Phylogenetic origins and adaptive evolution of avian and mammalian haemoglobin genes

Recent years have seen rapid growth in amino acid sequence data on globins and nucleotide sequence data on haemoglobin genes and pseudogenes, and cladistic analysis of these data continues to reveal new facets of globin evolution. Our present findings demonstrate: (1) avian and mammalian embryonic alpha genes (pi and xi, respectively) had a monophyletic origin involving an alpha locus duplication about 400 Myr ago soon after the duplication which separated alpha and beta genes; (2) much later in phylogeny, independent beta-gene duplications produced the embryonic rho locus of birds and embryonic epsilon and fetal gamma loci of mammals. This parallels the earlier finding that myoglobins evolved more than once from generalized globin ancestors. Here we support the view that such globin evolution resulted from natural selection acting on mutations in duplicated genes. Thus, our evidence contradicts the neutralist view in which almost all amino acid substitutions in descent to extant globins evaded positive selection.     

Genomic organization of the genes encoding mouse T-cell receptor alpha-chain

The vertebrate immune system uses two kinds of antigen-specific receptors, the immunoglobulin molecules of B cells and the antigen receptors of T cells. T-cell receptors are formed by a combination of two different polypeptide chains, alpha and beta (refs 1-3). Three related gene families are expressed in T cells, those encoding the T-cell receptor, alpha and beta, and a third, gamma (refs 4-6), whose function is unknown. Each of these polypeptide chains can be divided into variable (V) and constant (C) regions. The V beta regions are encoded by V beta, diversity (D beta) and joining (J beta) gene segments that rearrange in the differentiating T cell to generate V beta genes. The V gamma regions are encoded by V gamma, J gamma and, possibly, D gamma gene segments. Studies of alpha complementary DNA clones suggest that alpha-polypeptides have V alpha and C alpha regions and are encoded by V alpha and J alpha gene segments and a C alpha gene. Elsewhere in this issue we demonstrate that 18 of 19 J alpha sequences examined are distinct, indicating that the J alpha gene segment repertoire is much larger than those of the immunoglobulin (4-5) or beta (14) gene families. Here we report the germline structures of one V alpha and six J alpha mouse gene segments and demonstrate that the structures of the V alpha and J alpha gene segments and the alpha-recognition sequences for DNA rearrangement are similar to those of their immunoglobulin and beta-chain counterparts. We also show that the J alpha gene-segment organization is strikingly different from that of the other immunoglobulin and rearranging T-cell gene families. Eighteen J alpha gene segments map over 60 kilobases (kb) of DNA 5' to the C alpha gene.     

Human gamma-globin genes silenced independently of other genes in the beta-globin locus.

Erythropoiesis during human development is characterized by switches in expression of beta-like globin genes during the transition from the embryonic through fetal to adult stages. Activation and high-level expression of the genes is directed by the locus control region (LCR), located 5' to the epsilon gene. The location of the LCR and its role in directing high-level expression of the globin genes has led to the suggestion that competition from the beta gene for interaction with the LCR has a major role in silencing the fetal gamma genes during adult life. We have now constructed lines of transgenic mice containing the human A gamma globin gene linked to the LCR. We observe high-level expression of the transgene in the embryonic stages but silencing of the gene in adult animals. We conclude that the gamma gene is not deregulated by the presence of the LCR and that competition from the beta gene is not required for silencing of the gamma genes in adult life. The silencing is therefore likely to be mediated by stage-specific factors binding to sequences immediately flanking the genes.     

Rearrangement of two distinct T-cell gamma-chain variable-region genes in human DNA

Selective cloning procedures for T-cell-specific complementary DNAs have revealed the existence of a gene designated gamma as well as the main antigen receptor alpha- and beta-chain genes. The gamma-chain genes undergo rearrangement during T-cell differentiation but the patterns and complexity of such rearrangements differ markedly in mouse and human. In mouse, a panel of cytotoxic T-lymphocyte clones exhibit the same rearrangement pattern with a gamma-chain gene probe and a set of three gamma-chain variable (V) genes have been identified in the DNA. Clonal diversity in mouse seems to be confined to V-J (joining) regions. In contrast, human T-cell lines exhibit diverse rearrangements suggestive of a family of differing V gamma genes variously rearranging to the two gamma-chain constant (C) region genes. Here we report the cloning of two very different V gamma genes rearranged to J segments upstream of the two human C gamma genes. Both V gamma genes are rearranged productively but nucleotide sequence comparison shows that they possess very little homology with each other. This shows that human T-cell V gamma genes exist which differ significantly from each other at the nucleotide level and that such diverse genes can be usefully rearranged in different T cells.     

T-cell antigen receptor genes and T-cell recognition

The four distinct T-cell antigen receptor polypeptides (alpha, beta, gamma, delta) form two different heterodimers (alpha:beta and gamma:delta) that are very similar to immunoglobulins in primary sequence, gene organization and modes of rearrangement. Whereas antibodies have both soluble and membrane forms that can bind to antigens alone, T-cell receptors exist only on cell surfaces and recognize antigen fragments only when they are embedded in major histocompatibility complex (MHC) molecules. Patterns of diversity in T-cell receptor genes together with structural features of immunoglobulin and MHC molecules suggest a model for how this recognition might occur. This view of T-cell recognition has implications for how the receptors might be selected in the thymus and how they (and immunoglobulins) may have arisen during evolution.     

Deletion of immunoglobulin heavy chain genes from expressed allelic chromosome

We have studied the organization of immunoglobulin heavy-chain genes in a gamma 2b-chain (BALB/c allotype)-producing myeloma BKC F1 # 15 induced in a F1 mouse between C57BL and BALB/c. Southern blot hybridization studies using cloned mu, gamma 1 and gamma 2b-chain genes as probes demonstrate that the mu- and gamma 1-chain genes of the expressed chromosome are deleted while these genes of the unexpressed chromosome are retained. The gamma 2b-chain gene of the expressed allele is rearranged while that gene of the unexpressed allele seems unchanged, as do the gamma 2a-chain genes. These results support the allelic deletion mechanism in heavy-chain class switch and the order of H chain genes.     

Diversity of murine gamma genes and expression in fetal and adult T lymphocytes

The search for the genes encoding the T-cell receptor alpha and chains revealed a third gene, T gamma (ref. 1), which shares with t T alpha (refs 2-7) and T beta (refs 8-15) genes a number of structure features, including somatic rearrangement during T-cell development. T gamma gene expression appears to be unnecessary in son mature T cells and is at its greatest in fetal thymocytes encouraging speculation that T gamma has a role in T-cell development and may be involved in the recognition of polymorphic major histocompatibility complex (MHC) products during thymic education. One argument against the participation of T gamma in such a process has been its apparently limited diversity, due to the small number of gene segments available for rearrangement. We here describe the identification of additional T gamma V-gene segments and demonstrate that they can be rearranged to previously identified J- and C-gene segments and are expressed in fetal thymocytes. In addition we describe a variety of patterns of T gamma mRNA processing which may be significant for T gamma gene regulation.     

T gamma protein is expressed on murine fetal thymocytes as a disulphide-linked heterodimer

During the search for genes coding for the mouse alpha and beta subunits of the antigen-specific receptor of mouse T cells we encountered a third gene, subsequently designated gamma. This gene has many properties in common with the alpha and beta genes, somatic assembly from gene segments that resemble the gene segments for immunoglobulin variable (V), joining (J) and constant (C) regions; rearrangement and expression in T cells and not in B cells; low but distinct sequence homology to immunoglobulin V, J and C regions; other sequences that are reminiscent of the transmembrane and intracytoplasmic regions of integral membrane proteins; and a cysteine residue at the position expected for a disulphide bond linking two subunits of a dimeric membrane protein. Despite these similarities the gamma gene also shows some interesting unique features. These include a relatively limited repertoire of the germ-line gene segments, more pronounced expression at the RNA level in immature T cells such as fetal thymocytes and an apparent absence of in-frame RNA in some functional, alpha beta heterodimer-bearing T cells or cultured T clones and hybridomas. To understand the function of the putative gamma protein it is essential to define the cell population that expresses this protein. To this end we produced a fusion protein composed of Escherichia coli beta-galactosidase and the gamma-chain (hereafter referred to a beta-gal-gamma) using the phage expression vector lambda gt11 and raised rabbit antisera against the gamma determinants. Using the purified anti-gamma antibody we detected a polypeptide chain of relative molecular mass 35,000 (Mr 35K) on the surface of 16-day old fetal thymocytes. The gamma-chain is linked by a disulphide bridge to another component of 45K. No such heterodimer was detected on the surface of a cytotoxic T lymphocyte (CTL) clone 2C from which an in-phase gamma cDNA clone was originally isolated.     

Expression of the T-cell-specific gamma gene is unnecessary in T cells recognizing class II MHC determinants

Subtractive complementary DNA cloning combined with partial protein sequencing has allowed identification of the genes encoding the alpha and beta subunits of T-cell receptors. The subtractive cDNA library prepared from the cytotoxic T lymphocyte (Tc) clone 2C has been found to contain a third type of clone encoding the gamma chain. The gamma gene shares several features with the alpha and beta genes: (1) assembly from gene segments resembling immunoglobulin V, J and C (respectively variable, joining and constant region) DNA segments; (2) rearrangement and expression in T cells and not in B cells; (3) sequences reminiscent of transmembrane and intracytoplasmic regions of integral membrane proteins; (4) a cysteine residue at the position expected for an interchain disulphide bond. The alpha and beta genes are expressed at equivalent levels in both Tc cells and helper T cells (TH). The gamma gene, obtained from 2C, has been found to be expressed in all Tc cells studied. Here we present evidence that strongly suggests that TH cells do not require gamma gene expression.     

Abnormal tyrosine phosphorylation on T-cell receptor in lymphoproliferative disorders

The study of human autoimmune diseases has benefited greatly from analysis of animal models. Mice that are homozygous for either the lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) mutant genes develop a disease characterized by massive lymphadenopathy and autoantibody formation. With age, the lymphoid organs in these mice are replaced with a greatly expanded population of abnormal lymphocytes. Recent work has shown that these cells are likely to be in the T-cell lineage. They rearrange and transcribe the genes for the alpha and beta subunits of the T-cell receptor (TCR) and a third, T-cell receptor-like gene, T gamma. As determined by immunofluorescence with anti-receptor antibodies the cells also express TCR on the cell surface. The murine T-cell receptor consists of the alpha and beta chains, derived from the rearranged alpha and beta genes, in non-covalent association with seven other chains; the delta chain, of relative molecular mass (Mr) 26,000 (26K), the epsilon chain (25K), a glycosylated 21K chain (gp21) which is probably the homologue of the gamma chain of T3 (CD3), a 16K homodimer (zeta) and a 21K dimer (p21). This multichain complex is thought to be the murine analogue of the human T3 complex. After activation of normal T cells by antigen or lectin, p21 is phosphorylated on tyrosine residues and gp21 is phosphorylated on serine residues. In contrast, in the gld and lpr cells, p21 is phosphorylated even in the absence of antigen or lectin, whereas gp21 is not phosphorylated.     

Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases.

The high-affinity IgE receptor (Fc epsilon RI), which is expressed on the surface of mast cells and basophils, has a central role in immediate allergic responses. In the rat basophilic leukaemia cell line RBL-2H3, which is a model system for the analysis of Fc epsilon RI-mediated signal transduction, surface engagement of Fc epsilon RI induces histamine release and the tyrosine phosphorylation of several distinct proteins. Although the alpha, beta, and gamma subunits of Fc epsilon RI lack intrinsic tyrosine protein kinase (TPK) activity, a kinase that copurifies with Fc epsilon RI phosphorylates the beta and gamma subunits of the receptor on tyrosine residues. We report here that in RBL-2H3 cells, p56lyn and pp60c-src are activated after Fc epsilon RI crosslinking, and p56lyn coimmunoprecipitates with Fc epsilon RI. In the mouse mast-cell line PT-18, another cell type used to study FC epsilon RI-mediated signalling, tyrosine phosphorylation of proteins is also an immediate consequence of receptor crosslinking. Notably, the only detectable src protein-related TPK in PT-18 cells is p62c-yes, and it is this TPK that is activated on Fc epsilon RI engagement and coimmunoprecipitates with the receptor. Therefore, it seems that different src protein-related TPKs can associate with the same receptor and become activated after receptor engagement.     

Haemoglobin switching in human embryos: asynchrony of zeta----alpha and epsilon----gamma-globin switches in primitive and definite erythropoietic lineage

Haemoglobin switching in humans provides a unique model for investigating the mechanisms underlying expression of a developmentally regulated gene family. Numerous studies have focused on the switch from fetal to adult (that is, gamma----beta) globin, but little is known about the embryonic----fetal (that is, zeta----alpha and epsilon----gamma) switches, as well as the transition from 'primitive' yolk sac to 'definitive' liver erythropoiesis. Here we have studied the embryonic----fetal haemoglobin switches in yolk sac, liver and circulating blood erythroblasts from 25 embryos and 6 fetuses. Globin synthesis was also evaluated in purified 'primitive' and 'definitive' erythroblasts. Primitive erythroblasts synthesize essentially zeta and epsilon chains at 5 weeks and alpha- and epsilon-globin with a minor aliquot of zeta and gamma chains at 6-7 weeks, whereas definitive erythroblasts produce alpha and epsilon + gamma + beta-globin at 6 weeks but only alpha and gamma + beta chains from 8 weeks onward. In both lineages the zeta----alpha and the epsilon----gamma switches are asynchronous, the former preceding the latter. Furthermore, zeta- and beta-globin synthesis is restricted to primitive and definitive erythroblasts respectively. These findings are discussed in terms of a monoclonal model for haemoglobin switching in early human ontogeny.     

Unusual organization and diversity of T-cell receptor alpha-chain genes

T lymphocytes recognize cell-bound antigens in the molecular context of the self major histocompatibility complex (MHC) gene products through the surface T-cell receptor(s). The minimal component of the T-cell receptor is a heterodimer composed of alpha and beta subunits, each of relative molecular mass (Mr) approximately 45,000 (refs 1-3). Recently, complementary DNA clones encoding these subunits have been isolated and characterized along with that of a third subunit of unknown function, termed gamma (refs 4-9). These studies revealed a primary structure for each subunit that was clearly similar to that of immunoglobulin and indicated a somatic rearrangement of corresponding genes that are also immunoglobulin-like. Recently, the analysis of the sequence organization of the T-cell receptor beta-chain and T-cell-specific gamma-chain gene families has been reported. We now present an initial characterization of the murine T-cell receptor alpha-chain gene family, and conclude that although it is clearly related to the gene families encoding immunoglobulins, T-cell receptor beta-chains and also T-cell gamma-chains, it shows unique characteristics. There is only a single constant (C) region gene segment, which is an exceptionally large distance (approximately 20-40 kilobases (kb) in the cases studied here) from joining (J) gene segments. In addition, the J cluster and the variable (V) segment number seen to be very large. Finally, in the case studied here, a complete alpha-chain gene shows no somatic mutation and can be assembled directly from V alpha, J alpha and C alpha segments without inclusion of diversity (D alpha) segments.     

Complete structure and expression in transfected cells of high affinity IgE receptor

The high-affinity receptor for immunoglobulin E, Fc epsilon RI, is found exclusively on mast cells and basophils. When multivalent allergens bind to the receptor-bound IgE, the consequent aggregation of the receptors leads to the release of mediators responsible for allergic symptoms. In rodents Fc epsilon RI is a tetrameric complex of non-covalently attached subunits: one IgE-binding alpha subunit, one beta subunit and a dimer of disulphide-linked gamma subunits. Complementary DNA encoding the alpha and the beta subunits has recently been isolated, but expression of IgE-binding by transfected cells has not yet been achieved. Here we report the cloning of cDNA for the gamma subunit, and propose a model for the alpha beta gamma 2 tetramer which accounts for many of the structural features of the receptor. The rodent receptor on the surface of COS 7 cells was expressed only when the cDNAs for all three subunits were cotransfected. Successful expression of human IgE receptors should now be possible, eventually to permit the detailed analysis of the human IgE-receptor interaction and assist the search for therapeutically effective inhibitors.     

Extracellular domains mediating epsilon subunit interactions of muscle acetylcholine receptor.

Ligand-gated ion channels, a major class of cell-surface proteins, have a pseudosymmetric structure with five highly homologous subunits arranged around a central ion pore. The correct assembly of each channel, whose subunit composition varies with cell type and stage of development, requires specific recognition between the subunits. Assembly of the pentameric form of the acetylcholine receptor from adult muscle (AChR; alpha 2 beta epsilon delta) proceeds by a stepwise pathway starting with the formation of the heterodimers, alpha epsilon and alpha delta. The heterodimers than associate with the beta subunit and with each other to form the complete receptor. We have now determined which parts of the subunits mediate the interactions during assembly of the adult form of the receptor from mouse muscle by using a chimaeric subunit in which the N-terminal and C-terminal extracellular domains are derived from the epsilon subunit with the remainder from the beta subunit. The epsilon and beta subunits were chosen because the epsilon subunit forms a heterodimer with the alpha subunit in the pathway for assembly of the receptor, whereas the beta subunit does not. The epsilon beta chimera can substitute for the epsilon but not the beta subunit in the oligomeric receptor, indicating that the alpha subunit specifically recognizes an extracellular domain of the epsilon subunit.     

Phosphorylation and dephosphorylation of the high-affinity receptor for immunoglobulin E immediately after receptor engagement and disengagement.

Triggering of mast cells and basophils by immunoglobulin E (IgE) and antigen induces various biochemical signals, including tyrosine kinase activation, which lead to cell degranulation and the release of mediators of the allergic reaction. The high-affinity receptor for IgE (Fc epsilon RI) responsible for initiating these events is a complex structure composed of an IgE-binding alpha-chain, a beta-chain and a homodimer of gamma-chains. It has been assumed that beta and gamma, which have extensive cytoplasmic domains, play an important but undefined role in coupling Fc epsilon RI to signal transduction mechanisms. Here we show that Fc epsilon RI engagement induces immediate in vivo phosphorylation on beta (tyrosine and serine) and gamma (tyrosine and threonine) by at least two different non-receptor kinases. We take advantage of unique features of this receptor system to demonstrate that the phosphorylation signal is restricted to activated receptors and is immediately reversible upon receptor disengagement by undefined phosphatases. Rapid phosphorylation and dephosphorylation may be a general mechanism to couple and uncouple activated receptors to other effector molecules. This could be particularly relevant to other multimeric receptors containing Fc epsilon RI gamma-chains or the related zeta and eta chains such as the T-cell antigen receptor (TCR) and the low-affinity receptor for immunoglobulin G (Fc gamma RIII, CD16).