Diverse organization of immunoglobulin VH gene loci in a primitive vertebrate.

The immunoglobulin (Ig) heavy chain variable (VH) gene family of Heterodontus francisci (horned shark), a phylogenetically distant vertebrate, is unique in that VH, diversity (DH), joining (JH) and constant region (CH) gene segments are linked closely, in multiple individual clusters. The V regions of 12 genomic (liver and gonad) DNA clones have been sequenced completely and three organization patterns are evident: (i) VH-D1-D2-JH-CH with unique 12/22 and 12/12 spacers in the respective D recombination signal sequences (RSSs); VH and JH segments have 23 nucleotide (nt) spacers, (ii) VHDH-JH-CH, an unusual germline configuration with joined VH and DH segments and (iii) VHDHJH-CH, with all segmental elements being joined. The latter two configurations do not appear to be pseudogenes. Another VH-D1-D2-JH-CH gene possesses a D1 segment that is flanked by RSSs with 12 nt spacers and a D2 segment with 22/12 spacers. Based on the comparison of spleen, VH+ cDNA sequences to a germline consensus, it is evident that both DH segments as well as junctional and N-type diversity account for Ig variability. In this early vertebrate, the Ig genes share unique properties with higher vertebrate T-cell receptor as well as with Ig and may reflect the structure of a common ancestral antigen binding receptor gene.     

Intergenic exchange maintains identity between two human lambda light chain immunoglobulin gene intron sequences.

Evidence for gene conversion or unequal double crossover in the human lambda light chain immunoglobulin locus is presented. The high level of J2C2-J3C3 intron cross-hybridization, the identity of the J lambda and J lambda 3 coding and intron sequences, the presence of multiple base differences between the C lambda 2 and C lambda 3 coding regions, and the presence of both the unconverted and converted alleles in the normal gene pool, suggest that a recombinational event has resulted in the conversion of the J lambda 2 coding and intron sequences to those of J lambda 3 and its flanking sequences. Intergenic exchanges, such as the one described here, may provide a mechanism to maintain sequence homogeneity and functionality among the duplicated members of the human lambda gene family.     

Genomic organization of the serine protease light chain of mouse complement factor I gene

A portion of the mouse complement factor I (mCFI) gene encoding for the mCFI light chain was cloned from a mouse 129/SVJ1 bacterial artificial chromosome library. It contains five exons and four introns. The intron sizes are remarkably different from the human homolog. Several polymorphisms were found in exon 13. One polymorphism was in the coding region, which causes a threonine in the Balb/c mCFI to be replaced by an isoleucine in the 129/SVJ1 mCFI. The other two polymorphisms are located in the 3' untranslated region. The organization of the serine protease domain in mCFI is similar to that of trypsin but very different from that of the other complement serine proteases.     

Simple DNA sequences and dispersed repetitive elements in the vicinity of mouse immunoglobulin K light chain genes

The simple DNA sequences (T-G)20, (T-T-T-G-C)20 and (G-C-C-T-C-T)30 were found in the vicinity of mouse immunoglobulin genes and of dispersed repetitive elements as the R, B1 and B2 sequences. On the basis of sequence data, blot hybridizations with salmon and mouse DNA and with defined mouse DNA fragments, possible functional and evolutionary aspects of simple DNA sequences are discussed.     

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.     

Repetitive sequences in class-switch recombination regions of immunoglobulin heavy chain genes

Immunoglobulin class switch involves a unique recombination event that takes place at the region 5′ to each heavy chain constant region gene during B lymphocyte differentiation. Such regions that are responsible for the class-switch recombination are defined as S regions (Kataoka et al., Proc. Natl. Acad. Sci. USA 77, 919, 1980). We have cloned a rearranged γ2b gene from a mouse myeloma (MPC11) and compared its structure with the germ line counterparts. The rearranged γ2b gene contained the 5′ flanking region of the γ3 gene (S_γ3 region) which are linked to the 5′ flanking region of the γ2b gene (S_γ2b region). We have determined nucleotide sequences surrounding the recombination site of the rearranged and germ line γ2b genes, which include the S_γ2b and S_γ3 regions. Both _γ2b and S_γ3 regions comprise tandem repetition of conserved units of 49 bp. Similar 49 bp repeating units are also found in the previously determined sequence of the S_γ1 region in which class-switch recombination took place in MC101 myeloma. The nucleotide sequences of the S_γ1, S_γ2b and S_γ3 repeating units share significant homology with each other. The Sμ region, partial nucleotide sequence of which was previously determined, contains abundant short sequences such as AGCT, TGGG and AGCTGGGG which are shared in common by repeating sequences in S_γ regions. These results suggest that the recombination responsible for class switch from μ to γ or from a γ to another γ, may be facilitated directly or indirectly by homology of repeating sequences in S regions.     

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.     

Sequences of mouse immunoglobulin light chain genes before and after somatic changes.

We have determined the nucleotide sequences of the germ line gene as well as a corresponding somatically mutated and rearranged gene coding for a mouse immunoglobulin lambdaI type light chain. These sequencing studies were carried out on three Eco RI-DNA fragments which had been cloned from BALB/c mouse embryos or a lambdaI chainsecreting myeloma, H2020. The embryonic DNA clone Ig 99lambda contains two protein-encoding segments, one for the majority of the hydrophobic leader (L) and the other for the rest of the leader and the variable (V) region of the lambda0 chain (Cohn et al., 1974); these segments are separated by a 93 base pair (bp) intervening sequence (I-small). The coding of the V region ends with His at residue 97. The second embryonic DNA clone Ig 25lambda includes a 39 bp DNA segment (J) coding for the rest of the conventionally defined V region (that is, up to residue 110), and also contains the sequence coding for the constant (C) region approximately 1250 untranslated bp (I-large) away from the J sequence. The J sequence is directly linked with the V-coding sequence in the myeloma DNA clone, Ig 303lambda, which has the various DNA segments arranged in the following order: 5' untranslated region, L, l-small, V linked with J, l-large, C, 3' untranslated sequence. The lg 303lambda V DNA sequence codes for the V region synthesized by the H2020 myeloma and is different from the lg 99lambda V DNA sequence by only two bases. No silent base change was observed between the two DNA clones for the entire sequence spanning the 5' untranslated regions and the V-coding segments. These results confirm the previously drawn conclusion that an active complete lambdaI gene arises by somatic recombination that takes place at the ends of the V-coding DNA segment and the J sequence. No sequence homology was observed at or near the sites of the recombination.     

The physical organization of the human immunoglobulin heavy chain gene complex.

Two dimensional DNA electrophoresis (2D-DE) was used to map the variable (VH) region of the human heavy chain immunoglobulin gene cluster. Seventy-six VH gene segments were mapped to specific SfiI, BssHI and NotI fragments by 2D-DE. We have determined that a common insertion/deletion polymorphism of 80 kb, involving three VH gene segments, occurs in the VH region. The physical map suggests that the evolution of the human IGH gene complex involved duplication of blocks containing different VH families. This physical map will allow comparison of the usage of VH loci in human ontogeny with their proximity to the CH region. Knowledge of the germline repertoire of a particular DNA source studied in essential as the number of the dispersed VH gene segments of VH families, especially of the VH5 family, is variable. 2D-DE, as illustrated here for the IGH gene cluster, has general application in the development of large scale physical maps of gene and repeat families.     

Association of two different repetitive DNA elements near immunoglobulin light chain genes.

Sequence studies of repetitive DNA elements approximately 6 kb 3' of the mouse immunoglobulin CK region gene show that the R element located there (Gebhard et al. (1982) J. Mol. Biol. 157, 453-471) is adjacent to a 500 base pair long element which shows 80% homology to the BAM5 element sequenced by Fanning (Nuc. Acids Res. (1982), 10, 5003-5013). Neither the BAM5 element nor the R element itself is surrounded by a direct repeat, but the composite element (BAM5 + R) is surrounded by a 15 base pair direct repeat (with one mismatch). Direct repeats, consisting of target site sequences that surround a repetitive DNA element, are thought to arise during the insertion of the element at that site. It therefore appears that the BAM5 and R elements interacted and inserted as a linked entity. The existence of other BAM5/R composites throughout the mouse lambda chain locus indicates that BAM5-R cooperation is not a rare event.     

Refolding of the immunoglobulin light chain.

The kinetics of the refolding reactions of type lambda Bence Jones proteins from 4 M GuHCl were studied by CD, ultraviolet absorption, and fluorescence spectrophotometry. The kinetics were complex and consisted of at least three phases, an undetectable fast phase, a detectable fast phase, and a slow phase. The slow phase followed first-order kinetics and the three experimental methods used gave similar rate constants for all the Bence Jones proteins (about 3 X 10(-3) s-1). The refolding reaction of VL fragment was too fast to be measured in the present experiments. The refolding process of CL fragment was very similar to those of Bence Jones proteins except that the detectable fast phase was less significant. The rate constant of the slow phase observed for the CL fragment was similar to those of the slow phase observed for Bence Jones proteins. The activation energy of the slow phase was the same for a Bence Jones protein and its CL fragment. These results indicate that the refolding kinetics of the CL domain are very similar to those of isolated CL fragment and that refolding of the VL domain precedes refolding of the CL domain, even though both domains have similar immunoglobulin folds. However, the results of refolding experiments on Bence Jones proteins, and VL and CL fragments in the presence of ANS, as well as the other lines of evidence, indicate that the refolding kinetics of the Bence Jones protein molecule cannot be expressed as simple sum of the refolding reactions of isolated VL and CL fragments.     

Genomic organization of glycinin genes in soybean

Glycinin is the predominant seed storage protein in most soybean varieties. Previously, five major genes (designated Gy1 to Gy5) encoding glycinin subunits have been described. In this report two new genes are identified and mapped: a glycinin pseudogene, gy6, and a functional gene, Gy7. Messenger RNA for the gy6 pseudogene is not detected in developing seeds. While Gy7 mRNA was present at the midmaturation stage of seed development in the soybean variety Resnik, the steady state amount of this message was at least an order of magnitude less-prevalent than the mRNA encoding each of the other five glycinin subunits. Even though the amino-acid sequence of the glycinin subunit G7 is related to the other five soybean 11S subunits, it does not fit into either the Group-1 (G1, G2, G3) or the Group-2 (G4, G5) glycinin subunit families. The Gy7 gene is tandemly linked 3' to Gy3 on Linkage Group L (chromosome 19) of the public molecular linkage map. By contrast, the gy6 gene occupies a locus downstream from Gy2 on Linkage Group N (chromosome 3) in a region that is related to the position where Gy7 is located on chromosome 19.     

Genomic organization and gene function in Leishmania

Sequencing of the Leishmania major Friedlin genome is well underway with chromosome 1 (Chr1) and Chr3 having been completely sequenced, and Chr4 virtually complete. Sequencing of several other chromosomes is in progress and the complete genome sequence may be available as soon as 2003. A large proportion ( approximately 70%) of the newly identified genes remains unclassified, with many of these being potentially Leishmania- (or kinetoplastid-) specific. Most interestingly, the genes are organized into large (>100-300 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a 'divergent' manner, i. e. the mRNAs for the two sets of genes are both transcribed towards the telomeres. Chr3 contains two 'convergent' clusters, with a single 'divergent' gene at one telomere, with the two large clusters separated by a tRNA gene. We have characterized several genes from the LD1 (Leishmania DNA 1) region of Chr35. BT1 (formerly ORFG) encodes a biopterin transporter and ORFF encodes a nuclear protein of unknown function. Immunization of mice with recombinant antigens from these genes results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.     

Genomic organization of mouse gene zfp162.

We report the cloning and characterization of the alternatively spliced mouse gene zfp162, formerly termed mzfm, the homolog of the human ZFM1 gene encoding the splicing factor SF1 and a putative signal transduction and activation of RNA (STAR) protein. The zfp162 gene is about 14 kb long and consists of 14 exons and 13 introns. Comparison of zfp162 with the genomic sequences of ZFM1/SF1 revealed that the exon-intron structure and exon sequences are well conserved between the genes, whereas the introns differ in length and sequence composition. Using fluorescent in situ hybridization, the zfp162 gene was assigned to chromosome 19, region B. Screening of a genomic library integrated in lambda DASH II resulted in the identification of the 5'-flanking region of zfp162. Sequence analysis of this region showed that zfp162 is a TATA-less gene containing an initiator control element and two CCAAT boxes. The promoter exhibits the following motifs: AP-2, CRE, Ets, GRE, HNF5, MRE, SP-1, TRE, TCF1, and PU.1. The core promoter, from position -331 to -157, contains the motifs CRE, SP-1, MRE, and AP-2, as determined in transfected CHO-K1 cells and IC-21 cells by reporter gene assay using a secreted form of human placental alkaline phosphatase. The occurrence of PU.1/GRE supports the view that the zfp162 gene encodes a protein involved not only in nuclear RNA metabolism, as the human ZFM1/SF1, but also in as yet unknown macrophage-inherent functions.     

Evolutionary relationship between human and mouse immunoglobulin kappa light chain variable region genes

Similar to the Igh-V multigene family, the human or mouse Igk-V repertoirer is a distorted continuum of homologous genes that may be grouped into families displaying >80% nucleic acid sequence similarity among their members. systematic interspecies sequence comparisons reveal that most human Igk-V gene families exhibit clear homology to mouse Ogk-V families (sequence similarity >74%). A hypothetical phylogenetic tree of Igk-V genes predicts that a minimum of seven Igk-V genes/families predate mammalian radiation. In two cases, several interrelated mouse Igk-V families exhibit phylogenetic equidistance with just one human Igk-V family, implying a more pronounced divergence for the elevated number of Igk-V gene families in the mouse. Mouse-human Igk-V comaprisons, moreover, illustrate how expansion, contraction, and perhaps deletion of Igk-V gene families shape the Igk-V repertoire during mammalian evolution.     

Nucleotide sequences of two corn histone H3 genes. Genomic organization of the corn histone H3 and H4 genes

Summary Two histone H3 genes have been cloned from a gtWES.B corn genomic library. The nucleotide sequences show 96% homology and both encode the same protein, which differs from its counterpart in wheat and pea by one amino acid substitution. The 5-flanking regions of the two corn H3 genes contain the classical histone-gene-specific consensus sequences and possess several regions of extensive nucleotide homology. A conserved octanucleotide 5-CGCGGATC-3 occurs at approximately 200 nucleotides upstream from the initiation ATG codon. This octanucleotide was found to exist in all of the 7 plant histone genes sequenced so far. Codon usage is characterized by a very high frequency of C (67%) and G (28%) at the third position of the codons, those ending by A (1%) and T (4%) being practically excluded.Comparison of Southern blots of EcoRI, EcoRV and BamHI digested genomic DNA suggests that the corn H3 and H4 genes are not closely associated. The H3 genes exist as 60 to 80 copies and the H4 genes as 100 to 120 copies per diploid genome. re]19851002 rv]19851212 ac]19851216     

Genomic organization and DNA sequences of human 17 beta-hydroxysteroid dehydrogenase genes and flanking regions. Localization of multiple Alu sequences and putative cis-acting elements.

Genomic 17 beta-hydroxysteroid-dehydrogenase (17-HSD) clones were isolated from a human leucocyte genomic library using cDNA encoding human placental 17-HSD as a probe. The overlapping fragments spanned more than 21 kbp containing the duplications, 6.2 kbp of each, as well as 7 kbp upstream and 1.6 kbp downstream from the duplicated sequences. 17 complete and eight partial Alu elements were clustered in this area, covering about 30% of the region, including the borders of the duplications. Each duplication contained a 17-HSD gene and a conserved region of 1.56 kbp with 98% intercopy similarity. The exon structure of the 17-HSD gene II corresponded to the known cDNA species, but both genes contained a possible promoter region with TATA, GC and inverse CAAT boxes. The 5' flanking regions contained sequences similar to the consensus sequences of cis-acting elements, defined as regulators of 17-HSD gene expression. These putative sequences included estrogen and progesterone/glucocorticoid-response elements and a cyclic-AMP regulatory element.