Immunoglobulin genes of the kappa light chain type from two human lymphoid cell lines are closely related.

As a first step in our studies of functionally rearranged K genes of man we cloned the germline JK-CK region from placenta DNA employing a mouse JK clone as hybridization probe. Subclones of the human JK-CK region were then used to characterize and clone the rearranged K genes of the lymphoid cell lines Walker and Daudi. The Walker cell line contains one rearranged and one germline K allele (K+,KO; ref. 1). Only one K gene was found in Daudi cells (K+). Restriction mapping and DNA sequencing showed, that the rearranged K genes from both cell lines are closely related. These features make the two cell lines particularly suitable for studies on the chromatin structure of K light chain genes. The 5' flanks of the two genes (388 bp) are identical while there is a 12% divergence between the VK gene segments themselves. This situation may reflect somatic mutation processes and/or gene conversion like events.     

Isolation and characterization of endonuclease J: a sequence-specific endonuclease cleaving immunoglobulin genes.

An endonuclease activity which cleaves close to the recombination sites of the immunoglobulin JK segments was found in extracts of chicken bursa of Fabricius and characterized after partial purification. The enzyme preparation also cleaved a VK segment at its 3' end. A similar activity was found in mouse liver, mouse myelomas and Hela cells. The enzyme designated as endonuclease J introduces double-stranded cleavages preferentially at sequences containing G clusters of pBR322 as well as the JK segments. However, not all the G clusters were cleaved by endonuclease J, suggesting that the enzyme recognizes additional sequences. Deletion of the conserved nonamer (GGTTTTTGT) located immediately 5' to the JK4 segment drastically reduced the cleavage activity of its immediate downstream G cluster. Although biological function of endonuclease J is not clear at this stage, the possibilities of its involvement in the immunoglobulin gene recombination and general recombination were discussed.     

Deletion of human JK segments by site-specific recombination recognizing the conserved nonamer and heptamer sequences.

Mapping and partial sequencing of the productive K chain genomic DNA of FK-001 demonstrated a 1.8-kb deletion including the JK2, JK3, JK4, and JK5 segments. This deletion occurred between the heptamer recombination signal sequence of the JK2 segment and the heptamer-like sequence located 1.8 kb downstream of the JK2 segment. The recombination reaction kept the reciprocally joined signal sequences on the chromosome and deleted the intervening DNA segment. The cloned FK-001 K chain gene was expressed efficiently in mouse myeloma cells, demonstrating that the 1.8-kb deleted region conferred no functions for gene expression.     

Linking of the human immunoglobulin VK and JKCK regions by chromosomal walking.

The linking of the human VK and JKCK gene regions (abbreviations in ref. 1) by chromosomal walking is reported. Hybridization experiments with the DNA of a somatic cell hybrid containing the region between JKCK and the telomer show that none of the major VK gene clusters is located downstream of CK. The distance between the VK and JK genes was found to be 23 kb. The JK proximal VK gene is the B3 gene which is the only representative of subgroup IV in the genome. This gene and the neighbouring B2 gene (accompanying paper) are arranged in opposite orientation to JKCK and can therefore rearrange only by an inversion mechanism. This finding is used, together with previous data, to delineate the rearrangement processes in the Burkitt lymphoma derived cell line BL21 as comprising an inversion in the first and a deletion in the second step.     

Immunoglobulin genes of different subgroups are interdigitated within the VK locus

The variable regions of immunoglobulins are encoded by multigene families which are rearranged during B-cell differentiation. These families were classified in groups and subgroups based on their amino acid sequences. Genes belonging to a distinct subgroup are believed to occur in the genome within clusters. We are investigating the organization of human variable region genes of the kappa type (VK genes, ref. 1) in the germline and found now for the first time that VK sequences of three of the four different subgroups are interdigitated within the VK locus. We present evidence for the interspersion of two VKIII genes and a VKII pseudogene within an array of five VKI genes. All eight VK sequences are arranged in the same orientation. An evolutionary model for the generation of this 'mixed cluster' is discussed.     

Identification and cloning of peptide synthetase genes of thermostable bacilli using the polymerase chain reaction

Fourteen thermophilic and thermostable strains of the genus Bacillus were studied. Total DNA was isolated from these strains and used as a template to identify and clone peptide synthetase genes by means of polymerase chain reaction. Amplified DNA fragments were cloned into a phasmid vector, and nucleotide sequences of cloned fragments were determined. Stringent thermophilic strains were shown to lack genetic systems, which are responsible for the synthesis of secondary metabolites and homologous to the known peptide synthetase genes. On the contrary, thermostable strains had peptide synthetases and produced antimicrobial secondary metabolites. Analysis of nucleotide sequences and deduced amino acid sequences of cloned PCR fragments from B. licheniformis strains VK2, VK21, and VK2101 showed that they are absolutely identical. The cloned DNA fragment was found to be a portion of the open reading frame, which we termed ORF1. Data from analysis of a partial nucleotide sequence of the peptide synthetase gene of strain VK21 indicated that a 9.5-kb region of chromosomal DNA contains sequences of two genes homologous to the B. subtilis peptide synthetase genes dhbB and dhbF. Strains VK2, VK21, and VK2101 were shown to synthesize siderophores. A method for screening bacteria with peptide synthetase genes has been developed.     

Correlation between DNase I hypersensitive sites and putative regulatory sequences in human immunoglobulin genes of the kappa light chain type.

The human lymphoid cell lines Walker and Daudi constitute a particularly suitable system for studies on the chromatin structure of K light chain genes (see preceding paper). The rearranged and non-rearranged alleles of Walker cells were found to be about equally sensitive towards digestion with DNAase I. A DNAase I hypersensitive site was mapped 0.13 kb upstream of the leader segment of the rearranged VK genes; it comprises a region in which promoter-like regulatory elements were discovered recently. Additional hypersensitive sites are located further upstream. A hypersensitive site in the JK-CK intron coincides with a putative tissue specific enhancer element. A hypersensitive region down-stream of CK overlaps with the cleavage/polyadenylation recognition signal which is flanked by sequences related to the above mentioned putative regulatory sequences. The coincidence between DNAase I hypersensitive sites and those sequences may be functionally significant.     

An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH

We have determined the sequences of separate germline genetic elements which encode two parts of a mouse immunglobulin heavy chain variable region. These elements, termed gene segments, are heavy chain counterparts of the variable (V) and joining (J) gene segments of immunoglobulin light chains. The VH gene segment encodes amino acids 1-101 and the JH gene segment encodes amino acids 107-123 of the S107 phosphorylcholine-binding VH region. This JH gene segment and two other JH gene segments are located 5' to the mu constant region gene (Cmu) in germline DNA. We have also determined the sequence of a rearranged VH gene encoding a complete VH region, M603, which is closely related to S107. In addition, we have partially determined the VH coding sequences of the S107 and M167 heavy chain mRNAs. By comparing these sequences to the germline gene segments, we conclude that the germline VH and JH gene segments do not contain at least 13 nucleotides which are present in the rearranged VH genes. In S107, these nucleotides encode amino acids 102-106, which form part of the third hypervariable region and consequently influence the antigen-binding specificity of the immunoglobulin molecule. This portion of the variable region may be encoded by a separate germline gene segment which can be joined to the VH and JH gene segments. We term this postulated genetic element the D gene segment, referring to its role in the generation of heavy chain diversity. Essentially the same noncoding sequences are found 3' to the VH gene segment and as inverse complements 5' to two JH gene segments. These are the same conserved nucleotides previously found adjacent to light chain V and J gene segments. Each conserved sequence consists of blocks of seven and ten conserved nucleotides which are separated by a spacer of either 11 or 22 nonconserved nucleotides. The highly conserved spacing, corresponding to one or two turns of the DNA helix, maintains precise spatial orientations between blocks of conserved nucleotides. Gene segments which can join to one another (VK and JK, for example) always have spacers of different lengths. Based on these observations, we propose a model for variable region gene rearrangement mediated by proteins which recognize the same conserved sequences adjacent to both light and heavy chain immunoglobulin gene segments.     

Unusual joining sites in the H and L chains of an anti-lysozyme antibody

Nucleotide sequences of HyHEL-5, an antibody specific for chicken lysozyme (HEL), indicated unusual joins in the third complementarity-determining region of both the H and L chains. The VK-JK recombination site is unusual in that codon 96, normally derived from the JK gene segment, is deleted entirely, making the L3 one amino acid shorter than normal. Examination of the HyHEL-5 Fab-HEL x-ray structure suggests that the conformation of L3 is clearly important for Ag specificity. A comparison of the HyHEL-5 L3 with that of the structurally related antibody J539 indicates that the deleted residue significantly alters the conformation of the L3 turn. The H chain VH-DH join is also unusual; the VH junction site has probably occurred between the second and third nucleotides of codon 92, with the addition of five random nucleotides that encode for unusual amino acids Leu93 and His94. Although the conformation of H3 is different from what would be predicted from other H3 conformations and is clearly important to the complementarity of HyHEL-5 to HEL, the specific residues at the VH-DH join do not appear to directly contribute to Ag binding. It is not possible to attribute the main chain conformation of H3 to the particular sequence produced by the join; the structural features of H3 may be due to interactions with HEL and/or with other antibody residues.     

Functional analogues of the VKOx1 gene in different strains of mice: evolutionary conservation but diversity based on V-J joining

Monoclonal antibodies to the hapten phenyloxazolone were raised 7 days after immunization in mice of six strains (BALB/c, C57BL-Igha, DBA2, RF, A/J, and CE). Hybridomas were selected that produced 260 idiotype-positive antibodies, and their light chain mRNA were partially sequenced. (RF is an idiotype-negative strain, and sequencing was done without this selection.) All newly sequenced BALB/c, C57BL-Igha, DBA/2, A/J, or CE VK segments had a 100% nucleotide homology with the VKOx1 (H3) germline gene. This gene codes for one third of early BALB/c phenyloxazolone antibodies, and according to our results the same gene has a significant role in the early response of at least five strains of mice. Four RF hybridomas had identical nucleotide sequences, suggesting that they express a non-mutated nucleotide sequence of a new VK germ-line gene (VKOx2). This gene codes for a CDR1 which is two amino acids longer than the CDR1 coded by the VKOx1 gene, but otherwise the two genes are related (94.5% sequence homology). All but one of the 16 kappa chains studied had the J5 segment; this segment had the same sequence in all six strains. One RF antibody had the J4 segment the nucleotide sequence of which differs from the BALB/c J4 segment in two places. Three of the kappa chains had an extra long CDR3. Long and "normal" kappa chains were probably coded by the same pair of germ-line genes (VKOx1 and J5, or VKOx2 and J5). The length heterogeneity was probably caused by a lack of precision in VK-JK joining.     

A human immunoglobulin kappa orphon without sequence defects may be the product of a pericentric inversion.

The VK gene segments that have been transposed from the kappa locus on the short arm of chromosome 2 at 2p11-12 to other chromosomal sites are called orphons. The 18 VK orphons sequenced up to now carry defects and are to be considered pseudogenes. We now describe the VKI gene segment V108 whose sequence is without any defects and which was localized to the long arm of chromosome 2 at 2q12-14 by in situ hybridization. The V108 region may have been transposed from the short to the long arm of chromosome 2 by a pericentric inversion. Possible reasons for the conservation of its sequence are discussed. In spite of its bona fide sequence V108 is considered to be an unlikely candidate for a VK-JK rearrangement and subsequent functional expression.     

Identification and Cloning of Peptide Synthetase Genes of Thermostable Bacilli Using the Polymerase Chain Reaction

Fourteen thermophilic and thermostable strains of the genus Bacillus were studied. Total DNA was isolated from these strains and used as a template to identify and clone peptide synthetase genes by means of polymerase chain reaction. Amplified DNA fragments were cloned into a phasmid vector, and nucleotide sequences of cloned fragments were determined. Stringent thermophilic strains were shown to lack genetic systems, which are responsible for the synthesis of secondary metabolites and homologous to the known peptide synthetase genes. On the contrary, thermostable strains had peptide synthetases and produced antimicrobial secondary metabolites. Analysis of nucleotide sequences and deduced amino acid sequences of cloned PCR fragments from B. licheniformis strains VK2, VK21, and VK2101 showed that they are absolutely identical. The cloned DNA fragment was found to be a portion of the open reading frame, which we termed ORF1. Data from analysis of a partial nucleotide sequence of the peptide synthetase gene of strain VK21 indicated that a 9.5-kb region of chromosomal DNA contains sequences of two genes homologous to the B. subtilis peptide synthetase genesdhbB and dhbF. Strains VK2, VK21, and VK2101 were shown to synthesize siderophores. A method for screening bacteria with peptide synthetase genes has been developed.     

Simultaneously arising Ly-1(CD5) B cell lymphomas have identical expressed IgH and kappa-genes but different nonproductive heavy chain rearrangements.

A group of CD5(Ly-1) B cell lymphomas are described. They were derived from mice which received a common pool of syngeneic mouse spleen cells. Southern blot analysis revealed that the lymphomas exhibited an unusual set of Ig gene rearrangements. Six lymphomas analyzed had either of two rearrangement patterns. EcoRI restriction digests of tumor DNA probed for rearrangements in the JH region, resulted in restriction fragments of 4.7 and 5.6 kb or of 4.7 and 8.5 kb. Each had an identical HindIII restriction fragment identified when probed for kappa gene rearrangements. Inasmuch as several B cell lymphomas from mice receiving a common pool of spleen cells had identical kappa-rearrangements and one identical IgH rearrangement, it was important to determine the DNA sequence of expressed IgH and kappa-genes. Each tumor was found to have identical nucleotide sequences of VH-DH-JH and VK-JK. The nonproductive IgH rearrangements each consisted of incomplete DH-JH rearrangements. The 8.5-kb EcoRI fragment was generated from a DFL16 gene segment rearranged into JH3, and the 5.6-kb fragment was generated from DQ52 rearranged into JH)1. We conclude that these Ly-1 B tumors are most likely derived from a single clone of cells which underwent a secondary rearrangement on the nonproductive allele after kappa-rearrangement had occurred. The alternate possibility of independently arising lymphomas with identical expressed VH and VK sequences is discussed.     

Germline V genes encode viable motheaten mouse autoantibodies against thymocytes and red blood cells

Autoantibodies against thymocytes and RBC may contribute to the pathophysiology of homozygous viable motheaten (mev) autoimmune disease. Whether the production of these autoantibodies in mev mouse results from polyclonal nonspecific B cell activation or specific Ag-driven stimulation is not known. To understand the mechanisms involved in the induction of antithymocyte autoantibody response in mev mouse, we have studied the fine antigenic specificity, structure, and origin of three antithymocyte autoantibodies derived from mev splenic B cell hybridomas. Western blot analysis showed that these mAb bind to polypeptides of 33 and 105 kDa present in RBC and thymocytes, respectively. Additional specificities for the epitopes present in other polypeptides distinguished these three autoantibodies. Northern hybridization and flow microfluorimetry analysis indicated that these hybridomas are derived from the Ly1+ B cell subset. These autoreactive Ly-1 B cell hybridomas, chosen on the basis of their specificity, expressed L chain V genes from a single VK family (VK9) and VH genes from J606 and S107 families. Hybridomas UN34.11 and UN42.5 expressed the VK9 gene identical to that used by peritoneal Ly1+ B cells from various mouse strains and malignant B lymphoma cells secreting anti-mouse RBC treated with proteolytic enzyme bromelin and anti-SRBC antibodies. The third hybridoma, S2-14.2, used a VK9 gene identical to that expressed by MOPC41. None of the VK genes encoding these autoantibodies showed any somatic mutations. In the case of VH genes, the two hybridomas UN42.5 and S2-14.2 derived from two separate fusions, used identical VH genes from the J606 family. The third hybridoma UN34.11 used unmutated V11 germline VH gene, a member of the S107 family. Southern hybridizations, using oligonucleotide probes specific for CDR1 and CDR2, showed that the VH genes encoding the J606 autoantibodies were derived from a germline gene found in the 6.7-kb fragment of EcoRI-digested germline DNA. This germline VH gene is distinct from VH22.1 germline gene that codes for antigalactan antibodies. Sequence analysis of this gene showed perfect homology with the rearranged VH genes confirming the lack of somatic mutations. Thus, our data demonstrate that antithymocyte antibody response occurring in mev mouse is polyclonal and it involves Ly-1 B cells expressing unmutated germline VH and VK genes. These results indicate that antigen driven stimulation may not play an important role in the induction of anti-thymocyte antibody response in mev mouse.     

Fragile X syndrome: diagnosis using highly polymorphic microsatellite markers.

We describe two highly polymorphic microsatellite AC repeat sequences, VK23AC and VK14AC, which are closely linked to the fragile X at Xq27.3. Both VK23AC (DXS297) and VK14AC (DXS292) are proximal to the fragile site. Two-point linkage analysis in 31 fragile X families gave (a) a recombination frequency of 1% (range 0.00%-4%) with a maximum lod score of 32.04 for DXS297 and (b) a recombination frequency of 7% (range of 3%-15%) with a maximum lod score of 12.87 for DXS292. Both of these polymorphisms are applicable to diagnosis by linkage in families with fragile X syndrome. A multipoint linkage map of genetic markers at Xq27.3 was constructed from genotyping these polymorphisms in the CEPH pedigrees. The DXS292 marker is in the DXS98-DXS297 interval and in 3 cM proximal to DXS297.     

Distinct Requirements for Somatic and Germline Expression of a Generally Expressed Caernorhabditis Elegans Gene

In screening for embryonic-lethal mutations in Caenorhabditis elegans, we defined an essential gene (let-858) that encodes a nuclear protein rich in acidic and basic residues. We have named this product nucampholin. Closely homologous sequences in yeast, plants, and mammals demonstrate strong evolutionary conservation in eukaryotes. Nucampholin resides in all nuclei of C. elegans and is essential in early development and in differentiating tissue. Antisense-mediated depletion of LET-858 activity in early embryos causes a lethal phenotype similar to characterized treatments blocking embryonic gene expression. Using transgene-rescue, we demonstrated the additional requirement for let-858 in the larval germline. The broad requirements allowed investigation of soma-germline differences in gene expression. When introduced into standard transgene arrays, let-858 (like many other C. elegans genes) functions well in soma but poorly in germline. We observed incremental silencing of simple let-858 arrays in the first few generations following transformation and hypothesized that silencing might reflect recognition of arrays as repetitive or heterochromatin-like. To give the transgene a more physiological context, we included an excess of random genomic fragments with the injected DNA. The resulting transgenes show robust expression in both germline and soma. Our results suggest the possibility of concerted mechanisms for silencing unwanted germline expression of repetitive sequences.     

Sequences of the VH and VL regions of murine monoclonal antibodies against 3-fucosyllactosamine

Many mAb that bind the carbohydrate antigenic determinant 3-fucosyl-lactosamine (3-FL), Gal beta 1-4[Fuc alpha-3]GlcNAc-R have been raised in BALB/c mice, and we are studying the structure and regulation of these antibodies. In this report, we present the first information about their amino acid sequences and the Ig gene segments used to encode them. V regions of the H and L chains of three anti-3-FL antibodies, PMN6, PMN29, and PM81, were sequenced by a combination of mRNA and amino acid sequencing. The L chain sequences of PMN6 and PM81 antibodies indicate that their VK and JK regions are encoded by VK24B and JK1 germ-line genes, respectively. The nucleotide and amino acid sequences of the H chains suggest that the three anti-3-FL antibodies are encoded by the VH441 gene segment of the X24 VH family, and this conclusion was supported by Southern filter hybridization with VH441 and JH3-JH4 probes. PMN29 has at least 11 amino acid substitutions, which is an unusually large amount of somatic mutation for an IgM antibody. Previous analyses of BALB/c genomic libraries with VHX24 and VH441 probes make it unlikely that this VH family contains additional germ-line genes, but this possibility cannot be excluded. All three antibodies use the DQ52 and JH4 gene segments. The single VH and VL gene segments used to encode the anti-3-FL antibodies is in contrast to the multiple VH and VL segments used by antibodies against other carbohydrate Ag such as alpha 1-6 dextran and group A streptococcal carbohydrate. VH441 also encodes the VH regions of antibodies against galactan and levan (beta 2-6 fructosan). The similarities among VH segments of antibodies against 3-FL, levan, and galactan, and the striking differences in their CDR3 sequences, suggest that CDR3 plays an important role in the formation of the Ag binding site. The use of a single VH segment from the smallest VH gene family by antibodies against at least three different carbohydrate determinants is noteworthy. It raises the possibility that the amino acid sequence encoded by VH441 has some general structural features that make it particularly well adapted for binding to carbohydrate sequences.     

Sequences of herpes simplex virus type 1 that inhibit formation of stable TK+ transformants.

We have identified two regions of the herpes simplex virus type 1 (HSV-1) genome that inhibit DNA-mediated transformation of thymidine kinase-less L (Ltk-) cells by the cloned HSV-1 tk gene. When plasmids containing the EcoRI fragments EK or JK were mixed at 30 fmol/ml with the tk gene and transfected into Ltk- cells, the frequency of transformation was inhibited 80 to more than 90% relative to the control. Of the remaining 10 EcoRI fragments of the HSV-1 genome, 8 were inactive and 2 were weakly active. A 6.1-kilobase PstI subclone between 0.743 and 0.782 map units was isolated from pEK. This clone, pEK-P3P4, exhibited antitransformation activity toward HSV-1 tk and also the bacterial genes gpt and neo. pEK-P3P4 contains the alpha 27 gene, and restriction endonuclease inactivation and subcloning studies established that alpha 27 alone did not inhibit transformation. However, alpha 27 plus sequences both upstream and downstream of alpha 27 did inhibit transformation. In addition, alpha 0 or alpha 4 could substitute for alpha 27 in effecting antitransformation with these sequences. Therefore, an alpha gene and two additional loci in pEK-P3P4 are required for antitransformation. A second antitransforming locus in the reiterated sequences common to EK and JK and distinct from those in pEK-P3P4 was also identified but not characterized in detail. How antitransformation may be an expression of regulation of viral and host cell gene expression is discussed.