Lambda Gene Research Articles

Ig H and L Chain Variable Region Gene Sequence Analyses of Twelve Synovial Tissue-Derived B Cell Lines Producing IgA, IgG, and IgM Rheumatoid Factors Structure/Function Comparisons of Antigenic Specificity, V Gene Sequence, and IG Isotype

In the present study, the complete sequences of the Ig H and L chain variable region genes of twelve RF+ B cell lines from two patients with RA were analyzed. Seven of the RF-producing B cells used VH3 family genes, four used VH4 genes, and one a VH1 gene. All but two of the cell lines expressing VH3 genes utilized different family members; among the VH4-expressing cells, a more restricted pattern was noted. V kappa gene use was restricted to the V kappa I and III families; V lambda gene use was more diverse, involving five different families. Computer comparisons of the expressed VH genes with their presumed germline progenitors indicated significant differences in every instance; eight of the corresponding VL genes also were significantly different. In many cases, assignment of the germline D segment(s) incorporated into the rearranged VH genes was impossible. These differences from the germline gene segments indicated the extensive changes induced by rearrangement, enzymatic activities, and somatic mutation. In hopes of defining a structural reason for the disparate antigen specificities of these cells, the CDR3 amino acid sequences of the multi- vs. the mono-reactive RF-producers were compared. Although CDR3 length was not appreciably different between these two sets of mAb, a greater than two-fold increase in charged amino acids was found in the H chain CDR3 of the multireactive RF. This relationship did not exist for the L chain CDR3. Thus, these sequence data indicate the use of a broad base of Ig V gene segments that have undergone extensive diversification. Based on the localization of R substitutions in the CDR of most of the V genes studied, the diversification appears to be antigen driven and selected. The significance of these findings for the evolution of these B cell clones into isotype-switched producers that are heterogeneous for antigen specificity (mono- vs. multi-reactivity) is discussed.

The lambda terminase enzyme measures the point of its endonucleolytic attack 47 +/- 2 bp away from its site of specific DNA binding, the R site.

lambda terminase is an ATP-interactive, site-specific endonuclease comprising the products of lambda genes Nu1 and A. Terminase binds to cos, at the junction of two chromosomes in a concatemer, catalyzes cos cleavage and initiates the packaging of lambda DNA into proheads. cos consists of a nicking domain, cosN, where terminase cleaves to regenerate the 12 nucleotide cohesive ends of mature lambda chromosomes and a binding domain, cosB, where terminase binds to 16 bp repeat sequences called R3, R2 and R1. Evidence is presented that terminase is a single-strand endonuclease that can nick DNA by one of two mechanisms, both of which require ATP. (i) When bound to any R site, terminase nicks the strand which, within that R site, is purine-rich; the position of this nick is 47 +/- 2 nucleotides away from the mid-point of that R site, measured in the 3' direction; (ii) enzymes that are not bound to R sites nick DNA within certain specific sequences that resemble cosN half sites. These two modes of action are nicely combined for the R3-bound protomer that nicks the bottom strand at position N1 in cosN since the interval between N1 and the R3 midpoint is 47 nucleotides. Within cosN, the bottom and top strand nicks are generated by a rigid protein couple with a 2-fold rotational symmetry. The location of both of these nicks, however, is gauged asymmetrically from R3, 47 nucleotides away. Again, R1 and R2 are separated by 47 bp and orient bound protomers towards each other but, unless the DNA between these R sites is lengthened, the enzymes do not nick, indicating an inhibitory gpA-gpNu1 apposition.

Heterogeneous expression of human antibody lambda chains by concanavalin A-resistant hybridomas lead to changed antigen binding.

Human HB4C5 hybridoma cells produce a lung cancer-specific human monoclonal antibody that possesses a lambda light chain on which a N-linked carbohydrate chain is attached at the first complementary determining region. Up to six light chains of various sizes are secreted by the original and concanavalin A (ConA)-resistant HB4C5 clones. Two of these six light chains are derived directly from the HB4C5 original and are 30 and 32 kDa in size. The structural difference between these two light chains has been shown to be a N-linked carbohydrate located at a single site. The other four variants, which are derived from ConA-resistant variants, have light chain sizes ranging from about 26 to 29 kDa. No changes in the secretory forms were observed when glycosylation was inhibited by the addition of tunicamycin, indicating that the heterogeneous light chain forms produced by these ConA-resistant variants resulted from the size differences of core polypeptides and not from N-glycosylation. Sequence analysis from one of the variant light chains revealed that its V lambda gene segment differs markedly from identified human V lambda gene segments (at most, 60% homology with known human V lambda subgroups) and, furthermore, the variant light chain shares 80% homology with the rabbit V lambda gene. In addition, the antigen binding ability of these variant antibodies changed significantly in both specificity and affinity. These results suggest that heterogeneous light chains expression by ConA-resistant variants may provide a new way for developing an antigen-specific human monoclonal antibody repertoire.

Profile of lambda light chain variable region genes in graves' orbital tissue

Graves' ophthalmopathy, a human autoimmune disease of unknown etiology, is strongly associated with autoimmune hyperthyroidism. A major controversy is whether retro-ocular muscle or orbital fat/connective tissue is the target of the immune response. Previously, we observed preferential PCR amplification of lambda (relative to kappa) light chain DNA from cDNA of Graves' orbital tissue-infiltrating B cells/plasma cells. There is little information on V lambda gene usage in man and none in diseased tissue. To characterize the orbital lambda light chains, we constructed cDNA libraries using PCR-amplified DNA from three tissues and sequenced the variable region genes from randomly selected clones. Analysis of 27 clones from orbital fat/connective tissue libraries from two patients with acute inflammatory eye disease, and 15 clones from orbital muscle of one of these patients, revealed a diverse spectrum of lambda V region genes. The nucleotide sequences of these 42 clones were most homologous to 12 different germline genes: four family I (subfamilies I-a, -b and -c), three family II, two family III and one family VII germline genes. Each orbital tissue had a distinct profile of V lambda sequences. However, all clones used J lambda 2 3 and all three orbital tissues contained clones related to family II genes. Although some clones had V region sequences in near germline conformation, the majority differed from the closest germline gene in bot framework and complementarity determining regions. Whether or not these differences result from multiple germline gene usage or somatic mutation of a smaller number of germline genes cannot be determined until information on the V lambda repertoire and its polymorphisms is complete. However, the V lambda gene diversity we observed in both orbital muscle and orbital fat/connective tissue suggests a role for lambda autoantibodies in the pathogenesis of Graves' ophthalmopathy.

A dominant mutation in the bacteriophage lambda S gene causes premature lysis and an absolute defective plating phenotype.

The S and R genes of the bacteriophage lambda are required for lysis of the host. R encodes 'endolysin', a soluble transglycosylase which accumulates in the cytoplasm during late protein synthesis. S encodes a 'holin', a small membrane protein which, at a precisely scheduled time, terminates the vegetative cycle by forming a lethal lesion in the membrane through which gpR gains access to the peptidoglycan. A missense allele of S, Ala52Gly, causes lysis to occur prematurely at about 19-20 min after induction of a lysogen, compared to 45 min for the wild type. This allele has a severe plaque-forming defect which appears to be entirely a consequence of the early lysis and resultant severe reduction in particle burst size. The early-lysis phenotype is dominant and is aggravated, in terms of an even more reduced burst size, at both 30 degrees C and 42 degrees C. The mutation maps in the middle of a putative membrane-spanning helical domain of S, near the sites of other S- mutations with recessive non-lytic phenotypes. The mutation has no effect on S-protein accumulation or on the ratio of S107 and S105 products in the membrane. The mutation appears to affect the intrinsic timing function by which the S protein controls the lysis schedule.

Light chain isotype regulation in the horse. Characterization of Ig kappa genes.

Horse Ig kappa genes have been characterized to determine whether there may be a structural basis for the low level of kappa expression in this species. The overall organization of the J kappa-C kappa locus is remarkably similar to that of the mouse and human loci. A single C kappa exon is separated by 2.9 kb from five J kappa segments, four of which seem functional and three of which are associated with canonical recombination signal sequences. A highly conserved intron enhancer was identified upstream of the C kappa exon and a single restriction fragment in horse genomic DNA hybridized strongly with the mouse downstream kappa enhancer. Germ-line and splenic cDNA V kappa sequences were characterized and found to encode N-terminal amino acid sequences previously found by protein sequencing. Hybridization of horse genomic DNA with the horse V kappa-1 germ-line gene and a variety of mouse V kappa-region probes provided evidence for at least 20 V kappa gene segments. The results indicate that the horse possesses a functional kappa locus with a complement of variable region gene segments potentially as large as that previously found at the lambda locus although the exact numbers of functional variable region genes remains to be established for both loci. This finding suggests that the predominance of lambda-chains in horse Ig may not be simply a result of a lack of functional kappa genes or of a disproportionate number of lambda vs kappa variable region genes. The results are discussed in terms of various models of isotype regulation.

Preferential utilization of a novel V lambda 3 gene in monoclonal rheumatoid factors derived from the synovial cells of rheumatoid arthritis patients.

To further our understanding about the molecular genetics of rheumatoid factor (RF) in rheumatoid arthritis (RA). The heavy and light chain variable region (V) genes of 5 new human monoclonal IgM RFs were cloned and sequenced using the polymerase chain reaction and the dideoxynucleotide termination method. The results reveal the recurrent usage in two RA patients of a novel V lambda 3 germline gene, designated Humlv3c93. Specifically, in 2 of 3 RFs (C93 and D53) from one patient, the light chains in the V lambda gene-encoded region were identical to each other and to the light chain of an RF (H4) from another patient. Serologically, the light chains of these 3 RFs were classified as members of the V lambda 3b sub-subgroup. Each of the RFs was encoded by a different VH gene. Both C93 and D53 bound specifically with human and rabbit IgG, whereas H4 was monospecific for rabbit IgG. Since the lv3c93 gene is not homologous to any reported V lambda sequence from natural autoantibodies, it is possible that lv3c93 may represent a disease-specific RF-related V lambda gene. Moreover, the amino acid sequence CSGGSCY in the third complementarity-determining regions of 2 of the RF heavy chains is encoded by the DLR2 gene segment and has been found previously in 2 other RA-derived RFs, and thus may play a significant role in antigen binding.

Identification of a new human V lambda gene family--V lambda X.

Although approximately 40% of Ig light chains in human sera are of the Ig lambda isotype, until recently very little was known about the human Ig lambda gene locus. The genomic structure of the Ig lambda C region has been determined and consists of seven tandemly organized J-C lambda segments. The structure of the region containing the Ig lambda V gene segments is less well defined. Based on amino acid and nucleotide sequence homologies, the V lambda gene segments isolated have been grouped into nine families. Here we report on the isolation of a V lambda gene from the functional rearrangement of a human B lymphoma cell line that has less than 71% nucleotide sequence and 57% deduced amino acid sequence homology to any known V lambda gene. According to the current classification schemes, this gene does not belong to any existing V lambda family. Thus we suggest that this gene belongs to a new V lambda family, V lambda X. The genomic counterpart of the rearranged V lambda gene, and a second member of the family were isolated. The second member is a pseudogene. Southern analysis suggests that the V lambda X family is a small family. The functional V lambda X gene has a consensus heptamer and a nonamer that differs at two sites from the consensus recombination signal sequence. The promoter region contains a consensus TATA box and an octamer that diverge from the consensus sequence by two base pairs.

Analysis of Ig and T-cell receptor genes in 40 childhood acute lymphoblastic leukemias at diagnosis and subsequent relapse: implications for the detection of minimal residual disease by polymerase chain reaction analysis

The rearrangement patterns of Ig and T-cell receptor (TcR) genes were studied by Southern blot analysis in 30 precursor B-cell acute lymphoblastic leukemias (B-ALLs) and 10 T-ALLs at diagnosis and subsequent relapse. Eight precursor B-ALLs appeared to contain biclonal/oligoclonal Ig heavy-chain (IgH) gene rearrangements at diagnosis. Differences in rearrangement patterns between diagnosis and relapse were found in 67% (20 cases) of precursor B-ALLs (including all eight biclonal/oligoclonal cases) and 50% (five cases) of T-ALLs. In precursor B-ALLs, especially changes in IgH and/or TcR-delta gene rearrangements were found (17 cases), but also changes in TcR-beta, TcR- gamma, Ig kappa, and/or Ig lambda genes (11 cases) occurred. The changes in T-ALLs concerned the TcR-beta, TcR-gamma, TcR-delta, and/or IgH genes. Two precursor B-ALLs showed completely different Ig and TcR gene rearrangement patterns at relapse, suggesting the absence of a clonal relation between the leukemic cells at diagnosis and relapse and the development of a secondary leukemia. The clonal evolution in the other 23 ALL patients was based on continuing rearrangement processes and selection of subclones. The development of changes in Ig and TcR gene rearrangement patterns was related to remission duration, suggesting an increasing chance of continuing rearrangement processes with time. These immunogenotypic changes at relapse occurred in a hierarchical order, with changes in IgH and TcR-delta genes occurring after only 6 months of remission duration, whereas changes in other Ig and TcR genes were generally detectable after 1 to 2 years of remission duration. The heterogeneity reported here in Ig and/or TcR gene rearrangement patterns at diagnosis and relapse might hamper polymerase chain reaction (PCR)-mediated detection of minimal residual disease (MRD) using junctional regions of rearranged Ig or TcR genes as PCR targets. However, our data also indicate that in 75% to 90% of ALLs, at least one major rearranged IgH, TcR-gamma, or TcR-delta band (allele) remained stable at relapse. We conclude that two or more junctional regions of different genes (IgH, TcR-gamma, and/or TcR-delta) should be monitored during follow-up of ALL patients for MRD detection by use of PCR techniques. Especially in biclonal/oligoclonal precursor B-ALL cases, the monitoring should not be restricted to rearranged IgH genes, but TcR-gamma and/or TcR-delta genes should be monitored as well, because of the extensive changes in IgH gene rearrangement patterns in this ALL subgroup.

Cold-sensitive phenotype of Escherichia coli cells harboring a plasmid carrying the kil gene of phage lambda brought under control of cI857 gene promoters

A plasmid (pI-14), containing part of the phage λ control region ( EcoRI-D fragment of λ cJ857) with a large deletion between genes rex and kil, confers a cold-sensitive (cs) phenotype on the host bacteria, whereas the parent plasmid (pMM200) without the deletion made the host bacteria ( Escherichia coli strain DOO) high-temperature sensitive. This phenomenon could be explained on the basis of the sequence analysis of the deletion. Upon this deletion, the λ kil gene, which was originally under the control of the p L promoter, was brought under the control of the λ cI promoters, resulting in the reversal of the host cell response to temperature. This example shows that gene circuits showing diametrically opposite responses to environmental factors (in this case, temperature) can be constructed from the same elements when the effector gene (here, kil) is connected in different ways to the sensor gene (here, cI857). This cold-dependent killing activity was also dependent on the crp + state of the host.

Deletion and editing of B cells that express antibodies to DNA.

We previously demonstrated that in mice transgenic for genes coding for an anti-ssDNA autoantibody B cells were functionally inactivated but not physically deleted. We have now extended this model by introducing an arginine into the CDR2 of the heavy chain transgene. This change alters the specificity of the Ab from anti-ssDNA to anti-dsDNA and increases the affinity for ssDNA. Mice carrying this transgene displayed a significant reduction of peripheral B cells and anti-dsDNA B cells were not recovered from the spleens. The remaining B cells escape deletion by revising their Ag receptors in several ways: 1) elimination of the transgenic heavy chain gene via intrachromosomal recombination, followed by rearrangement and expression of endogenous VH genes; 2) ongoing rearrangement of endogenous kappa light chain genes to generate a non-dsDNA-binding Ab; and 3) expression of a rare V lambda gene, V lambda x, to generate a non-DNA-binding Ab.

Utilization of a Potentially Universal Downstream Primer in the Rapid Identification and Characterization of Vβ Genes from Two New Human Vλ Gene Families

Polymerase chain reaction (PCR) has increased dramatically the speed of cloning and characterizing numerous genes. However, its application to identifying and analysing new germline Ig-variable (V) gene families has been hampered by the lack of sequence information in the downstream flanking regions of the concerned V genes, which are deleted during V(D)J rearrangements. To circumvent this problem, the possibility was explored that a degenerate downstream primer may be used in conjunction with a specific upstream primer, to clone members of new V lambda gene families, as much less is known about V lambda genes than Vh and Vk genes in humans. Firstly the feasibility and the specificity of a degenerate primer was examined by comparing it with an established downstream primer in amplifying known V lambda 1 genes. The results were positive. Thus, the degenerate primer was used to clone and characterize germline V lambda genes of the recently defined V lambda 8 and V lambda 9 gene families. This current strategy may help speed up the identification and characterization of all human V lambda genes. Moreover, a similar strategy can be applied to identify and characterize rapidly new V genes of either known or unknown Ig and T-cell receptor V gene families.

Transforming growth factor-beta regulates normal human pre-B cell differentiation.

Light chain gene rearrangement during mammalian pre-B differentiation generally occurs in an orderly manner, beginning with kappa genes and proceeding through lambda genes. We have previously shown that human pre-B cell differentiation in vitro leads to a skewing toward lambda expression, resulting in a higher percentage of lambda+ cells than kappa+ cells. We now report that the multifunctional polypeptide transforming growth factor-beta (TGF-beta) exerts a selective inhibitory effect on the acquisition of cell surface lambda light chains during in vitro differentiation of normal human pre-B cells, giving rise to a balanced ratio (approximately 1:1) of kappa+ to lambda+ cells that resembles what normally exists in vivo. The TGF-beta effect was ablated using a neutralizing anti-TGF-beta antiserum and TGF-beta had no significant effect on the acquisition of kappa or surrogate light chains. Experiments using highly enriched pre-B cells (90-95% cytoplasmic mu+) suggested that the TGF-beta effect was directly on the pre-B cell or the pre-B cell to mu+/lambda+ immature B cell transition. The following peptides, cytokines, and antibodies had no effect on light chain acquisition or expression: substance P, vasoactive intestinal peptide, leu/met enkephalin, IL-1, IL-4, IL-7, anti-class II MHC, anti-CD24, anti-CD40, and the CD10 inhibitor phosphoramidon. A selective regulatory role for TGF-beta on normal human B cell development in the bone marrow microenvironment is suggested by these results.

Site-specific targeting of psoralen photoadducts with a triple helix-forming oligonucleotide: characterization of psoralen monoadduct and crosslink formation.

A polypurine tract in the supF gene of bacteriophage lambda (base pairs 167-176) was selected as the target for triple helix formation and targeted mutagenesis by an oligopurine (5'-AGGAAGGGGG-3') containing a chemically linked psoralen derivative (4'-hydroxymethyl-4,5',8-trimethylpsoralen) at its 5' terminus (psoAG10). The thymines at base pairs 166 and 167, a 5'ApT site, were targeted for photomodification. Exposure of the triple helical complex to long wavelength ultraviolet radiation led to the covalent binding of psoAG10 to the targeted region in the supF gene and to the induction of site-specific mutations. We report here experiments to characterize the photomodification of the targeted region of the supF gene in the context of triple helix formation. An electrophoretic mobility-shift assay showed that, at low radiation doses, monoadducts at base pair 166 were the major photoadducts. At higher doses the monoadducts were converted to crosslinks between base pairs 166 and 167. HPLC analysis of enzymatically hydrolyzed photoreaction mixtures was used to confirm the electrophoresis results. A strong strand preference for specific photoadduct formation was also detected.

Immunoglobulin somatic variation; studies of receptor editing in a human B cell lymphoma.

In order to study mechanisms of immunoglobulin somatic variation in committed immunoglobulin (Ig) expressing B cells, we used the fluorescent activated cell sorter to isolate rare variants from a surface Ig positive (sIg+) diffuse large cell B lymphoma cell line (mu lambda+). These variants were either negative for sIg expression (sIg-) or expressed sIg which differed from the original parental tumor cell line, both in idiotypes and Ig lambda isotypes (sIg+Id-). In the following report we review the results from the studies of these variants. DNA analysis showed that all variants had new Ig lambda gene rearrangements, which had occurred either on a previously excluded allele, or on the productively rearranged allele of the parental cell line. The sIg- variants, which had undergone nonfunctional Ig lambda rearrangements on the expressed parental allele, and thereby deleted the productive rearrangement, continued to functionally rearrange the same allele and regenerated sIg expression. While the parental cell line expressed low levels of the recombination activating genes, RAG1 and RAG2, expression of these genes were considerably upregulated in both the immunoglobulin negative and the idiotypic variants. Somatic immunoglobulin V gene hypermutation did not contribute to the observed immunoglobulin somatic variation. These results demonstrate that, through differential expression of the RAG genes, sIg+ B cells are able to somatically alter their sIg receptors through secondary Ig lambda gene rearrangements. This mechanism may allow B cells with non-selectable, or auto-reactive, antigen receptors to alter these receptors (receptor editing). Ongoing Ig gene rearrangement may limit the usefulness of immunotherapeutic approaches directed at the antigen receptor in some diffuse large cell lymphomas.

Genetic and molecular analyses of the C-terminal region of the recE gene from the Rac prophage of Escherichia coli K-12 reveal the recT gene

The nucleotide sequence of the C-terminal region of the recE gene of the Rac prophage of Escherichia coli K-12 reveals the presence of a partially overlapping reading frame we call recT. Deletion mutations show that recT is required for the RecE pathway of conjugational recombination. By cloning recT with a plasmid vector compatible with pBR322, we showed by cis-trans tests that the portion of the recE gene encoding ExoVIII DNA nuclease activity is also required for RecE pathway conjugational recombination. The recT gene can replace the redB gene of lambda for recA-independent plasmid recombination. A Tn10 insertion mutation previously thought to be in recE is located in recT and is renamed recT101::Tn10. Discrepancies between the molecular mass estimates of wild-type ExoVIII protein determined from mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and calculated from the predicted amino acid sequence are discussed. The hypothesis that wild-type ExoVIII protein results from fusion of RecE and RecT proteins is disproved genetically, thus supporting a previous hypothesis that the discrepancies are due to abnormal protein mobility in SDS-PAGE. A computer-performed scan of the bacteriophage nucleotide sequence data base of GenBank revealed substantial similarity between most of recE and a 2.5-kb portion of the b2 region of lambda. This suggests interesting speculations concerning the evolutionary relationship of lambda and Rac prophages.

Molecular selection of human antibodies with an unconventional bacterial B cell antigen.

Unconventional Ag for B cells that are comparable to known superantigens for T lymphocytes have not been well characterized. However, the bacterial membrane protein, Staphylococcal protein A (SpA), has sites that interact with the Fab of many IgM, IgA, IgG, and IgE, and in recent reports we have provided evidence of VH restriction in Fab that bind SpA. To investigate the molecular basis for this Fab binding specificity, we have selected monoclonal Fab from a phage-display combinatorial Ig library, based on the ability to bind SpA. By this approach, in an unselected human polyclonal IgG Fab library, about 17% of antibody-expressing clones were found to bind SpA. SpA binding was completely restricted to Fab with VH3 H chains, and about 60% of VH3 Fab in the unselected library had SpA binding capacity. Analysis of 21 VH sequences and 6 VL sequences demonstrated that Fab that bind SpA use diverse VH3 genes, while the L chains derive from a variety of V kappa and V lambda gene families. By creation of antibodies with differential H-L chain pairing, the global capacity to bind SpA was shown to be dictated by VH3 usage, but different L chain usage could result in up to a fourfold change in binding affinity. The apparent KD of the SpA binding by different Fab ranged from 2.5 x 10(-7) to > 10(-5) M. Furthermore, repeated rounds of in vitro panning selected for antibodies based on higher binding affinity. These data indicate that the pattern of VH family restriction of Ig reactive with SpA is comparable to known superantigens, and there is a hierarchy within the binding affinities of VH3 Fab based on V gene usage.

Superinfection exclusion (sieB) genes of bacteriophages P22 and lambda.

The superinfection exclusion gene (sieB) of Salmonella phage P22 was mapped with phage deletion mutants. The DNA sequence in the region was reexamined in order to find an open reading frame consistent with the deletion mapping. Several discrepancies with the previously published sequence were discovered. The revised sequence revealed a single open reading frame of 242 codons with six likely translation initiation codons. On the basis of deletion and amber mutant phenotypes, the second of these six sites was inferred to be the translation initiation site of the sieB gene. The sieB gene encodes a polypeptide with 192 amino acid residues with a calculated molecular weight of 22,442, which is in reasonable agreement with that estimated from polyacrylamide gels. The transcription start site of sieB was identified by the use of an RNase protection assay. The sieB promoter thus identified was inactivated by a 2-base substitution in its -10 hexamer. The sieB gene of coliphage lambda was also identified. The promoter for lambda sieB was identified by homology to that of P22 sieB.

Expression of the Rz gene and the overlapping Rz1 reading frame present at the right end of the bacteriophage lambda genome

The Rz lysis gene of bacteriophage lambda was cloned into the expression vectors, pT7-3 and pT7-7. The recombinant plasmids expressed either a protein of an unexpected 6.5-kDa size (pT7-3H and pSB54) or two proteins of 6.5 and 17.2 kDa (pBH21). The 6.5-kDa protein alone did not complement the lysis defect of the lambda Rz mutant; hence, this protein was not the Rz gene product. Complementation observed as a result of pBH21 expression thus can be ascribed to the 17.2-kDa protein, which agrees with the size based on the nucleotide sequence of Rz. The 6.5 kDa is a product of an open reading frame entirely encompassed within the Rz sequence and denoted by us Rz1. Both proteins were detectable only by autoradiography, which may mean that the genes are expressed at low rates. Polyclonal anti-Rz antibodies (Ab) were obtained by rabbit immunization with a synthetic polypeptide corresponding to an antigenic determinant of Rz defined by a computer program. The Ab reacted with the 17.2-kDa protein resulting from pBH21 expression, as well as with the 17.2-kDa protein present in the induced Escherichia coli W3350(lambda cI857Sam7) lysate.

Generation and molecular analyses of two rheumatoid synovial fluid–derived IgG rheumatoid factors

To study the Ig genes that encode IgG rheumatoid factor (IgG-RF) from rheumatoid synovial fluid. We used rheumatoid synovial fluid B cells to generate IgG-RF-secreting hybridomas. We then characterized their binding properties and determined their nucleotide sequences. Two monospecific IgG-RFs were obtained. Sequence analysis of the RFs revealed a new V lambda gene family (designated V lambda 9) and extensive somatic diversification, including a duplication-insertion of 18 nucleotides (6 amino acid residues) into a hypervariable region. The data provide further support for an antigen-driven response in the sustained production of potentially pathogenic IgG-RFs in rheumatoid synovium.