The origin of the very variable haemolytic activities of the common human complement component C4 allotypes including C4-A6.

The human complement component C4 occurs in many different forms which show big differences in their haemolytic activities. This phenomenon seems likely to be of considerable importance both physiologically and pathologically. C4 is coded by duplicated genes between HLA-D and HLA-B loci in the major histocompatibility complex in man. Several fold differences in haemolytic activity between products of the two loci C4-A and C4-B have been correlated with changes of six amino acid residues in this large protein of 1722 residues and with differences of several fold in the covalent binding of C4 to antibody-antigen aggregates. Some allotypes of one locus also differ markedly, notably C4-A6 which has 1/10th the haemolytic activity of other C4-A allotypes. A monoclonal antibody affinity column has been prepared which is able to separate C4-A from C4-B proteins and, using serum from an individual expressing only the C4-A6 allele at the C4-A locus, C4-A6 protein has been prepared. Investigation has shown C4-A6 to have the same reactivity as other C4-A allotypes except in the formation of the complex protease, C5 convertase. This protease is formed from C4, C2 and C3 and if C4-A6 is used it has approximately 1/5th the catalytic activity compared with other C4-A allotype. Allelic differences in sequence identified in C4 proteins so far are few and it is probable that the big difference in catalytic activity of C5 convertase is caused by very small changes in structure.     

Rhesus macaque KIR bind human MHC class I with broad specificity and recognize HLA-C more effectively than HLA-A and HLA-B

Human killer cell immunoglobulin-like receptors (KIR) recognize A3/11, Bw4, C1, and C2 epitopes carried by mutually exclusive subsets of human leukocyte antigen (HLA)-A, -B, and -C allotypes. Chimpanzee and orangutan have counterparts to HLA-A, -B, and -C, and KIR that recognize the A3/11, Bw4, C1, and C2 epitopes, either individually or in combination. Because rhesus macaque has counterparts of HLA-A and -B, but not HLA-C, we expected that rhesus KIR would better recognize HLA-A and -B, than HLA-C. Comparison of the interactions of nine rhesus KIR3D with 95 HLA isoforms, showed the KIR have broad specificity for HLA-A, -B, and -C, but vary in avidity. Considering both the strength and breadth of reaction, HLA-C was the major target for rhesus KIR, followed by HLA-B, then HLA-A. Strong reactions with HLA-A were restricted to the minority of allotypes carrying the Bw4 epitope, whereas strong reactions with HLA-B partitioned between allotypes having and lacking Bw4. Contrasting to HLA-A and -B, every HLA-C allotype bound to the nine rhesus KIR. Sequence comparison of high- and low-binding HLA allotypes revealed the importance of polymorphism in the helix of the α₁ domain and the peptide-binding pockets. At peptide position 9, nonpolar residues favor binding to rhesus KIR, whereas charged residues do not. Contrary to expectation, rhesus KIR bind more effectively to HLA-C, than to HLA-A and -B. This property is consistent with major histocompatibility complex (MHC)-C having evolved in hominids to be a generally superior ligand for KIR than MHC-A and MHC-B.     

Differential expression of the p65 gene family

The genome of the marine ray Discopyge ommata contains at least three p65-related genes. o-p65-A is 84% identical, o-p65-B is 78% identical, and o-p65-C is only 41% identical to a previously characterized rat p65. The cytoplasmic domain, particularly the two regions that are similar to the regulatory domain of protein kinase C, are most highly conserved. The three genes are expressed in different but overlapping patterns in the central nervous system. o-p65-A immunoreactivity is found predominantly in forebrain, cerebellum, and neuroendocrine cells, while o-p65-B immunoreactivity is predominantly localized to the spinal cord, brainstem, and midbrain. Many synaptic vesicle proteins are members of small gene families that are differentially expressed, resulting in several unique combinations of these molecules in specific brain regions.     

Mammalian cells express two VPS4 proteins both of which are involved in intracellular protein trafficking

The yeast Vps4 protein (Vps4p) is a member of the AAA protein family (ATPases associated with diverse cellular activities) and a key player in the transport of proteins out of a prevacuolar endosomal compartment. In human cells, we identified two non-allelic orthologous proteins (VPS4-A and VPS4-B) of yeast Vps4p. The human VPS4-A and VPS4-B proteins display a high degree of sequence identity to each other (80 %) and to the yeast Vps4 protein (59 and 60 %, respectively). Yeast cells lacking a functional VPS4 gene exhibit a temperature-sensitive growth defect and mislocalise a carboxypeptidase Y-invertase fusion protein to the cell surface. Heterologous expression of human VPS4 genes in vps4 mutant yeast strains led, in the case of human VPS4-A, to a partial and, in the case of human VPS4-B, to a complete suppression of the temperature-sensitive growth defect. The vacuolar protein sorting defect of vps4 mutant yeast cells was complemented completely by heterologous expressed human VPS4-B protein, and partially by the human VPS4-A protein. Expression of mutant human VPS4-A (E228Q) and VPS4-B (E235Q) proteins, harbouring single amino acid exchanges in their AAA domains, induced dominant-negative vacuolar protein sorting defects in wild-type yeast cells in both cases. Two-hybrid experiments suggest that the human VPS4-A and VPS4-B proteins can form heteromeric complexes, and subcellular localisation experiments indicate that both human VPS4 proteins associate with endosomal compartments in yeast. Based on these results, we conclude that both human VPS4 proteins are involved in intracellular protein trafficking, presumably at a late endosomal protein transport step, similar to the Vps4p in yeast.     

RNA binding specificity of a Drosophila snRNP protein that shares sequence homology with mammalian U1-A and U2-B" proteins.

We have characterized a recombinant Drosophila melanogaster RNA binding protein, D25, by virtue of its antigenic relationship to mammalian U1 and U2 small nuclear ribonucleoprotein (U snRNP) proteins. Sequence analysis revealed that D25 bears strong similarity to both the human U1 snRNP-A (U1-A) and U2 snRNP-B" (U2-B") proteins. However, at residues known to be critical for the RNA binding specificities of U1-A and U2-B" D25 sequence is more similar to U2-B". Using direct RNA binding assays D25 selected U1 RNA from either HeLa or Drosophila Kc cell total RNA. Furthermore, D25 bound U1 RNA when transfected into mammalian cells. Thus, D25 appears to be a Drosophila homolog of the mammalian U1-A protein, despite its sequence similarity to U2-B".     

Promiscuous binding of invariant chain-derived CLIP peptide to distinct HLA-I molecules revealed in leukemic cells

Antigen presentation by HLA class I (HLA-I) and HLA class II (HLA-II) complexes is achieved by proteins that are specific for their respective processing pathway. The invariant chain (Ii)-derived peptide CLIP is required for HLA-II-mediated antigen presentation by stabilizing HLA-II molecules before antigen loading through transient and promiscuous binding to different HLA-II peptide grooves. Here, we demonstrate alternative binding of CLIP to surface HLA-I molecules on leukemic cells. In HLA-II-negative AML cells, we found plasma membrane display of the CLIP peptide. Silencing Ii in AML cells resulted in reduced HLA-I cell surface display, which indicated a direct role of CLIP in the HLA-I antigen presentation pathway. In HLA-I-specific peptide eluates from B-LCLs, five Ii-derived peptides were identified, of which two were from the CLIP region. In vitro peptide binding assays strikingly revealed that the eluted CLIP peptide RMATPLLMQALPM efficiently bound to four distinct HLA-I supertypes (-A2, -B7, -A3, -B40). Furthermore, shorter length variants of this CLIP peptide also bound to these four supertypes, although in silico algorithms only predicted binding to HLA-A2 or -B7. Immunization of HLA-A2 transgenic mice with these peptides did not induce CTL responses. Together these data show a remarkable promiscuity of CLIP for binding to a wide variety of HLA-I molecules. The found participation of CLIP in the HLA-I antigen presentation pathway could reflect an aberrant mechanism in leukemic cells, but might also lead to elucidation of novel processing pathways or immune escape mechanisms.     

A neural network model approach to the study of human TAP transporter

We used an artificial neural network (ANN) computer model to study peptide binding to the human transporter associated with antigen processing (TAP). After validation, an ANN model of TAP-peptide binding was used to mine a database of HLA-binding peptides to elucidate patterns of TAP binding. The affinity of HLA-binding peptides for TAP was found to differ according to the HLA supertype concerned: HLA-B27, -A3 or -A24 binding peptides had high, whereas HLA-A2, -B7 or -B8 binding peptides had low affinity for TAP. These results support the idea that TAP and particular HLA molecules may have co-evolved for efficient peptide processing and presentation. The strong similarity between the sets of peptides bound by TAP or HLA-B27 suggests functional co-evolution whereas the lack of a relationship between the sets of peptides bound by TAP or HLA-A2 is against these particular molecules having co-evolved. In support of these conclusions, the affinities of HLA-A2 and HLA-B7 binding peptides for TAP show similar distributions to that of randomly generated peptides. On the basis of these results we propose that HLA alleles constitute two separate classes: those that are TAP-efficient for peptide loading (HLA-B27, -A3 and -A24) and those that are TAP-inefficient (HLA-A2, -B7 and -B8). Computer modelling can be used to complement laboratory experiments and thereby speed up knowledge discovery in biology. In particular, we provide evidence that large-scale experiments can be avoided by combining initial experimental data with limited laboratory experiments sufficient to develop and validate appropriate computer models. These models can then be used to perform large-scale simulated experiments the results of which can then be validated by further small-scale laboratory experiments.     

Structure, chromosomal localization and evolutionary conservation of the gene encoding human U1 snRNP-specific A protein.

Three specific proteins, called A, 70K and C, are present in the U1 small nuclear ribonucleoprotein (snRNP) particle, in addition to the common proteins. The human U1 snRNP-specific A protein is, apart from a proline-rich region, highly similar to the U2 snRNP-specific protein B". To examine the homologous regions at the genomic level, we isolated and characterized the human U1-A gene. The human U1-A protein appears to be encoded by a single-copy gene and its locus has been mapped to the q arm of chromosome 19. The gene, about 14-16 kb in length, consists of six exons. The regions homologous to the U2-B" gene are not limited to single exons and are mostly not confined by exon-exon junctions in the corresponding U1-A mRNA. However, the proline-rich region of U1-A, absent in U2-B", is encoded by a single exon, suggesting a specific function for this domain of U1-A. The region of the cap site and upstream sequences contain interesting similarities to the promoter region of other snRNP protein-encoding genes and several housekeeping genes, in particular the vertebrate ribosomal protein-encoding genes. Hybridization experiments with various vertebrate genomic DNAs revealed that U1-A sequences are evolutionarily conserved in all tested vertebrate genomes, except for chicken, duck and pigeon. The divergence of these avian genomes is probably typical for the class of birds.     

Preparation of VT1 and VT2 hybrid toxins from their purified dissociated subunits. Evidence for B subunit modulation of a subunit function.

Verocytotoxins comprise a family of closely related subunit proteins. Two members of this group, VT1 and the immunologically distinct VT2, have been found to share similar physical properties, and yet several differences in their biological activities have been noted. The subunits of these toxins were separated using urea and isolated by high performance liquid chromatography gel filtration. Reconstituted VT1 and VT2 as well as VT1-A:VT2-B and VT2-A:VT1-B hybrid toxins were then prepared. The B subunit was found to determine cell culture specificity, cytotoxic titer, and antibody neutralizability as determined on Vero and MRC-5 cells. Cross-linking isolated B chains revealed 5 species upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis for both VT1-B and VT2-B. Using an in vitro translation system, both toxin A subunits inhibited protein synthesis at concentrations as low as 4 pM. In glycolipid binding assays, VT1 and VT1-B subunits competed equally on a molar basis with 125I-VT1 for the receptor, globotriaosylceramide, however, a 1000-fold excess of VT2 was required. Ligand analysis of direct VT1 and VT2 receptor binding assays revealed a difference in binding affinity constants (Kd of VT1 = 4.6 x 10(-8) M; VT2 = 3.7 x 10(-7) M).     

The clinical immunogenetics of viral hepatitis C in a Caucasoid population of western Siberia

The analysis of the immunogenetic studies on hepatitis C patients among the Caucasoid population of western Siberia has revealed a significant increase in the detection rate of antigens HLA-A10 and HLA-DR5, the combinations of DR2-DR5, DR5-DR7, DR1-B27 and the complete absence of antigen HLA-DR4, which is indicative of the fact that susceptibility and resistance to the development of the disease is associated with the genes of the main histocompatibility complex. In hepatitis of mixed etiology, B and C, a significant increase in the occurrence of HLA antigens: -A1, -B8, -DR1 and -DR3, as well as the combinations of A1-DR1, A1-DR3, A3-DR3, A9-A10, DR1-DR3, B8-DR3 is noted; at the same time a decrease in the occurrence of antigen DR4 and its combination with antigen HLA-A2 is observed.     

Stabilization of the amplification convertase of complement by monoclonal antibodies directed against human factor B

Three IgM mouse monoclonal antibodies (MoAb), 5B5-A, 2D2-B, and 5B12-A, were prepared by fusion of spleen cells from mice immunized with human B with the SP 2/0 myeloma cell line. They were assessed for their effect on cell-bound and fluid-phase amplification convertases of complement (C) with purified proteins in vitro. 5B5-A and 2D2-B were similar in their effects on cell-bound preformed C3bBb in that they bound to cell-bound C3bBb, stabilized the C3bBb convertase, and rendered the C3bBb convertase relatively resistant to the plasma protein H. These two MoAb were also able to enhance C3 consumption in vitro in reaction mixtures containing C3b, C3, B, and D. At the same time, they presumably stabilized the C3 convertase and caused relative sparing of B hemolytic activity in the reaction mixtures. In contrast, 5B12-A, which also bound to Bb and C3bBb, did not induce any stabilization, but rather caused accelerated decay of cell-bound C3bBb. These results indicate that MoAb against B can have C3NeF-like activity. On the other hand, not all MoAb against B have stabilizing activity on the C3bBb convertase.     

Evidence That Two Distinct Populations of Rabbit Anti-Idiotypic Antibodies Are Induced by Three Monoclonal Antibodies Specific for Bluetongue Virus Core Protein VP7

Three groups of anti-idiotypic antibodies (anti-Id or Ab2), designated RAb2-A, RAb2-B1, and RAb2-B2, were isolated from rabbit antiserum raised against three monoclonal antibodies (mAbs) (M1875, M1877, and M1886) specific for the bluetongue virus core protein, VP7. RAb2-A was specific for the idiotype of M1875. RAb2-B1 and RAb2-B2, isolated through the M1877 and M1886 affinity columns, respectively, were directed against the common idiotype that is shared by M1877 and M1886 and therefore classified in the same population (RAb2-B). Further characterization revealed that the two anti-Id populations, RAb2-A and RAb2-B, were significantly different. RAb2-A was an Ab2β type of anti-Id since (i) its reaction with M1875 was inhibited by the antigen; (ii) it inhibited the M1875-VP7 interaction; and (iii) it elicited anti-VP7 antibody response in Balb/c mice. In contrast, RAb2-B may represent an Ab2α type of anti-Id since its reactions with M1877 or M1886 were not inhibited by the antigen, even though it inhibited mAbs from binding to the antigen. These results indicated that RAb2-A and RAb2-B represent two distinct populations of anti-Ids to anti-VP7 mAbs with similar epitope specificity.     

A single arginine to tryptophan interchange at beta-chain residue 458 of human complement component C4 accounts for the defect in classical pathway C5 convertase activity of allotype C4A6. Implications for the location of a C5 binding site in C4.

In general, C4A allotypes of human C4 show one-fourth to one-third the hemolytic activity of C4B allotypes. An exception to this rule is C4A6 which is almost totally deficient in hemolytic activity. Previous studies have localized the defect in C4A6 to the C5 convertase stage. Of the two critical events required for C5 cleavage, namely formation of a covalent adduct between C3b and the C4b subunit of the C3 convertase (C4b2a), and binding of C5 to this C4b-C3b complex, it is a defect in the latter step that accounts for the aberrant activity of C4A6. DNA sequencing studies described in a companion paper have suggested that the sole C4A6-specific difference was a Trp for Arg replacement at beta-chain residue 458. To directly ascertain whether this single substitution was responsible for the hemolytic defect in C4A6, we have used site-directed mutagenesis to introduce this change into both C4A and C4B cDNA expression plasmids. We found that the R to W replacement totally abrogated hemolytic activity. However, irrespective of the amino acid at residue 458, the mutant proteins behaved like their wild-type counterparts with respect to covalent binding to C1-bearing targets, i.e., the C4B recombinants displayed higher binding to sheep and human red cells than did the C4A counterparts. Furthermore, the mutants were able to form covalent C4b-C3b adducts. There was, however, substantially less C5 cleavage produced by cell-bound C4boxy23b complexes made with R458W mutant C4B than with wild-type C4B. These results are consistent with the sole defect in the mutants being at the C5 binding stage and strongly suggest that Arg 458 of the C4 beta-chain contributes to the C5 binding site of the molecule.     

Structural and functional characterization of disulfide isoforms of the human IgG2 subclass

In the accompanying report ( Wypych, J., Li, M., Guo, A., Zhang, Z., Martinez, T., Allen, M. J., Fodor, S., Kelner, D. N., Flynn, G. C., Liu, Y. D., Bondarenko, P. V., Ricci, M. S., Dillon, T. M., and Balland, A. (2008) J. Biol. Chem. 283, 16194-16205 ), we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1beta ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 m guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 > IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2lambda composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "blood-like" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.     

DNA Polymorphism of the major histocompatibility class I genes and their association with serologically defined haplotypes

DNA of unrelated persons as well as members of families that were totally or partially homozygous or completely heterozygous on the loci of the major histocompatibility class I genes has been isolated from peripheral blood lymphocytes and blot hybridized with the class I pseudogene pHLA 12.4 probe. The autoradiographic DNA patterns were discussed and compared with well-defined serological features. Positive associations with serologically typed alleles had been demonstrated for HLA-A1,11 ; -A2; -A3; -B7; -B14; -B35;-Bw41; and -Cw5.     

小麦tae-MIR156前体基因的克隆及其靶基因TaSPL17多态性分析

Squamosa-promoter binding protein (SBP)-box基因是植物特有的一类转录因子, 广泛参与植物生长发育, 其部分成员受miR156调控。文章克隆了小麦(Triticum aestivum) tae-MIR156前体基因, 转录后能够形成茎环结构。小麦10个SBP-box基因中, 仅TaSPL3和TaSPL17在编码区存在tae-miR156识别位点。SPL17在普通小麦的A基因组供体种乌拉尔图小麦(Triticum urartu, AA) UR209和B基因组供体种拟斯卑尔脱山羊草(Aegilops speltoides, BB) Y2001中均为多拷贝(SPL17-A1、SPL17-A2和SPL17-A3; SPL17-B1、SPL17-B2和SPL17-B3), 在D基因组供体种粗山羊草(Aegilops tauschii, DD) Ae38中仅检测到一种序列(SPL17-D); SPL17-A2与SPL17-B2, SPL17-A3与SPL17-B3、SPL17-D两两之间序列的一致性程度均大于99%, 且与普通小麦(中国春、衡观35和双丰收)的TaSPL17序列具有较高的一致性, 提示它们可能来源于共同的祖先基因, 并且在进化过程中高度保守。靶基因TaSPL17中的tae-miR156识别位点非常保守, 在根据单株穗数和基因型多样性挑选的SubP1和SubP2群体中均未检测到tae-miR156识别位点存在变异碱基。     

Murine sex-limited protein (Slp) antigenic sites in human complement component C4

Human complement component C4 is encoded by two HLA-linked loci, A and B. In the mouse, the H-2 region contains structural genes for two serum proteins that react with antibodies to human C4, but one of these proteins (Slp) has no C4 hemolytic activity. Because the product of C4-A locus in man has low hemolytic activity, a previous report suggests it may be the homologue of murine Slp. We show here that Slp antigenic determinants are found in human C4. However, they are expressed in the products of both loci A and B, that is, C4A and C4B, since both proteins were specifically immunoprecipitated by the IgG fraction of alloantisera to mouse Slp. Therefore, Slp-associated structural features are preserved in evolution, although they do not seem to be relevant to the hemolytic properties of C4.     

Investigation of the antibiotic complex produced by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> 194, Variant K

Phase variation in the culture of the environmental strain Lactococcus lactis subsp. lactis 194 resulted in the formation of two types of colonies differing by 15% in antibiotic activity. The active variant 194-K produced an antibiotic complex with a broad spectrum of antibacterial and antifungal activity. Five components (194-A, B, C, D, and E) were isolated from the complex by solid-phase extraction and thin-layer chromatography. Components 194-A and 194-B were hydrophobic neutral compounds soluble in organic solvents. Component 194-A possessed fungicidal activity, whereas component 194-B exhibited only bactericidal activity. Physicochemical studies of the isolated components 194-A and 194-B revealed that they had no analogs in the Berdy database of biologically active substances (BNPD) and appeared to be novel antibiotics. Component 194-C was a hydrophilic polar compound inhibiting growth of gram-positive and gramnegative bacteria. Component 194-D belonged to peptide antibiotics; it inhibited growth of only gram-positive bacteria and was similar to nisin A in biological properties but differed in electrophoretic mobility and molecular mass.     

Downregulation of major histocompatibility complex class I molecules by Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins

The T-cell-mediated immune response plays a central role in the defense against intracellular pathogens. To avoid this immune response, viruses have evolved elaborate mechanisms that target and modulate many different aspects of the host's immune system. A target common to many of these viruses is the major histocompatibility complex (MHC) class I molecules. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes K3 and K5 zinc finger membrane proteins which remove MHC class I molecules from the cell surface. K3 and K5 exhibit 40% amino acid identity to each other and localize primarily near the plasma membrane. While K3 and K5 dramatically downregulated class I molecules, they displayed different specificities in downregulation of HLA allotypes. K5 significantly downregulated HLA-A and -B and downregulated HLA-C only weakly, but not HLA-E, whereas K3 downregulated all four HLA allotypes. This selective downregulation of HLA allotypes by K5 was partly due to differences in amino acid sequences in their transmembrane regions. Biochemical analyses demonstrated that while K3 and K5 did not affect expression and intracellular transport of class I molecules, their expression induced rapid endocytosis of the molecules. These results demonstrate that KSHV has evolved a novel immune evasion mechanism by harboring similar but distinct genes, K3 and K5, which target MHC class I molecules in different ways.     

Studies of HLA, factor B (Bf), complement C2 and C4 haplotypes in type 1 diabetic and control families from northern Sweden

The HLA-A,-B,-C,-DR antigens and the complement factors C2, C4 and Bf were determined in 30 insulin-dependent diabetes mellitus (IDDM) patients and 30 healthy controls from northern Sweden. Family studies allowed the deduction of extended haplotypes in the HLA and complement systems. Phenotype studies revealed significant associations between IDDM and HLA-DR4 (p less than 0.001), HLA-DR3 (p less than 0.05), HLA-DR3/4 (p less than 0.025), C4-B3 (p less than 0.001) and Bf-S (p less than 0.025). Haplotype studies showed that the extended haplotype [HLA-B15, C2-1, C4-A3B3, Bf-S, HLA-DR4] had a particularly strong association to IDDM. This haplotype was found in 10 out of 30 IDDM probands but in none of 30 control children and accounts for practically all the C4-B3 allotypes among the 30 IDDM probands. The C4-B3 gene therefore seems to be a valuable marker for IDDM. No haplotype containing HLA-DR3 was increased in frequency among the IDDM probands. The extended haplotype [HLA-B7, C2-1, C4-A3B1, Bf-S, HLA-DR2] present among the controls was absent in the IDDM probands. The frequency of the extended haplotype [HLA-B15, C2-1, C4-A3B3, Bf-S, HLA-DR4] was increased also among the parents to the IDDM probands compared to those of the control parents, whereas the frequency of [HLA-B7, C2-1, C4-A3B1, Bf-S, HLA-DR2] was decreased. The extended haplotype [HLA-B8, C2-1, C4-B1, Bf-S, HLA-DR3] was more common among the males (p less than 0.05) compared to the females in the total material. The family analysis showed that 3 out of 5 affected sibs shared both haplotypes with their IDDM proband. This was the case for only 3 out of 35 unaffected sibs.