Two new Ig VH gene families in Oncorhynchus mykiss

Genes encoding the immunoglobulin heavy-chain variable region (Ig VH) in rainbow trout (Oncorhynchus mykiss) have been grouped into 11 families. While obtaining a baseline assessment of the various gene families utilized by trout in the production of secreted antibody, we discovered two new families. These proposed Ig VH families, Families XII and XIII, were rarely observed; only two VH sequence types were detected for each new family, suggesting that they may not be commonly used in response to antigens, or that the captive environment may not lead to typical exposures seen in the wild. Additionally, unlike preceding studies, we found at least one representative gene sequence for each of the 11 reported Ig VH gene families, possibly indicating that the repertoire of trout Ig VH gene families may be more universal among different stocks than previously realized. GenBank accession numbers: Family XII—DQ453185 and DQ453150; Family XIII—DQ453153 and DQ453146; others DQ453143, DQ453156, DQ831723, DQ831825.     

The smaller human VH gene families display remarkably little polymorphism.

We report the nucleotide sequence of 30 distinct human VH gene segments from the VHIV, VHV and VHVI gene families. When these sequences were compared to previously published sequences from these smaller human VH families a surprisingly low level of polymorphism was noted. Two VHIV gene segments from unrelated individuals were identical to two previously published VHIV sequences. Five VHV sequences were identical and seven VHVI gene segments were identical. Where differences were found between the sequences, allele specific oligonucleotide probes were used to verify the germline nature of the change and to test for segregation in several large kindreds. These data provide evidence that at least some human VH gene segments are remarkably stable.     

Strain-dependent restricted VH and VL usage by anti-bacterial levan monoclonal antibodies.

The immune response to polysaccharides is highly regulated and has several distinguishing features, including restricted clonotype and isotype expression. The basis for this highly restricted response is not fully understood. To address these questions in a systematic manner, we have generated a panel of 102 mAb from CBA/CaHN (CBA/Ca) and BALB/cAnN (BALB/c) mice after one and two injections of bacterial levan (BL), a beta(2----6)-linked polyfructosan with beta(2----1)-linked fructose branch points (inulin determinant, In). This panel of mAb was examined for isotype, fine specificity, VH and VL region gene family usage and relationships between these parameters. After one or two injections of BL in both strains, mAb were IgM and IgG3. Fine specificity and VH/VL gene family usage differed markedly, however, between the two strains. Only 4% (2/51) of CBA/Ca mAb recognized the In determinant, whereas 77% (40/51) of BALB/c mAb recognized this epitope. In both strains, VH usage was restricted and certain families were overrepresented. In CBA/Ca mice, the overall response to BL was dominated by VHJ558 (45%, 23/51), the largest VH family, but VH36-60 (27%, 14/51) and VHJ606 (25%, 13/51) were also highly utilized and overrepresented. In BALB/c mice, the overall response to BL was dominated by VHJ606 (79%, 39/49 designated), a relatively small VH family. More importantly, after a single immunization with BL one particular VH/VL pair (VHJ606/ kappa 11) was used by 88% (36/41) of BALB/c mAb and was associated with In reactivity. In summary, BALB/c and CBA/Ca responses to BL differ in fine specificity and VH/VL usage.     

Developmentally controlled and tissue-specific expression of unrearranged VH gene segments

It is generally accepted that unrearranged immunoglobulin VH gene segments are not expressed and that assembly of a complete heavy chain gene is required to activate a previously silent VH promoter. We report that unrearranged VH gene segments are indeed expressed at a high level, but only in a developmentally controlled and tissue-specific manner. Unrearranged VH expression is limited to the very early stages of the B-lymphocyte differentiation pathway, and it is most prominent in cells undergoing VH to DJH rearrangement. Germ-line VH expression is independent of the heavy chain enhancer, may be controlled by 5' sequence elements, and is repressible by LPS. In contrast to earlier interpretations, our results demonstrate that the lack of unrearranged VH segment expression in mature, Ig-secreting cells is due to the inactivation of a previously active locus. These findings may provide insight into the mechanisms that control ordered rearrangement and allelic exclusion.     

Genetic mechanisms for dominant VH gene expression. The VHB512 gene.

A total of 37 mAb with reactivity for dextran B512 have been studied; 30 of them were products of independent rearrangements and 21 made use of the same VH gene, the VHB512 gene. These results unambiguously established that the immune response to dextran in the high responder mouse strain C57BL/6 was restricted. Idiotypic determinants are located all over the Ig V region. Many but not all Id described so far can be ascribed to protein structures encoded by VH or VL gene segments. The expression of the major Id, 17-9 Id, in C57BL/6 was not absolutely correlated with the expression of the dominant VHB512 gene in the same mouse strain. Inspection of amino acid sequences of the CDR3 of idiotypic positive and negative clones suggested that idiotypic structures may be associated with the expression of Tyr at position 95 and Phe or Leu at position 96 in the H and L chains, respectively. Therefore the indiscriminate use of idiotypic markers to characterize VH genes and the relevance of idiotypic regulation in VH gene expression are questioned. Id-positive and Id-negative clones displayed similar affinity values for dextran, indicating that idiotypic and binding structures were probably separated. The exchange of Asp65 for Gly65 in one of the clones reduced affinity for dextran, suggesting the involvement of CDR2 in dextran binding. The dominant expression of VH genes can be explained by somatic and/or genetic mechanisms. Because somatic mechanisms such as idiotypic regulation or selection based on affinity for dextran did not seem to influence the expression of the VHB512 gene we favor a genetic alternative. We discuss a model based on the distance between VH genes and D and JH elements. This model is compatible with somatic and genetic regulation in other systems and provides a new theoretical approach to the understanding of immune VH dominance and low responsiveness.     

VH gene expression by nontransformed pre-B cells upon differentiation in vitro

Mice have more than 1000 VH gene segments, and each pre-B cell must choose a single one for rearrangement to encode the V portion of the antibody H chain. Presumably, all or most of the functional VH gene segments must be chosen by the population of B lymphocytes if the organism is to express the diversity that is observed in the immune system. Control of the selection of a VH gene segment for expression is not understood. We have found that the members of the VH gene family closest to the constant genes, the 7183 family, are transcribed in a manner that is specific for the stage of B cell development after pre-B cells derived from spleens of 6- to 8-wk-old nude mice are induced to differentiate in vitro by a mixture of dendritic cells and mitogen-activated T lymphocytes (DC-T). DC-T from spleens and lymph nodes induce transient high levels of synthesis of RNA from the 7183 VH family, whereas DC-T from Peyer's patches of mice of the same age as those from which spleen and lymph node DC-T were prepared did not induce the expression of RNA from that gene family. Spleen and Peyer's patch DC-T induce secretion of similar total amounts of antibody. Therefore, the RNA synthesis from members of at least one VH gene family is specific both for the lymphoid tissue in which B cell differentiation occurs and for the developmental stage of the B lymphoid cells.     

Eleven MRL-lpr/lpr anti-DNA autoantibodies are encoded by genes from four VH gene families: a potentially biased usage of VH genes

The genes encoding 11 independently derived anti-DNA autoantibodies from the lupus-prone mouse strain, MRL-lpr/lpr, were examined with VH, D, and JH gene probes. These autoantibodies do not define new VH gene families, since all of the autoantibodies were encoded by VH genes from four of the nine known gene families. A minimum of nine different VH genes encoded this panel of 11 anti-DNA autoantibodies. These results are consistent with the stochastic use of the VH gene repertoire and the expression of multiple VH genes. However, the data is also consistent with a biased usage of the VH gene repertoire. First, two pairs of autoantibodies, one from the J558 family and one from the 7183 family, appear to express identical or closely related VH genes as determined by the position of two restriction enzyme sites 5' of the expressed VH genes. In addition, three autoantibodies that appear to be sister clones might define a third VH gene that is used repeatedly. Secondly, about 45% of the panel is encoded by the Q52 and 7183 families, which are the 3' most families. These families have been shown to be preferentially rearranged early in B cell ontogeny. This suggests that some anti-DNA autoantibodies might originate from a population of B cells that predominate early in ontogeny. An alternative hypothesis is that the potential bias in VH gene and gene family usage could be due to antigen selection. All four JH genes are expressed, although the JH1 gene appears to be underutilized in both expressed and unexpressed rearrangements. Two members of the panel that bind double-stranded DNA were encoded by two different VH gene families, the S107 family and the J558 family.     

Members of novel VH gene families are found in VDJ regions of polyclonally activated B-lymphocytes.

Four potentially productive and two non-productive VDJ gene segments were isolated from the DNA of mouse B-lymphocytes which had been polyclonally activated by bacterial lipopolysaccharide (LPS). Three VDJ regions exhibit VH genes which stem from two novel VH gene families. The complexity of these families is 5-9 genes. One of the non-productive VDJ regions exhibits a D segment which may have been generated by joining of two DSP2 segments. Both non-productive VDJ regions appear to contain rearranged pseudo VH genes. Three potential somatic mutations distributed over two productive VDJ regions are observed.     

Interspersion of the VHQ52 and VH7183 gene families in the NFS/N mouse

Deletion mapping analysis has shown that members of the VH7183 and VHQ52 gene families are interspersed in the NFS/N mouse. To obtain direct evidence that members of these gene families are physically linked, an NFS/N liver library was constructed and genomic clones were analyzed for hybridization to both VHQ52 and VH7183 gene probes. Four clones were identified which contained both VHQ52 and VH7183 hybridizable restriction fragments. Two clones containing rearranged VHQ52 genes were also found to hybridize with the VH7183 gene probe. Sequence analysis of three of the VH7183-containing restriction fragments indicate that all are pseudogenes which contain interruptions at either the 5' and/or 3' ends of the VH coding region. Given the D-proximal location of at least a portion of the VHQ52 gene family relative to VH7183 in NFS/N mice, and the known correlation between D proximity and the frequency of VH gene utilization, 22 NFS/N-derived pre-B cell lines were analyzed for VHQ52 gene utilization. More than 40% of the identified H chain (VHDJH) rearrangements in this survey used members of this gene family. Furthermore, analysis of poly(A)+ RNA from NFS/N fetal liver and adult spleen also indicates preferential utilization of VHQ52 family in fetal liver. Kinetic studies show, however, that there are no changes in relative utilization throughout fetal ontogeny. The implications of these findings for the expression and randomization of the VH repertoire are discussed.     

Nucleotide sequences of eight human natural autoantibody VH regions reveals apparent restricted use of VH families

Eight full length cDNA were isolated from EBV transformed human PBL derived from different normal individuals. Five were derived from antibodies with the characteristics of natural polyreactive antibodies. Three were either monoreactive or bireactive. The most striking feature of the structure of these molecules was their utilization of VH families. Although three used the large VHIII family and one used the large VHI family, the other four used genes derived from two of the recently defined small human VH families VHIV and VHV. Three of the molecules represent VHIV expressed sequences and one is the first example of a VHV gene used in an antibody of defined specificity. The nucleotide sequences of some of the molecules were remarkably similar in their VH gene segments to previously described VH genes. The data suggest that natural autoantibodies may use a restricted portion of the VH repertoire, and, in addition, that some polyreactive antibodies may be germ line encoded. The implication of these findings for the origin and diversity of the human B cell repertoire is discussed.     

The organization and expression of histone gene families

The reiteration frequency of the genes that encode the structural proteins of the mammalian ribosome was studied with a set of cloned cDNA probes containing several different mouse r-protein mRNA sequences. Results from a reassociation kinetics analysis, Southern blotting experiments and gene cloning studies collectively indicate that each individual r-protein species is represented by multiple genes in mammals. Among the examples studied, the multiplicity of mouse r-protein genes varied from about 7 to 20, a striking contrast to the low copy numbers observed in less evolutionarily advanced eucaryotes. The multiplicity of individual r-protein genes in humans and rodents is similar.     

Structure, map position, and evolution of two newly diverged mouse Ig VH gene families.

We have characterized two novel mouse VH gene families, VH3609N and VHSM7. These VH families have recently diverged from previously defined VH families. The VH3609N family, which may contain only one member in most inbred strains of mice, shares sequence similarity with the VHJ606 family and is located to the 3' side of VHJ606. VHSM7, with at least three members, is related to the VHJ558 family but maps 3' of VHJ558. These findings suggest that physical displacement of VH sequences may facilitate their subsequent divergence. During the early stages of VH gene family evolution that are exemplified by these new families, amino acid replacements have been selected against in frame-work regions and selected for in complementarity-determining regions. This pattern of nucleotide substitution appears to reflect evolutionary pressures to maintain germ-line VH diversity and, possibly, to select for new antibody specificities, as well as to select against mutations resulting in aberrant Ig. The classification of VH sequences with borderline similarity to previously defined VH families is discussed.     

Strain-dependent expression of metabolic proteins in the mouse hippocampus

Individual mouse strains differ significantly in terms of behavior and cognitive function. Strain-specific variation of metabolic protein levels in the hippocampus among various commonly used mouse strains, however, has not been investigated yet. A proteomic approach based on two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry [high capacity ion trap (HCT)] has been chosen to address this question by determining strain-dependent levels of metabolic proteins in hippocampal tissue of four inbred and one outbred mouse strain. Statistical analysis of protein spots on 2-DE gels of the individual strains (n = 10) revealed significant strain-dependent differences in densities of 39 spots. Subsequent HCT analysis led to the identification of 22 different metabolic proteins presenting with differential protein levels among the five mouse strains investigated. Among those are proteins concerned with the metabolism of amino acid, nucleic acid, carbohydrate and energy. Moreover, proteins known to play a pivotal role in the processes of learning and memory, such as calcium/calmodulin-dependent protein kinase type II alpha chain, were found to present with significant inter-strain variability, which is also in agreement with our previous reports. Strain-specific protein levels of metabolic proteins in the mouse hippocampus may provide some insight into the molecular underpinnings and genetic determination of strain-dependent neuronal function. Furthermore, data presented herein emphasize the significance of the genetic background for the analysis of metabolic pathways in the hippocampus in wild-type mice as well as in gene-targeting experiments.     

The generation of major and minor idiotype-bearing families of anti-p-azophenylarsonate antibodies; stochastic utilization of VH gene segments

An average of 50% of anti-p-azophenylarsonate (Ars) antibodies bear a cross-reactive idiotype, IdCR, and an average of 15% bear a relatively minor idiotype, Id, in A/J mice. To begin to investigate the processes that influence the expressed levels of these idiotype-bearing antibodies in serum, we have examined the frequency among preimmune B cells of cells that utilize the heavy chain variable region gene segment (VH) needed for IdCR and that which is needed for Id anti-Ars antibody expression. Our results indicate these VH gene segments are functionally rearranged at frequencies one would expect for random usage. The frequency of VH gene segment utilization is similar to, if not higher than, that of VHCR, arguing that the predominance of IdCR-over Id-bearing antibodies is not due to preferential usage of the VHCR gene segment. In addition to the analysis of Ars-immune sera pooled from several mice, we have examined 20 individual A/J mice to determine whether the relative serum levels of IdCR- and Id-bearing antibodies are strictly regulated relative to each other. Among individuals, we find that IdCR and Id antibody levels fluctuate over a 28-fold and a 120-fold range, respectively. The ratio of IdCR to Id antibody levels was found not to be strictly regulated, varying over a 300-fold range. Linear regression analysis of IdCR relative to Id concentrations shows a correlation coefficient of only 0.093. Indeed, rare mice can be found that generate greater levels of Id-bearing antibodies than those bearing IdCR. These results are indicative of a stochastic process involved during the generation of these IdCR-and Id-bearing antibody families. Models accounting for the generation of this highly variable serologic response derived from a preimmune repertoire in which VH gene segments are equivalently utilized are discussed.     

A new murine Ig VH gene family

A novel murine VH gene family, termed VH10, has been found and characterized. Based on RFLP analysis, this family exhibits extensive polymorphism among inbred strains of mice and encompasses two to five members, depending on the Igh haplotype. Analyses of recombinant inbred strains suggest a map position of this family 5' to the 7183 and Q52 VH gene families. A VH10 gene has been found to encode anti-DNA autoantibodies from lupus mice; another one may be a pseudogene.     

Concerted evolution of the immunoglobulin VH gene family

With the aim of understanding the concerted evolution of the immunoglobulin VH multigene family, a phylogenetic tree for the DNA sequences of 16 mouse and five human germ line genes was constructed. This tree indicates that all genes in this family have undergone substantial evolutionary divergence. The most closely related genes so far identified in the mouse genome seem to have diverged about 6 million years (MY) ago, whereas the most distantly related genes diverged about 300 MY ago. This suggests that gene duplication caused by unequal crossing-over or gene conversion occurs very slowly in this gene family. The rate of occurrence of gene duplication in the VH gene family has been estimated to be 5 x 10(-7) per gene per year, which seems to be at least about 100 times lower than that for the rRNA gene family. This low rate of concerted evolution in the VH gene family helps retain intergenic genetic variability that in turn contributes to antibody diversity. Because of accumulation of destructive mutations, however, about one-third of the mouse and human VH genes seem to have become nonfunctional. Many of these pseudogenes have apparently originated recently, but some of them seem to have existed in the genome for more than 10 MY. The rate of nucleotide substitution for the complementarity-determining regions (CDRs) is as high as that of pseudogenes. This suggests that there is virtually no purifying selection operating in the CDRs and that germ line mutations are effectively used for generating antibody diversity.      

葡萄生长素响应基因家族生物信息学鉴定和表达分析

生长素响应基因家族能调节植物体内生长素平衡和生长素信号途径。文章采用生物信息学方法检索获得葡萄(Vitisvinifera L.)基因组数据库中的生长素响应基因,通过分析其染色体定位、基因共线性和系统进化,发现葡萄基因组含有25个AUX_IAA基因、19个ARF基因、9个GH3基因、42个LBD基因。这些生长素响应基因不均匀分布在葡萄的19条染色体上,部分家族基因在染色体上形成基因簇。葡萄芯片数据结果表明,生长素响应基因在葡萄不同时期的果实和叶芽中均有表达,尤其在果实转色期、叶芽萌发或休眠期表达量急剧变化。研究结果为葡萄生长素响应基因在叶片和果实发育过程中的功能研究提供参考。     

Factors affecting gene expression of patatin and proteinase-inhibitor-II gene families in detached potato leaves:

In whole intact potato (Solanum tuberosum L.) plants, the gene families of class-I patatin and proteinase inhibitor II (Pin 2) are constitutively expressed in the tubers. However, they are also induced in detached potato leaves in the presence of light. To further characterize this light action, the detached leaves were subjected to monochromatic light of different wavelengths and to darkness in the presence of metabolites and inhibitors. Patatin genes could be induced by the simultaneous application of sucrose (sugars) and glutamine in darkness. Neither of these metabolites was active when supplied alone. When photosynthesis was blocked by 3-(3,4-Di-chlorophenyl)-1, 1-dimethylurea (DCMU) in the light, patatin genes were not expressed; however, the inhibition was overcome in the presence of sucrose. This indicates that besides its role in photosynthetic carbohydrate production, light may be essential for the supply of amino acids (or reduced nitrogen). Unlike patatin, Pin 2 genes were, to a small extent, also active in darkness, and sucrose weakly enhanced this expression. However, DCMU did not affect Pin 2 expression in the light. Both abscisic acid and methyl jasmonate strongly promoted the accumulation of Pin 2 mRNA independent of the light conditions, indicating that the gene family is probably under hormonal control. The phytohormones did not affect patatin gene expression. Inhibitors of cytosolic (cycloheximide) and organellar (chloramphenicol) translation had opposite effects on the two gene families. Careful evaluation of the inhibitors' action indicates that protein synthesis (cytosol) is required for the expression of Pin 2 genes but not for the patatin genes. These results clearly demonstrate that, although in situ both gene families are constitutively expressed in the same plant organ (tuber) in intact plants, their expression is mediated by different factors.Abbreviations ABA cis-abscisic acid - DCMU 3-(3,4-dichlorphenyl)-1,1-dimethylurea - GUS -glucuronidase activity - MeJA methyl jasmonate - Pin 2 proteinase inhibitor II We thank Beate Küsgen and Regina Breitfeld for the greenhouse work. This work was supported by a grant from the Bundesministerium für Forschung und Technologie.