A strong,preferential response of mice to polymorphic antigenic determinants of the chicken MHC,analyzed with mouse hybridoma (monoclonal) antibodies

CBA/J mice were immunized with B²/B² chicken RBC's (theB locus is the major histocompatibility complex (MHC) of the chicken). Spleen cells from these mice gave a much higher plaque-forming cell response when tested with RBC's bearing B² alloantigens, than with RBC's bearing any other MHC alloantigens. Similarly, immunization with chicken RBC's of other genotypes also produced responses that were highest when tested on the genotype used for immunization. Spleen cells from mice immunized 3 days previously with B²/B² RBC's were fused with mouse myeloma cells and hybrid clones which secreted anti-B² antibodies were selected. These monoclonal antibodies could be divided into three groups: (1) those which react with all genotypes of RBC's (panreactive); (2) those which react with only certain genotypes of RBC's and detect “public” MHC antigens; and (3) those which react with only B²/B² RBC's and detect “private” MHC antigens. Monoclonal antibodies which detect a private B² alloantigen were shown to be excellent typing reagents, as all birds of an outbred population which possessed the B² allele were easily detected using a simple one-step direct agglutination assay. No “false positives” were seen. The high and preferential response of the mouse to chicken MHC alloantigens suggested that mice might possess preexisting immunity to these antigens. In agreement with this hypothesis, normal mouse serum was found to have high titres of “natural” antibody against chicken MHC antigens.     

Monoclonal antibody differentiates chicken A system alloantigens

A monoclonal antibody (ISU-cA) was produced that recognized certain alloantigens of the chicken A blood group locus. Antigens produced by alleles A3, A4 and A8 were positive, and those produced by A2 and A5 were negative, by haemagglutination. The specificity of ISU-cA for chicken A blood group antigens was demonstrated by serologic analyses, genetic crosses and competitive inhibition of binding by anti-A alloantisera. To our knowledge, this is the first reported monoclonal antibody against a chicken blood group alloantigen system other than the B complex.     

MHC genotype predicts mate choice in the ring-necked pheasant Phasianus colchicus

Females of several vertebrate species selectively mate with males on the basis of the major histocompatibility complex (MHC) genes. As androgen-mediated maternal effects have long-lasting consequences for the adult phenotype, both mating and reproductive success may depend on the combined effect of MHC genotype and exposure to androgens during early ontogeny. We studied how MHC-based mate choice in ring-necked pheasants (Phasianus colchicus) was influenced by an experimental in ovo testosterone (T) increase. There was no conclusive evidence of in ovo T treatment differentially affecting mate choice in relation to MHC genotype. However, females avoided mating with males with a wholly different MHC genotype compared with males sharing at least one MHC allele. Females also tended to avoid mating with MHC-identical males, though not significantly so. These findings suggest that female pheasants preferred males with intermediate MHC dissimilarity. Male MHC heterozygosity or diversity did not predict the expression of ornaments or male dominance rank. Thus, MHC-based mating preferences in the ring-necked pheasant do not seem to be mediated by ornaments' expression and may have evolved mainly to reduce the costs of high heterozygosity at MHC loci for the progeny, such as increased risk of autoimmune diseases or disruption of coadapted gene pools.     

Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant

The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.     

Studies on murine natural killer (NK) cells. V. Genetic analysis of NK cell markers

This paper attempts to clarify the number and nomenclature of murine natural killer (NK) cell specific alloantigens by defining the genetic relationships between them, that is, are they coded by loci which are independent, allelic, or linked. Strain typing and F2 analyses using five alloantisera (C3H X BALB/c)F1 anti-CE, CE anti-CBA, NZB anti-BALB/c, C3H anti-ST, and BALB/c anti-DBA/2 revealed that (a) the alloantigens NK-1.1 and NK-3.1 are determined by distinct loci which are linked on the same chromosomes, (b) the alloantigen NK-2.1 is determined by an independently segregating locus to those coding for NK-1.1 and NK-3.1, (c) the alloantisera, CE anti-CBA and NZB anti-BALB/c, which have been designated anti-NK-2.1 alloantisera recognize different alloantigens coded by independent genetic loci. Thus, these five alloantisera detect four NK cell specific alloantigens which, based on the chronology of their discovery, have been designated NK-1.1-(C3H X BALB/c)F1 anti-CE, NK-2.1-CE anti-CBA, NK-3.1-C3H anti-ST, and BALB/c anti-DBA/2 and NK-4.1-NZB anti-BALB/c.     

秦岭北坡雉类种群密度和群落结构的初步研究

本研究将秦岭北坡周至自然保护区划分为4个生态地理类型带,在以Gates截线法调查雉类种群密度的基础上,对不同生态地理类型雉类群落结构进行了研究。结果表明,秦岭北坡雉类的物种多样性与植被和海拔有一定关系,环境的稳定性对雉类物种多样性有着一定影响。调查中还发现红腹锦鸡已分布到海拔2700 m的山坡上。     

The use of a clonal assay for cytotoxic T lymphocytes to determine the Ly phenotypes of the cytotoxic precursor and effector cells to alloantigens and trinitrophenyl-modified self antigens

Considerable controversy has arisen regarding the Ly phenotypes of cytotoxic T lymphocytes (CTL) and their precursors (CLP) to alloantigens and modified-self antigens. Although there is general agreement that all CTL and their pregenitors express the Ly2 alloantigen, the presence of the LY1 alloantigen on either CTL or CLP is debated. Clonal assays for CLP, capable of detecting single CLP in the absence of accessory cells, have recently been developed. This assay system provides a sensitive means of determining the Ly phenotypes of CLP to alloantigens or trinitrophenyl- (TNP) modified self antigens. Lymph node cells from C57BL/6 (Ly-1.2, 2.2, 3.2) or CBA (Ly-1.1, 2.1, 3.2) mice were treated with anti-Ly serum and complement (C), and the frequencies of CLP of the treated populations to alloantigens or TNP-modified self antigens were determined. We found that the number of CLP reactive to alloantigens or TNP-modified self antigens were greatly reduced after treatment with either anti-Ly-1 or anti-Ly-2 serum and C in both C57BL/6 and CBA mice. In other words, the CLP to alloantigens or TNP-modified self antigens in these 2 strains of mice are Ly 1+2+. We also found that the CTL derived from the Ly1+2+ CLP were also Ly1+2+. The significance of this finding with respect to the cytotoxic repertoire for alloantigens and modified self antigens is discussed.     

Influence of genotypes at a non-MHC B lymphocyte alloantigen locus (Bu-1) on expression of Ia (B-L) antigen on chicken bursal lymphocytes

Two inbred lines of chickens known to be identical at the MHC differ in their expression of Ia antigen on cells of the bursa. Line 6 bursas had 23% of intensely staining Ia+ cells while line 7 bursas had a much higher level, 85%. Studies of F4 progeny of line 6(3) X 100 crosses showed that genetic control of the high bursal proportion of Ia+ cells was determined by the Bu-1 alloantigen system. Line 100 is identical to line 7 for the lymphocyte alloantigens identified by the T and B cell reagents used in this study. Tests of F4 heterozygotes at the Bu-1 locus show a dominant effect of the Bu-1b gene in control of a high proportion of strongly staining Ia+ cells in the bursa.     

Comparison of the replication of influenza A viruses in Chinese ring-necked pheasants and chukar partridges

We investigated the replication and transmission of avian influenza A viruses in two species thought to be intermediate hosts in the spread of influenza A viruses in live poultry markets: Chinese ring-necked pheasants and chukar partridges. All 15 hemagglutinin subtypes replicated in pheasants, and most subtypes transmitted to na?ve contact pheasants, primarily via the fecal-oral route. Many viruses were shed from the gastrointestinal tract of experimentally inoculated pheasants for 14 days or longer. Virus was isolated from the cloacal swabs of one contact pheasant for an unprecedented 45 days. Chukar partridges were less susceptible to infection with avian influenza viruses. The viruses that replicated in chukar partridges were isolated for 7 days after experimental inoculation, predominantly from the respiratory tract. We detected high neutralizing antibody titers with correspondingly low levels of serum hemagglutination inhibition antibody titers in pheasants and chukar partridges when chicken red blood cells were used in serological analyses. When horse erythrocytes were used, antibody titers were comparable to those obtained by using the neutralization assay. More importantly, the results suggested that pheasants can serve as a reservoir of influenza virus. Because of their continuous asymptomatic infection and longer stay in the markets, pheasants are ideal "carriers" of influenza A viruses. Their continued presence in live markets contributes to the perpetuation and genetic interaction of influenza viruses there. On the basis of our findings, it does not make good sense to ban quail but not pheasants from the live markets.     

The phylogenetic relationships of immunoglobulin allotypes and 7S immunoglobulin isotypes of chickens and other phasianoids (turkey,pheasant, quail)

Pheasants, quail and turkeys from different geographical locations were surveyed for the presence of eight 7S Ig and four IgM chicken allotypes. No IgM and only two 7S Ig allotypes were detected. Chicken 7S Ig allotypic specificity G-1.7 cross-reacted with pheasant and turkey isotypic specificities, and was absent in quail. The other determinant (G-1.9) cross-reacted with an allotype found only in turkeys and golden pheasants. These data suggest that G-1.7 and G-1.9 are probably phylogenetically ancient determinants and that polymorphism of chicken immunoglobulins arose after divergence of chickens from other phasianoid birds. Based on the allotypic and isotypic analysis of the 7S Ig antigenic determinants, turkey 7S Ig was as closely related to chicken 7S Ig as was pheasant 7S Ig. Jungle fowl, the ancestor of chickens, had most of the chicken 7S Ig and IgM allotypes present as polymorphic markers.     

The phylogenetic relationships of immunoglobulin allotypes and 7S immunoglobulin isotypes of chickens and other phasianoids (turkey, pheasant, quail)

Pheasants, quail and turkeys from different geographical locations were surveyed for the presence of eight 7S Ig and four IgM chicken allotypes. No IgM and only two 7S Ig allotypes were detected. Chicken 7S Ig allotypic specificity G-1.7 cross-reacted with pheasant and turkey isotypic specificities, and was absent in quail. The other determinant (G-1.9) cross-reacted with an allotype found only in turkeys and golden pheasants. These data suggest that G-1.7 and G-1.9 are probably phylogenetically ancient determinants and that polymorphism of chicken immunoglobulins arose after divergence of chickens from other phasianoid birds. Based on the allotypic and isotypic analysis of the 7S Ig antigenic determinants, turkey 7S Ig was as closely related to chicken 7S Ig as was pheasant 7S Ig. Jungle fowl, the ancestor of chickens, had most of the chicken 7S Ig and IgM allotypes present as polymorphic markers.     

MHC variation in birds and reptiles

The major histocompatibility complex (MHC) has been studied in a multitude of mammals by now, but much less is known about its organisation and variation in other vertebrate species. The mammalian MHC is organised as a single gene cluster, but recent studies on birds suggest that this paradigm of MHC organisation has to be supplemented. The domestic chicken thus possesses two separate gene clusters which both contain MHC class I and class II B genes, and we have shown that the ring‐necked pheasant Phasianus colchicus also has two unlinked clusters of class II B genes. We are studying the effect of the MHC on mate choice, survival and reproductive success in natural populations of birds and reptiles. For this reason, we are developing DNA techniques to determine the animals' MHC genotype. The amplification of the hypervariable exon 3 of the class I gene from songbirds and reptiles has provided us with species specific probes that can be used in Southern blot analysis. The first results indicate very extensive variation in all studied species, that is starlings Sturnus vulgaris, great reed warblers Acrocephalus arundinaceus and water pythons Liasis fuscus. The restriction fragment length polymorphism (RFLP) analysis also suggests that the number of MHC genes is significantly larger in these species than in pheasants and domestic chickens. This revised version was published online in July 2006 with corrections to the Cover Date.     

Reproduction of wild birds via interspecies germ cell transplantation

The present study was conducted to apply an interspecies germ cell transfer technique to wild bird reproduction. Pheasant (Phasianus colchicus) primordial germ cells (PGCs) retrieved from the gonads of 7-day-old embryos were transferred to the bloodstream of 2.5-day-old chicken (Gallus gallus) embryos. Pheasant-to-chicken germline chimeras hatched from the recipient embryos, and 10 pheasants were derived from testcross reproduction of the male chimeras with female pheasants. Gonadal migration of the transferred PGCs, their involvement in spermatogenesis, and production of chimeric semen were confirmed. The phenotype of pheasant progenies derived from the interspecies transfer was identical to that of wild pheasants. The average efficiency of reproduction estimated from the percentage of pheasants to total progenies was 17.5%. In conclusion, interspecies germ cell transfer into a developing embryo can be used for wild bird reproduction, and this reproductive technology may be applicable in conserving endangered bird species.     

Cytological evidence of maternal meiotic errors in a line of chickens with a high incidence of triploidy.

Direct evidence of the nature of maternal meiotic errors in a selected line of chickens with a high incidence of triploidy was obtained by using cytologically marked paternal gametes derived from a closely related avian species. Matings were made by artificial insemination of female chickens of the selection line and a control line with semen from ring-necked male pheasants. A total of five triploid, one pentaploid, and 21 diploid hybrid embryos were karyotyped. Each triploid hybrid embryo contained one set of paternal pheasant chromosomes and two sets of maternal chicken chromosomes, providing irrefutable cytological evidence that the triploids were derived from diploid ova produced by females of the selection line. The pentaploid hybrid contained one set of paternal pheasant chromosomes and four sets of maternal chicken chromosomes, indicating that it had been derived from a tetraploid ovum. Females of the selection line are thought to have a genetically mediated susceptibility to nondisjunction which is responsible for the high incidence of meiotic errors. Evidence is provided that the non-disjunction occurs at both meiosis I and meiosis II.     

Helper T lymphocytes which recognize the MHC class I alloantigens in vivo are CD4+CD8-1

To determine the CD4 or CD8 phenotype of the Th lymphocyte which recognizes in vivo the MHC class I alloantigens, B10 recombinant mice were treated with anti-CD8 or anti-CD4 mAb and immunized with lymphoid cells from donors differing in the K or D region of the MHC. Alloantibodies were evaluated by a 51Cr-release assay or by indirect immunofluorescence. The production of IgG anti-Dd and anti-Kk alloantibodies was increased by the deletion of the CD8+ and absent in mice depleted of the CD4+ subset. These experiments indicate that the helper influence elicited by the recognition of a MHC class I alloantigen in vivo is due to cells of the CD4+CD8- phenotype.     

Tolerance to antigen-presenting cell-depleted islet allografts is CD4 T cell dependent

Pretreatment of pancreatic islets in 95% oxygen culture depletes graft-associated APCs and leads to indefinite allograft acceptance in immunocompetent recipients. As such, the APC-depleted allograft represents a model of peripheral alloantigen presentation in the absence of donor-derived costimulation. Over time, a state of donor-specific tolerance develops in which recipients are resistant to donor APC-induced graft rejection. Thus, persistence of the graft is sufficient to induce tolerance independent of other immune interventions. Donor-specific tolerance could be adoptively transferred to immune-deficient SCID recipient mice transplanted with fresh immunogenic islet allografts, indicating that the original recipient was not simply "ignorant" of donor antigens. Interestingly, despite the fact that the original islet allograft presented only MHC class I alloantigens, CD8+ T cells obtained from tolerant animals readily collaborated with naive CD4+ T cells to reject donor-type islet grafts. Conversely, tolerant CD4+ T cells failed to collaborate effectively with naive CD8+ T cells for the rejection of donor-type grafts. In conclusion, the MHC class I+, II- islet allograft paradoxically leads to a change in the donor-reactive CD4 T cell subset and not in the CD8 subset. We hypothesize that the tolerant state is not due to direct class I alloantigen presentation to CD8 T cells but, rather, occurs via the indirect pathway of donor Ag presentation to CD4 T cells in the context of host MHC class II molecules.     

L3T4+ T-cell-independent reactivity of Lyt2+ T cells in vivo

The aim of this study was to analyze in vivo the L3T4+ T-cell-subset-independent reactivity of Lyt2+ T cells toward transplantation alloantigens. To this end, we depleted normal mice of L3T4+ T cells by injection of monoclonal antibodies to the L3T4 antigen. This procedure not only led phenotypically to a disappearance of L3T4+ T cells, but also effectively abolished reactivity toward class II MHC antigens in vitro and in vivo. However, L3T4+ T-cell-depleted mice still reacted to class I MHC alloantigens in vivo: after immunization with class I MHC alloantigens Il-2 receptor-bearing T cells appeared in the draining lymph nodes, and developed antigen-specific cytolytic activity. Moreover, upon in vivo priming the frequencies of class I MHC-specific precursors of Il-2-producing and cytolytic Lyt2+ T lymphocytes increased up to 20-fold. L3T4+ T-cell-depleted mice rejected class I MHC-bearing skin grafts promptly. We conclude that not only in vitro but also in vivo Lyt2+ T cells remain reactive toward class I MHC antigens in the absence of L3T4+ T helper cells.     

U2 RNA shares a structural domain with U1, U4, and U5 RNAs.

We previously reported common structural features within the 3'-terminal regions of U1, U4, and U5 RNAs. To check whether these features also exist in U2 RNA, the primary and secondary structures of the 3'-terminal regions of chicken, pheasant, and rat U2 RNAs were examined. Whereas no difference was observed between pheasant and chicken, the chicken and rat sequences were only 82.5% homologous. Such divergence allowed us to propose a unique model of secondary structure based on maximum base-pairing and secondary structure conservation. The same model was obtained from the results of limited digestion of U2 RNA with various nucleases. Comparison of this structure with those of U1, U4, and U5 RNAs shows that the four RNAs share a common structure designated as domain A, and consisting of a free single-stranded region with the sequence Pu-A-(U)n-G-Pup flanked by two hairpins. The hairpin on the 3' side is very stable and has the sequence Py-N-Py-Gp in the loop. The presence of this common domain is discussed in connection with relationships among U RNAs and common protein binding sites.