Organization, complexity and allelic diversity of the porcine (Sus scrofa domestica) immunoglobulin lambda locus

We have characterized the organization, complexity, and expression of the porcine (Sus scrofa domestica) immunoglobulin lambda (IGL) light chain locus, which accounts for about half of antibody light chain usage in swine, yet is nearly totally unknown. Twenty-two IGL variable (IGLV) genes were identified that belong to seven subgroups. Nine genes appear to be functional. Eight possess stop codons, frameshifts, or both, and one is missing the V-EXON. Two additional genes are missing an essential cysteine residue and are classified as ORF (open reading frame). The IGLV genes are organized in two distinct clusters, a constant (C)-proximal cluster dominated by genes similar to the human IGLV3 subgroup, and a C-distal cluster dominated by genes most similar to the human IGLV8 and IGLV5 subgroups. Phylogenetic analysis reveals that the porcine IGLV8 subgroup genes have recently expanded, suggesting a particularly effective role in immunity to porcine-specific pathogens. Moreover, expression of IGLV genes is nearly exclusively restricted to the IGLV3 and IGLV8 genes. The constant locus comprises three tandem cassettes comprised of a joining (IGLJ) gene and a constant (IGLC) gene, whereas a fourth downstream IGLJ gene has no corresponding associated IGLC gene. Comparison of individual BACs generated from the same individual revealed polymorphisms in IGLC2 and several IGLV genes, indicating that allelic variation in IGLV further expands the porcine antibody light chain repertoire.     

T cell receptor-gamma allele-specific selection of V gamma 1/V delta 4 cells in the intestinal epithelium

Previous genetic analyses have shown that the relative representation of subsets of gammadelta intestinal intraepithelial lymphocytes (i-IELs) is influenced by genes linked to the TCRgamma, TCRdelta, and MHC loci. Here, we have analyzed V-gene use in gammadelta i-IELs from C57BL/6 (B6) and C57BL/10 (B10) mice and from their F(1) and F(2) progenies with a larger panel of Vgamma- and Vdelta-specific mAbs and have shown that the influence of TCRgamma-linked genes operates at two levels: one influencing the representation of Vgamma1 (or Vgamma7) i-IELs and other acting specifically on the Vgamma1/Vdelta4 i-IEL subset, which represents 3% and 15% of the gammadelta i-IELs in B6 and B10 mice, respectively. Analysis of mice transgenic for a rearranged Vgamma1Jgamma4Cgamma4 chain of B6 origin demonstrated that the TCRgamma-linked genes influencing the representation of the Vgamma1/Vdelta4 i-IEL subset are the structural genes of TCRgamma chains. This influence is allele specific and cell autonomous, as evidenced by the different behavior of Vgamma1/Vdelta4 cells bearing either parental allele in F(1) mice. The representation of Vgamma1/Vdelta4 cells among gammadelta thymocytes is similar in B6 and B10 mice, demonstrating that the Vdelta4 chain can pair well with both alleles of the Vgamma1Jgamma4Cgamma4 chain and strongly suggesting that a cellular selection mechanism is responsible for the observed differences. The Vgamma1-Jgamma4 junctional amino acid sequences of B6 Vgamma1/Vdelta4 i-IELs are diverse but display less variation in length than those found in similar cells from B10 mice, indicating that B6 Vgamma1/Vdelta4 cells are the target of this cellular selection event.     

Essential contribution of germline-encoded lysine residues in Jgamma1.2 segment to the recognition of nonpeptide antigens by human gammadelta T cells.

Human gammadelta T cells display unique repertoires of Ag specificities largely imposed by selective usages of distinct Vgamma and Vdelta genes. Among them, Vgamma2/Vdelta2(+) T cells predominate in the circulation of healthy adults and respond to various microbial small molecular mass nonpeptide Ags. The present results indicate that the primary Vgamma2/Vdelta2(+) T cells stimulated with the distinct groups of nonpeptide Ags, including monoethyl pyrophosphate, isobutyl amine, and aminobisphosphonate, invariably exhibit Jgamma1.2 in the Vgamma2(+) TCR-gamma chains. Gene transfer studies revealed that most of the randomly cloned Vgamma2/Jgamma1.2(+) TCR-gamma genes bearing diverse Vgamma/Jgamma junctional sequences could confer the responsiveness to all these nonpeptide Ags, while none of the Vgamma2/Jgamma1.1(+) or Vgamma2/Jgamma1.3(+) TCR-gamma genes could do so. Furthermore, mutation of the lysine residues encoded by the Jgamma1.2 gene, which are unique in human Jgamma1.2 and absent in other human or mouse Jgamma segments, completely abrogated the responsiveness to all the nonpeptide Ags without affecting the response to anti-CD3 mAb. These results strongly suggested that the positively charged lysine residues in the TCR-gamma chain CDR3 region encoded by the germline Jgamma1.2 gene play a key role in the recognition of diverse small molecular mass nonpeptide Ags.     

Evidence that Xenopus laevis contains two different nonallelic insulin-like growth factor-I genes

Using the polymerase chain reaction (PCR), we have amplified and characterized partial nucleotide sequences of two distinct insulin-like growth factor-I genes (designated IGF-I' and IGF-I") from the amphibian, Xenopus laevis. The amplified fragments encoded much of the coding region of the mature peptide (exon III in mammalian IGF-I genes), and exhibited 93% similarity to each other, and 68-82% similarity to mammalian IGF-I amino acid sequences. Southern blot analysis using genomic DNA from a homozygous frog revealed that these two genes are nonallelic in a single organism, like the two nonallelic genes encoding Xenopus insulins that we have characterized previously. Furthermore, both IGF-I mRNAs are expressed in similar quantities in adult liver.     

Genomic<Emphasis Type="Italic"> Eco</Emphasis>RI polymorphism and cosmid sequencing reveal an insertion/deletion and a new<Emphasis Type="Italic"> IGLV5</Emphasis> allele in the human immunoglobulin lambda variable locus (22q11.2/IGLV)

The human immunoglobulin lambda locus ( IGL) is mapped at Chromosome 22q11.2, spanning about 1 Mb of DNA, and directs the synthesis of lambda-type immunoglobulin light chains. The positions of about 73-74 germline V-lambda genes, depending on the haplotypes, are known, with 29-33 of them being functional IGLV genes. These genes were divided into 11 subgroups ( IGLV1 to IGLV11) distributed into three gene clusters ( VA, VB, and VC). We constructed a high-resolution restriction map of a 37-kb cosmid clone (cosmid 8.3) harboring genes of the IGLV1, IGLV7, and IGLV5 families and the non-coding sequences IGLV(I)-42 and IGLV(VII)-41-1, located at cluster VB of the IGL locus. These IGLV genes were associated with unique EcoRI fragments detectable in Southern blots of genomic DNA. Population RFLP has revealed new IGLV alleles and haplotypes. We used the restriction map of cosmid 8.3 and the IMGT database as a reference for RFLP studies. EcoRI Southern blot hybridizations with subgroup-specific probes of the functional and open reading frame sequences present in cosmid 8.3 revealed different frequencies of IGLV gene fragments, as well as deletions of IGLV1-50 and IGLV5-39 genes and RFLP involving IGLV5-45 and IGLV5-48 genes. All members of the IGLV7 subgroup were monomorphic. Sequencing of the genes present in cosmid 8.3 revealed a new allelic variant of the IGLV5 subgroup. These data contribute to a better understanding of the contribution of the germline IGLV genes to the human genetic background and polymorphism.     

Structure and diversity of T-lymphocyte antigen receptors alpha and gamma in Xenopus

Short primer PCR directed at conserved regions of amino acid sequence within the T-cell antigen receptor (TCR) and immunoglobulin (Ig) light chain variable ( V) regions was used to amplify putative TCRgamma V region amplicons from stage 45 Xenopus laevis (African clawed frog) mRNA (cDNA). An adult Xenopus spleen cDNA library was screened using the Vgamma and a putative TCRValpha amplicon. Full copy length cDNAs containing the specific PCR-derived Valpha and Vgamma amplicons were recovered at relatively low frequency. Probes complementing the TCRalpha and TCRgamma constant ( C) regions were employed to isolate equivalent numbers of additional TCR alpha and TCR gamma cDNAs in an unbiased (non- V-based) manner. Few Vgamma genes appear to be expressed relative to the highly diverse expression of V alpha genes in equivalent numbers of cDNAs that were analyzed. Two TCRgamma C regions differ at only two positions; whereas two TCRalpha C regions differ at 33 coding positions as well as in their respective 3' untranslated regions, consistent with two independent loci. However, genomic Southern blots revealed considerably higher numbers of hybridizing bands when probed with C gamma than with C alpha. A potential novel mechanism of diversification is suggested by an unusual TCR alpha cDNA in which the V region can be translated in two frames through utilization of two closely linked V genes and an alternative splicing process. This process produces a translatable cDNA that is not readily predictable from the genomic locus utilizing normal recombination and splicing mechanisms.     

Structural and biochemical characterization of the lungfish (Lepidosiren paradoxa) liver basic fatty acid binding protein

Only one fatty acid-binding protein (FABP) from the liver of the lungfish (Lepidosiren paradoxa) was isolated and characterized. The sequence comparison of lungfish FABP with that of the known members of the liver FABP (L-FABP) and liver basic FABP (Lb-FABP) subfamilies indicates that it is more closely related to chicken, iguana, frog, axolotl, catfish, and shark Lb-FABPs than to mammalian and axolotl L-FABPs. Lungfish liver expression of this single Lb-FABP contrasts with the other fish studied so far which coexpress an Lb-FABP with heart-adipocyte and/or intestinal FABP types. The lungfish liver FABP expression pattern resembles that of tetrapods, which only expresses liver type FABPs. Lungfish Lb-FABP is one of the two FABPs reported to have a disulfide bridge. The molecular modeling of lungfish Lb-FABP predicts that nine of the conserved residues of Lb-FABPs are oriented toward the binding cavity, thus suggesting they are related to the protein binding characteristics.     

Structure and diversity of the T-cell receptor α chain in the Mexican axolotl

 Polymerase chain reaction was used to isolate cDNA clones encoding putative T-cell receptor (TCR) α chains in an amphibian, the Mexican axolotl (Ambystoma mexicanum). Five TCRα-V chain-encoding segments were identified, each belonging to a separate family. The best identity scores for these axolotl TCRα-V segments were all provided by sequences belonging to the human TCRα-V1 family and the mouse TCRα-V3 and TCRα-V8 families. A total of 14 different TCRA-J segments were identified from 44 TCRA-V/TCRA-J regions sequenced, suggesting that a large repertoire of TCRA-J segments is a characteristic of most vertebrates. The structure of the axolotl CDR3 α chain loop is in good agreement with that of mammals, including a majority of small hydrophobic residues at position 92 and of charged, hydrophilic, or polar residues at positions 93 and 94, which are highly variable and correspond to the TCRA-V/J junction. This suggests that some positions of the axolotl CDR3 α chain loop are positively selected during T-cell differentiation, particularly around residue 93 that could be selected for its ability to makes contacts with major histocompatibility complex-associated antigenic peptides, as in mammals. The axolotl Cα domain had the typical structure of mammalian and avian Cα domains, including the charged residues in the TM segment that are thought to interact with other proteins in the membrane, as well as most of the residues forming the conserved antigen receptor transmembrane motif. Received: 12 June 1996 / Revised: 11 September 1996     

Structure and diversity of the heavy chain VDJ junctions in the developing Mexican axolotl

 The immune capacity of young and adult axolotls (Ambystoma mexicanum) was evaluated by examining the combinatorial and junctional diversity of the VH chain. A large number of VDJ rearrangements isolated from 2.5-, 3.5-, 10-, and 24-month-old animals were sequenced. Six JH segments were identified with the canonical structure of all known vertebrate JHs, including the conserved Trp103-Gly104-X-Gly106 motif. Four core DH-like sequences were used by most (80%) of the VDJ junctions. These G-rich sequences had structures reminiscent of the TCRB DB sequences, and were equally used in their three reading frames. About 25% of the Igh, VDJ junctions from 3.5-month-old axolotls were out of frame, but most rearrangements were in frame at 10 and 24 months, suggesting that there is active selection of the productively rearranged Igh chains in the developing animals. There was no significant difference between the size of CDR3 in young (3.5 months) and subadult (10 months) axolotls (mean: 8.5 amino acids). However, the CDR3 loop was 1 amino acid longer in 2-year-old adult animals (mean: 9.5 residues). Several pairs of identical VDJ/CDR3 sequences were shared between 3.5-month-old individually analyzed axolotls, or between groups of axolotl of different ages. These identical rearrangements might be provided by the selection of some B-cell clones important for species survival, although the probability that different 3.5-month-old axolotl larvae would produce identical junctions seems very low, considering their limited number of B cells (less than 10⁵). The high frequency of tyrosine residues and the paucity of charged residues in the axolotl CDR3 loops may explain the polyreactivity of natural antibodies, and also clarify why it is so difficult to raise specific antibodies against soluble antigens. Received: 11 March 1997 / Revised: 1 May 1997     

Comparison of nonerythroid alpha-spectrin genes reveals strict homology among diverse species.

The spectrins are a family of widely distributed filamentous proteins. In association with actin, spectrins form a supporting and organizing scaffold for cell membranes. Using antibodies specific for human brain alpha-spectrin (alpha-fodrin), we have cloned a rat brain alpha-spectrin cDNA from an expression library. Several closely related human clones were also isolated by hybridization. Comparison of sequences of these and other overlapping nonerythroid and erythroid alpha-spectrin genes demonstrated that the nonerythroid genes are strictly conserved across species, while the mammalian erythroid genes have diverged rapidly. Peptide sequences deduced from these cDNAs revealed that the nonerythroid alpha-spectrin chain, like the erythroid spectrin, is composed of multiple 106-amino-acid repeating units, with the characteristic invariant tryptophan as well as other charged and hydrophobic residues in conserved locations. However, the carboxy-terminal sequence varies markedly from this internal repeat pattern and may represent a specialized functional site. The nonerythroid alpha-spectrin gene was mapped to human chromosome 9, in contrast to the erythroid alpha-spectrin gene, which has previously been assigned to a locus on chromosome 1.     

Expression of intermediate filament proteins during development of Xenopus laevis. I. cDNA clones encoding different forms of vimentin

To provide a basis for studies of the expression of genes encoding the diverse kinds of intermediate-filament (IF) proteins during embryogenesis of Xenopus laevis we have isolated and characterized IF protein cDNA clones. Here we report the identification of two types of Xenopus vimentin, Vim1 and Vim4, with their complete amino acid sequences as deduced from the cloned cDNAs, both of which are expressed during early embryogenesis. In addition, we have obtained two further vimentin cDNAs (Vim2 and 3) which are sequence variants of closely related Vim1. The high evolutionary conservation of the amino acid sequences (Vim1: 458 residues; Mr approximately 52,800; Vim4: 463 residues; Mr approximately 53,500) to avian and mammalian vimentin and, to a lesser degree, to desmin from the same and higher vertebrate species, is emphasized, including conserved oligopeptide motifs in their head domains. Using these cDNAs in RNA blot and ribonuclease protection assays of various embryonic stages, we observed a dramatic increase of vimentin RNA at stage 14, in agreement with immunocytochemical results obtained with antibody VIM-3B4. The significance of very weak mRNA signals detected in earlier stages is discussed in relation to negative immunocytochemical results obtained in these stages. The first appearance of vimentin has been localized to a distinct mesenchymal cell layer underlying the neural plate or tube, respectively. The results are discussed in relation to programs of de novo synthesis of other cytoskeletal proteins in amphibian and mammalian development.     

cDNA cloning, expression pattern, genomic structure and chromosomal location of RAB6KIFL, a human kinesin-like gene

Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal 35 kDa, plant 55 kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known examples from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.     

Comparison of mammalian, chicken and Xenopus brain-derived neurotrophic factor coding sequences

We have used the polymerase chain reaction (PCR) to amplify and clone coding sequences of the mature region of brain-derived neurotrophic factor (BDNF) from monkey, rat, chicken and Xenopus genomic DNA. Consistent with previous reports, the predicted amino acid sequences obtained in this manner from monkey and rat were identical to other mammalian BDNF sequences. The chicken and Xenopus BDNF sequences are also highly conserved, but contain 7 and 8 amino acid substitutions, respectively, compared to mammalian BDNF. Comparison of these sequences with the homologous NGF and NT3 coding regions provides further insight into amino acid residues that may be responsible for the different receptor specificities of these factors.     

Genes encoding Xenopus laevis Ig L chains. Implications for the evolution of kappa and lambda chains.

Xenopus laevis Ig contain two distinct types of L chains, designated rho or L1 and sigma or L2. We have analyzed Xenopus genomic DNA by Southern blotting with cDNA probes specific for L1 V and C regions. Many fragments hybridized to the V probe, but only one or two fragments hybridized to the C probe. Corresponding C, J, and V gene segments were identified on clones isolated from a genomic library prepared from the same DNA. One clone contains a C gene segment separated from a J gene segment by an intron of 3.4 kb. The J and C gene segments are nearly identical in sequence to cDNA clones analyzed previously. The C segment is somewhat more similar and the J segment considerably more similar in sequence to the corresponding segments of mammalian kappa chains than to those of mammalian lambda chains. Upstream of the J segment is a typical recombination signal sequence with a spacer of 23 bp, as in J kappa. A second clone from the library contains four V gene segments, separated by 2.1 to 3.6 kb. Two of these, V1 and V3, have the expected structural and regulatory features of V genes, and are very similar in sequence to each other and to mammalian V kappa. A third gene segment, V2, resembles V1 and V3 in its coding region and nearby 5'-flanking region, but diverges in sequence 5' to position -95 with loss of the octamer promoter element. The fourth V-like segment is similar to the others at the 3'-end, but upstream of codon 64 bears no resemblance in sequence to any Ig V region. All four V segments have typical recombination signal sequences with 12-bp spacers at their 3'-ends, as in V kappa. Taken together, the data suggest that Xenopus L1 L chain genes are members of the kappa gene family.     

Primary structure from amino acid and cDNA sequences of two Cu,Zn superoxide dismutase variants from Xenopus laevis

A mixture of two different amino acid sequences was discovered in Cu,Zn superoxide dismutase purified from the amphibian Xenopus laevis. No N-terminal post-translational modification was found. The high number of substitutions in the sequence suggested that protein heterogeneity was a product of gene duplication. This was confirmed by isolation of two different cDNA clones. Nucleotide sequence analysis allowed the primary structure of the two peptide chains to be unambiguously assigned. The observed changes (19 in 150 residues) are distributed along the peptide chain to give similar protein net charges although substitutions of the same polarity and/or charge were the exception rather than the rule. The degree of diversity between the two Xenopus variants is comparable to that between mammalian sequences and shows that the putative increase of the rate of mutation for Cu,Zn superoxide dismutase at later evolution stages (Y. M. Lee et al., 1985, Arch. Biochem. Biophys. 241, 577-589; G. J. Steffens et al., 1986, Biol. Chem. Hoppe-Seyler 367, 1017-1024) is observed in amphibians. This is the first time complete sequences for Cu,Zn superoxide dismutase variants from the same organism have been found to be products of divergent genes and not simply allelic mutations.     

Amino acid sequence microheterogeneities of basic (type II) cytokeratins of Xenopus laevis epidermis and evolutionary conservativity of helical and non-helical domains

Three clones coding for carboxy-terminal portions of type II cytokeratins have been isolated from a cDNA library constructed from the epidermis of the frog Xenopus laevis. These clones have been identified by hybridization-selection-translation and Northern blot analysis, and contain sequences complementary to mRNAs of similar size that code for three different polypeptides of the Mr 64,000 group, Ia-c, i.e. the only major type II cytokeratins expressed in this tissue. A comparison of the corresponding nucleotide sequences and the amino acid sequences deduced therefrom shows only minor differences in these polypeptides, most of which occur as isolated point mutations. This indicates that coding sequences of the different type II cytokeratin genes in epidermis of Xenopus are very similar, in contrast to the more extended differences of type II cytokeratin genes expressed in mammalian epidermis, which probably reflects a lower degree of evolutionary divergence of members of this protein family in amphibia. A comparison of the Xenopus sequences with those of mammalian type II cytokeratins reveals the same characteristic features, i.e. an alpha-helical domain ending with the familiar consensus sequence T Y R (X Y) L E G E, followed by a non-helical domain Cl enriched in hydroxyamino acids. Both domains are remarkably conserved in sequence between Xenopus and mammals. The following glycine-rich domain (C2) displays similar oligopeptide repeats (mostly of the type G G G M in the frog keratins), and the terminal C3 domain is characterized by a region exceptionally rich in hydroxyamino acids, which immediately precedes a cluster of basic amino acids at the carboxy terminus. Our results show that the typical features of the domain of type II cytokeratins are already established in amphibia and that these homologies are not restricted to the alpha-helical rod of these proteins but, in principle, extend to the other domains located in the so-called hypervariable tail portion. This suggests that the hypervariable regions are not subject to random variability but contain functionally important domains that have been well conserved during evolution.     

Human 18 S ribosomal RNA sequence inferred from DNA sequence. Variations in 18 S sequences and secondary modification patterns between vertebrates.

We have determined the DNA sequences encoding 18 S ribosomal RNA in man and in the frog, Xenopus borealis. We have also corrected the Xenopus laevis 18 S sequence: an A residue follows G-684 in the sequence. These and other available data provide a number of representative examples of variation in primary structure and secondary modification of 18 S ribosomal RNA between different groups of vertebrates. First, Xenopus laevis and Xenopus borealis 18 S ribosomal genes differ from each other by only two base substitutions, and we have found no evidence of intraspecies heterogeneity within the 18 S ribosomal DNA of Xenopus (in contrast to the Xenopus transcribed spacers). Second, the human 18 S sequence differs from that of Xenopus by approx. 6.5%. About 4% of the differences are single base changes; the remainder comprise insertions in the human sequence and other changes affecting several nucleotides. Most of these more extensive changes are clustered in a relatively short region between nucleotides 190 and 280 in the human sequence. Third, the human 18 S sequence differs from non-primate mammalian sequences by only about 1%. Fourth, nearly all of the 47 methyl groups in mammalian 18 S ribosomal RNA can be located in the sequence. The methyl group distribution corresponds closely to that in Xenopus, but there are several extra methyl groups in mammalian 18 S ribosomal RNA. Finally, minor revisions are made to the estimated numbers of pseudouridines in human and Xenopus 18 S ribosomal RNA.     

Evolutionary relationships of class II major-histocompatibility-complex genes in mammals

The major histocompatibility complex (MHC) class II molecule consists of noncovalently associated alpha and beta chains. In mammals studied so far, the class II MHC can be divided into a number of regions, each containing one or more alpha-chain genes (A genes) and beta-chain genes (B genes), and it has been known for some time that orthologous relationships exist between genes in corresponding regions from different mammalian species. A phylogenetic analysis of DNA sequences of class II A and B genes confirmed these relationships; but no such orthologous relationship was observed between the B genes of mammals and those of birds. Thus, the class II regions have diverged since the separation of birds and mammals (approximately 300 Mya) but before the radiation of the placental mammalian orders (60-80 Mya). Comparison of the phylogenetic trees for A and B genes revealed an unexpected characteristic of DP-region genes: DPB genes are most closely related to DQB genes, whereas DPA chain genes are most closely related to DRA-chain genes. Thus, the DP region seems to have originated through a recombinational event which brought together a DQB gene and a DRA gene (perhaps approximately 120 Mya). The 5' untranslated region of all class II genes includes sequences which are believed to be important in regulating class II gene expression but which are not conserved in known pseudogenes. These sequences are conserved to an extraordinary degree in the human DQB1 gene and its mouse homologue A beta 1, suggesting that regulation of expression of this locus may play a key role in expression of the entire class II MHC.