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.     

Gene replacement with one-sided homologous recombination.

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.     

Composite human VK genes and a model of their evolution.

A phage library and two cosmid libraries were screened for human VK genes. Two recombinant phage and four cosmid clones were analysed in detail by restriction mapping and sequencing. Each one contained a single VKI sequence. Two of these six sequences are potentially functional VK genes and four are pseudogenes. Two pseudogenes derived from different genomic DNAs are highly homologous and are therefore either allelic variants or the products of a recent duplication event. Comparisons of our sequences with all fully determined human VKI amino acid and DNA sequences reveal identical segments which at first sight appear like minigenes. But these segments do not coincide with the subregions and some of the segments include both, framework and complementarity determining regions (FR, CDR, ref. 2). The findings may be explained by an evolutionary model generating composite genes by gene conversion and selection.     

The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor.

The relationship between platelet-derived growth factor (PDGF) and the proto-oncogene c-sis has been determined by amino acid sequence analysis of PDGF and nucleotide sequence analysis of c-sis genomic clones. The nucleotide sequences of five regions of the human c-sis gene which are homologous to sequences of the transforming region (v-sis) of simian sarcoma virus (SSV) were determined. By alignment of the c-sis and v-sis nucleotide sequences the predicted amino acid sequence of a polypeptide homologous to the putative transforming protein p28sis of SSV was deduced. Both predicted sequences use the same termination codon and additional coding sequences may lie 5' to the homologous regions. Amino acid sequence analysis of the PDGF B chain shows identity to the amino acid sequence predicted from the c-sis sequences over 109 amino acid residues. Polymorphism may exist at two amino acid residues. These results suggest that c-sis encodes a polypeptide precursor of the B chain. A partial amino acid sequence of the PDGF A chain is also described. This chain is 60% homologous to the B chain and cannot be encoded by that part of c-sis which has been sequenced but could be encoded by sequences which lie 5' to the five regions of v-sis homology in c-sis, or at a separate locus.     

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.     

Evolution of human immunoglobulin kappa J region genes

Immunoglobulin kappa chain variable region genes are assembled from two discontinuous DNA segments, a V and a J gene. The J region genes, in addition to encoding amino acid positions 96-108 of the kappa polypeptide chain, also provide sequences required for both DNA and RNA splicing reactions. For purposes of evolutionary comparison and to establish the complexity of the kappa J region locus in man, we have determined an approximately 3000 basepair nucleotide sequence in a cloned human DNA fragment that encodes the germline distinct J region segments. Significant blocks of homology have been tightly maintained between this region and an analogous segment of the mouse genome. In particular, the short sequences, GGTTTTTGT and CACTGTG, thought to be involved in V-J recombination, are the most highly conserved regions (97% homology). In addition, from heteroduplex data and computer analysis of the nucleotide sequences, it is clear that the mouse J3 sequence, a pseudogene, is not present in the human cluster. This can be explained by a duplication event in the mouse J region gene cluster that may have been the result of unequal crossing over between homologous chromosomes.     

Stage-specific keratins in Xenopus laevis embryos and tadpoles: the XK81 gene family

This report describes the isolation and characterization of genomic and cDNA clones which define a subfamily of type I keratins in Xenopus laevis whose expression is restricted to embryonic and larval stages. The XK81 subfamily, named after the prototype cDNA clone DG81, contains four members arranged in two pairs of closely homologous loci; they were named 81A1, A2, B1, and B2. Genomic clones were obtained representing all of these regions. The A1 gene has been completely sequenced together with approximately 1 kb of flanking sequences at each end; this gene corresponds to the previously reported cDNA clone 8128 (Jonas, E., T. D. Sargent, and I. B. Dawid, 1985, Proc. Natl. Acad. Sci. USA, 82:5413-5417). The B2 gene is represented by a partial cDNA clone, DG118. Upstream sequences and about half of the coding regions have been sequenced for the B1 and B2 genes, whereas the A2 locus has been identified on the basis of hybridization data and could be a gene or pseudogene. Genomic Southern blotting indicates that all members of the subfamily have been isolated. The keratin proteins encoded by the B1 and B2 genes are 96% homologous in the central rod domain, whereas A/B gene homology in this region is 81%. During development mRNAs derived from A and B genes accumulate coordinately during gastrula and neurula stages; in the tadpole, 81A mRNA decays rapidly, whereas 81B mRNA shows a second abundance peak, persists for most of tadpole life, and decays by metamorphosis. RNAs derived from the XK81 keratin subfamily are undetectable in the adult, where different type I keratin genes are expressed.     

Plasmid pColBM-Cl139 does not encode a colicin lysis protein but contains sequences highly homologous to the D protein (resolvase) and the oriV region of the miniF plasmid

Colicins are usually released from producing cells by so-called lysis proteins. No sequence homologous to the structurally very similar colicin lysis genes was found in the gene cluster cmi cma cbi cba, which determines the activity and immunity proteins of colicin B and M on pColBM-Cl139. Instead, the region upstream of cmi contained sequences that showed 91% homology to the structural gene of protein D (resolvase) and 75.5% homology to the rfsF sequence of the Escherichia coli miniF plasmid. It is concluded that colicins B and M are not released via the activity of lysis proteins and that the highly homologous regions encode a resolvase and its target respectively.     

The mouse c-abl locus: molecular cloning and characterization

The mouse c-abl gene, part of the sequence of which was captured in Moloney murine leukemia virus to generate the transforming gene (v-abl) of the Abelson murine leukemia virus, has been isolated and characterized. The c-abl locus spans 40 kb in the mouse genome with the v-abl homologies distributed in no less than ten clusters along 25 kb of the cloned DNA. Partial sequence of the v-abl homologous regions indicates that v-abl derived from c-abl mainly by splicing of multiple exons of the c-abl gene. The c-abl sequences can be subdivided into two regions: a tyrosine kinase coding sequence distributed among eight small clusters on the 5' end of the gene and a C-terminal portion consisting of one small and one large cluster, which are needed neither for the tyrosine kinase activity nor for the transforming ability of v-abl. Apparent exon/intron boundaries in the homologous kinase-coding regions of c-abl and c-src are at different locations.     

Gene Conversions Can Generate Sequence Variants in the Late Chorion Multigene Families of Bombyx Mori

The 140-kbp late chorion locus of Bombyx mori strain 703 contains 15 divergently oriented gene pairs encoding the high cysteine (Hc) eggshell proteins. Sequence homology is approximately 91% for the 2-kb region of each gene pair, including the 5' flanking region, intron and exons. The homology rapidly disappears within a few hundred basepairs of the 3' end of most genes. Here we present the results of the nucleotide sequence and genomic blot comparison of Hc genes from different races of B. mori. Comparison of the nucleotide sequences of the same gene pair in two different races reveals that most of the nucleotide differences occur in clusters or patches and correspond to sequences present in other Hc genes in the locus. The number of nucleotide differences that have accumulated in the highly conserved regions of the gene pair (2.3/100 bp), most of which are attributable to patchwork exchanges, is significantly higher than the number of differences in the poorly conserved 3' flanking regions (0.6/100 bp), due primarily to new mutations. These data are consistent with a gene conversion process, which in the short-term generates new combinations of sequence variants, but in the long-term results in concerted evolution. Genomic blot analyses of different geographical races of B. mori reveal that there is variation in the number of Hc gene pairs (14-19 gene pairs), indicating that unequal crossovers also occur in the locus.     

Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes.

Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome. The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in lambda EMBL3. Genomic maps were drawn for three ORS571 Nif gene loci. Forty-five Nif::Vi mutants in genomic Nif locus 1 defined two gene clusters separated by 8 kilobase pairs (kb) of DNA. In the first cluster, 36 Nif::Vi mutants mapped to a 7-kb DNA segment that showed DNA homology with Klebsiella pneumoniae nifHDKE and encoded at least two Nif operons. In the other cluster, nine Nif::Vi mutants mapped to a 1.5-kb DNA segment that showed homology with K. pneumoniae and Rhizobium meliloti nifA; this DNA segment encoded a separate Nif operon. Fifteen Nif::Vi mutants mapped to a 3.5-kb DNA segment defined as Nif locus 2 and showed DNA homology with the R. meliloti P2 fixABC operon. Nif locus 2 carries a second nifH (nifH2) gene. Four Nif::Vi mutants mapped to a 2-kb DNA segment defined as Nif locus 3 and showed DNA homology with K. pneumoniae nifB. DNA from lambda Nif phages comprising all three genomic Nif loci was subcloned in plasmid vectors able to stably replicate in ORS571. These plasmid subclones were introduced into ORS571 strains carrying physically mapped Nif::Vi insertions, and genetic complementations were conducted. With the exception of certain mutants mapping to the nifDK genes, all mutants could be complemented to Nif+ when they carried plasmid subclones of defined genomic DNA regions. Conversely, most nifDK mutants behaved as pseudodominant alleles.     

Structural characterization of the dihydroflavonol 4-reductase B (DFR-B) gene in the sweet potato.

A genomic fragment containing the dihydroflavonol 4-reductase B (DFR-B) gene was cloned from the sweet potato (Ipomoea batatas) and its nucleotide sequence was analyzed. The exons and flanking regions were highly homologous to those of previously reported DFR-B genes of the Japanese morning glory, whereas the introns and the intergenic region were less conserved. In addition to the sequences of three miniature inverted-repeat transposable elements (MITEs) and one direct repeat previously reported in the DFR-B gene of Japanese morning glory, two mobile element-like sequences were newly identified in the sweet potato DFR-B gene. At least four allelic sequences were found to exist by amplification of the DFR-B gene from various sweet potato cultivars. One of these allelic sequences had a 2-kb deletion in the intergenic region and was observed in the cultivars with high anthocyanin content in their storage roots.     

Use of long sequence alignments to study the evolution and regulation of mammalian globin gene clusters

The determination of long segments of DNA sequences encompassing the beta- and alpha-globin gene clusters has provided an unprecedented data base for analysis of genome evolution and regulation of gene clusters. A newly developed computer tool kit generates local alignments between such long sequences in a space-efficient manner, helps the user analyze the alignments effectively, and finds consistently aligning blocks of sequences in multiple pairwise comparisons. Such sequence analyses among the beta-like globin gene clusters of human, galago, rabbit, and mouse have revealed the general patterns of evolution of this gene cluster. Alignments in the flanking regions are very useful in assigning orthologous relationships. Investigation of such matches between the mouse and human beta-like globin gene clusters has led to a reassessment of some orthologous assignments in mouse and to a revision of the proposed pathway for evolution of this gene cluster. In general, the interspersed repetitive elements have inserted independently, presumably via a retrotransposition mechanism, in the different mammalian lineages. However, some examples of ancient L1 repeats are found, including one between the epsilon- and gamma-globin genes that appears to have been in the ancestral eutherian gene cluster. Prominent matching sequences are found in a long region 5' to the epsilon-globin gene, the locus control region (LCR) that is a positive regulator of the entire gene cluster. Three-way alignments among the human, goat, and rabbit sequences can extend for > or = 3 kb in part of the LCR (DNase hypersensitive site 3), indicating that the cis-acting components of this complex regulatory region cover a long segment of DNA. In contrast to the beta-like globin gene clusters, the alpha-like globin gene clusters of many mammals occur in very G+C-rich isochores and contain prominent CpG islands. The regions between the alpha-like globin genes are evolving faster than the intergenic regions of the beta-like globin gene clusters. The contrasts between the two gene clusters can be attributed to differences in DNA metabolism in the isochore. The proximal control elements of the rabbit alpha-globin gene are located both 5' to and within the gene. All of this region is part of a prominent CpG island that may be acting as an extended, enhancer- independent promoter. One can hypothesize that the analogue to the LCR in the alpha-globin gene cluster may interface with the distinctive alpha-globin promoter in ways different from the interaction between the beta LCR and the promoters of beta-like globin genes.(ABSTRACT TRUNCATED AT 400 WORDS)      

Location of mouse and human genes corresponding to conserved canine olfactory receptor gene subfamilies

Olfactory receptors are G protein-coupled, seven-transmembrane-domain proteins that are responsible for binding odorants in the nasal epithelium. They are encoded by a large gene family, members of which are organized in several clusters scattered throughout the genomes of mammalian species. Here we describe the mapping of mouse sequences corresponding to four conserved olfactory receptor genes, each representing separate, recently identified canine gene subfamilies. Three of the four canine genes detected related gene clusters in regions of mouse Chromosomes (Chrs) 2, 9, and 10, near previously mapped mouse olfactory genes, while one detected a formerly unidentified gene cluster located on mouse Chr 6. In addition, we have localized two human gene clusters with homology to the canine gene, CfOLF4, within the established physical map of Chr 19p. Combined with recently published studies, these data link the four conserved olfactory gene subfamilies to homologous regions of the human, dog, and mouse genomes. Received: 10 September 1997 / Accepted: 29 December 1997     

The rabbit kappa 1 b5 immunoglobulin gene: another J region gene cluster with only one functional J gene segment?

Our previous analysis of an immunoglobulin gene encoding the rabbit kappa chain of b4 allotype revealed that of the five J-like sequences in the J kappa cluster of this gene, only one, J2, appeared to be functional. This unusual ratio of J pseudogenes to functional J genes is unique among all J clusters of light and heavy chain genes of all species examined to date, including the cluster from the rabbit kappa 2 isotype, and must have consequences for diversity generation of b4 immunoglobulins. The fact that the only two known b5 J kappa sequences are different from the functional J2 of the b4 allotype prompted investigation of the b5 J kappa cluster to determine whether it resembled the b4 cluster, or the more typical mouse or human J kappa clusters. Our analysis of the b5 gene reveals a J kappa cluster strikingly similar to that of b4; apparent defects occur in all J sequences except J2. Although J2 is apparently functional, it differs from the J2 of the b4 locus by four nucleotide and three amino acid substitutions. The unusually high degree of sequence similarity previously observed between the b4 and b5 loci in the noncoding (vs the coding) regions extends through the newly sequenced DNA segment and remains an enigma.     

The wheat D-genome HMW-glutenin locus: BAC sequencing,gene distribution,and retrotransposon clusters

A bacterial-artificial-chromosome (BAC) clone from the genome of Triticum tauschii, the D-genome ancestor of hexaploid bread wheat, was sequenced and the presence of the two paralogous x- and y-type high-molecular-weight (HMW) glutenin genes of the Glu-D1 locus was confirmed. These two genes occur in the same orientation, are 51,893 bp apart, and the separating DNA includes a 31,000-bp cluster of retrotransposons. A second retrotransposon cluster of 32,000 bp follows the x-type HMW-glutenin gene region. Each HMW-glutenin gene is found within a region of mainly unique DNA sequence which includes multiple additional genes including an active endosperm globulin gene not previously reported in the Triticeae family, a leucine-rich-repeat (LRR) type gene truncated at the 5′ end of the BAC, a kinase gene of unknown activity, remnants of a paralogous second globulin gene, and genes similar to two hypothetical rice genes. The newly identified globulin genes are assigned to a locus designated Glo-2. Comparison to available orthologous regions of the wheat A and B genomes show rapid sequence divergences flanking the HMW-glutenin genes, and the absence of two hypothetical and unknown genes found 5′ to the B-genome x-type ortholog. The region surrounding the Glu-D1 locus is similar to other reported Triticeae BAC sequences; i.e. small gene islands separated by retrotransposon clusters. Electronic Publication