Indirect and direct evidence for DNA double-strand breaks in hypermutating immunoglobulin genes.

The generation of a diverse antigen receptor repertoire is fundamental for the functionality of the adaptive immune system. While the V(D)J recombination process that generates the primary antigen receptor repertoire is understood in great detail, it is still unclear by which mechanism immunoglobulin (Ig) genes are further diversified by somatic hypermutation. Using mouse strains that carry a non-functional, pre-defined V(H)D(H)J(H) gene segment in their IgH locus we demonstrate DNA double-strand breaks (DSBs) in and around V(H)D(H)J(H) in B cells undergoing somatic hypermutation. The generation of these DSBs depends on transcriptional activity, and their distribution along the V(H)D(H)J(H) segment parallels that of point mutations in the hypermutation domain. Furthermore, similar to hot spots of somatic hypermutation, 50-60% of all DSBs occur preferentially at RGYW motifs. DSBs may transiently dissociate the Ig promoter from the intronic enhancer to block further transcription and to initiate an error-prone non-homologous DSB repair pathway. In accord with this model large deletions are frequently produced, along with point mutations, in a V(H)D(H)J(H) segment inserted together with its promoter into the IgH locus in inverted orientation. Our data suggest that DSBs are reaction intermediates of the mechanism underlying somatic hypermutation.

DNA double-strand breaks in immunoglobulin genes undergoing somatic hypermutation.

How rearranged immunoglobulin (Ig) genes are further diversified by somatic hypermutation is unknown. Using VDJ passenger Ig heavy chain (IgH) knockin mouse strains, we now demonstrate a high frequency of DNA double-strand breaks (DSBs) in the targeted VDJ passenger gene of germinal center (GC) B cells. These DSBs parallel the distribution of mutations in the targeted hypermutation domain and are found preferentially at RGYW motifs, the intrinsic hot spots of somatic hypermutation. The introduction of DSBs appears to depend on transcriptional activity. Thus, secondary diversification of rearranged V gene segments relates to an error-prone nonhomologous DSB repair system acting in B cells of the GC.

Tuning somatic hypermutation by transcription.

The dependence of somatic hypermutation on transcription was studied in three mutant immunoglobulin heavy chain (IgH) insertion mice in which a targeted non-functional VHB1-8 passenger transgene was either placed under the transcriptional control of a truncated DQ52 promoter (p delta), its own RNA polymerase II dependent IgH promoter (pII) or a RNA polymerase I dependent promoter (pI). The relative mutation-frequency of the VHB1-8 passenger transgene in memory B cells of p delta, pI and pII mice (7%, 60% and 100%) correlated with the relative levels of transgene-specific pre-mRNA expressed in germinal center B cells isolated from the mutant mice (8%, 72% and 100%, respectively). These data indicate that the mutation load of rearranged Ig genes can be tuned by transcription. The question, whether somatic hypermutation requires transcription per se or a specific component of the RNA polymerase II complex, is under investigation.

Somatic hypermutation in the heavy chain locus correlates with transcription.

Three mutant immunoglobulin heavy chain (IgH) insertion mice were generated in which a targeted nonfunctional IgH passenger transgene was either devoid of promoter (pdelta) or was placed under the transcriptional control of either its own RNA polymerase II-dependent IgH promoter (pII) or a RNA polymerase I-dependent promoter (pI). While the transgene mutation-frequency (0.85%) in memory B cells of pI mice was reduced compared to that in pII mice (1.4%), the distribution and the base exchange pattern of point mutations were comparable. In pdelta mice, the mutation frequency was drastically reduced (0.09%). The mutation frequencies correlated with the levels of transgene-specific pre-mRNA expressed in germinal center B cells isolated from the mutant mice.

Hypermutation of immunoglobulin genes in memory B cells of DNA repair-deficient mice.

To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vlambda1 light chain genes from naive and memory B cells in DNA repair-deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand-break repair, and AAG-mediated base excision repair.

Recent translocation of variable and diversity segments of the human immunoglobulin heavy chain from chromosome 14 to chromosomes 15 and 16.

We studied the organization and origin of three orphon regions, VH-F, D5-a, and D5-b, of the human immunoglobulin heavy-chain gene using yeast artificial chromosomes. VH-F and two D5 regions were mapped to chromosome bands 16p11 and 15q11-q12, respectively, by using human/rodent somatic cell hybrids and fluorescence in situ hybridization. No D5 segments were found on chromosome 14, in contradiction to previous reports. The VH-F region consists of 7 VH segments and encompasses 160 kb of DNA. A cluster of VH segments homologous to the VH-F region orphons was found in the region 245-430 kb (V2-26 to V3-11) upstream of the JH cluster on chromosome 14. Comparison of VH sequences between the VH-F and the chromosome 14 loci indicates that the translocation of the VH-F region took place, at the earliest, 20 x 10(6) years ago. The D5-a and D5-b regions were obtained in two independent contigs. The former contains only D segments in 140 kb of DNA, while the latter carries 3 VH segments downstream of D segments in 110 kb of DNA. V54, one of these VH orphon segments, is about 95% homologous to V1-18, which is located within the putative ancestor of the VH-F region on chromosome 14. Furthermore, the region detected by two DNA probes flanking the V54 segment was found only in the proximity of V1-18 within the 0.8-Mb VH region on chromosome 14. These results suggest that the two orphon loci on chromosomes 15 and 16 may have been translocated simultaneously.

Structural analysis of the human VH locus using nonrepetitive intergenic probes and repetitive sequence probes. Evidence for recent reshuffling.

The organization and evolution of the 0.8-Mb JH-proximal region in the human Ig VH locus were studied by mapping DNA fragments hybridized to non-repetitive intergenic probes and by determination of the content and distribution of repetitive sequences. Southern blot analysis of cloned DNA covering the 0.8-Mb region with intergenic probes allowed us to map two to seven cross-hybridizing fragments by each probe. Clusters of fragments detected by an identical set of probes appeared repeatedly within the 0.8-Mb JH-proximal region. Distantly located VH segments flanked by a cluster of DNA fragments hybridized by the same set of probes were highly homologous to each other, providing evidence for recent frequent duplication and translocation throughout the locus. DNA fragments detected by the same set of probes were orientated with the same 5' to 3' order within the cluster, suggesting little involvement of inversion upon recombination in the locus. The content of interspersed Alu and L1 sequences in the VH locus were not significantly greater than the average in the genome.

Comparison and evolution of human immunoglobulin VH segments located in the 3' 0.8-megabase region. Evidence for unidirectional transfer of segmental gene sequences.

Nucleotide sequences of 64 VH segments within the 3' 0.8-megabase region of the human immunoglobulin germ line VH locus were compared with trace evolution of human VH segments. Based on alignment of the deduced amino acid sequences of 37 functional germ line VH segments, a phylogenetic tree was generated using the neighbor-joining method. The phylogenetic tree clearly supports the previous classification of human VH segments into six families, which correlate roughly with mouse VH families with varying conservation. The human VH-III family is most homologous to mouse VH segments, suggesting that members of the VH-III family may be conserved by some functional constraint. The 5'-flanking region of each family has a family-specific structure. The sequenced 64 VH segments include 31 pseudogenes, of which 24 were highly conserved. Unidirectional transfer of segmental sequences was identified within the VH-III and VH-IV families, providing clear examples of germ line gene conversion. Such gene conversion may contribute to conserve structures of pseudo-VH segments. Comparison of the VH-IV family members indicates that recent repeated duplications and frequent gene conversions are responsible for strong conservation of this family, although functional selection is not completely excluded.

The human S mu bp-2, a DNA-binding protein specific to the single-stranded guanine-rich sequence related to the immunoglobulin mu chain switch region.

We have cloned the cDNA encoding the human homologue of S mu bp-2, which binds to single-stranded DNA with 5'-phosphorylated guanine-rich sequences related to the immunoglobulin mu chain switch (S mu) region. The deduced amino acid sequences of the mouse and human S mu bp-2 are 76.5% homologous and contain motifs conserved among helicases. We have identified a domain essential for DNA binding at residues 638-786. The binding domain is less conserved (63% homologous) than the putative catalytic domain of N-terminal half containing most of the helicase motifs (85% homologous). The human and mouse S mu bp-2 have similar, although slightly different, binding specificities. Although the mouse S mu bp-2 preferentially binds to the mouse S mu motif (GGGGT), the human S mu bp-2 binds equally well to the human (GGGCT) and mouse S mu motifs. The human S mu bp-2 gene was mapped to chromosome 11 q13.2-q13.4 by in situ hybridization.

Isolation of cDNA encoding a binding protein specific to 5'-phosphorylated single-stranded DNA with G-rich sequences.

We have isolated the cDNA encoding a binding protein to the sequence motif of the immunoglobulin S mu region by the southwestern method. The binding protein designated S mu bp-2 specifically binds to 5'-phosphorylated single-stranded DNA containing 5'-G and GGGG stretches. The amino acid sequence deduced from the cDNA sequence showed that the S mu bp-2 belongs to the putative helicase superfamily which is involved in replication, recombination and repair. Expression of S mu bp-2 mRNA is ubiquitous and augmented in spleen cells stimulated with lipopolysaccharide and interleukin 4 which also induce class switching. The S mu bp-2 gene is conserved among vertebrates. Possible involvement of S mu bp-2 in class switching is discussed.

Structure and physical map of 64 variable segments in the 3'0.8-megabase region of the human immunoglobulin heavy-chain locus.

We have constructed the physical map of the 0.8 megabase DNA fragment which contains the 3' 64 variable region (V) gene segments of the human immunoglobulin heavy chain (H) locus. The organization of the VH locus showed several features that indicate dynamic reshuffling of this locus. The sequenced 64 VH segments include 31 pseudogenes, of which 24 are highly conserved except for a few point mutations. Comparison of the 64 germline VH sequences shows that each VH family has conserved sequences, suggesting that there might be some genetic or selection mechanisms involved in maintenance of each family. The total number of the human VH segments was estimated to be about 120, including at least 7 orphons.

Physical map of the 3' region of the human immunoglobulin heavy chain locus: clustering of autoantibody-related variable segments in one haplotype.

We have constructed the physical map of the 3' region of the human immunoglobulin heavy chain variable region (VH) genes. DNA segments extending to 200 kb upstream of the JH segment were isolated in two YAC clones. Five VH segments were identified in this region in the 5' to 3' order, V(II-5), V(IV-4), V(I-3), V(I-2), and V(VI-1) segments which were all structurally normal and orientated in the same direction as the JH segments. From DNA of a different cell line we have isolated a cosmid contig containing the same DNA region which has extraordinary polymorphism. The YAC and cosmid DNAs were called haplotypes A and B, respectively. Haplotype B contained an additional VH-I segment (V(I-4.1b)) between the V(II-5) and V(IV-4) segments. V(I-4.1b) segment is almost identical to a previously published VH sequence encoding a rheumatoid factor. Another VH segment in the B haplotype (V(I-3b)) corresponding to the V(I-3) segment also showed 99.7% nucleotide sequence homology with an anti-DNA autoantibody VH sequence. However, none of the VH sequences in haplotype A showed such strong homology with autoantibody VH sequences. The results suggest that VH haplotypes may have linkage with autoantibody production.

A null cell adenoma of the pituitary detected seven years after removal of a prolactinoma. Recurrence or de novo tumourigenesis?

We report an extremely unusual case of prolactinoma which emerged at recurrence as a null cell adenoma. A 53-year-old woman sought medical attention for progressive visual loss and headache. A pituitary tumour was detected by a computed tomographic scan, and hyperprolactinemia was noted. The tumour, removed by a transfrontal surgery, was a chromophobe adenoma, and immunohistochemically the adenoma cells were selectively positive for PRL, which indicated a prolactinoma. Postoperatively, her plasma PRL level was normalized. Seven years later, she noted blurred vision and again sought medical attention. A CT scan demonstrated recurrence of a pituitary tumour. On this occasion, however, she was not hyperprolactinemic. She underwent again a transfrontal resection of the pituitary tumour. Its histology was again a chromophobe adenoma, but the adenoma cells showed no positive immunostaining for any anterior pituitary hormone including PRL, which indicated a null cell adenoma. We have no clear explanation of the pathogenesis underlying her very unusual course. However, null cells (assuming that the original tumour was a mixed adenoma) left behind at the first surgery, or unidentified hypothalamic and/or pituitary derangements might possibly have been responsible for the recurrence. We learned from this patient that recurrent pituitary adenomas may not necessarily have the same endocrine features as did the original tumours. This information appears to make a valid clinical point, because if hormone levels alone are followed after pituitary surgery, recurrent pituitary tumours may be overlooked.

Antitumor activity of streptococcal acid glycoprotein produced by Streptococcus pyogenes Su.

Streptococcal acid glycoprotein (SAGP) was purified from the cultured cells of Streptococcus pyogenes Su, and its in vitro and in vivo antitumor activities were investigated in comparison with those of OK-432, a cell preparation of S. pyogenes Su which is used clinically as a potent antitumor agent. SAGP inhibited the growth of several tumor cell lines in vitro at less than 0.1 microgram/ml, while it did not affect the growth of the other tumor and normal cell lines even at 10 micrograms/ml. This selective cytotoxicity is a unique characteristic of SAGP. OK-432 did not show cytotoxicity in vitro. SAGP also showed a considerable life-span-prolonging effect on mice bearing Meth A tumor and inhibited the growth of sarcoma 180 tumor implanted im. The comparison of antitumor activities between SAGP and OK-432 definitely suggested a difference in the mechanisms of their actions, even though they were derived from the same bacterial strain.

Basal and postprotein insulin and glucagon levels during a high and low carbohydrate intake and their relationships to plasma triglycerides.

The effects of acute and chronic differences in the carbohydrate content of the diet on plasma insulin, glucagon, insulin-glucagon molar ratio (I/G), and triglycerides were studied. Acute effects were studied by varying the carbohydrate content of a single test meal, while chronic effects were determined by varying the carbohydrate content of the diet for a week. A test meal containing 0.6 gm of gelatin per kilogram plus 0.6 gm. per kilogram of glucose resulted in much higher levels of insulin and I/G (p smaller than 0.005), lower glucagon levels (p smaller than 0.05), and slightly higher triglycerides (N.S.) than did a meal of 1.2 gm, per kilogram of gelatin alone. One week of a 12 gm. carbohydrate, 2870-calorie diet lowered insulin (p smaller than 0.001), I/G (p smaller than 0.05), and triglycerides (p smaller than 0.001) and increased glucagon (N.S.), whereas a 390-gm. carbohydrate, 2784-calorie intake significantly increased insulin, I/G, and triglycerides (p smaller than 0.005) and lowered glucagon (p smaller than 0.02) within two days; even greater changes in hormones were observed on a 510-gm. carbohydrate intake. Of those patients in whom a high carbohydrate intake induced a triglyceride rise of at least 40 mg. per deciliter, a significant correlation between the change in I/G and the change in triglycerides was noted (r equals 0.85; p smaller than 0.01). The results are compatible with but do not prove the proposal that pancreatic alpha and beta cells play a mediating role in carbohydrate induction of hyperlipidemia.