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.     

Organization of variable region segments of the human immunoglobulin heavy chain: duplication of the D5 cluster within the locus and interchromosomal translocation of variable region segments.

We have studied the organization of variable region (V) genes of the human immunoglobulin heavy chain (H) by cosmid cloning. We isolated two independent immunoglobulin D5 clusters (D5-a and D5-b) from cosmid libraries of the human genome. Restriction maps of these two regions showed that downstream 15 kb portions of the 55 kb overlap were different although upstream 40 kb portions were almost identical. Four more D segments, (DM, DXP, DA and DK) were found around the D5 segment in the conserved region of each cluster. Nucleotide sequences of the corresponding D segments from each cluster were almost identical and they encoded potentially functional D regions. Analysis using human-rodent somatic cell hybrids demonstrated that both clusters were located in the immunoglobulin heavy chain (H) locus on chromosome 14, suggesting that the D5-a and D5-b regions evolved by internal duplication within this locus. We also isolated a 60 kb DNA region carrying four VH segments, designated as VH-F region, which was located on chromosome 16. Nucleotide sequences of the four VH segments were determined. Two of them encoded potentially functional VH segments, and the other two were pseudogenes. Some more VH segments were found to be located outside chromosome 14, by Southern blot hybridization of human-rodent hybrid cell DNAs. These results provide further evidence that the human VH locus has undergone recent reorganization.     

Molecular footprints of human immunoglobulin gene evolution: a new sequence family.

Analysis of the human VK (ref. 2) gene locus led to the detection of a new sequence family (L sequences). Its copy number is in the range of 10(2). The L sequences, which are about 500 bp long, are found as part of the 3' flanking regions of a clustered set of human VKI genes but they occur also separate from the genes. Models are discussed in which L sequences are viewed as molecular footprints of amplification and transposition processes of VK genes.     

Organization and evolution of variable region genes of the human immunoglobulin heavy chain

We have isolated 23 different cosmid clones of the heavy-chain variable region genes (VH) of human immunoglobulin. These clones encompass about 1000 X 10(3) base-pairs of DNA containing 61 VH genes. Characterization of the 23 clones by Southern blot hybridization showed that VH genes belonging to different families were physically linked in many regions. Cluster 71, which was analyzed in detail, comprised seven VH segments arranged in the same orientation with different intervals. This clone contained internal homology regions, each carrying two VH segments of different families. Comparison of the nucleotide sequences of VH segments within each family showed that profiles of accumulation of mutations in framework (FR) and complementarity-determining (CDR) regions were different. CDR had more mutations at amino-acid-substituting positions than at silent positions, whereas FR had the reverse distribution of mutations. Five out of seven VH segments of this cluster were pseudogenes containing various mutations. VH pseudogenes were classified into two distinct groups; one with a few replacement mutations (conserved pseudogenes), and the other with rather extensive mutations (diverged pseudogenes). The possibility that conserved pseudogenes serve as a reservoir of VH segments is discussed.     

The human T cell receptor beta-chain gene complex contains at least 57 variable gene segments. Identification of six V beta genes in four new gene families.

The human TCR beta-chain gene complex includes at least 57 variable (V) gene segments, a number estimated using a combination of Southern blots of conventional and pulsed field gels, sequence analysis of cDNA clones, and from the analysis of genomic cosmid and phage clones. This number includes six TCR beta-chain V genes in four new families identified here by sequence analysis of clones derived from a human TCR beta-chain specific cDNA library. Comparison of the sequences of the new V beta genes with previously reported V beta sequences reveals predicted similarities but less than 75% nucleic acid identity that establishes them as new V beta families. One of the new V beta gene families includes three genes and the other three are single member families. Identification of these six new V beta genes falling into four V beta families brings the total number of transcribed human V beta families to 24 and makes it possible to refine the estimate of the total number of human TCR V beta genes to 57.     

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.     

Class II genes of the human major histocompatibility complex. Organization and evolutionary relationship of the DR beta genes

The genes of the polymorphic HLA-DR molecules are located within the human major histocompatibility complex. We have studied the HLA-DR genes of an HLA homozygous individual typed to be DR4, Dw4, and DRw53. Fourteen cosmid and phage clones from genomic libraries were isolated and grouped into three clusters comprising a total of 165 kilobases. These clusters contain four DR beta genes. Nucleotide sequence determination showed that two of the genes encode beta chains that carry the DR4 and DRw53 specificities, respectively, while the other two genes are presumably pseudogenes. Comparisons of the nucleotide sequences of all four DR beta genes of the DR4 haplotype show that the genes are extensively similar, approximately 90% in both exons and introns. All four genes are equally similar to each other. These observations are consistent with the notion that the genes arose by duplications that were followed by homogenization through gene conversion. The existence of more than one DR beta gene homologue but only a single DR alpha gene homologue in mouse, rabbit, and cattle suggests that the DR beta gene duplications occurred at or early during mammalian speciation.     

Evolution of human cytoplasmic actin gene sequences: chromosome mapping and structural characterizations of three cytoplasmic actin-like pseudogenes including one on the Y chromosome

Eight recombinant phage clones containing cytoplasmic actin-like gene sequences have been isolated from a human genomic library for structural characterization. Kpn I family repeat sequences flank six of these actin genes isolated, and Alu family repeats are scattered throughout the DNA inserts of all eight phage clones. Three of these genes are γ actin-like, and the other five are β actin-like. The complete nucleotide sequence analysis of one β and one γ actin-like genes and their flanking regions demonstrates that they both are processed pseudogenes. Using unique DNA sequences flanking these two pseudogenes as hybridization probes for human-mouse somatic cell hybrid DNAs, we have mapped the two actin pseudogenes on human chromosomes 8 and 3, respectively. We have also determined the DNA sequence of a human Y chromosome-linked, processed actin pseudogene. The different values of sequence divergence of these processed pseudogenes and their functional counterparts allow us to estimate the time of generation of the pseudogenes. The results suggest that the cDNA insertion events generating the human cytoplasmic actin-like pseudogenes have occurred at significantly different times during the evolution of primates, after their separation from other mammalian species.     

Human immunoglobulin kappa light chain genes of subgroups II and III.

The first complete sequences of functionally rearranged VK genes (abbreviations ref. 1) of subgroups II and III are reported. The genes have been cloned from lymphoid cell lines synthesizing KII or KIII light chains as evidenced from immunochemical analyses with anti-VK subgroup-specific antisera. These data, together with the sequence of a KIV gene (described in the accompanying paper) and those of previously published KI genes make possible a comparison of genes representative of the four known V region subgroups of human K light chains. The VKII gene is distinguished from the VKI, VKIII, and VKIV genes by a much longer intron within the leader sequence: 426 bp vs ca. 120-220 bp. Blot hybridization experiments with human DNA digests using probes from the KII and KIII genes and from the respective upstream regions help to define subgroup specific probes and hybridization conditions.     

SB subregion of the human major histocompatibility complex: gene organization, allelic polymorphism and expression in transformed cells

The SB region of the human major histocompatibility complex (MHC) has been cloned from cosmid and lambda phage libraries made from the human B-lymphoblastoid cell line Priess (DR4/4, DC4/4, SB3/4). Two alpha genes and two beta genes are encoded in the 100 kb long SB region in the order SB alpha-SB beta-SX alpha-SX beta. The SB alpha and SB beta genes encode the alpha and beta subunits of the SB subset of class II MHC molecules. Both the SX alpha and the SX beta genes are pseudogenes in the haplotype examined. From the isolated clones, the two haplotypes of the Priess cell line, SB3 and SB4, are distinguished by nucleotide sequencing and blot hybridization analyses. Restriction site polymorphisms between the SB3 and SB4 clones were observed only in relatively small regions of the SB beta and SX beta genes. A mouse macrophage cell line was transfected with one of the cosmid clones containing both SB alpha and SB beta genes. Expression of the alpha and beta genes was detected by fluorescene-activated cell sorting (FACS) and two-dimensional gel electrophoresis using SB-specific monoclonal antibodies.     

Structural relationship of human interferon alpha genes and pseudogenes

We have isolated and characterized DNA segments containing IFN-alpha-related sequences from human lambda and cosmid clone banks. We describe six linkage groups comprising 18 distinct IFN-alpha-related loci, and report the nucleotide sequences of nine chromosomal IFN-alpha-genes with intact reading frames, as well as of five pseudogenes. Taking into account as yet unsequenced genes as well as clones described by others, there are now seven linkage groups and 23 loci, of which 15 correspond to potentially functional genes and six to non-functional genes; two loci remain unsequenced. Eighteen additional sequences are likely to be allelic to the above. The finding that at least two IFN-alpha genes appear to be natural hybrids of other IFN-alpha genes, and that two distinct IFN-alpha loci have completely identical coding sequences, although their flanking regions are different, is evidence for information exchange between the individual genes.     

Human U1 small nuclear RNA genes: extensive conservation of flanking sequences suggests cycles of gene amplification and transposition.

The DNA immediately flanking the 164-base-pair U1 RNA coding region is highly conserved among the approximately 30 human U1 genes. The U1 multigene family also contains many U1 pseudogenes (designated class I) with striking although imperfect flanking homology to the true U1 genes. Using cosmid vectors, we now have cloned, characterized, and partially sequenced three 35-kilobase (kb) regions of the human genome spanning U1 homologies. Two clones contain one true U1 gene each, and the third bears two class I pseudogenes 9 kb apart in the opposite orientation. We show by genomic blotting and by direct DNA sequence determination that the conserved sequences surrounding U1 genes are much more extensive than previously estimated: nearly perfect sequence homology between many true U1 genes extends for at least 24 kb upstream and at least 20 kb downstream from the U1 coding region. In addition, the sequences of the two new pseudogenes provide evidence that class I U1 pseudogenes are more closely related to each other than to true genes. Finally, it is demonstrated elsewhere (Lindgren et al., Mol. Cell. Biol. 5:2190-2196, 1985) that both true U1 genes and class I U1 pseudogenes map to chromosome 1, but in separate clusters located far apart on opposite sides of the centromere. Taken together, these results suggest a model for the evolution of the U1 multigene family. We speculate that the contemporary family of true U1 genes was derived from a more ancient family of U1 genes (now class I U1 pseudogenes) by gene amplification and transposition. Gene amplification provides the simplest explanation for the clustering of both U1 genes and class I pseudogenes and for the conservation of at least 44 kb of DNA flanking the U1 coding region in a large fraction of the 30 true U1 genes.     

Structural analysis of gene loci for rat U1 small nuclear RNA.

Four phage clones which hybridize with U1 small nuclear RNA were obtained from a rat gene library. Two clones contain a presumed pseudogene. A third clone includes two gene candidates that are co-linear with the rat U1-RNA, 3.6kb apart and in the opposite orientation. The two genes are surrounded by identical sequences of 491bp upstream and 178bp downstream. The upstream sequences do not contain a TATA box, but share many block homologies with those for the human U1-RNA gene(1-3). A 101bp "identifier (ID) sequence", which was reported to be specifically expressed in rat brain (4), is inserted immediately after the shared sequence downstream of one of the genes. In the fourth clone, there are two putative pseudogenes, which have one or three nucleotide changes, 3kb apart and in the same orientation. Southern blot analysis of total rat DNA reveals about 50 U1-RNA genes/pseudogenes in the genome.     

Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences.

Two functional cytosolic thymidine kinase (tk) cDNA clones were isolated from a mouse L-cell library. An RNA blot analysis indicated that one of these clones contains a nearly full-length tk sequence and that LTK- cells contain little or no TK message. The nucleotide sequences of both clones were determined, and the functional mouse tk cDNA contains 1,156 base pairs. An analysis of the sequence implied that there is an untranslated 32-nucleotide region at the 5' end of the mRNA, followed by an open reading frame of 699 nucleotides. The 3' untranslated region is 422 nucleotides long. Thus, the gene codes for a protein containing 233 amino acids, with a molecular weight of 25,873. A comparison of the coding sequences of the mouse tk cDNA with the human and chicken tk genes revealed about 86 and 70% homology, respectively. We also isolated the tk gene from a mouse C57BL/10J cosmid library. The structural organization was determined by restriction mapping, Southern blotting, and heteroduplex analysis of the cloned sequences, in combination with a mouse tk cDNA. The tk gene spans approximately 11 kilobases and contains at least five introns. Southern blot analysis revealed that this gene is deleted in mouse LTK- cells, consistent with the inability of these cells to synthesize TK message. This analysis also showed that tk-related sequences are present in the genomes of several mouse strains, as well as in LTK- cells. These segments may represent pseudogenes.     

Three transposed elements in the intron of a human VK immunoglobulin gene.

Two gene segments coding for the variable region of human immunoglobulin light chains of the kappa type (VK genes, ref. 2) were found to have unusual structures. The two genes which are called A6 and A22 are located in duplicated gene clusters. Their restriction maps are very similar. About 4 kb of the A22 gene region were sequenced. It turned out that the intron contains an insert with the characteristics of a transposed element. The inserted DNA of 1.2 kb length contains imperfect direct and inverted repeats at its ends; at the insertion site a duplication of five nucleotides was found. Within the inserted DNA one copy each of an Alu element and of the simple sequence motif (T-G)17 were identified. Also these two repetitive sequences are themselves flanked by short direct repeats. The major inserted DNA has no significant homology to published human nucleic acid sequences. The whole structure is interpreted best by assuming a sequential insertion of the three elements. The coding region of the VK gene itself has several mutations which by themselves would render it a pseudogene; we assume that the insertion event(s) occurred prior to the mutations. According to mapping and hybridization data A6 is very similar to A22.     

Molecular analysis of the human interferon-alpha gene family

Fifteen DNA clones containing sequences related to human interferon-alpha cDNA were isolated from a human chromosomal gene bank (Nagata et al., Nature 287 (1980) 401-408) and characterized by restriction mapping, R-loop and heteroduplex analysis. Nine distinct DNA segments hybridized strongly with interferon-alpha 1 cDNA and formed R-loops with poly(A) RNA from interferon-producing human leukocytes; most if not all of these segments represent functional interferon genes. Five segments hybridized weakly with the probe and did not form R-loops with the poly(A) RNA; one of these was characterized as an interferon-alpha pseudogene. Several DNA segments overlap and define a region of 36 kilobase pairs (kb) that contains three strongly and three weakly hybridizing sequences. From our data and those of Goeddel et al. (Nature 290 (1981) 20-25) we conclude that there exist at least 11 distinct genes of gene-like sequences of the interferon-alpha type in the human genome, of which most likely represents an allelic variant, and at least five pseudogenes distantly related to the interferon-alpha genes.     

抗破伤风类毒素单抗可变区基因的克隆及在大肠杆菌中表达的三种工程抗体

The heavy and light chain variable region genes of anti-tetanus toxoid (TT) antibody and the heavy chain Fd genes were amplified and cloned through RT-PCR from mouse hybridoma cells. The sequences of VH and VK were determined. Fd gene fragments were expressed in E. coli. The ELISA results indicated that the expressed Fd showed antigen binding activity but was nonspecific. Furthermore, through SOE and PCR techniques, the VH and VK gene fragments together with ScFv linker were assembled into single chain antibody (ScFv) gene fragment. While together with human heavy chain CH 1 gene fragment and Fab linker, they were assembled into chimeric Fab gene fragment. The two assembled gene fragments were separately inserted into phagemid pHEN 1, which was a fd-based vector containing gene 3 encoding the minor coat protein. In presence of helper phage M 13-VCS the anti-TT phage-ScFv or phage-Fab were displayed on the surface of phage particles respectively. Results from phage-ELISA indicated that both phage antibodies were TT-specific.