cDNA clones encoding mink immunoglobulin lambda chains.

Screening of a mink cDNA library with an antibody probe resulted in the isolation of clone pIGL-2 containing an Ig lambda chain coding sequence. The sequence comprised almost the entire V segment as well as J, C, and 3'-untranslated sequences. A second clone, pIGL-10, was isolated by rescreening the cDNA library with the use of pIGL-2 as a probe. pIGL-10 was found to contain a frameshift deletion of a single nucleotide in the C region. pIGL-2 and pIGL-10 were 81% homologous to each other in the FR3 of the V segment, and 95% of homology was found in their C regions. The J segments of the two clones differed in only one nucleotide position. Comparison of cloned lambda chain sequences with those of other mammals revealed that mink V lambda and C lambda genes have the highest homology with their human counterparts. The V lambda sequence of clone pIGL-2 appears to be a homologue of human subgroup III V lambda genes. Southern blot hybridization of mink DNA with the C lambda and V lambda probes derived from pIGL-2 revealed five or six hybridizing C lambda fragments and at least 11 hybridizing V lambda fragments. This suggested that the lambda genes in carnivores, like those in primates, have duplicated extensively during evolution.

Structure of mink immunoglobulin gamma chain cDNA.

Two cDNA clones, encoding mink Ig gamma chains were characterized. The pIGG47 clone contains a part of the leader segment, VDJ and C regions, and pIGG14 contains a part of the J and a complete C region. The clones differ by only four nucleotides in the C region, and they most probably represent allelic variants of the same gene. The V gene segment of pIGG47 was found to be highly similar to human VHIII subgroup sequences; there was 86-87% similarity for the whole V gene segment and 91% for the VHIII specific regions (codons 65-87). Southern blot analysis demonstrated that a high proportion of mink VH genes is VHIII related. The V gene segment used as a probe revealed 19-23 bands in mink DNA under stringent conditions. This is in agreement with our previous data showing that a high proportion of mink Ig contains an 'alternative' binding site for protein A, a feature common to VHIII-related molecules. According to Southern blot analysis there may be 5-7 C gamma genes at the mink IgH locus.

Cloning of a CXCR4 homolog in chondrostean fish and characterization of the CXCR4-specific structural features.

The chemokine receptor CXCR4 and its ligand SDF-1 are essential components of hematopoiesis, organogenesis and immunomodulation in mammalian species. We cloned a cDNA encoding CXCR4 homolog of sterlet (Acipenser ruthenus), a representative of chondrostean fishes. The deduced amino acid sequence of sterlet CXCR4 is almost equally distant from mammalian and teleost CXCR4 (66-68% identical residues). Percent identity with the other chemokine receptors varies in the 30-35% range. The CXCR4 sequences from the three phylogenetically diverged lineages were compared with the sequences of the other chemokine receptors to determine the CXCR4-specific structural elements that were conserved during vertebrate evolution. The characteristic residues and/or motifs are located predominantly in the intracellular and extracellular regions and in the third, fourth and fifth transmembrane domains. The data presented may be helpful for structure-function analysis of the CXCR4 ligand binding and signal transduction.

Cloning and characterization of the human FCRL2 gene.

We report cloning and characterization of FCRL2, a novel human gene that belongs to the FcR family. The gene is closely linked and structurally similar to the recently identified FCRL/FREB/FcRX gene. The encoded protein is composed of three Ig-like domains and a C-terminal mucin-like domain containing a conserved alpha-helical motif with dileucine signals. Intraexonic splicing may generate two alternative transcripts, coding for isoforms with the third and fourth domains replaced by entirely different amino acid sequences. Like FCRL, the full-length isoform of FCRL2 is expressed intracellularly in transfected 293T cells. Expression analysis revealed FCRL2 mRNA only in placenta. The gene transcripts were not detected in lymphoid tissues or in the main leukocyte subsets isolated from peripheral blood. However, we found that FCRL2 is differentially expressed by transformed B cell lines. Of interest is also the finding that the gene expression may be up-regulated in the progression of melanocytic tumors.

Identification of an IL-8 homolog in lamprey (Lampetra fluviatilis): early evolutionary divergence of chemokines.

Subtractive hybridization was used to study river lamprey (Lampetra fluviatilis) leukocyte-specific cDNA. A clone representing the most abundant component (12%) of the leukocyte library subtracted with liver cDNA was isolated and characterized. The cDNA encodes a presumably secreted polypeptide of 101 residues. The 3' untranslated region of the cDNA contains motifs characteristic of the transiently expressing genes. Comparison of the deduced amino acid sequence with known protein sequences revealed its homology to the members of the chemokine superfamily. Designated as LFCA-1, the lamprey protein contains four conserved cysteines, of which the first two are separated by a residue, and a number of other CXC family characteristic residues. LFCA-1 has the highest similarity to the chicken EMF-1 (40%) and to the mammalian IL-8 (32-33%). However, it lacks the ELR motif essential for the function of the mammalian IL-8-related chemokines. Based on the phylogenetic analysis of the LFCA-1 relationship to the higher vertebrate chemokines, it is concluded that the evolutionary origin of the chemokine superfamily is ancient, and that the divergence of the CXC and CC families most likely occurred at the time or before the first vertebrates emerged.

CD3epsilon homologues in the chondrostean fish Acipenser ruthenus.

CD3epsilon is an essential component of the T-cell receptor (TCR) complex for antigen. We report here molecular cloning and characterization of cDNAs encoding the CD3epsilon homologues in sterlet (Acipenser ruthenus), a representative of primitive chondrostean fishes. Sequence analysis of the cDNA clones demonstrated unexpectedly high CD3epsilon gene heterogeneity in this species. While some cDNAs encoded proteins with the structure typical of mammalian CD3epsilon, others coded for proteins lacking the membrane-proximal half of the extracellular domain. Two cDNAs contained in-frame stop codons in the region encoding the cytoplasmic domain. Based on genomic blot analysis and RT-PCR typing of individual spleen RNAs, we suggest that sterlet may possess two highly polymorphic CD3epsilon loci, of which one can produce alternatively spliced transcripts. The structural elements shown to be functionally important in the mammalian CD3epsilon are strongly conserved in the sterlet CD3epsilon. The cytoplasmic region contains an immunoreceptor tyrosine-based activation motif (ITAM) with YEPI and YSGL tyrosine-containing sequences that are characteristic of only this TCR subunit. The pattern of sequence conservation indicates also that strong selection pressure was imposed on a motif VYYW at the C-end of the transmembrane domain and on a CD3epsilon-specific proline-rich motif RXPPVP juxtaposed to the N-terminus of the ITAM. Weak similarity of the sterlet CD3epsilon with the chicken and Xenopus CD3gamma/delta indicates that these two TCR subunits diverged before radiation of bony fishes and tetrapods. While the role of CD3epsilon heterogeneity in sterlet remains to be elucidated, the data obtained show that the basic mechanisms of TCR signaling have ancient evolutionary origin.

The mink gene for the lambda light immunoglobulin chain: characterization of cDNA and chromosomal localization.

A cDNA library from mink spleen was constructed by use of the phage lambda gt11. The library was screened using polyvalent serum raised against the mink immunoglobulin lambda chain. As a result, several clones expressing mink immunoglobulin lambda light chains were identified. Sequencing of one of the clones with an 803 bp insert was performed. The insert comprised nearly the entire coding region for the mature lambda light immunoglobulin gene with the exception of the leader polypeptide and several amino acids of the FR1 region of the V segment. Compared with the rabbit, mouse and human lambda light immunoglobulin genes, the homology of the cloned sequence was found to be highest relative to the rabbit gene. With the cloned mink cDNA containing the C-region only as a probe, the DNAs from mink-Chinese hamster hybrid clones were studied. The results of segregation analysis of this mink cDNA sequence and mink chromosomes in the mink-Chinese hamster clone panel allowed us to assign the gene for the lambda light immunoglobulin constant polypeptide (IGLC) to mink Chromosome (Chr) 4.