Scott J. Strong

α, β, γ, and δ T Cell Antigen Receptor Genes Arose Early in Vertebrate Phylogeny

Abstract A series of products were amplified using a PCR strategy based on short minimally degenerate primers and R. eglanteria (clearnose skate) spleen cDNA as template. These products were used as probes to select corresponding cDNAs from a spleen cDNA library. The cDNA sequences exhibit significant identity with prototypic α, β, γ, and δ T cell antigen receptor (TCR) genes. Characterization of cDNAs reveals extensive variable region diversity, putative diversity segments, and varying degrees of junctional diversification. This demonstrates expression of both α/β and γ/δ TCR genes at an early level of vertebrate phylogeny and indicates that the three major known classes of rearranging antigen receptors were present in the common ancestor of the present-day jawed vertebrates.

Extreme variation in X-linked agammaglobulinemia phenotype in a three-generation family

BACKGROUND X-linked agammaglobulinemia is typically a severe life-threatening disease characterized by the failure of B-cell differentiation and antibody production, which manifests in infancy and early childhood. Recently, we reported a novel mutation (Cys145-->STOP) in Brutons tyrosine kinase in a 51-year-old man who was referred for evaluation because of chronic nasal congestion, recurrent sinusitis, sporadic pneumonia, and a family history suggestive of an X-linked immunodeficiency disease. He had not been treated with gammaglobulin. OBJECTIVE This study was performed to investigate the clinical and immunologic phenotypes of this patients other affected male family members. METHODS A detailed family history and comprehensive review of medical records was carried out. Genetic mutation analysis of the gene encoding Brutons tyrosine kinase was carried out in the probands brother and nephew. RESULTS Clinically affected male family members exhibit marked phenotypic variation with manifestations ranging from extremely mild to severe recurrent infections. Immunologic evaluation revealed extreme variation in immunoglobulin levels, B-cell numbers, and functional antibody titers. Genetic analysis documented a novel mutation in the gene encoding Brutons tyrosine kinase in the proband, his brother, and his nephew. CONCLUSIONS Despite their sharing the same genetic abnormality, extreme variation was noted in the immunologic findings and phenotypic expression of affected family members. This family study is extraordinary in that clinically affected male members who did not receive aggressive medical treatment died of the disease in childhood or survived into late adulthood.

Distinct patterns of IgH structure and organization in a divergent lineage of chrondrichthyan fishes

Abstract Immunoglobulin heavy chain (IgH) genes in representative chondrichthyan fishes (sharks and skates) consist of independently functioning clusters, containing separate variable (VH), diversity (DH), and joining (JH) region elements and constant (CH) region exons. IgH loci have been characterized in Hydrolagus colliei (spotted ratfish), a modern representative of a major independent chondrichthyan lineage. Three distinct families of IgH gene clusters were identified. The most numerous genes consist of unjoined VH-D1-D2-JH segments that correspond to the most abundant Hydrolagus spleen (cDNA) transcripts which apparently arise from a diversified gene family. In the second cluster type, VH, DH, and JH segments are germline-joined, whereas the CH exons exhibit typical organization. This gene type is found in only a few copies per haploid genome and both transmembrane and secretory transcripts have been identified. A third cluster type has been identified that consists of unjoined VH elements but lacks a typical CH1 exon, which is substituted with a second CH2-like exon. Transcripts from this third cluster type also appear to derive from a diversified gene family. Genomic D regions of the two unjoined clone types exhibit structural differences that are consistent with incorporation of recombination machinery-mediated events. Genomic library screening indicates that 90% of VH+ clones are truncated, nearly identical pseudogenes (lacking JH and CH). These studies demonstrate an early phylogenetic origin for the cluster type of gene organization and document extensive organizational diversification within an apparent single class of IgH genes.

A long form of the skate IgX gene exhibits a striking resemblance to the new shark IgW and IgNARC genes

Abstract Differential screening has been used to identify cDNAs encoding a long form of IgX in Raja eglanteria (clearnose skate). Comparisons of the IgX long form with the previously described short-form IgX cDNAs and the genomic IgX locus indicate that the V and two 5′C regions of the short and long forms of IgX are >90% identical at the nucleotide level. Differences between the V sequences of the long- and short-form IgX genes are concentrated in complementarity determining regions, suggesting that these forms are derived through alternative splicing of the same genomic loci or transcription of highly related loci. The extreme conservation of nucleotide sequence, including third position codons, among different cDNAs as well as the near identity of nucleotide sequence in the intervening sequences of germline IgX, IgX short-form sterile transcripts and IgX long-form sterile transcripts indicate that the multiple IgX loci are recently diverged from one another and/or are under intense gene correction. Phylogenetic analyses of the known cartilaginous fish immunoglobulin loci demonstrate that the long form of IgX is orthologous to IgW/IgNARC (NARC) and is most consistent with: 1) the divergence of the IgX/IgW/NARC and IgM-like loci from a common ancestral locus prior to the divergence of the cartilaginous/bony fish lineages and 2) the divergence of the NAR locus from the IgX/IgW/NARC gene(s) after the cartilaginous/bony fish split but prior to the shark/skate split, approximately 220 million years ago.

Identification and characterization of a homologue of Bruton's tyrosine kinase, a Tec kinase involved in B-cell development, in a modern representative of a phylogenetically ancient vertebrate.

As the most phylogenetically distant (from mammals) of the jawed vertebrates, the cartilaginous fish are of considerable importance in terms of our understanding of the immune system evolution. Antigen binding receptors associated with both B(Litman et al. 1985) and T (Rast and Litman 1994; Rast et al. 1995) -cell lineages in higher vertebrates have been identified in these species. Although immunoglobulin (Ig) genes in cartilaginous fish are restricted in terms of diversity relative to mammalian prototypes (Hinds-Frey et al. 1993), T-cell antigen receptors may more closely resemble forms found in higher vertebrates (Rast et al. 1997). Furthermore, the unusual organization of Ig and Ig-like genes in cartilaginous fish suggest that B-cell development is not associated with isotype switching such as that seen in mammals (Hinds and Litman 1986). Despite the fairly extensive understanding of antigen receptor gene loci in cartilaginous fish, relatively little is known with regard to development of lymphocytes in these species, which lack an anatomical equivalent of bone marrow and possess lymphoid tissues not found in higher vertebrates (Luer et al. 1995). In terms of the genes involved in Blymphocyte development in higher vertebrates, Bruton 9s tyrosine kinase ( Btk) is the best understood (Satterthwaite and Witte 1996). Identification of homologues of such genes would represent an important first step in characterizing the mechanism(s) of B-cell development in lower vertebrate species. We herein describe the structure of a homologue ofBtk that has been identified in a representative cartilaginous fish,Raja eglanteria(clearnose skate). R. eglanteriawere obtained from local sources. Standard procedures were employed for isolation of genomic DNA, RNA, and mRNA. Cloning of polymerase chain reaction (PCR) products, cDNA library screening, DNA sequencing, and DNA sequence analyses were as described (Rast et al. 1997). PCR primers complementing Tec family kinases were based on the conserved regions of exon 17 of humanBtk, and the corresponding exons of Txk and Tec (Ohta et al. 1994, 1996) as well as the equivalent sequences inferred fromEmt (Gibson et al. 1993) and BMX (Tamagnone et al. 1994) cDNAs. The structures of the primers and with cloning sites underlined are: forward ( Pst I), 59-TTCTGCAGCTGGATGAT(CG)A(AG)TA-39; reverse ( Hin dIII), 59-CCAAGCTTTC(AGT)GA(CT)TT(AG)CTGCT-39. PCR amplification (1 min, 94°C; 1 min, 53°C; and 1 min, 72°C) utilizing Raja genomic DNA and the Tec family-specific primers yielded a ~120 base pair (bp) product which was digested withPstI andHin dIII and cloned into M13 mp18/ 19. The DNA sequence was found to consist of a continuous open reading frame with sequence identity to mammalian Tec kinases. The 120 bp product was used as a probe to screen a Raja spleen cDNA library (2×105 pfu) and 12 positively hybridizing clones were identified. All 12 clones were partially sequenced and appear to derive from the same gene, referred to as skate PTK (protein tyrosine kinase). Human DNA was amplified as a control utilizing the same primers and gene segments corresponding to four Tec family kinases ( Tec, Emt, Btk, andTxk) were identified. The sequence of skate PTK run against GenBank using FASTDB (Brutlag et al. 1990; IntelliGenetics, Inc., Mountain View, CA) indicated the greatest sequence identity with human (Vetrie et al. 1993; Tsukada et al. 1993) and mouse (Sideras et al. 1994) Btk and somewhat less identity to BMX. The overall identity of functionally equivalent amino acids of skate PTK to human Btk is 82% (Fig. 1), not including the three short sequence gaps discussed below, within the identity range of any two mammalian Tec family kinases to each other. The overall length of the skate cDNA corresponds to 627 amino acid residues as compared with 659 in mammalian Btk. This difference can be largely accounted for by an absence of the DNA segment corresponding to residues 81–101 in the pleckstrin similarity domain and encoded for The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U85659

Tec-family non-receptor tyrosine kinase expressed in zebrafish kidney

Tec-family tyrosine kinases comprise a family of cytoplasmic kinases that are related to, but are structurally distinct from the Src family of kinases. The Tec-family genes: Btk, Itk, Tec, Txk, and Bmx (Desiderio and Siliciano 1994; Rawlings and Witte 1995) are expressed primarily in hematopoietic lineages. Tec-family kinases contain a pleckstrin homology, a tec homology, SH3, SH2, and kinase domains; only Txk lacks the amino terminal pleckstrin homology domain (Haire et al. 1994). The genomic structures of several Tec-family kinases have been resolved (Ohta et al. 1994, 1996). Btk, the most extensively studied Tec-family kinase, is the affected gene in X-linked agammaglobulinemia in human and Xid in mouse. This report describes a new Tec-family non-receptor tyrosine kinase that has been isolated from a zebrafish kidney cDNA library. Primers based on a kinase region exon of mammalian Tec-family kinases, corresponding to exon 15 in Btk, were used to amplify zebrafish genomic DNA. The structures of the primers are: 59-TTCTGCAGGTGCA(GA)(TC)T(TG)TATGG-39 (Pst I-linkered) and 59-CCAAGCTTACCA(AG)(AG)TC(TC)C-39 (Hin dIII linkered). As predicted, the polymerase chain reaction generated a 210 base pair product that was used as a probe to screen a zebrafish kidney cDNA library cloned in ZAP Express (Stratagene, La Jolla, Calif.). Twenty-four hybridizing plaques were isolated and eight were sequenced on a LICOR 4000L

Genetic analysis of theexed region in mouse

Summary: The extraembryonic ectoderm development (exed) mutant phenotype was described in mice homozygous for the c6H deletion, a radiation‐induced deletion in the tyrosinase region of mouse Chromosome 7. These mutants fail to gastrulate and die around embryonic day 8.0. Several genes including, for example, embryonic ectoderm development (eed), are deleted in the c6H mutants; however, the portion of the chromosome responsible for the more severe exed phenotype is localized to a 20‐kb region called the “exed‐critical region.” To understand the genetics behind the exed phenotype, we analyzed this region in two ways. First, to determine whether the 20‐kb exed‐critical region alone causes the mutant phenotype, we removed it from a wild‐type chromosome. The resulting mice homozygous for this deletion were viable and fertile, indicating that the 20‐kb exed‐critical region by itself is not sufficient to cause the phenotype when deleted. We then sequenced the 20‐kb exed‐critical region and no expressed exons were found. Several short matches to GenBank Expressed Sequence Tag (EST) databases were identified; however, none of these ESTs mapped to the region. Taken together, these results indicate that the exed phenotype may either be a position effect on a distal gene caused by the c6H breakpoint or the result of composite effects of nullizygosity of multiple genes in the deletion homozygotes. genesis 27:174–179, 2000.