Eleven distinct VH gene families and additional patterns of sequence variation suggest a high degree of immunoglobulin gene complexity in a lower vertebrate, Xenopus laevis.

Lower vertebrate species, including Xenopus laevis, exhibit restricted antibody diversity relative to higher vertebrates. We have analyzed more than 180 VH gene-containing recombinant clones from an unamplified spleen cDNA library by selective sequencing of JH and CH positive clones following iterative hybridization screening with family-specific VH probes, 11 unique families of VH genes, each associated with a unique genomic Southern blot hybridization pattern, are described and compared. Considerable variation in the number of hybridizing components detected by each probe is evident. The nucleotide sequence difference between VH families is as great as, if not more than, that reported in other systems, including representatives of the mammalian, avian, and elasmobranch lineages. Some Xenopus Ig gene families encode alternative amino acids at positions that are otherwise invariant or very rarely substituted in known Igs. Furthermore, variations in complementarity determining region sequences among members of the same gene family and high degrees of DH and JH region complexity are described, suggesting that in at least this lower vertebrate species, the diversity of expressed Ig VH genes is not restricted.

Diversification, not use, of the immunoglobulin VH gene repertoire is restricted in DiGeorge syndrome.

Immunoglobulin (Ig) genes were isolated from unamplified conventional as well as polymerase chain reaction-generated cDNA libraries constructed from the peripheral blood cells of a patient with complete DiGeorge syndrome. Comparison of the sequences of 36 heavy chain clones to the recently expanded database of human VH genes permitted identification of the germline VH genes that are expressed in this patient as well as placement of 19 of these genes in a partially resolved 0.8-mb region of the human VH locus. The pattern of VH gene use does not resemble the fetal (early) repertoire. However, as in the fetal repertoire, there are a number of cDNAs derived from germline genes that previously have been identified as autoantibodies. Two D mu sequences also were identified, as was another sequence resulting from a unique recombination event linking JH to an unidentified sequence containing a recombination signal sequence-like heptamer. All of the DiGeorge cDNAs are closely related to germline VH genes, showing little or no evidence of somatic mutation. In contrast, comparably selected IgM VH sequences derived from normal adult and age-matched human libraries, and from a second DiGeorge syndrome patient in whom the degree of thymic dysfunction is much less severe, exhibit considerable evidence of somatic mutation. The absence of somatic mutation is consistent with the atypical development of functional antibody responses associated with complete DiGeorge syndrome and implicates a role for T cells in the generation of diversity within the B cell repertoire.

Mitogen activation induces the enhanced synthesis of two heat-shock proteins in human lymphocytes.

We have used mitogenic lectin (PHA) and a monoclonal antibody (OKT3) to stimulate human peripheral blood (G0) lymphocytes, in the presence of monocytes, and have found two major preferentially synthesized proteins, 73 and 95 kD, which are induced by the mitogens. The elevated synthesis of both proteins begins approximately 4-6 h after mitogen addition (early to mid G0/G1) before entry into first S phase. Maximum synthesis of both proteins is reached by 12 h after mitogen addition when P95 synthesis represents approximately 4%, and P73 approximately 2%, of the total protein synthesis, compared with less than 0.5% for each protein in cells cultured without mitogen. Thus, the proteins appear to be major components of activated cells. We find that both P73 and P95 are induced by heat stress as well as mitogenic stimulation. The induction of the proteins is not affected by either deleting glucose from the culture media or, alternatively, by supplementing it. Using polyclonal antibodies prepared to each of the proteins isolated from mitogen activated cells and monoclonal antibodies that were raised to heat shock proteins, we are able to show that P95 is electrophoretically and immunologically identical to the HSP 90 induced by heat stress. P73 is one of the 70 kD HSPs, (termed HSC 70; Pelham, H. R. B. 1986. Cell. 46: 959-961), but is different from the most strongly heat inducible form of HSP 70 (72 kD). The distribution of both proteins in subcellular fractions of mitogen activated lymphocytes is similar to the reported localization of the respective HSP's in other cell types. The results suggest that HSP 90 and HSC 70 may have functional roles in stress response and growth processes of human lymphocytes.

Human Txk: genomic organization, structure and contiguous physical linkage with the Tec gene.

Txk is a Tec-family tyrosine kinase expressed in mouse and human T lymphocytes. Among the Tec kinases, Txk is unique in that its amino terminal region does not include a pleckstrin homology domain or other known extended functional region. Txk is encoded at human chromosome 4p12 and at a recognized region of conserved synteny on mouse chromosome 5. The genomic organization of Txk consists of 15 exons with strong exon-intron organizational homology to Btk, the only other Tec-family kinase for which the genomic structure is fully known. The human Tec gene also maps to 4p12 and, based on limited studies reported here, possesses organizational homology with Btk and Txk. We have sequenced a continuous region of DNA that contains 3' Tec and 5' Txk exons separated by only a approximately 1.5 kb intergenic region containing the putative promoter region of Txk. The close physical linkage of these Tec-family tyrosine kinases, which are expressed in different hematopoetic cell lineages, suggests their potential for coordinate cis-regulation.

Characterization of in vivo effects of dilithium carbamyl phosphate on dog hemoglobin structure and function.

The effect of oral administration of dilithium carbamyl phosphate on adult beagle dogs is dose related. The levels of carbamylation as mol valine hydantoin/mol hemoglobin tetramer reaches a plateau of 0.3-0.38 with daily doses of 100 mg/kg body weight. Related changes in oxygen binding by whole blood and hemoglobin amount to a 5-15% left shift in oxygen isotherms. After discontinuing the administration of carbamyl phosphate, the disappearance of modified hemoglobin with a return to normal oxygen binding values follows the gradual replacement of old cells by new cells in the circulation. When the level of in vivo carbamylation in dog blood is greater than 0.16, it is similar to the levels of in vitro carbamylation of hemoglobin SS which result in the interference with sickling of erythrocytes at low levelsof oxygen.

Thermodynamic aspects of the linkage between binding of chloride and oxygen to human hemoglobin.

Oxygen isotherms of human hemoglobin measured in distilled water and in solutions of sodium chloride in the concentration range from 0.02 to 3.0 M indicate that the oxygen affinity decreases up to about 1 M salt and then begins to increase. The isotherms obtained in the range from 0.02 to 0.6 M sodium chloride, at 37 degrees and pH 7.4, have been analyzed in terms of changes in Gibbs free energy of heme ligation, resulting from the differential interaction between the chloride ion and the two forms of hemoglobin. The maximal theoretical change in Gibbs free energy that chloride ion can exert on the oxygen binding of hemoglobin amounts to 4.9 +/- 0.2 kcal/mol (21 +/- 0.8 kJ/mol) of hemoglobin tetramer. A plot of the logarithm of oxygen concentration at half saturation versus the logarithm of the chloride concentration has a slope of 0.40, suggesting 1.6 apparent chloride sites per hemoglobin tetramer. Because the interaction between chloride and hemoglobin is dependent on pH, the apparent thermodynamic linkage between chloride and oxygen binding will also include the salt dependence of the Bohr effect at pH 7.4. The fractional change in Gibbs free energy, measured as a function of the chloride concentration, can be approximated by the binding isotherm between a protein and a ligand, using an association constant of 11 M(-1). Thus, if the number of oxygen-linked chloride sites is more than one per hemoglobin tetramer, these sites must be considered independent.

Hemoglobin function in the water-ethylene glycol cosolvent system: linkage between oxygen binding and hydration.

The effects of ethylene glycol (EG) on the oxygen binding properties of human hemoglobin are described in this report. Under the conditions used, the hemoglobin molecule remains in the intact tetrameric form in up to 70% (w/w) EG, corresponding to a mole fraction of EG of 0.4. Interaction between the cosolvent and the hemoglobin is quite weak. Only at high concentrations of EG are the effects on the oxygen binding curve detectable. In the range of mole fraction of EG up to 0.2, oxygen affinity is decreased. In the range of mole fraction of EG between 0.2 and 0.4 (corresponding to molar concentrations of 8-12 M EG), hemoglobin oxygen affinity increases, eventually becoming higher than the value obtained in the absence of EG. Experiments were carried out in the presence of 0.013, 0.10, and 1.0 M NaCl to evaluate the linkage between EG and chloride as allosteric effectors and the possible general effect of ionic strength on oxygen binding properties of hemoglobin in the presence of cosolvent. The effects of EG on hemoglobin ligation are discussed in terms of a model in which EG interacts with hemoglobin in a weak allosteric fashion at the lower concentration range (less than mole fraction of 0.2) while at the higher range (mole fraction of 0.2-0.4) perturbations of protein hydration lead to stabilization of the high-affinity form of hemoglobin.

Mitogen-induced preferential synthesis of proteins during the G0 to S phase transition in human lymphocytes.

We have followed the induction of protein synthesis in mitogen-activated human peripheral blood mononuclear cells during the transition from quiescence, or G0, through the prereplicative phase and into first S phase. Doses of mitogens optimal for proliferative response preferentially enhance the synthesis of a subset of intracellular proteins during the approximately 24-h lag interval. The mitogenic lectin phytohemagglutinin (PHA) and OKT3, a mitogenic monoclonal antibody to the CD3 component of the T cell antigen receptor, preferentially enhance bands of the same molecular weight in one-dimensional SDS-PAGE. The proteins are low detergent soluble (0.1% Triton X-100) "cytoplasmic" cellular components and some have been identified as single spots on two-dimensional gels. Bands of 51 and 66 kDa are induced early in lag phase (4 h after stimulation) but are transiently synthesized, decreasing later in lag phase. The majority of the mitogen-induced proteins, 39, 51, 55, 60, 73, and 95 kDa are enhanced by mid lag phase (12 h after stimulation). With the exception of the 55-kDa band, five of these proteins are clearly enhanced in T cells purified after mitogen stimulation. The same five bands show sustained synthesis in actively cycling cells 42-48 h after stimulation and are major synthesized proteins, and corresponding bands are synthesized in a transformed T cell line, MOLT-4. Two of the proteins in this group that are most prominently synthesized during the lag interval have been previously identified as the heat shock proteins, HSP 90 (95-kDa band) and HSC 70 (73-kDa band). We speculate that this group of five proteins, including HSP 90 and HSC 70, may be coordinately expressed in actively replicating T cells and may have some common structural or functional role in sustaining the replicative state.

Determination of the apparent thermodynamic activities of saturated protein solutions.

Although the solubility of a protein is a particularly informative solution parameter, little is known about the thermodynamics of protein solubilization. In these experiments, polyethylene glycol (PEG) is used to decrease the solubility of a number of proteins in a quantifiable manner. Simple thermodynamic considerations show that if the chemical potential of the PEG-induced solid phase is constant and plots of log protein solubility versus PEG concentration are linear, a valid extrapolation of the apparent solubility to zero PEG content can be made. Given the validity of these assumptions, extrapolated values should represent the activity of the protein in saturated solution. Evidence for the validity of this extrapolation includes (a) the experimentally observed linearity of log solubility versus PEG concentration plots, (b) the extrapolation of such plots to correct activities in the situation where protein activities can be experimentally determined, and (c) the independence of the extrapolated activities on protein concentration over a wide range. The utility of the PEG-determined activities, when applied in a comparative manner, is illustrated by application to various hemoglobin solutions. It is found that saturated solutions of the various hemoglobin forms, with the exception of deoxyhemoglobin S, manifest similar activities. In addition, all of the solutions demonstrate an apparent, surprising thermodynamic ideality.

Characterization of three isotypes of immunoglobulin light chains and T-cell antigen receptor alpha in zebrafish.

The zebrafish (Danio rerio) has become a significant model for understanding the developmental regulation of gene expression and holds considerable potential for characterizing the development of the immune system. Using a number of different approaches, including heterologous hybridization and short-primer PCR, cDNAs for three different classes of light-chain genes were identified and characterized. The zebrafish light chains are similar to trout type 1, trout type 2, and catfish type F, respectively. T-cell antigen receptor alpha (TCRalpha) was also identified and characterized. A high proportion of unusual transcripts including sterile transcripts, germline VJC transcripts, aberrant splice forms, and V-V transcripts were encountered in the immunoglobulin and TCR cDNAs examined. The light-chain and TCRalpha loci each consist of multiple families of V gene segments, apparent even from the small numbers of cDNAs of each isotype sequenced. The gene sequences reported provide an essential set of markers of both B- and T-cell lineages that will facilitate investigations of immune system development.

The genomic organization of immunoglobulin VH genes in Xenopus laevis shows evidence for interspersion of families.

The complete genomic sequences of Xenopus laevis immunoglobulin heavy chain variable region (VH) genes comprising families IV-XI are reported. Using VH family-specific probes, linkage relationships for Xenopus VHI-VHXI have been determined. With the possible exceptions of VHIII and VHVII, Xenopus VH genes appear to be interspersed. When from two to five VH segments are identified in individual clones, the elements are found to be in the same relative transcriptional orientation. Although the relationships of promoter sequences, including the regulatory octamer, resemble those seen in other vertebrate VH genes, several Xenopus VH families are associated with additional 5' octamer sequences and octamer-like motifs. The similarities between the genomic organization of VH genes in Xenopus and higher vertebrates, indicate an early phylogenetic emergence of the mammalian-type of gene organization and regulation.

Members of the Ikaros gene family are present in early representative vertebrates.

Members of the Ikaros multigene family of zinc finger proteins are expressed in a tissue-specific manner and most are critical determinants in the development of both the B and T lymphocytes as well as NK and dendritic APC lineages. A PCR amplification strategy that is based on regions of shared sequence identity in Ikaros multigene family members found in mammals and several other vertebrates has led to the recovery of cDNAs that represent the orthologues of Ikaros, Aiolos, Helios, and Eos in Raja eglanteria (clearnose skate), a cartilaginous fish that is representative of an early divergence event in the phylogenetic diversification of the vertebrates. The tissue-specific patterns of expression for at least two of the four Ikaros family members in skate resemble the patterns observed in mammals, i.e., in hematopoietic tissues. Prominent expression of Ikaros in skate also is found in the lymphoid Leydig organ and epigonal tissues, which are unique to cartilaginous fish. An Ikaros-related gene has been identified in Petromyzon marinus (sea lamprey), a jawless vertebrate species, in which neither Ig nor TCRs have been identified. In addition to establishing a high degree of evolutionary conservation of the Ikaros multigene family from cartilaginous fish through mammals, these studies define a possible link between factors that regulate the differentiation of immune-type cells in the jawed vertebrates and related factors of unknown function in jawless vertebrates.

Identification and characterization of T-cell antigen receptor-related genes in phylogenetically diverse vertebrate species.

Characterization of the structure, multiplicity, organization, and cell lineage-specific expression of T-cell receptor (TCR) genes of nonmammalian vertebrate species is central to the understanding of the evolutionary origins of rearranging genes of the vertebrate immune system. We recently described a polymerase chain reaction (PCR) strategy that relies on short sequence similarities shared by nearly all vertebrate TCR and immunoglobulin (Ig) variable (V) regions and have used this approach to isolate a TCR beta (TCRB) homolog from a cartilaginous fish. Using these short PCR products as probes in spleen cDNA and genomic libraries, we were able to isolate a variety of unique TCR and TCR-like genes. Here we report the identification and characterization of a chicken TCR gamma (TCRG) homolog, apparent Xenopus and pufferfish TCR alpha (TCRA) homologs, and two horned shark TCR delta (TCRD)-like genes. In addition, we have identified what could be a novel representative of the Ig gene superfamily in the pufferfish. This method of using short, minimally degenerate PCR primers should speed progress in the phylogenetic investigations of the TCR and related genes and lend important insights into both the origins and functions of these unique gene systems.

Polyphasic linkage between protein solubility and ligand binding in the hemoglobin-polyethylene glycol system.

Dilute hemoglobin A, hemoglobin S, and horse hemoglobins can be made to precipitate by addition of polyethylene glycol (PEG). PEG itself has no effect on the oxygen affinity of the soluble hemoglobin. In the resulting two-phase system the amount of precipitation is dependent on the oxygen saturation and, conversely, thw two-phase mixtures exhibit altered oxygen affinity such as is seen with gelled hemoglobin S. A decreased oxygen affinity results when the deoxy form is the less soluble as in the case of hemoglobin A or S. When the oxy form is the less soluble, as in the case of horse hemoglobin, an affinity increase results. These reciprocal shifts in solubility and oxygen affinity are seen as thermodynamically linked processes, independent of solid phase structure. However, the structure, as seen by electron microscopy, reveals that deoxyhemoglobin S gelled in the absence of PEG is identical with deoxyhemoglobin S solid phase formed in the presence of PEG.

VH gene organization in a relict species, the coelacanth Latimeria chalumnae: evolutionary implications.

The living coelacanth Latimeria chalumnae is a relict species whose higher-level phylogenetic relationships have not been resolved clearly by traditional systematic approaches. Previous studies show that major differences in immunoglobulin gene structure and organization typify different phylogenetic lineages. To date, mammalian-, avian-, and elasmobranch-type gene organizations have been identified in representatives of these different phylads. A fourth form or organization is found in Latimeria, which possesses immunoglobulin heavy-chain variable region (VH) elements separated by approximately 190 nucleotides from diversity (D) elements. Adjacency of VH and D elements is characteristic of the elasmobranch "clustered" arrangement, although many other features of coelacanth VH gene organization and structure are more similar to those of bony fishes and tetrapods. These observations strongly support a phylogenetic hypothesis in which Latimeria occupies a sister-group relationship with teleosts and tetrapods.

TXK, a novel human tyrosine kinase expressed in T cells shares sequence identity with Tec family kinases and maps to 4p12.

A gene for a novel, putative cytoplasmic tyrosine kinase, TXK has been isolated from a human peripheral blood cDNA library. The complete nucleotide sequence of the cDNA indicates that it is related most closely to EMT, a tyrosine kinase of T cells and to the B-cell tyrosine kinase Btk, which is mutated in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency disease (XID) in mouse. TXK, like BTK, is a member of the Tec sub-family of Src-type (non-receptor) tyrosine kinases. Like similar Tec sub-family members, and unlike the other Src kinases, TXK lacks both the N-terminal myristylation signal and the C-terminal regulatory tyrosine. TXK expression is detected primarily in T cells and some myeloid cell lines but not in a number of other cell types. TXK shares 60% amino acid homology with EMT and 57% with BTK over the SH3, SH2 (Src-homology) and catalytic domains but unlike BTK, EMT and tec, it lacks Gap 1 homology and steroid hormone receptor homology in the N-terminal region. Genomic clones containing TXK have been isolated and hybridize to chromosome position 4p12.

Unusual patterns of exon skipping in Bruton tyrosine kinase are associated with mutations involving the intron 17 3' splice site.

Seven individuals with the diagnosis of X-linked agammaglobulinemia were analyzed for mutations in Bruton tyrosine kinase (Btk) gene at both the cDNA transcript and genomic DNA levels. In addition, maternal carrier status was determined in six of the seven families by examining X chromosome-inactivation patterns for B cells in comparison with other types of blood cells. Three categories of mutations were identified: (1) three patients have missense mutations in either the pleckstrin or SH2 domains of Btk; (2) three patients exhibit mutations at or near intron/exon splice sites, two of which represent inherited mutations within the kinase domain; and (3) one patient has inherited a 2.5-kb deletion with the loss of a DNA segment encoding three exons of the kinase domain. Variation in the lengths of Btk transcripts was evident in two patients with splice-site mutations and in the patient with the DNA deletion. Sequences of the different cDNA transcripts from the patients with 3' splice-site mutations reveal complex patterns of exon skipping involving from one to four exons of the kinase domain. These findings implicate 3' splice sites of the penultimate exon in the recognition or processing of upstream exons.