Nuclear factors binding to the human immunoglobulin heavy-chain gene enhancer.

The human immunoglobulin heavy chain (IgH) gene contains at least two tissue-specific regulatory regions, which are similar to the mouse IgH gene. One is the J-C enhancer and another is located in the 5' promoter region. Using an electrophoretic mobility shift assay and DNase I footprint, we have examined the interaction of factors in B cell nuclear extracts with the two regulatory regions of the human IgH gene. We have identified a nuclear factor in mouse B cell nuclear extracts which bound to specific sequence in the human IgH enhancer. This factor is apparently not present in mouse fibroblast nuclear extracts. We also found factor(s) which bound to the highly conserved octanucleotide sequence within the human IgH enhancer and 5' promoter regions.     

Ets proteins: new factors that regulate immunoglobulin heavy-chain gene expression.

We used a DNA-protein interaction screening method to isolate a cDNA, Erg-3, whose product binds to a site, designated pi, present in the immunoglobulin (Ig) heavy-chain gene enhancer. Erg-3 is an alternatively spliced product of the erg gene and contains an Ets DNA-binding domain. Fli-1 and PU.1, related Ets proteins, also bind to the same site. In addition, PU.1 binds to a second site, designated microB, in the Ig heavy-chain enhancer. We demonstrate that the pi binding site is crucial for Ig heavy-chain gene enhancer function. In addition, we show that Erg-3 and Fli.1, but not PU.1, can activate a reporter construct containing a multimer of protein-binding sites, synergistically with helix-loop-helix protein E12. We discuss how combinatorial interactions between members of the helix-loop-helix and Ets families may account for the tissue specificity of these proteins.     

Evolution of the rat immunoglobulin gamma heavy-chain gene family

The sequences of the four immunoglobulin gamma heavy chains of the rat (gamma 1, gamma 2a, gamma 2b, gamma 2c) have been determined. These sequences reveal that the rat genes have evolved differently from the closely related mouse gamma genes (gamma 1, gamma 2a, gamma 2b, gamma 3): in rat two of the four genes (gamma 2a and gamma 1) are 94% homologous to each other and best resemble the single mouse gamma 1 gene. Rat gamma 2b is equivalent to the mouse gamma 2a/gamma 2b pair as regards both nucleotide sequence and antibody effector functions whilst rat gamma 2c resembles mouse gamma 3. In evolutionary terms this suggests the existence of a set of three common C gamma genes before separation of rat and mouse as individual species. In addition, two independent duplication events must have occurred after species separation affecting different constant regions; this yielded rat gamma 2a and gamma 1 as a recently evolved pair and mouse gamma 2a and gamma 2b as a different pair. Furthermore, the sequence comparisons reveal several other features of interest; rat IgG2b lacks two amino acids in CH1 which are conserved in all other sequenced gamma chains. Residues believed to be essential for monocyte interaction (FcRI) are retained only in rat gamma 2b and not in the other rat gamma genes whilst a particular motif involved in C1q interaction shows a variation in both rat IgG1 and rat IgG2a which has not been observed previously.     

IgG heavy-chain (Gm) allotypes in familial polyposis coli.

Serum samples from 40 Danish patients with familial polyposis coli and 105 normal blood donors were typed for eight Gm and one Km markers. The distribution of all Gm phenotypes as a group was significantly different in the patient population as compared to the controls. Examination of individual Gm phenotypes showed an increased frequency of Gm3;5,13 in the patients.     

Polymorphisms of the immunoglobulin heavy-chain delta gene and association with other constant-region genes.

Polymorphisms have previously been reported for the C mu, C alpha, C epsilon, and C gamma genes of the immunoglobulin heavy-chain (IGH) gene cluster. Here we report polymorphisms of the IGH C delta gene region, observed using the enzymes ApaI, AvaII, TaqI, and XbaI. The TaqI and XbaI polymorphisms were used in an investigation of linkage disequilibrium throughout the cluster of constant-region genes. The TaqI polymorphism, located 5' to the C delta gene, is in linkage disequilibrium with a polymorphism of the C mu switch region. The XbaI polymorphism, which is in the vicinity of the C delta 2 exon, is not strongly associated with any other polymorphisms, including the TaqI polymorphism and the Gm polymorphism of C gamma 3. Although there is a high degree of association between most genes of the IGH region, there is a lack of association between C delta and C gamma 3, which may indicate a hot spot for recombination.     

Genetic analysis of eight linked polymorphisms within the human immunoglobulin heavy-chain region.

Genetic analyses of multiple restriction fragment length polymorphisms, revealed by a single DNA probe containing the switch region of the immunoglobulin constant heavy-chain (IgCH) mu gene, are presented here in detail. Five of the polymorphic loci segregate in complete linkage with IgCH allotypic markers, while one appears to be located at more than 10 centimorgans from the IgCH region. A study of over 100 random haplotypes typed at eight linked loci, including the Ig switch polymorphisms and the classical Gm-Am allotypes, allowed us to construct an evolutionary tree by which each haplotypic variant can be derived one from the other either by single-step mutation or by recombination. A few of the recombinant haplotypes appeared to carry large DNA duplications that could be explained by unequal crossing over; others might postulate gene-conversion events. Linkage disequilibria observed between the IgCH-linked loci were compared with expected ones. A heterogeneous distribution of recombination rates is clearly documented, a "hot" region of recombination being present between the gamma 2 and switch alpha 2 loci.     

B-lymphocyte targeting of gene expression in transgenic mice with the immunoglobulin heavy-chain enhancer.

A hybrid gene containing rabbit beta-globin structural sequences (-9 to +1650), and a chicken conalbumin gene promoter (+62 to -102) in the place of the beta-globin promoter (upstream from -9), was inactive in 5 different transgenic mouse line. Adding the mouse immunoglobulin heavy-chain (IgH) enhancer to this construction specifically stimulated expression in B-cells. These results show that IgH enhancer is specifically active in B-cells. Expression of the hybrid gene was low compared to the endogenous immunoglobulin heavy and light-chain genes. Substituting the mouse immunoglobulin kappa light-chain gene (Ig kappa) promoter (+4 to -800) for the heterologous conalbumin promoter was not sufficient to restore gene expression to level of the endogenous genes. In addition to the reproducible B cell expression, we also found inheritable unexpected expression in certain tissues, which varied from line to line.     

Functional analysis of defined mutations in the immunoglobulin heavy-chain enhancer in transgenic mice.

We have analyzed the effect of defined mutations in the mouse immunoglobulin heavy-chain enhancer after introduction into the germline of transgenic mice. We have tested a mutation of the enhancer octamer motif, a double mutation of the octamer motif and the microB-site, and a triple mutation in the microE2, microE3 and microE4-sites. All constructs are expressed in the spleen of transgenic mice. Furthermore, expression is exclusively detectable in lymphoid organs and not in several nonlymphoid tissues. Whereas mutations in the microE-sites have a more pronounced effect on transgene activity in thymocytes as compared to bone marrow and spleen cells, the octamer/microB double mutation shows significantly reduced expression levels only in B-cells. Finally, our results demonstrate that the intronic heavy-chain enhancer element does not contribute to the increase steady state levels of heavy-chain mRNA after stimulation of spleen cells with LPS.     

Copy choice mechanism of immunoglobulin heavy-chain switch recombination.

The immunoglobulin heavy-chain switch is mediated by a recombination event between DNA switch regions associated with donor and recipient constant-region genes. We have determined that the mutations which can be found in some switch regions after recombination appear to arise on only one strand of DNA. This result suggests that switch recombination involves error-prone synthesis of one DNA strand and ligation of the other strand from preexisting DNA.     

Many human immunoglobulin heavy-chain IGHV gene polymorphisms have been reported in error

The identification of the genes that make up rearranged immunoglobulin genes is critical to many studies. For example, the enumeration of mutations in immunoglobulin genes is important for the prognosis of chronic lymphocytic leukemia, and this requires the accurate identification of the germline genes from which a particular sequence is derived. The immunoglobulin heavy-chain variable (IGHV) gene repertoire is generally considered to be highly polymorphic. In this report, we describe a bioinformatic analysis of germline and rearranged immunoglobulin gene sequences which casts doubt on the existence of a substantial proportion of reported germline polymorphisms. We report a five-level classification system for IGHV genes, which indicates the likelihood that the genes have been reported accurately. The classification scheme also reflects the likelihood that germline genes could be incorrectly identified in mutated VDJ rearrangements, because of similarities to other alleles. Of the 226 IGHV alleles that have previously been reported, our analysis suggests that 104 of these alleles almost certainly include sequence errors, and should be removed from the available repertoire. The analysis also highlights the presence of common mismatches, with respect to the germline, in many rearranged heavy-chain sequences, suggesting the existence of twelve previously unreported alleles. Sequencing of IGHV genes from six individuals in this study confirmed the existence of three of these alleles, which we designate IGHV3-49*04, IGHV3-49*05 and IGHV4-39*07. We therefore present a revised repertoire of expressed IGHV genes, which should substantially improve the accuracy of immunoglobulin gene analysis.     

The D-JH complex is an intermediate to the complete immunoglobulin heavy-chain V-region gene

We have examined the organization of the immunoglobulin JH segments in three clones derived from a single Abelson murine leukemia virus-transformed cell. Cloning and nucleotide sequence analyses of the JH-containing fragments have revealed the rearrangement from the preformed D-JH complex to the complete VH-D-JH gene, which was accompanied by the expression of the intra-cytoplasmic mu chain. In one case a JH segment downstream to the preformed D-JH was used to create a new VH-D-JH gene. Upon the D-JH and VH-D-JH rearrangements the intervening D segments were deleted from the chromosome. One of the expressed VH genes suffered from a large deletion of the 3' portion (including the 95th cysteine residue) of the VH segment. We discuss the possible mechanism of the allelic exclusion.     

Complex protein binding within the mouse immunoglobulin heavy-chain enhancer.

We have begun to purify and characterize several proteins which bind to the mouse immunoglobulin heavy-chain enhancer to understand the molecular interactions important for enhancer activity. Three proteins which bind to different sites on the immunoglobulin heavy-chain enhancer have been chromatographically separated and partially purified. One protein binds a site which has not been reported previously and does not bind to other reported protein-binding sites on the immunoglobulin heavy-chain enhancer. Binding-site boundaries for the three partially purified proteins have been precisely mapped by methylation interference, DNase I footprinting, and orthophenanthroline/copper chemical nuclease footprinting. We have also characterized these three proteins with respect to dissociation rate constants.     

Reconsidering the human immunoglobulin heavy-chain locus:

We have used a bioinformatics approach to evaluate the completeness and functionality of the reported human immunoglobulin heavy-chain IGHD gene repertoire. Using the hidden Markov-model-based iHMMune-align program, 1,080 relatively unmutated heavy-chain sequences were aligned against the reported repertoire. These alignments were compared with alignments to 1,639 more highly mutated sequences. Comparisons of the frequencies of gene utilization in the two databases, and analysis of features of aligned IGHD gene segments, including their length, the frequency with which they appear to mutate, and the frequency with which specific mutations were seen, were used to determine the reliability of alignments to the less commonly seen IGHD genes. Analysis demonstrates that IGHD4-23 and IGHD5-24, which have been reported to be open reading frames of uncertain functionality, are represented in the expressed gene repertoire; however, the functionality of IGHD6-25 must be questioned. Sequence similarities make the unequivocal identification of members of the IGHD1 gene family problematic, although all genes except IGHD1-14*01 appear to be functional. On the other hand, reported allelic variants of IGHD2-2 and of the IGHD3 gene family appear to be nonfunctional, very rare, or nonexistent. Analysis also suggests that the reported repertoire is relatively complete, although one new putative polymorphism (IGHD3-10*p03) was identified. This study therefore confirms a surprising lack of diversity in the available IGHD gene repertoire, and restriction of the germline sequence databases to the functional set described here will substantially improve the accuracy of IGHD gene alignments and therefore the accuracy of analysis of the V–D–J junction.Electronic Supplementary Material Supplementary material is available for this article at     

Gm and Km immunoglobulin allotypes in Sicily

The aim of this study was to evaluate the intra- and inter-population variability of the Gm/Km system in the Madonie Mountains, one of the main geographical barriers in north-central Sicily. We analysed 392 samples: 145 from Alia, 128 from Valledolmo, 25 from Cerda and 94 from Palermo. Serum samples were tested for G1m (1,2,3,17), G2m (23), G3m (5,6,10,11,13,14,15,16,21,24,28) and Km (1) allotypes by the standard agglutination-inhibition method. We found the typical genetic patterns of populations in peripheral areas of the Mediterranean basin, with a high frequency of haplotypes Gm5*;3;23 and Gm5*;3;... The frequency of Gm21,28;1,17;... (about 16%) is rather high compared with other southern areas. Of great importance is the presence of the common African haplotype Gm 5*;1,17;..., ranging in frequency from 1.56% at Valledolmo to 5.5% at Alia. The presence of this haplotype suggests past contacts with peoples from North Africa. The introduction of African markers could be due to the Phoenician colonization at the end of the 2nd millennium b.c. or to the more recent Arab conquest (8th–9th centuries a.d.).     

Gm(1) and Gm(2) immunoglobulin allotypes in patients with malignant melanoma.

Gm allotype markers were determined in sera from 71 melanoma patients and 400 control persons. There was no significant difference between both groups in Gm distribution. The results were compared to a recent report. Furthermore, in 25 malanoma patients the capacity of serum to interfere with cell-mediated cytotoxicity (CMC) of autologous lymphocytes was determined and related to the Gm allotype.