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PubMed1991

Molecular characterization of a supratypic cross-reactive idiotype associated with IgM autoantibodies.

Neoplastic B cells from patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphomas (SLL) frequently express surface Ig reactive with the mouse mAb, Lc1. Raised against a human monoclonal IgM with rheumatoid factor activity, Lc1 detects a major cross-reactive Id (CRI) present on the H chain of many monoclonal IgM autoantibodies. In contrast to other major autoantibody-CRI investigated to date, we note that the Lc1-CRI is expressed by subpopulation of cells in the germinal centers, as well as in the mantle zones, of secondary human B cell follicles. To examine the molecular basis for Lc1 expression, we used the polymerase chain reaction to isolate the functionally rearranged Ig VH genes of monoclonal Lc1-reactive B cell populations from six unrelated patients with CLL or SLL. Although the neoplastic B cells from most patients with CLL or SLL express the CD5 surface differentiation Ag, the lymphoma cells from one patient with SLL were CD5-negative. We find that the Lc1-reactive cells from each cell population have Ig rearrangements involving a VH gene of the VH4 subgroup. However, the VH4 genes rearranged in different Lc1-reactive tumor populations may originate from at least two disparate germ-line VH4 genes. Also, in contrast to the CD5-positive tumor populations, we find evidence for intraclonal diversity in the functionally rearranged VH4 genes of the CD5-negative SLL. Collectively, this study discerns a degeneracy in the VH4 genes that can encode the Lc1 CRI, indicating the term "supratypic cross-reactive idiotype" may best describe the specificity of the Lc1 mAb. Also, this study suggests that expression of CD5 may delineate categories of B cell SLL that differ in their relative rates of constitutive Ig V gene somatic mutation.

Amino Acid Sequence↗

PubMed1990

Structure of rearranged and germ-line rabbit V kappa genes indicated that the CDR3 is encoded by the V kappa gene.

The third complementary-determining regions (CDR3) of rabbit kappa chains are unusually long and the length is more heterogeneous when compared to those of the mouse and the human kappa chains. To study how the rabbit kappa light (L) chain genes create diversity and generate CDR3, we analyzed the structure of a rearranged variable kappa gene (Vr) and the variable (Vg) and joining (Jg) regions of the putative precursor genes. Alignment of the Vr gene sequence with that of the Vg and Jg regions allowed precise determination of the recombination event. Five nucleotides between the recombination point and the J2 heptamer were deleted, indicating flexibility in the recombination producing rabbit kappa chains. The entire Vg is contained in the rearranged product demonstrating that neither a D element nor an N sequence addition are required for the CDR3 formation. Comparison of the Vr and the Vg gene sequences show base substitutions suggesting that somatic mutations may contribute to rabbit kappa L chain diversity.

Amino Acid Sequence↗

PubMed1992

Sequencing heavy- and light-chain variable genes of single B-hybridoma cells by total enzymatic amplification.

We have devised a protocol to obtain accurate and complete sequences of the immunoglobulin heavy- and light-chain variable-region (VH and VL) genes of single B-hybridoma cells that express defined V genes. The amplification achieved ranges from 2 x 10(13)- to 1 x 10(14)-fold. Only one potential Taq DNA polymerase error was observed in 7590 nucleotides of sequence, thus permitting the identification of naturally occurring somatic mutations. The two-step nature of the amplification protocol provides sufficient DNA for a minimum of 160 sets of sequencing reactions of both the VH and VL genes from one cell without cloning. The amplification of relatively long segments of DNA in the first step of the protocol permits second-step amplification and sequencing of regions that flank VH and VL codons. Fractionating cellular lysates prior to the first step of amplification permits the separate amplification of V genes on opposite sister chromatids and possibly on opposite strands of the same DNA duplex. Accurate sequencing of VH and VL genes of defined germ-line origin that are expressed by single B cells taken directly from the animal is thus made feasible by this approach.

Animals↗

PubMedDate not supplied

The complete sequence of the murine monoclonal immunoglobulin MOPC 173 (IgG2a): genetic implications.

The complete amino acid sequence of the murine monoclonal immunoglobulin MOPC 173 (IgG2a, kappa) is reported. The heavy chain contains 447 amino-acid residues, and one carbohydrate prosthetic group attached to the ASX residue 299. The kappa light chain is composed of 214 residues. The H chains are covalently linked by 3 interchain disulfide bridges. The H-L bond-forming cysteine of the H chain is between the VH and the CH1 domain. Intrachain bridges are disposed linearly, according to the classical model. There is no simple relationship between the primary structure and any given function of a particular domain. This is presumably due to the fact that the selection pressure exerts itself on the three-dimensional structure which may retain a conserved general organization as a result of balanced multiple mutations. Selection seems to act in two ways: --horizontally, in a multigene system such as the immunoglobulin classes (C domains of the heavy chains), leading to interclass homologies which are particularly marked for all the COOH-terminal domains of H and L chains which have, in addition a fair degree of homology with human beta2 microglobulin (about 30% identities); --vertically, in which case strictly homologous domains appear extremely conserved between distinct animal species. Conservation of the VH domains seems just as high as conservation of the CH domains. The VH region contains 3 types of positions: invariant and subgroup characteristic ("framework") which may be accounted for by a rather small number of germ-line genes, and hypervariable for which the origin of diversity, somatic or germinal, cannot be decided from sequence data alone. Murine VK domains, although basically built according to the same pattern, show a much more marked polymorphism of the framework, which might necessitate a higher number of basic germ-line genes. Finally, a hypothetical model of the switch mechanism is proposed. Rotational symmetry regions can be deduced at the DNA level from the known amino acid sequences of the switch peptides for the three translocational systems: H, kappa and lambda. These would provide recognition signals for restriction-like enzymes such as those which operate in prokaryotes. An implication of this model is the definition of an exact limit between the V and the C regions of all immunoglobulin chains.

Amino Acid Sequence↗

PubMed1990

A molecular and structural analysis of the VH and VK regions of monoclonal antibodies bearing the A48 regulatory idiotype.

The results presented in this paper explore the molecular basis for expression of the A48 regulatory Id (RI). A48 RI+ mAb derived from idiotypically manipulated mice molecularly resembled the A48 and UPC 10 prototypes of this system by utilizing a VHX24-Vk10 combination. Id expression by these antibodies was not restricted by a particular D region sequence, JH, or JK segment, but quantitative differences in Id expression were associated with utilization of different members of the VK10 germ-line gene families. The VL sequences of these A48 RI+ mAb has identified amino acid residues lying in four different idiotope-determining regions which may contribute to the structural correlate of this Id. A comparative sequence analysis of the VH regions of these VHX24 utilizing A48 RI+ mAb with several A48 RI+ mAb utilizing VHJ558 or VH7183 VH genes as well as a hybrid transfectoma antibody derived from two A48 RI-, VHJ558 utilizing hybridomas, all suggested that four nonconsecutive positions which lie outside the idiotope-determining regions may contribute structural elements toward expression of this Id. The VH and VL regions of the A48RI+, VHX24-Vk 10+ mAb showed low to moderate levels of somatic mutation which showed different patterns of distribution between the complementary determining region (CDR) and framework regions in the H and L chains. Although the VK sequences contained 50% of the replacement mutations in the CDR, with a replacement/silent mutation ratio of 10, the CDR of the VH sequences contained only 31% of the replacement mutations with a replacement/silent mutation ratio of 0.69.

Amino Acid Sequence↗

PubMed1990

Estimation of D segment usage in initial D to JH joinings in a murine immature B cell line. Preferential usage of DFL16.1, the most 5' D segment and DQ52, the most JH-proximal D segment.

We established SPL2-1-2, a murine immature B cell line transformed by tsOS-59, a temperature-sensitive mutant of Abelson murine leukemia virus. SPL2-1-2 has a VHDJH-/G (germ-line) configuration, and can continuously generate D to JH joinings from the germ-line allele during culture. The D to JH joinings are strongly promoted by the shift of the culture temperature from permissive (35 degrees C) to non-permissive temperature (39 degrees C). Using this H chain gene rearrangement system, we succeeded in the estimation of the frequencies of D segment usage in initial D to JH joinings by deletion-mapping analysis. The results demonstrated the preferential utilization of DFL16.1, the most 5' D segment and DQ52, the most JH-proximal D segment in initial D to JH joinings.

Alleles↗

PubMed1992

The forces driving autoimmune disease.

There are two classes of autoimmune disease, organ-specific and non-organ specific or systemic. That cells producing autoantibodies are selected by antigen is strongly suggested by the presence of mutations and high affinity antibody. T-cells are pivotal in all forms of autoimmunity as evidenced by the therapeutic benefit of anti-T-cell monoclonals such as anti-CD4, and the frequent development of high affinity IgG autoantibodies. The production of anergic T-cells by the use of non-depleting anti-CD4 in the presence of antigen is discussed with particular reference to its potential for immunological intervention in autoimmune disease. It is possible to identify T-cell epitopes in organ-specific autoimmunity using pathogenic T-cell clones or hybridomas to identify the peptide sequences which are reactive. Antigen-specific therapy may ultimately be based on such peptide epitopes. The specificity of the T-cells in systemic autoimmunity is still obscure, but there is some evidence that reactivity with certain germ-line idiotypes can lead to the development of systemic autoimmunity. The possibility of stimulating B-cells specific for auto-antigens such as DNA becomes feasible if a complex of antibody and DNA is taken up by these specific B-cells and processed idiotype is presented to T-helpers specific for those idiotype epitopes. Evidence is presented that there may be pre-existing defects in the target organ in certain organ-specific disorders, and the evidence for a glycosylation defect in the IgG in patients with rheumatoid arthritis is explored. It is noted that the spouses of probands with rheumatoid arthritis is explored. It is noted that the spouses of probands with rheumatoid arthritis also tend to have this glycosylation defect and this raises the possibility of an effect due to an environmental factor, such as a microbial infection. Molecular mimicry of autoantigens by microbes can stimulate autoreactive cells by their cross-reactivity. It is emphasized that cross-reaction which gives rise to the priming of autoreactive T-cells could give rise to the establishment of a chronic autoimmune state. In animals with normal regulatory immune systems, such induced autoimmunity is ultimately corrected and it is only in animals where there are defects in regulation, that autoimmunity persists. Thus, there are many factors giving rise to autoimmunity, and the diseases are rightly regarded as multifactorial in origin.

Amino Acid Sequence↗

PubMed1991

tudor, a posterior-group gene of Drosophila melanogaster, encodes a novel protein and an mRNA localized during mid-oogenesis.

The tudor (tud) locus of Drosophila melanogaster is required during oogenesis for the formation of primordial germ cells and for normal abdominal segmentation. The tud locus was cloned, and its product was identified by Northern analysis of wild-type and tud mutant RNAs. The locus encodes a single mRNA of approximately 8.0 kb that is expressed throughout the life cycle, beginning in the early stages of germ-line development in the female. During oogenesis, tud mRNA appears to be present in the oocyte precursor within the germarial cysts, and in stages 1-3 it accumulates within the developing oocyte. The transcript is localized to the posterior half of the oocyte during oogenetic stages 4-7 but is not detectable within the ooplasm by egg deposition and throughout early embryogenesis. The tud protein has a predicted molecular mass of 285,000 daltons and has no distinctive sequence similarity to known proteins or protein structural motifs. Taken together, these results indicate that the tud product is a novel protein required during oogenesis for establishment of a functional center of morphogenetic activity in the posterior tip of the Drosophila embryo.

Amino Acid Sequence↗

PubMed1992

Chorion gene cis-regulatory DNA restricts tissue specificity of reporter gene expression in transformed Drosophila.

P element mediated germ-line transformation was used to study the developmental specificity of Drosophila chorion gene regulatory sequences directing expression of the bacterial reporter genes for chloramphenicol acetyltransferase (CAT) and beta-galactosidase (lacZ). DNA fragments containing 5' flanking plus the entire 5' untranslated and the beginning of the coding region of either the s36 or the s15 chorion gene are able to confer on the reporter genes normal tissue as well as temporal specificity of expression, exclusively in the ovary of transformed female flies. However, if 5' untranslated and coding regions are omitted, normal ovarian expression is maintained but tissue specificity is relaxed: expression of the reporter gene is detected both in the ovary and in specific non-ovarian tissues of transformed females and males. The evidence suggests that the missing 5' untranslated and coding sequences may include negative elements that normally suppress expression in non-ovarian tissues, and that these putative elements are distinct from those that prevent premature expression in the ovarian follicles. The exact location of ectopic lacZ expression within the internal male genitalia depends on the constellation of 5' flanking chorion regulatory sequences included in the P element constructs. Ectopic expression of the CAT gene in the male genitalia under s15 promoter control can be abolished by mutating the hexamer TCACGT, a sequence previously shown to be essential for the normal expression of this chorion gene in the ovary.

Animals↗

PubMed1992

Intracellular selection, conversion bias, and the expected substitution rate of organelle genes.

A key step in the substitution of a new organelle mutant throughout a population is the generation of germ-line cells homoplasmic for that mutant. Given that each cell typically contains multiple copies of organelles, each of which in turn contains multiple copies of the organelle genome, processes akin to drift and selection in a population are responsible for producing homoplasmic cells. This paper examines the expected substitution rate of new mutants by obtaining the probability that a new mutant is fixed throughout a cell, allowing for arbitrary rates of genome turnover within an organelle and organelle turnover within the cell, as well as (possibly biased) gene conversion and genetic differences in genome and/or organelle replication rates. Analysis is based on a variation of Moran's model for drift in a haploid population. One interesting result is that if the rate of unbiased conversion is sufficiently strong, it creates enough intracellular drift to overcome even strong differences in the replication rates of wild-type and mutant genomes. Thus, organelles with very high conversion rates are more resistant to intracellular selection based on differences in genome replication and/or degradation rates. It is found that the amount of genetic exchange between organelles within the cell greatly influences the probability of fixation. In the absence of exchange, biased gene conversion and/or differences in genome replication rates do not influence the probability of fixation beyond the initial fixation within a single organelle. With exchange, both these processes influence the probability of fixation throughout the entire cell. Generally speaking, exchange between organelles accentuates the effects of directional intracellular forces.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Replication↗

PubMed1991

V lambda and J lambda-C lambda gene segments of the human immunoglobulin lambda light chain locus are separated by 14 kb and rearrange by a deletion mechanism.

We have cloned a region of 124 kb of the human immunoglobulin lambda light chain locus on chromosome 22 encompassing seven V lambda and seven J-C lambda gene segments. No further C lambda gene segment was found in a region of 35 kb downstream of C lambda 7, which encodes the Ke+Oz- isotype. The C lambda proximal V lambda gene segment V lambda III. 1 is located 14.5 kb upstream of C lambda 1. The five sequenced V lambda genes have the same transcriptional orientation as the J-C lambda gene segments which is likely to be true for the majority of the V lambda gene segments in the human lambda locus and which suggests a deletion mechanism for DNA rearrangement. This is supported by hybridization of V lambda gene probes to germ-line and rearranged DNA from lambda light chain-producing cell lines. Sequences of 23 cDNA clones allow to establish a V lambda subgroup classification based on nucleic acid sequence data and an estimate of the J-C lambda usage.

Amino Acid Sequence↗

PubMedDate not supplied

Primary B-cell response to neuropeptide Y and bovine pancreatic polypeptide.

An analysis of the murine primary response to protein epitopes has been made with two small highly structured proteins, neuropeptide Y (NPY) and bovine pancreatic polypeptide (BPP), both of 36-amino acid residue length and containing helical structures. A group of cell lines producing monoclonal IgM antibody have been prepared consisting of six anti-NPY and two anti-BPP. The VH nucleotide sequences have been determined and characterized as germ-line either by identity to established germ-line sequences or by inference from the germ-line character of the D and JH segments. The intrinsic association constants for the homologous ligands have been estimated to range from 10(4) to 10(7) M-1 based on competitive ELISA. No severe restriction in the utilization of VH families, D segments or JH segments appears to be involved in this response. Among the eight cell lines, three VH families were represented as well as all three families of D segments and all of the JH segments, although some preference for JH3 was indicated. The length of the N(D)N sequences was also not subject to restriction, ranging from 9 to 29. Two unusual features of the CRD3s were noted, one involving the utilization of an uncommon DSP2 segment and the other the apparent occurrence of a D-D fusion.

Amino Acid Sequence↗

PubMed1992

Identification of a genomic DNA fragment containing the Drosophila melanogaster ovarian tumor gene (otu) and localization of regions governing its expression.

We have identified a genomic DNA fragment which restores fertility to mutants of the ovarian tumor locus (otu) of Drosophila melanogaster. Germ-line transformants bearing this fragment express otu mRNA with the same tissue specificity as, and at levels comparable to, the wild-type otu gene. Transcription from the otu promoter, P(otu), which lacks a TATA element, appears to be initiated at multiple transcription start points (tsp) within an 80-bp region. Deletion of sequences upstream of the tsp indicates that a region between nucleotides -190 and -310 is required for proper expression from the otu gene. A DNA fragment containing 452 bp upstream and 126 bp downstream from the tsp is able to direct expression of the Escherichia coli lacZ gene in the germ cells of the ovary and testis, indicating that cis-acting regulatory elements governing these expression patterns are located in a 578-bp region surrounding the multiple tsp.

Animals↗

PubMed1991

Effects of the steel gene product on mouse primordial germ cells in culture.

Mutations at the steel (sl) and dominant white spotting (W) loci in the mouse affect primordial germ cells (PGC), melanoblasts and haemopoietic stem cells. The W gene encodes a cell-surface receptor of the tyrosine kinase family, the proto-oncogene c-kit. In situ analysis has shown c-kit messenger RNA expression in PGC in the early genital ridges. The Sl gene encodes the ligand for this receptor, a peptide growth factor, called here stem cell factor (SCF). SCF mRNA is expressed in many regions of the early mouse embryo, including the areas of migration of these cell types. It is important now to identify the role of the Sl-W interaction in the development of these migratory embryonic stem cell populations. Using an in vitro assay system, we show that SCF increases both the overall numbers and colony sizes of migratory PGC isolated from wild-type mouse embryos, and cultured on irradiated feeder layers of STO cells (a mouse embryonic fibroblast line). In the absence of feeder cells, SCF causes a large increase in the initial survival and apparent motility of PGC in culture. But labelling with bromodeoxyuridine shows that SCF is not, by itself, a mitogen for PGC. SCF does not exert a chemotropic effect on PGC in in vitro assays. These results suggest that SCF in vivo is an essential requirement for PGC survival. This demonstrates the control of the early germ-line population by a specific trophic factor.

Animals↗

PubMed1979

Demonstration of low density lipoprotein receptors in mouse teratocarcinoma stem cells and description of a method for producing receptor-deficient mutant mice.

Familial hypercholesterolemia, a widespread human genetic disorder implicated in vascular and coronary disease, has had no laboratory animal counterpart that would enable the pathogenesis to be analyzed and drugs to be tested in vivo. The primary lesion in some patients is known to occur in the cells' initial handling of the major cholesterol-carrying lipoprotein of plasma. It entails a deficiency in the specific cell surface receptor that binds low density lipoprotein (LDL), with a consequent alteration in the control of cholesterol metabolism. The present study was undertaken to devise a practical scheme for producing, from developmentally versatile mouse teratocarcinoma stem cells, whole-animal models with a comparable genetic lesion. This requires first learning whether the tumor stem cells in culture express LDL receptors, and next establishing a selection or screening procedure to identify receptor-deficient mutants in mutagenized cell cultures. The results show that the teratocarcinoma cells do in fact have specific high-affinity LDL receptors which are similar to those reported for fibroblasts and the parenchymal cells of specialized tissues and different from those of phagocytic cells. Sterols suppressed the otherwise efficient binding, internalization, and degradation of LDL ((125)I-labeled) by the cells. Acetylation of LDL blocked the binding. Only LDL and not high density lipoprotein (HDL) was bound. After LDL uptake and degradation, the liberated cholesterol led, as expected, to increased cholesteryl ester formation; it also suppressed activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase [HMG CoA reductase; mevalonate: NADP(+) oxidoreductase (CoA-acylating), EC 1.1.1.34], the rate-limiting step in cholesterol biosynthesis. Cells with LDL receptors were readily visualized by administering a fluorescent derivative of LDL; in the fluorescence microscope, labeling was seen in all cells. Cells with experimentally depressed receptors, yielding little fluorescence, were separable from those with normal fluorescence in the fluorescence-activated cell sorter. Thus, two methods for isolating receptor-deficient cells from mutagenized cultures are now available, either by visual recognition of low-fluorescing or nonfluorescing colonies in culture plates or by electronic cell sorting. Such mutants in an appropriate line of teratocarcinoma cells can then be passaged into blastocysts for full somatic tissue differentiation and germ-line development into mice.

Animals↗

PubMed1992

Selective disruption of genes expressed in totipotent embryonal stem cells.

Two retrovirus promoter trap vectors (U3His and U3Neo) have been used to disrupt genes expressed in totipotent murine embryonal stem (ES) cells. Selection in L-histidinol or G418 produced clones in which the coding sequences for histidinol-dehydrogenase or neomycin-phosphotransferase were fused to sequences in or near the 5' exons of expressed genes, including one in the developmentally regulated REX-1 gene. Five of seven histidinol-resistant clones and three of three G418-resistant clones generated germ-line chimeras. A total of four disrupted genes have been passed to the germ line, of which two resulted in embryonic lethalities when bred to homozygosity. The ability to screen large numbers of recombinant ES cell clones for significant mutations, both in vitro and in vivo, circumvents genetic limitations imposed by the size and long generation time of mice and will facilitate a functional analysis of the mouse genome.

Alcohol Oxidoreductases↗

PubMed1976

The biological origin of antibody diversity.

Antibody diversity has a compelling fascination for many scientists and over the years speculations have sometimes seemed more numerous than facts. Now the structural basis of antibody specificity is well defined. Amino acid sequences and recently three-dimensional structures of various immunoglobulins provide the most solid basis for discussing the origin of diversity. The novel pattern of variable (V) and Constant (C) regions of amino acid sequence has been resolved further to show the functional pattern of variability. Inheritance of separate V and C genes is accepted, but attempts to define more than one gene coding for each V region are considered here to be unnecessary. The pattern of variability is still best understood in terms of mutation and the presence or absence of various selective pressures. The major area of debate still hinges around the extent to which mutation and selection operate during evolution or somatically. Sequence data have now been generally interpreted to require multiple V genes carried in the germ line. A few individual VH genes have been mapped in close linkage to CH genes in the mouse. The apparent existence of three VH alleles in rabbits was a strong argument against multiple V genes. Now the three phenotypes have been shown to be due to alleles controlling the expression of three sets of VH genes all present on the same chromosome. That V-gene expression requires rejoining of V and C genes at the DNA level is now almost certain. Models for the joining process can draw on the precedents of transposable genetic elements, which are widespread in Nature. The total extent of antibody diversity remains a philosophical point. Estimates of the number of antibody molecules required for observed diversity are reduced by two recently documented proposals. Each antibody combining site apparently has many (estimated at 100) different specificities and most combinations of VH and VL regions probably form a viable site. A given combining site can be defined by its pattern of shared specificities. Several specific antibody repertoires have been measured and the size in each case is consistent with the stringency with which the specificity is selected. Repertoire size appears to be under genetic control, but there are problems in viewing the genotype through the veil of clonal selection. Molecular hybridization has been used recently in an attempt to count V and C genes directly. C genes are seen in DNA having nonreiterated sequences, as formal genetics predicts. Each V-region probe hybridizes at a similar rate to C-region probes. Interpretation of this result depends on the extent to which one V-region probe will reveal nonhomologous V genes. Previous estimates that many cross-hybridizing genes should have been seen if present are possibly exaggerated. It is argued here that the data are compatible with a germ-line gene for each probe studied. Maximum estimates for the number of germ-line genes are sufficient to account for antibody diversity...

Amino Acid Sequence↗

PubMed1991

Influence of a V kappa 8 L chain transgene on endogenous rearrangements and the immune response to the HA(Sb) determinant on influenza virus.

A rearranged murine V kappa 8/J kappa 5 L chain gene that codes for the L chain of most antibodies generated in the primary response of BALB/c mice to the antigenic site, Sb, of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8) has been cloned. Three transgenic lines were generated by microinjecting the gene. Lines Ga and L each contain a single copy of the transgene whereas line Gb contains three complete copies. Mice of the Ga lineage showed increased V kappa 8-specific mRNA levels only in spleen, but not in nonlymphoid organs and therefore displayed apparently normal lymphoid-specific regulation of the Ig transgene. B cell hybridomas generated from these mice were analyzed for rearrangements of endogenous V kappa genes. Greater than 90% of the C kappa alleles were retained in germ-line configuration in the Ga line, compared with only 0 to 18% in the L line. Thus, a wide variation in the frequency of endogenous rearrangements is seen among mice of different lineages using the same transgene construct. None of more than 150 hybridomas derived from LPS-stimulated splenic B cells of Ga mice exhibited HA-binding activity although they expressed the transgene and, in most cases, excluded endogenous V kappa rearrangements. In contrast, a large fraction of hybridomas isolated after primary immunization with PR8 were HA(Sb)-specific. This indicated that the transgene was functional but formed HA-specific antibodies with a more restricted set of H chains than previously hypothesized. The primary anti-HA response to immunization with PR8 was diminished in all lines compared with normal mice except for a slightly accelerated but transient burst of anti-HA antibody formation in two out of three lines (Ga and Gb). This early response in G lineage mice was largely specific for HA(Sb) and thus appeared to be composed of transgene-expressing antibodies. No differences in serum titers were observed in the secondary anti-HA responses to booster inoculation with PR8 between transgenic and normal mice.

Animals↗