c-myc Gene rearrangements involving gamma immunoglobulin heavy chain gene switch regions in murine plasmacytomas

In murine plasmacytomas, the c-myc gene has frequently been found to undergo rearrangement by virtue of a T(12;15) chromosome translocation. The immunoglobulin heavy chain gene switch region (S alpha) constitutes the target for most of these recombinations particularly in IgA producing plasmacytomas. We sought to identify non-S alpha myc target sites in several IgG producing tumors. The c-myc target in MPC-11 (a BALB/c IgG2b producing plasmacytoma) has been cloned, localized to the Igh-C locus and identified as the gamma 2a heavy chain gene switch region (S gamma 2a). Furthermore, by Southern blot hybridization, we have determined that the S gamma 2b region is the c-myc target in two NZB IgG2b producing plasmacytomas. The potential relation between Ig class expressed and c-myc translocation target is discussed.     

The immunoglobulin heavy chain class switch

Conclusions While much has been learned concerning the molecular structural basis for the heavy chain class switch, many questions relating to the regulation of the switch remain unanswered, or at least controversial. Identification of the enzyme system which mediates the class switch, as well as other regulatory, possibly X-linked, genes should provide the necessary key to our understanding of this unique process.AbbreviationsB cell lymphocyte derived from the bone marrow in adult mammals or the bursa of Fabricius in chickens - bp base pair - C immunoglobulin constant region - CDR complementarity-determining region of the immunoglobulin variable region - D diversity gene segment of the immunoglobulin heavy chain variable region gene - H immunoglobulin heavy chain - Ig immunoglobulin - J joining region gene segment of the immunoglobulin variable region gene - kb kilobase - L immunoglobulin light chain - LPS lipopolysaccharide - Pyr pyrimidine - S-, s-site, s-region switch rearrangement site - SCE sister chromatid exchange - sIg surface immunoglobulin - T cell lymphocyte derived from the thymus - USCE unequal sister chromatid exchange - V immunoglobulin variable region     

Nucleotide sequence and properties of the murine gamma 3 immunoglobulin heavy chain gene switch region: implications for successive C gamma gene switching

During B lymphocyte differentiation, immunoglobulin heavy chain constant region (CH) genes undergo a unique series of DNA recombination events culminating in the CH class switch. CH switch (S) regions are located 2 kb 5' of each CH gene except delta (i.e. mu, gamma 3, gamma 1, gamma 2b, gamma 2a, epsilon and alpha). We describe the structural features of the gamma 3 switch region. Hybridization experiments show that S gamma 3 has remarkable homology to both S mu and other S gamma regions while S mu possesses limited homology to the other S gamma sequences. However, S mu possesses extensive sequence homology with S epsilon and S alpha. The nucleotide sequence of S gamma 3 reveals higher densities of S mu repetitive sequences (GAGCT and GGGGT) and another S region common sequence (YAGGTTG) than observed for S gamma 1, S gamma 2b or S gamma 2a. In addition, the conservation of S mu like repetitive sequences in S gamma regions is correlated with the 5' leads to 3' gamma gene order (i.e. S gamma 3 greater than S gamma 1 greater than S gamma 2b greater than S gamma 2a). A model is presented which suggests that the unique features of S gamma 3 may allow for successive switches from C mu to any C gamma gene.     

The structural heterogeneity among degraded gamma heavy chain disease proteins.

The amino terminal sequence of seven newly studied and three previously reported gamma Heavy Chain disease proteins show that they begin at several sites ranging from position 215--234 in the hinge region. The presence of more than one species of molecule in several of the sera and the susceptibility of this region to proteolytic cleavage, taken together with the finding of a blocked Heavy Chain disease protein in the serum of one and the cells of another patient support the concept that these molecules are the result of proteolytic cleavage of typical Heavy Chain disease proteins.     

Primate immunoglobulin heavy chain constant gamma genes: an hypothesis of their evolution

Four Immunoglobulin gamma heavy chain isotypes are present in humans; the true phylogenetic relationship between the genes are not known because of the complex concerted evolution of the Ig multigene locus. Here we present data obtained from Southern blot analysis of the gamma genes in several primate species including prosimians (Lemur catta), New World Monkeys (Saguinus oedipus) and Old World Monkeys (Cercopitecus aethiops andMacaca fascicularis). Our data show the presence of a single IgG gene inLemur and probably inS.l oedipus, and of multiple genes in the two Cercopithecinae. These findings suggest that a single IgG gene was present in the ancestor of primates: we suppose that this IgG gene was later duplicated several times during primate evolution.     

Duplication of the human immunoglobulin heavy chain gamma 2 gene.

The five C gamma genes in the human immunoglobulin heavy chain region show nonrandom association and segregation as haplotypes. From the study of genetic variation in C gamma genes of 58 healthy Caucasian volunteers, we have identified a haplotype that involves a duplication of C gamma 2. This haplotype contains both the 13.5-kilobase (kb) and 25-kb BamHI fragment alleles of C gamma 2. In addition, the patterns and relative intensity of BamHI fragments containing C gamma genes were those expected for genomic DNA containing three copies of C gamma 2 for every two copies of the four other C gamma genes. A new EcoRI polymorphism in C gamma 4 was useful in defining the haplotype containing the duplication. Alleles of the C gamma genes in the duplication haplotype, including Gm markers of C gamma 1 and C gamma 3 and DNA polymorphisms of C psi gamma, C gamma 2, and C gamma 4, were consistent with its origin from an unequal crossover between the two common C gamma haplotypes, H1 and H2. This recombinant haplotype, which has been designated H2;1(gamma 2 dup) to reflect its origin, occurred with a frequency of .043 in a random sample of 116 chromosomes.     

Repetitive sequences in class-switch recombination regions of immunoglobulin heavy chain genes

Immunoglobulin class switch involves a unique recombination event that takes place at the region 5′ to each heavy chain constant region gene during B lymphocyte differentiation. Such regions that are responsible for the class-switch recombination are defined as S regions (Kataoka et al., Proc. Natl. Acad. Sci. USA 77, 919, 1980). We have cloned a rearranged γ2b gene from a mouse myeloma (MPC11) and compared its structure with the germ line counterparts. The rearranged γ2b gene contained the 5′ flanking region of the γ3 gene (S_γ3 region) which are linked to the 5′ flanking region of the γ2b gene (S_γ2b region). We have determined nucleotide sequences surrounding the recombination site of the rearranged and germ line γ2b genes, which include the S_γ2b and S_γ3 regions. Both _γ2b and S_γ3 regions comprise tandem repetition of conserved units of 49 bp. Similar 49 bp repeating units are also found in the previously determined sequence of the S_γ1 region in which class-switch recombination took place in MC101 myeloma. The nucleotide sequences of the S_γ1, S_γ2b and S_γ3 repeating units share significant homology with each other. The Sμ region, partial nucleotide sequence of which was previously determined, contains abundant short sequences such as AGCT, TGGG and AGCTGGGG which are shared in common by repeating sequences in S_γ regions. These results suggest that the recombination responsible for class switch from μ to γ or from a γ to another γ, may be facilitated directly or indirectly by homology of repeating sequences in S regions.     

Restriction fragment length polymorphisms associated with immunoglobulin heavy chain gamma genes in Tunisians

Summary DNA polymorphisms in the human immunoglobulin gamma () region have been studied in random Arabo-Berber Tunisians and in a large Tunisian Berber kindred. Haplotypes have then been designated, based on variation in the BamHI restriction fragments containing the C1, C2, C4, and C genes. Two new haplotypes, in addition to the four previously described, have been observed. These new haplotypes, designated H5 and H6, were confirmed by family studies. The H5 haplotype was associated with black African Gm haplotypes · (Gm^{1,17;..;5,6,11} and Gm^1,17;..;5,11) (Gm^{a,z;..;blc3bo} and Gm^a,z;..;blbo) and probably represents a common haplotype in the black population. The haplotype H6 may be derived from H5. One of 39 random Tunisians was homozygous for a multigene deletion. DNA polymorphisms of the C genes, in conjuction with Gm markers, provide highly variable genetic markers important for the characterization of human populations.     

Nucleotide sequences of switch regions of immunoglobulin C epsilon and C gamma genes and their comparison

Immunoglobulin class switch involves a unique recombination event that takes place at the switch (S) region which is located 5' to each constant region (C) gene of the heavy (H) chain. For example, differentiation of the B lymphocyte from a mu-chain producer to an epsilon-chain producer is mediated by the switch recombination between the S mu and S epsilon regions. In order to elucidate the molecular mechanism for the switch recombination, we have determined nucleotide sequences surrounding the class switch recombination sites of the C epsilon and C gamma 3 genes and those in the 5' flanking regions of the C gamma 2a and C delta genes. The results indicate that the 5' flanking regions of all the CH genes except for the C delta gene contain the S regions which comprise tandem repetition of short unit sequences in agreement with the previous analyses of the S gamma 1, S gamma 2b, S mu, and S alpha regions. Comparison of the nucleotide sequences of all the S regions revealed that length as well as nucleotide sequences of the S regions vary among different classes of the CH gene, but they share short common sequences, (G)AGCT and TGGG(G). The nucleotide sequence of the S mu region is homologous to those of the other S regions in the decreasing order of the S epsilon, S alpha, S gamma 3, and (S gamma 1, S gamma 2b, s gamma 2a) regions. We have compared the nucleotide sequences immediately adjacent to the recombination sites of seven rearranged genes and have always fund tetranucleotides TGAG and/or TGGG, except for one case. Such tetranucleotides may constitute a part of the recognition sequence of a putative recombinase. These results provide further support for our previous proposal that the switch recombination may be facilitated by short common sequences dispersed in all the S regions.     

Origins of immunoglobulin heavy chain domains

Summary Using computer programs that analyze the evolutionary history and probability of relationship of protein sequences, we have investigated the gene duplication events that led to the present configuration of immunoglobulin C regions, with particular attention to the origins of the homology regions (domains) of the heavy chains. We conclude that all of the sequenced heavy chains share a common ancestor consisting of four domains and that the two shorter heavy chains, alpha and gamma, have independently lost most of the second domain. These conclusions allow us to align corresponding regions of these sequences for the purpose of deriving evolutionary trees. Three independent internal gene duplications are postulated to explain the observed pattern of relationships among the four domains: first a duplication of the ancestral single domain C region, followed by independent duplications of the resulting first and last domains. In these studies there was no evidence of crossing-over and recombination between ancestral chains of different classes; however, certain types of recombinations would not be detectable from the available sequence data.     

On immunoglobulin heavy chain gene switching: two gamma 2b genes are rearranged via switch sequences in MPC-11 cells but only one is expressed

During B lymphocytes differentiation, switches in the expression of heavy chain immunoglobulin constant region (CH) genes occur by a novel DNA recombination mechanism. We have investigated the requirements of the CH gene switch by characterizing two rearranged gamma 2b genes from a gamma 2b producing mouse myeloma (MPC-11). One of the two gamma 2b genes is present in 2-3 copies per cell (gamma 2b strong hybridizer) while the other is present in approximately 1 copy per cell (gamma 2b weak hybridizer). Genomic clones of the gamma 2b strongly hybridizing gene indicate that this is an abortive switch event between the S gamma 3 and S gamma 2b regions. However, clones of the gamma 2b weakly hybridizing gene suggest a functional rearrangement due to the presence of VH, JH and S mu sequences. The switch-recombination sites of these rearranged gamma 2b genes and those of other CH genes show a high degree of preference for the sequence AGGTTG 5' of either the S mu donor site or the appropriate CH S acceptor site. AGGTTG and its analogs are rare in the S mu region, are somewhat prevalent in s alpha and in the case of S mu are found 5' of a tandemly repeated DNA sequence (GAGCT, GGGGT) comprising most of S mu.     

Rat immunoglobulin E heavy chain locus

A 2100 base-pair long sequence has been established which covers all four constant domains of the rat epsilon-chain. An analysis of messenger RNA from an immunoglobulin E producing rat immunocytoma revealed two separate epsilon-chain mRNA species, 2.3 X 10(3) and 2.8 X 10(3) base-pairs long. The latter mRNA encodes the membrane binding form of the epsilon-chain. The membrane exons which are located approximately 2 X 10(3) base-pairs away from the 3'-side of the CH4 exon were also sequenced. A comparison between the rat and mouse epsilon-chains at the protein sequence level revealed an overall homology of 80% which, as expected, is considerably higher than the homology found between rat and human epsilon-chains. The fourth constant domain together with the two membrane exons exhibited the highest degree of homology, 81 to 89%. Only two differences were found when the epsilon-chains from LOU and Sprague Dawley rats were compared. The most striking difference at the nucleotide sequence level between the rat, mouse, and human epsilon genes was found within the first intron. The mouse genome contains a unique 366 base-pair long sequence in this region. The inserted sequence is repetitive and present in approximately 100 copies in the mouse genome. It is flanked by 22 base-pair long direct repeats and contains also 14 base-pair long inverted repeats, thus having properties in common with transposable elements.     

Replacement recombinant events targeted at immunoglobulin heavy chain DNA sequences in mouse myeloma cells

The rate of gene conversions and double crossovers between transfected and integrated mu heavy chain immunoglobulin genes was measured in myeloma cells. The assay relies on correction of an integrated and defective mu heavy chain expression unit, present in a repeated head to tail array in the genome of the myeloma cell line J558L. Following electroporation of these cells with restriction fragments containing normal immunoglobulin sequences, targeted recombination events are identified by a complement-mediated haemolytic plaque assay measuring production of functional IgM. Recombination results in replacement of a segment of the target sequence with the exogenous sequence. Different crossover positions are possible, giving rise to alternative rearrangements of the target site. In the case of one of the recombinants we analysed, more than one of the repeated targets had undergone a conversion event. The efficiency of homologous recombination was shown to depend on the extent of homology between transfected and target DNA. A targeting efficiency of 1 x 10(-5) to 2 x 10(-5) was achieved when the exogenous DNA contained 10,000 bases of sequence homologous with the target.     

Sequence of a human immunoglobulin gamma 3 heavy chain constant region gene: comparison with the other human C gamma genes.

We report the first and complete nucleotide sequence of a human gamma 3 heavy chain constant region gene (C gamma 3). This gene displays the same organization than the others C gamma genes and exhibits normal RNA splice and polyadenylation sites. A comparison of its primary sequence with those of C gamma 1, C gamma 2 and C gamma 4 genes confirms the high degree of homology (95%) of the human family in both coding and non-coding regions, and the divergence of the hinge region. The C gamma 3 gene we sequenced codes for a Gm(b) gamma 3 chain (EZZ). Comparison with other known protein sequences reveals that only two specific aminoacids are involved in the Gm(b) and Gm(g) allotypes, which suggests an important part of the spatial configuration in the allotypic specificities.