Identification and structural characterization of an LYT-2/3 homolog in tunicates.

1. A serologic and structural homolog to murine Lyt-2/3 molecular complex was sought in tunicate hemocytes by using a monoclonal antibody specific to Lyt-2 framework determinants (mAb 53-6.7). 2. This antibody labeled a distinct population of tunicate hemocytes, as determined in indirect immunofluorescence and FACS analysis, and immunoprecipitated disulfide-bonded subunits from hemocytes equivalent to the 38 kDa (alpha), 34 kDa (alpha') and 30 kDa (beta) subunits of murine Lyt-2/3 molecules. 3. As in mice, tunicate alpha- and alpha' -subunits each appeared to bear three N-linked oligosaccharides, one high mannose- and two complex-type glycans and focused as a number of heterogeneous spots on IEF gels. 4. In contrast, beta-subunits of both species were associated with a single N-linked glycan of the complex type and focused as basic components of limited charge heterogeneity. 5. Based on tryptic peptide patterns, alpha and alpha' -subunits, are likely to be structurally similar in both tunicate and mouse complexes. 6. However, CNBr cleavage patterns indicated that the alpha-subunit of both species may differ in the size-location of the intrachain disulfide bridge. 7. Collectively our observations suggest the phylogenetic emergence of an Lyt-2/3 homolog at an early level of evolution.     

Serological characterization and partial purification of an Lyt-1 homolog in tunicate hemocytes.

A panel of alloantisera and monoclonal antibodies specific to murine Lyt-1 allotypic and framework determinants was used in indirect immunofluorescence and FACS analysis to investigate the occurrence of an Lyt-1 homolog in tunicate (protochordate) hemocytes. Binding assays and quantitative absorption experiments established the expression of Lyt-1 cross-reacting determinants on a distinct population of tunicate hemocytes. These determinants were expressed exclusively by cells with the morphological characteristics of hemoblasts and lymphocytes. In a rapid two-step purification procedure, Lyt-1 glycoproteins from tunicate hemocytes and C57B1/6 mouse thymocytes were solubilized and partially purified by affinity chromatography using a mAb anti-Lyt-1 frame-work determinant. In both cell types, antigenic activities were associated with a major 67-kDa component. Our findings suggest an early phylogenetic emergence of an Lyt-1 homolog at this level of evolution.     

Two Lyt-2 polypeptides arise from a single gene by alternative splicing patterns of mRNA

The Lyt-2/3 molecule is a glycoprotein expressed on T lymphocytes and has classically been considered a marker for the cytotoxic/suppressor T cell subset. It has been postulated to be a receptor for class I major histocompatibility complex proteins. We have used a cDNA clone encoding the analogous human protein, Leu-2/T8, to isolate mouse cDNA clones, which were used as probes to isolate mouse genomic clones. By transfection we have shown that the mouse homologue of Leu-2/T8 is Lyt-2 and not Lyt-3. We have further demonstrated that two Lyt-2 polypeptide chains are encoded by a single gene and result from alternative modes of mRNA splicing. The nucleotide sequence of cDNA clones encoding each of these polypeptide chains has been determined and shows the difference between the two Lyt-2 polypeptide chains to be in the lengths of their cytoplasmic tails.     

The organization of immunoglobulin variable kappa chain genes on mouse chromosome 6

One mouse with a known recombination (NAK) at the Igk locus on chromosome 6 and two new recombinants [B6.PL (7 NS) and B6.PL (85NS)] were examined using a series of probes, each of which is specific for a set of immunoglobulin (Ig) Vk genes. Under high stringency conditions, each probe detects from 1 to 19 Bam HI restriction endonuclease fragments (REFs) in genomic DNA by Southern transfer hybridization techniques. Analysis of the REF patterns indicate that the NAK recombination event occurred within the variable region of Igk. The REF patterns of the two B6.PL congenic mice provided two additional recombination events which could be examined. Although some of the REFs had shared mobility among the parental strains, at least 1 and up to 13 polymorphic REFs were present for a given probe among the NZB and AKR parental strains. The results from the NAK mouse indicate that at least some members of Vk4, Vk8, Vk10, and Vk21 were on one side of the recombination event linked to the Lyt-2 ^a and Igk-Efl ^a alleles of AKR, while the Vk9, Vk11, and Vk24 REF patterns came from the NZB parental strain linked to the Igk-Ef2 ^b (Vk1) allele. The two B6.PL congenics produced a refined map on the Lyt-2, Lyt-3 side of the Vk region. The B6.PL (85NS) mice retained the Vk21 REF pattern of the Lyt-2 ^a, Lyt-3 ^a donor strain PL/J, while displaying the C57BL/6 REF pattern for the other Vk gene groups tested. The B6.PL (75NS) mice retained the REF patterns of PL/J for Vk21 and Ef-1, indicating a third recombination. This indicates the Vk gene order is (Lyt-2; Vk21); Ef-1; (Vk4; Vk8; Vk10); and (Vk9; Vk11; Vk24; Ef-2).     

Structure and expression of the Lyt-3 agene of C.AKR mice

The mouse Lyt-3 ^agene, which encodes the Lyt-3.1 T-cell surface alloantigen of the C.AKR strain, has been cloned, and the nucleotide sequence of its exons and more than 2 kb of 5 flanking sequence have been determined. The gene extends over approximately 16 kb of DNA and consists of six exons encoding leader, leader plus V-like domain, membrane-proximal, transmembrane, and cytoplasmic domains. The only difference between the coding region of the Lyt-3 ^agene and the cDNA sequences reported for Lyt-3 ^b(Nakauchi et al. 1987, Panaccio et al. 1987) is at position 77 of the mature protein where Lyt-3 ^aencodes serine and Lyt-3 ^bencodes arginine. This substitution must therefore be the basis for the serological distinction between the Lyt-3.1 and Lyt-3.2 alloantigens. Potential TATA and CAAT sequences, two Sp1 protein binding sites, two extended repeats of the dinucleotide, CA, a number of short inverted repeats, and an inverted segment of the mouse B1 repetitive sequence are found 5 to the Lyt-3 ^agene. Two consensus poly-A addition signals and a complete copy of the mouse B1 sequence are found 3 to the gene. Both B1-related regions are flanked by short direct repeats suggesting that they arose by an insertional mechanism. Cotransfection of the Lyt-3 ^agene together with a cloned Lyt-2 ^agene resulted in expression of both Lyt-2 and Lyt-3.1 on the surface of Ltk^– and BW5147 cells. Transfection of the Lyt-3 ^agene without Lyt-2 ^aled to expression of Lyt-3-related cellular RNA but did not result in surface expression of Lyt-3.1, suggesting that the Lyt-3 glycoprotein is not expressed on the cell surface in the absence of Lyt-2.     

Two genes controlling the expression of extended globoglycolipids in mouse kidney are closely linked to each other on chromosome 19

The gene (Gsl-5) controlling the expression of GL-Y (Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6(Gal beta 1-3)Gb4Cer) in mouse kidney was suggested to be located near Ea-4 on mouse chromosome 19 by the results of glycolipid analysis of BXD/Ty recombinant inbred strains (Sekine et al. [1987] J. Biochem. 101, 563-568). In this study, Gsl-5 was mapped on mouse chromosome 19. Among 133 backcross progeny produced on mating between DBA/2 mice and (WHT/Ht x DBA/2)F1 mice, 10 recombinants between Lyt-1 and Gsl-5 were detected, indicating that Gsl-5 is located at 7.5 +/- 2.3 centimorgans (cM) from Lyt-1. While among 154 backcross progeny produced on mating between DBA/2 and (DBA/2 x Mus musculus castaneus)F1 mice, 39 recombinants between Got-1 and Gsl-5 were obtained, indicating that the distance between Got-1 and Gsl-5 is 25.3 +/- 3.5 cM and that Gsl-5 is telomeric to Lyt-1. In the latter mating experiment, we detected 3 recombinants between Gsl-5 and the gene (Gsl-6) controlling the expression of the Z1 ganglioside (NeuGc alpha 2-3Gal beta 1-3Gb4Cer) among the 154 backcross mice. These results indicate that these two genes, Gsl-5 and Gsl-6, are closely linked to each other, being 1.9 +/- 1.1 cM apart. This is the report of evidence that two genes controlling the expression of carbohydrates in glycoconjugates are closely linked and the first to suggest that some genes controlling the expression of carbohydrates may be clustered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hemocytes/coelomocytes DNA content in five marine invertebrates: cell cycles and genome sizes

The hemocytes/coelomocytes DNA content in five selected marine invertebrates (sea mouse Aphrodita aculeata, spiny crab Maja crispata, sea star Echinaster sepositus, sea urchin Paracentrotus lividus, and tunicate Phallusia mammillata) was investigated by flow cytometry. The cell cycle analyses identified sea mouse coelomocytes as proliferating cells and revealed that spiny crab hemocytes and sea urchin coelomocytes complete their division in the hemolymph and coelom, respectively. The genome sizes of sea mouse and spiny crab are reported for the first time. The diploid DNA content (2C) in sea mouse A. aculeate was 1.24 pg, spiny crab M. crispata 7.76 pg, red starfish E. sepositus 1.52 pg and sea urchin P. lividus 1.08 pg. The mean diploid DNA content in tunicate P. mammillata was 0.11 pg with a high interindividual variability (45%). The presented results provide a useful database for future studies in the field of invertebrate physiology, ecotoxicology, biodiversity, species conservation and phylogeny.     

Properties of isolated human alpha1-antitrypsins of Pi types M, S and Z.

1. alpha1-Antitrypsin contains a single thiol group partly blocked in native plasma and reactive after mild reduction. 2. Human alpha1-antitrypsins of Pi types F, M, S and Z have been isolated with native microheterogeneity using thiol-disulfide (SH-SS) interchange reactions utilizing the reactive thiol group. 3. The pI of the various microheterogeneous fractions are given for protein M. Stepwise desialylation of alpha1-antitrypsin indicates that the charge difference between the major fractions is one sialic acid residue between each. This is further supported by the pI changes obtained on substitution of the single thiol with positively or negatively charged compounds. 4. Desialyation of purified proteins from each Pi type converts the individual microheterogeneous fractions to one major fraction. The pI shift for the variants studied indicate a difference of plus or minus one or two charge units between protein M and the variants. 5. A difference of one sialic acid residue was obtained for proteins M and Z by the thiobarbituric assay, but stepwise removal of sialic acid with neuraminidase revealed almost identical stepwise change of pattern of both proteins indicating the same number of sialic acid residues. 6. Electrofocusing has been used to identify CNBr fragments from proteins M, S and Z. 7. An amino acid substitution has been found to be located in one of the eight CNBr fragments, glutamic acid in protein M is substituted by lysine in protein Z. 8. The average concentration of alpha1-antityprsin in plasma from healthy males was found to be 1.32 g/1.     

Myelin basic protein is affected by reduced synthesis of myelin proteolipid protein in the jimpy mouse.

Myelin basic proteins (MBPs) from 6-day-old, 10-day-old, 20-day-old and adult normal mouse brain were compared with those from 20-day-old jimpy (dysmyelinating mutant) mouse brain to determine the effect of reduced levels of proteolipid protein (PLP) on MBPs. Alkaline-urea-gel electrophoresis showed that 6-day-old and 10-day-old normal and jimpy MBPs lacked charge microheterogeneity, since C8 (the least cationic of the components; not be confused with complement component C8) was the only charge isomer present. In contrast, MBPs from 20-day-old and adult normal mouse brain displayed extensive charge microheterogeneity, having at least eight components. A 32 kDa MBP was the major isoform observed on immunoblots of acid-soluble protein from 6-day-old and 10-day-old normal and 20-day-old jimpy mouse brain. There were eight bands present in 20-day-old and adult normal mouse brain. Purified human MBP charge heteromers C1, C2, C3 and C4 reacted strongly with rat 14 kDa MBP antiserum, whereas the reaction with human C8 was weak. This suggested that MBPs from early-myelinating and jimpy mice did not react to MBP antisera because C8 was the major charge isomer in these animals. Purification of MBPs from normal and jimpy brain by alkaline-gel electrophoresis showed that both normal and jimpy MBPs have size heterogeneity when subjected to SDS/PAGE. However, the size isoforms in normal mouse brain (32, 21, 18.5, 17 and 14 kDa) differed from those in jimpy brain (32, 21, 20, 17, 15 and 14 kDa) in both size and relative amounts. Amino acid analyses of MBPs from jimpy brain showed an increase in glutamic acid, alanine and ornithine, and a decrease in histidine, arginine and proline. The changes in glutamic acid, ornithine and arginine are characteristic of the differences observed in human C8 when compared with C1.     

Differences between macrophage migration inhibitions by lymphokines and muramyl dipeptide (MDP) or lipopolysaccharide (LPS): migration enhancement by lymphokines

To investigate the repertoire of molecules which are associated with cytolytic T-lymphocyte (CTL)-mediated killing, function-blocking monoclonal antibodies (MAb) have been selected and characterized. Spleen cells from rats immunized with secondary mouse CTL were fused with mouse myeloma cells. Antibodies secreted by 2400 hybrid cultures were selected solely by their ability to block CTL-mediated killing in a mouse anti-rat xenogeneic system. Fifteen cultures with antibodies which blocked CTL-mediated killing were chosen for cloning and further characterized by immunoprecipitation and immunofluorescence flow cytometry. One group of five monoclonal antibodies recognized the Lyt-2,3 molecule of 35,000 M_r. The second group of six MAb recognized the LFA-1 antigen containing two subunits of 180,000 and 95,000 M_r. One MAb giving only partial inhibition of killing was an IgM anti-Thy-1. It strongly agglutinated CTL. The target antigens defined by three other MAb were not definitively identified. Competition in cell binding between anti-Lyt-2,3 and anti-LFA-1 MAb suggested that their blocking effect in cytolysis is due to binding to distinct and spatially separate molecules on effector cells. The results of direct screening for functional blockade support the important role of Lyt-2,3 and LFA-1 molecules in T-cell-mediated cytolysis.     

Granulocyte/macrophage colony-stimulating factor from mouse lung conditioned medium. Purification of multiple forms and radioiodination.

Four forms of mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) were purified 100,000-fold from mouse lung conditioned medium. Each of the CSF species stimulated the formation of both granulocyte and macrophage colonies, and half-maximal stimulation in the semi-solid mouse bone-marrow colony assay occurred at 1 pm. The four GM-CSF species exhibited similar charge microheterogeneity, focusing between pH 4.2 and pH 5.2. On SDS/polyacrylamide gels two of the GM-CSF sub-species had apparent Mr values of 23,000, and the other two, 21, 000. Treatment with neuraminidase decreased the Mr values of these two sets to 21,000 and 19,000 respectively. Incubation with endoglucosidase F decreased the charge heterogeneity and the Mr of all species to 16,500. A gas-phase radioiodination procedure was used to incorporate 2-3 atoms of 125I/molecule into purified GM-CSF without any loss of biological activity. The 125I-labelled GM-CSF was analysed on a microbore reversed-phase h.p.l.c. column to determine its specific radioactivity directly. This 125I-labelled GM-CSF molecule is suitable for cell-surface receptor-binding studies.     

Effect of bovine basic protein charge microheterogeneity on protein-induced aggregation of unilamellar vesicles containing a mixture of acidic and neutral phospholipids

Two of the charge isomers (components 1 and 2) normally found as microheteromers of myelin basic protein were isolated, and their abilities to aggregate vesicles consisting of mixed phospholipids were studied. Component 1 (the most cationic of the microheteromers) aggregated phosphatidylcholine (PC) vesicles containing 7.8 mol% phosphatidylserine (PS) more rapidly and at lower protein concentrations than component 2, which differs from component 1 by 1 net positive charge. Modification of components 1 and 2 in vitro by phosphorylation with rabbit muscle protein kinase decreased the ability of both components to aggregate vesicles. The greater the extent of phosphorylation, the less effective were the isomers at inducing aggregation. Decreasing the charge of either component 1 or component 2 by removal of the two C-terminal arginyl residues also decreased the ability of the isomers to induce aggregation. Therefore, charge microheterogeneity, whether arising in vivo or generated in vitro, markedly affected the ability of these microheteromers to aggregate PC vesicles containing 7.8 mol% PS. Because a small difference in the charge of the protein had a marked effect on vesicle aggregation, we propose that charge microheterogeneity may play an important and dynamic role in the structure and function of normal myelin.     

Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins

Physico-chemical characterization of the sex steroid-binding protein, SBP, of rabbit plasma reveals that it is a dimer of mol. wt 85,800 composed of similar subunits of mol. wt 43,000. These data confirm our original proposal for a dimeric structure. The protein contains 9% carbohydrate, comprised of mannose, galactose, N-acetylglucosamine and sialic acid. It is devoid of N-acetylgalactosamine and fucose. The protein binds one molecule of 5 alpha-dihydrotestosterone per dimer with a Kd of 0.89 nM (12 degrees C). Comparison with the human, monkey and baboon SBPs indicates that all these proteins have the same dimeric molecular organization and exhibit microheterogeneity in SDS-PAGE and isoelectricfocusing. Rabbit SBP, however, contains less carbohydrate and has a higher polypeptide molecular weight than all the other SBPs. Spectrophotometric data also indicate that some tryptophan residues are in a different chemical environment than those in other SBPs. The observed microheterogeneity in all four SBP species is due for the most part to variable glycosylation of the subunit and variability at the amino-terminal region of the subunit. Combination of these and other phenomena will generate a significant number of isomeric forms of the SBP subunit which will then interact stoichiometrically to yield active dimeric SBP molecules. These differ slightly from each other depending upon the charge and size of the subunit comprising the dimeric structure, and will result in the observed microheterogeneity of pure SBP preparations. Based on these results along with more recent amino acid sequence data, we conclude that all four SBPs are dimers composed of identical polypeptide chains.     

Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). V. T cell proliferative response and cellular interactions

We previously demonstrated that in vivo antibody production to HBsAg in the mouse is regulated by at least two immune response (Ir) genes mapping in the I-A (HBs-Ir-1) and I-C (HBs-Ir-2) subregions of the H-2 locus. To confirm that H-2-linked Ir genes regulate the immune response to HBsAg at the T cell level and to determine if the same Ir genes function in T cell activation as in B cell activation, the HBsAg-specific T cell responses of H-2 congenic and intra-H-2 recombinant strains were analyzed. HBsAg-specific T cell proliferation, IL 2 production, and the surface marker phenotype of the proliferating T cells were evaluated. Additionally, T cell-antigen-presenting cell (APC) interactions were examined with respect to genetic restriction and the role of Ia molecules in HBsAg presentation. The HBsAg-specific T cell proliferative responses of H-2 congenic and intra-H-2 recombinant strains generally paralleled in vivo anti-HBs production in terms of the Ir genes involved, the hierarchy of responses status among H-2 haplotypes, antigen specificity, and kinetics. However, the correlation was not absolute in that several strains capable of producing group-specific anti-HBs in vivo did not demonstrate a group-specific T cell proliferative response to HBsAg. The proliferative responses to subtype- and group-specific determinants of HBsAg were mediated by Thy-1+, Lyt-1+2- T cells, and a possible suppressive role for Lyt-1-2+ T cells was observed. In addition to T cell proliferation, HBsAg-specific T cell activation could be measured in terms of IL 2 production, because anti-HBs responder but not nonresponder HBs-Ag-primed T cells quantitatively produced Il 2 in vitro. Finally, the T cell proliferative response to HBsAg was APC dependent and genetically restricted in that responder but not nonresponder parental APC could reconstitute the T cell response of (responder X nonresponder)F1 mice, and Ia molecules encoded in both the I-A and I-E subregion are involved in HBsAg-presenting cell function.     

Sequential precipitation of mouse thymocyte extracts with anti-Lyt-2 and anti-Lyt-3 sera. I. Lyt-2.1 and Lyt-3.1 antigenic determinants reside on separable molecular species.

Anti-Lyt-2.1 and anti-Lyt-3.1 sera were employed for sequential precipitation of NP-40 extracts of 125I-labeled C57BL/6-Lyt-2a, Lyt-3a thymocytes (Lyt-2.1, Lyt-3.1) to determine whether these alloantigenic determinants are present on the same or different molecular species. Treatment of extracts with anti-Lyt-3.1 serum and SaCI completely precipitated both Lyt-3.1 and Lyt-2.1-specific components, whereas treatment with anti-Lyt-2.1 serum reduced by approximately 37% the quantity of labeled species subsequently precipitable by anti-Lyt-3.1 serum. When 125I-labeled thymocytes were subjected to mild trypsinization before NP-40 extraction, the quantity of radioactive components precipitated by anti-Lyt-2.1 serum was essentially unchanged, but that of anti-Lyt-3.1-precipitable components was greatly reduced. Moreover, sequential precipitation of extracts of trypsinized thymocytes with anti-Lyt-2.1 and anti-Lyt-3.1 sera demonstrated that these molecular species were precipitated independently. Thus 1) Lyt-2.1 and Lyt-3.1 antigenic determinants appear to reside on different molecular species; 2) some Lyt-2.1- and Lyt-3.1-positive molecules appear to be complexed with each other in the NP-40 extract; and 3) this association of Lyt-2.1- and Lyt-3.1-positive species was dependent upon components that were labile to trypsinization of intact thymocytes.     

Recombination between kappa chain genetic markers and the Lyt-3 locus

Recombination has been detected for the first time between chromosome 6 loci controlling kappa chain expression in normal mouse serum immunoglobulin and the Lyt-3 locus. The recombination event occurred at the 26th or 27th backcross generation during the derivation of the Lyt-2a, Lyt-3a-congenic line B6.PL(85NS). The line is now homozygous for the Lyt-2a, Lyt-3a allele(s) at N30F13 and homozygous animals express the Igk-Ef1b allele derived from C57BL/6. The frequency of recombination has been estimated to be 0.30% based on the present results and previous studies in which no recombination was detected. The results rule out the hypothesis that the Lyt-3 locus itself controls the light chain phenotype observed in normal serum immunoglobulin.     

Cell age dependent decay of human erythrocytes glucose-6-phosphate isomerase

Glucose-6-phosphate isomerase shows a biphasic decay pattern during red blood cell aging, which is very fast during the first part of cell's life span in circulation. This decay is not due to accumulation of inactive enzyme molecules, as shown by immunological studies, but is accompanied by the formation of secondary isozymes (i.e., chemically modified forms). Electrophoretic and ion-exchange chromatographic experiments show that glucose-6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) consists of only one isozymic form in young erythrocytes but is present in two components, with different electric charge, in mature and old cells. This secondary isozyme is more stable to heat treatment and is inactivated by IgG anti-glucose-6-phosphate isomerase with a lower affinity than the native isozyme. In vitro incubation of homogeneous human glucose-6-phosphate isomerase under conditions known to produce enzyme deamination does not reproduce the isozymic pattern found in erythrocytes, suggesting that one or more mechanisms other than those previously described to explain glucose-6-phosphate isomerase microheterogeneity occur in the human erythrocyte.     

Isolation, purification, and amino acid composition of the tunicate hemocyte Thy-1 homolog

A serologic cross-reacting homolog to rodent Thy-1 glycoproteins has been isolated from hemocyte cell surfaces of the advanced invertebrate group of tunicates. The Thy-1.1 cross-reacting antigenic activity was followed during purification by inhibiting the binding of MRC OX7 monoclonal antibody to pure rat brain Thy-1 in a soluble phase radioimmunoassay. After solubilization in deoxycholate, tunicate hemocyte Thy-1.1 antigenic activity was purified by affinity chromatography using an MRC OX7 monoclonal antibody affinity column, followed by gel filtration. A 602-fold enrichment in the Thy-1.1 antigenic activity, with a yield of 55.6% compared to the starting crude membrane fraction, was obtained. The antigenic activity was associated with a single glycoprotein of molecular size of 3.1 nm and molecular weight estimated at 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15% gels). Amino acid composition of the purified molecule was compared by the S delta Q index of differences in composition to mammalian and non-mammalian Thy-1 glycoproteins, Ig, major histocompatibility complex-encoded polypeptides, beta 2-microglobulin, and other recognition molecules. With this parameter, the tunicate hemocyte Thy-1 homology revealed significant relatedness to avian and mammalian Thy-1 molecules and was interestingly more related to mu chains of primitive vertebrates and to HLA class I and II encoded polypeptides than to Thy-1 molecules of higher vertebrates. Based upon these observations, the tunicate hemocyte Thy-1 homolog seems to represent an ancestral Thy-1 molecule which, in structural terms, may represent an invertebrate member of the Ig superfamily.     

Structure, sequence, and polymorphism of the Lyt-2 T cell differentiation antigen gene

We have determined the nucleotide sequence and genomic organization of the mouse Lyt-2 T lymphocyte differentiation antigen gene. This gene consists of five exons and four introns, and the organization roughly parallels the protein domains. Alternative splicing to include or exclude exon IV (encoding part of the cytoplasmic tail) results in two forms of mRNA and accounts for the difference in size between the alpha- and alpha'-chains of Lyt-2. The gene structure provides further evidence for the evolutionary relationship between Lyt-2 and immunoglobulin genes. Comparison of the nucleotide sequence of the Lyt-2.1 and Lyt-2.2 alleles shows a high degree of conservation, but indicates that a single nucleotide change and consequent amino acid substitution in the variable region-like domain accounts for the serologic difference between these two alleles.     

Microheterogeneity of the major grass group 6 allergen Phl p 6: Analysis by mass spectrometry

The precise structural characterization of allergens is a basic requirement to improve diagnostics and to find therapeutic strategies against allergic disorders. Natural grass pollen allergens exhibit a wide variety of isoforms and it is still unknown whether this microheterogeneity is essential for the allergic reaction or has a functional effect on sensitization. Well-defined recombinant allergens are considered to replace natural allergens for clinical trials. For the major timothy grass pollen allergen Phl p 6 (approximately 12 kDa) and a recombinant rPhl p 6 we determined the structural microheterogeneity by two-dimensional electrophoresis (2-DE), high-resolution electrospray ionization-Fourier transform-mass spectrometry (ESI-FT-MS) of the intact molecules, and by tryptic peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Natural Phl p 6 is a mixture of mainly two isoforms that differ by two amino acids leading to a mass difference of 5 Da. For each of this two isoforms six variants were identified with modifications at the C- and/or N-terminus. The recombinant Phl p 6 comprises the same structure as one of the main isoforms indicating that it represents a major part of the natural Phl p 6.