLA-B antigens. HLA-C antigens could not be detected in this assay system. The polymorphism detected by IEF banding patterns was as extensive as the serologically detected polymorphism identified by classical HLA serology. Moreover, variants for some HLA allotypes could be detected by this method. In addition to previously recognized A2 variants, new variants were identified for HLA-A1, A26, and Bw44. Each A1 and Bw44 variant was associated with particular haplotypes. The HLA-A2 antigens occurred on 43 HLA haplotypes in the unrelated Caucasian population. Only one of each A2 variants was identified in this population.     

DNA sequence heterogeneity within the Epstein-Barr virus family of repeats in the latent origin of replication

To detect the presence of variability in the tandemly repeated sequences of the Epstein-Barr virus latent origin of replication, we analyzed the length of the family of repeats in 14 lymphoblastoid and Burkitt's lymphoma cell lines by PCR amplification. The gel electrophoresis analysis of the PCR products revealed a broad banding pattern, characteristic of each line, consisting of several fragments, sometimes smeared, of variable length. This finding was interpreted as a result of the hairpin-like structures generated by the palindrome within the family of repeats, able to originate artefacts. Since the banding pattern was different only in strictly non-correlated cell lines, we supposed that the sequence of the repeat units was polymorphic. We therefore sequenced the family of repeats in three healthy bone marrow derived lymphoblastoid cell lines carrying an endogenous EBV as well as in a B95-8 infected cell line as control. The sequence analysis revealed that each line is different both in the number and in the sequence of repeats. At the 3' end of the family of repeats the B95-8 virus was found to have a 252 bp region missing in the GenBank standard sequence. This one is probably a partial sequence since it was shorter than the control specimens obtained from different sources of B95-8 DNA analyzed by Southern blot hybridization. The length analysis of the family of repeats can be used to characterize EBV strains by PCR.     

The Purification and properties of some less common allotypes of the fourth component of human complement

Human complement component C4 is coded by two genes situated between HLA-D and HLA-B. Both genes are highly polymorphic; C4-A gene products normally carry the blood group antigen Rodgers and C4-B proteins usually carry the Chido antigen. Using a monoclonal antibody which binds Rodgers-positive and Chido-positive proteins with different affinities, we have purified a number of less common C4 allotypes and compared their properties. All C4-B allotypes tested have similar specific hemolytic activities and binding efficiencies to small molecules. All C4-A proteins tested had similar binding to small molecules and hemolytic activities except for the C4-A6 proteins from two individuals with different extended haplotypes, both of which had identical hemolytic activities and much lower ones than other C4-A allotypes. Two allotypes, C4 Al, Rodgers-negative but Chido-positive, and C4-B5, Chido-negative but probably Rodgers-positive, were found to behave as typical C4A and C4-B proteins, respectively, apart from the switch in their antigenic properties.Deceased     

Characterization of a de novo conversion in human complement C4 gene producing a C4B5-like protein

Complement C4 is a highly polymorphic protein essential for the activation of the classical complement pathway. Most of the allelic variation of C4 resides in the C4d region. Four polymorphic amino acid residues specify the isotype and an additional four specify the Rodgers and Chido determinants of the protein. Rare C4 allotypes have been postulated to originate from recombination between highly homologous C4 genes through gene conversions. Here we describe the development of a de novo C4 hybrid protein with allotypic and antigenic diversity resulting from nonhomologous intra or interchromosomal recombination of the maternal chromosomes. A conversion was observed between maternal C4A3a and C4B1b genes producing a functional hybrid gene in one of the children. The codons determining the isotype, Asp(1054), Leu(1101), Ser(1102), Ile(1105) and His(1106), were characteristic of C4B gene, whereas the polymorphic sites in exon and intron 28 were indicative of C4A3a sequence. The protein produced by this hybrid gene was electrophoretically similar to C4B5 allotype. It also possesses reversed antigenicity being Rodgers 1, 2, 3 and Chido-1, -2, -3, 4, -5, and -6. Our case describes the development of a rare bimodular C4B-C4B haplotype containing a functional de novo C4 hybrid gene arisen through gene conversion from C4A to C4B. Overall the data supports the hypothesis of gene conversions as an ongoing process increasing allelic diversity in the C4 locus.     

Rapid identification of environmental isolates of Pseudomonas aeruginosa, P. fluorescens and P. putida by SDS-PAGE analysis of whole-cell protein patterns

Abstract Environmental isolates of fluorescent pseudomonads grown to early stationary phase in glucose-enriched Luria broth were treated with proteinase K in sodium dodecylsulphate (SDS) lysis buffer and subsequently analyzed by polyacrylamide gel electrophoresis (PAGE). Four silver-staining protein-fragment bands could be used for rapid identification at the species level. Pseudomonas aeruginosa isolates were easily recognized by a unique banding pattern. Isolates considered to be P. fluorescen from biochemical and physiological tests (classical biotypes I, II, III, IV and V) also had a characteristic banding pattern, which in turn was different from that of P. putida isolates (classical biotype A). A residual group representing intermediate isolates of P. fluorescens (new biotype VI of Barrett et al., J. Gen. Microbiol. 132, 1986) or P. putida (biotype B) had a banding pattern similar to that of classical P. fluorescens biotypes. On the other hand, a group representing other intermediate isolates of P. putida (new biotype C of Barrett et al., J. Gen. Microbiol. 132, 1986) had a unique banding pattern resembling that of classical P. putida biotype A. A small number of protein fragment bands appearing in SDS-PAGE analysis of whole-cell lysates seems adequate for a rapid identification at the species level of P. aeruginosa, P. fluorescens and P. putida isolated from natural environments.     

Karyotype of the midge Chironomus heterodentatus Konstantinov from the group Obtusidens (Diptera, Chironomidae)

The karyotype of Chironomus heterodentatus, belonging to the obtusidens-group described by Konstantinov from the Volga in 1956, was studied in detail. Combinations of chromosomal arms are AB, CD, EF and G (cytocomplex thummi). The Ch. heterodentatus karyotype cleary differs from those of other members of the obtusidens-group with a species-specific banding pattern in arms A, B, C and D. Chromosomal polymorphism on homo- and heterozygous inversions was found in arms, A, B, D, E and G. 19 inversion banding sequences and their 27 genotypic combinations have been recorded. The shortest arm G is highly polymorphic. Heterozygotes on the Balbiani ring activity were found in arm G along with homo- and heterozygotes on inversions. B-chromosomes with a frequency equal to 2.7-25.0% were recorded in some Volga populations.     

Allotypes associated with B apolipoproteins in rabbits

Western blot analysis of the alloantisera (i.e., anti-Lpq1, anti-Lpq2, anti-Lpq3, and anti-Lpq4) which defined the three lpq genes of rabbit linkage group VIII showed that they reacted strongly with an apolipoprotein of molecular weight 320,000. They also cross-reacted with an apolipoprotein of molecular weight 220,000. The two apolipoproteins that reacted with the alloantisera were found by SDS-polyacrylamide gel electrophoresis to be present in very low density (VLDL), intermediate density (IDL), and low density (LDL) lipoprotein fractions and by Western blot analysis to react with an anti-apolipoprotein B antiserum. These results support the conclusion that the alloantisera react with allotypes associated with the B apolipoproteins. The distribution of the four allotypes among different lipoprotein fractions, however, differed. The quantitative competitive Enzyme Linked Immunosorbant Assay (ELISA) showed that the Lpq1, Lpq2, and Lpq4 allotypes were found in the highest concentration in VLDL, IDL, and LDL, and in significantly lower concentrations in plasma chylomicrons. The concentrations of these allotypes in high density lipoproteins (HDL) as measured in the ELISA were about 1% of the concentrations found in LDL. The Lpq3 allotype, on the other hand, was present in the highest concentrations only in IDL and LDL and in significantly lower concentrations in VLDL and plasma chylomicrons. Surprisingly, the concentration of the Lpq3 allotype in HDL was 20% of the level found in LDL.     

Polymorphism of the fourth component of complement in Turks

An analysis of polymorphism in the fourth component of human complement (C4) was performed on EDTA-plasma from 142 unrelated, randomly selected Turks without collagen-vascular disease or recurrent infections. Plasma samples treated with neuraminidase and carboxypeptidase-B were subjected to high-voltage agarose gel electrophoresis followed by immunofixation. C4B allotypes were further detected in some samples by Western blots with monoclonal antibody 1228 (anti-C4B/Ch1 reactivity). The frequencies of C4A and C4B alleles were determined. Allele C4B*5, which has been found to be relatively common in Asian (Oriental) populations, was not detected in this study. No specific predilection could be noted among the rare variants. C4A*3-C4B*1 was the most common haplotype (n = 40/142, or 28%) but was found less frequently than in Caucasian populations. This finding may be the result of the limited number of samples examined. C4A and/or C4B null allotypes were seen in 49 of 142 (34.6%) subjects. The most frequent C4 null allotype seen was C4B null (37/142, or 26%): 28 subjects had one C4B null allele; 1 had a homozygous deficiency of C4B (C4B*QO, *QO) and 7 had C4A*QO C4B*QO, a double heterozygous haplotype. Frequencies of homozygous haplotype C4A*Q0-C4B*Q0 in the population studied were found to be 0.007. The results of this study demonstrate that the genetic composition of the Turkish population exhibits both similarities and differences with the European population, and ranges between Caucasian and Mongoloid (Asian) populations.     

Antibiotic susceptibility and molecular characterisation of Proteus mirabilis isolates in hospitals from the West Pomeranian area of Poland.

Proteus mirabilis isolates (n = 177), collected between 1996 and 2000 in four hospitals in the West Pomeranian area of Poland, were characterized by antibiotype and pulsed-field gel electrophoresis (PFGE). The selected isolates were collected from different wards (intensive care unit, surgery, internal medicine, and urology). The strains were cultured from various specimen types, mostly from urine, wound samples, bronchial exudates and sputa. The identification was done by biochemical test ID 32E ATB (bioMerieux). Analysis of PFGE patterns was based on comparison of the banding patterns obtained by PFGE of chromosomal DNA digested with SfiI enzyme. Among all P. mirabilis isolates tested three major genotypes A (A1-A7), B (B1-B4), C (C1-C5) and 71 unique patterns were identified. The same genotypes were obtained from different patients, treated in different wards and hospitals during a 5-year period. The strains which belonged to the genotypes A and B were multiresistant and most of them produced ESBL; genotype C was more sensitive to antibiotics.     

The location ofC2, C4, andBF relative toHLA-B andHLA-D

The loci forHLA-A, B, C, D, andDR are known to be closely linked to the structural loci for the complement components C2, BF, and the duplicated loci for C4, C4A and C4B. Conflicting evidence has been presented for the order of these genes. However, new techniques have made possible identification of markers in theHLA-D andC4 region for nearly all identified haplotypes. In our population we have confirmed fiveHLA-B-D crossovers and in each case informative allotypes of C2, BF, or C4A and C4B segregated withHLA-D orDR suggesting that the loci for these proteins lie close toHLA-D andDR. These findings may be of importance for resolving problems encountered in the assignment ofHLA-D alleles.     

Mass Spectrometry Detection of G3m and IGHG3 Alleles and Follow-Up of Differential Mother and Neonate IgG3

Mass spectrometry (MS) analysis for detection of immunoglobulins (IG) of the human IgG3 subclass is described that relies on polymorphic amino acids of the heavy gamma3 chains. IgG3 is the most polymorphic human IgG subclass with thirteen G3m allotypes located on the constant CH2 and CH3 domains of the gamma3 chain, the combination of which leads to six major G3m alleles. Amino acid changes resulting of extensive sequencing previously led to the definition of 19 IGHG3 alleles that have been correlated to the G3m alleles. As a proof of concept, MS proteotypic peptides were defined which encompass discriminatory amino acids for the identification of the G3m and IGHG3 alleles. Plasma samples originating from ten individuals either homozygous or heterozygous for different G3m alleles, and including one mother and her baby (drawn sequentially from birth to 9 months of age), were analyzed. Total IgG3 were purified using affinity chromatography and then digested by a combination of AspN and trypsin proteases, and peptides of interest were detected by mass spectrometry. The sensitivity of the method was assessed by mixing variable amounts of two plasma samples bearing distinct G3m allotypes. A label-free approach using the high-performance liquid chromatography (HPLC) retention time of peptides and their MS mass analyzer peak intensity gave semi-quantitative information. Quantification was realized by selected reaction monitoring (SRM) using synthetic peptides as internal standards. The possibility offered by this new methodology to detect and quantify neo-synthesized IgG in newborns will improve knowledge on the first acquisition of antibodies in infants and constitutes a promising diagnostic tool for vertically-transmitted diseases.     

Human C81 (alpha-gamma) polymorphism: detection in the alpha-gamma subunit on SDS-PAGE, formal genetics and linkage relationship.

The molecular basis of human C81 (alpha-gamma) polymorphism could be elucidated by immunoprecipitation of human C81 allotypes and separation of the alpha-gamma and beta subunits on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. If the C8 molecules were completely reduced, C81 polymorphism was no longer detectable on SDS-PAGE. It is concluded that C81 variation depends on charge rather than molecular weight differences. Four C81 allotypes, the common A and B and two rare allotypes provisionally named A2 and B1, could be distinguished. The rare allotype A1 as detected by isoelectric focusing with subsequent C8 (alpha-gamma)-dependent functional overlay could no longer be visualized on SDS-PAGE. This allotype may therefore be elicited only in the intact C8 molecule. The beta-chain polymorphism named C82, probably also reflecting charge variation of the C8 molecule, could not be detected yet on SDS-PAGE. The distributions of C81 phenotypes and their respective allele frequencies were in good agreement with previously reported data. In the study of 30 families with 100 offspring, no deviation from the rule of at least four codominant alleles at one genetic locus was found. Linkage between C81 gene(s) and PGM1a encoded on chromosome 1 could be confirmed. The following estimates were obtained: (formula; see text) with S theta being the standard error of the maximum likelihood estimate theta. The new technique for allotyping human C81 at the subunit may provide a new tool for the differentiation of qualitative and quantitative variation of the eighth component of human complement.     

Identification of cultivars and validation of genetic relationships in Mangifera indica L. using RAPD markers

Twenty-five accessions of mango were examined for random amplified polymorphic DNA (RAPD) genetic markers with 80 10-mer random primers. Of the 80 primers screened, 33 did not amplify, 19 were monomorphic, and 28 gave reproducible, polymorphic DNA amplification patterns. Eleven primers were selected from the 28 for the study. The number of bands generated was primer- and genotype-dependent, and ranged from 1 to 10. No primer gave unique banding patterns for each of the 25 accessions; however, ten different combinations of 2 primer banding patterns produced unique fingerprints for each accession. A maternal half-sib (MHS) family was included among the 25 accessions to see if genetic relationships could be detected. RAPD data were used to generate simple matching coefficients, which were analyzed phenetically and by means of principal coordinate analysis (PCA). The MHS clustered together in both the phenetic and the PCA while the randomly selected accessions were scattered with no apparent pattern. The uses of RAPD analysis for Mangifera germ plasm classification and clonal identification are discussed.     

Differentiation of Legionella species by soluble protein patterns in polyacrylamide slab gels

Water-soluble protein extracts were obtained from representative strains of eleven species of Legionella and examined by polyacrylamide slab gel electrophoresis (PAGE). The eleven species included: L. pneumophila (serogroups 1-9), L. micdadei, L. longbeachae (serogroups 1 and 2), L. gormanii, L. dumoffii, L. bozemanii, L. jordanis, L. oakridgensis, L. wadsworthii, L. feeleii and L. maceachernii. Species identification was verified by gas-liquid chromatography of the non-hydroxy, monohydroxy and dihydroxy fatty acids. Strains within each species shared common patterns of protein banding. Each species had a distinctive protein banding pattern. Results indicate that PAGE of water soluble protein extracts is a useful technique for identification of Legionella isolates.