The expression of Mls c determinants on Mls a,Mls b,and Mls x prototypic strains

In the mouse sytem, specific determinants other than major histocompatibility complex (MHC) gene products are capable of inducing strong primary proliferative responses in naive T cells. These determinants are encoded by at least two gene loci designated as minor lymphocyte stimulatory (Mls) loci. In order to elucidate the biological role of the Mls system, an effort has been initiated to clarify the fundamental immunogenetic characteristics of the Mls system. In this report, we describe the unexpected finding that Mls ^c determinants are expressed on splenocytes of strains including those which have been used as prototypic examples of three other Mls types: Mls ^a (DBA/2, DBA/1), Mls ^b , (BALB/c), and Mls ^x (PL/J). The expression of Mls ^c by these strains was demonstrated both by the response patterns of unprimed T cells from MHC-identical inbred or F₁ hybrid strains and by the responses of a panel of Mls-specific T-cell clones. The experimental results reported here also suggest that the expression of Mls determinants may be influenced by multiple other genes, including MHC-linked genes.Abbreviations used in this paper MHC major histocompatibility complex - MLR mixed lymphocyte reaction - Mls minor lymphocyte stimulating locus antigen - MMC mitomycin C - NNT nylon wool nonadherent T cells     

Multi-gene/allele control of Mls b of CBA/H

Festenstein originally described the Mls locus as a single dominant autosomal gene with four alleles which mapped in the 13th linkage group of chromosome 1. We subsequently presented evidence indicating that the mixed leukocyte reaction (MLR) stimulatory products of DBA/2 and CBA/J were controlled by two independently segregating Mls loci and that Mls of C3H was in fact a composite of three independently segregating loci. Recently, Mls ^d of CBA/J was shown to be composed of Mls ^a of AKR and a product on C3H, which was presumed to be Mls ^c . Based on strain distributions, this product cannot be encoded by the Mls ^c originally defined by Festenstein. In the present report, three Mls specificities of CBA/H (Mls ^b ) are defined. Based on the strain distribution, we postulate that these specificities are controlled by three loci, three alleles/locus, or by some combination of the preceding two possibilities.     

Mls genes and self-superantigens.

The Mls gene products, which have long been known for their potent T-cell stimulatory function, have recently come of age through two pivotal discoveries, revealing that they act as superantigens and originate from retroviruses. In addition, recent experiments suggest that two retroviruses, the murine B-type mammary tumor virus and the human lentivirus HIV, make use of the T-cell stimulatory capacity of a virally encoded superantigen for facilitating viral replication.     

Genetic analysis of the Mls system. Formal Mls typing of the commonly used inbred strains

In order to elucidate the biological role of minor lymphocyte stimulating (Mls) gene products, we have been investigating the fundamental immunogenetic characteristics of the Mls system. In this report, describe the distribution of stimulatory Mls products, Mls^a and Mls^c, in a panel of laboratory inbred strains based on the response pattern of H-2-compatible naive T-cell populations as well as monospecific Mls^a- or Mls^c-reactive T-cell clones. In addition, the expression of four different T-cell receptor (Tcr) b-V segment Tcrb-V3, –V6, –V8.1, and –V9, which were recently reported to be associated with T-cell recognition of Mls gene products in these strains, was examined. The results indicate that the majority of commonly used laboratory strains including those originally typed as Mls ^aare also expressing Mls^c determinants and that very few independent inbred strains are non-Mls ^c. Moreover, the pattern of Tcrb-V expression in spleen as well as in thymus suggests that the association between Mls expression and clonal deletion of self Mls-reactive T cells appears to be the general rule in inbred strains. Based on these results, implications for the nondetectable Mls-like gene products in other species besides the mouse are discussed.     

Specific neonatal induction of functional tolerance to allogeneic Mls determinants occurs intrathymically and the tolerant state is Mls haplotype-specific

The studies described show that functional Mls specific tolerance, which we previously reported in peripheral spleen cells of mice injected within 24 h of birth with Mls incompatible spleen cells, is observable in the thymus on day 6. At this time a significant positive response is not detectable in spleen cells of normal mice. In the limiting dilution assay, we are able to detect a more profound depletion than others have found with anti-TCR antibodies. The tolerance in the thymus is not due to active suppression or simple dilution of responders by nonresponsive cells of the neonatal inoculum. By tolerizing BALB/c (Mls(b,c] mice with spleen cells from Mls(a) congenic mice, we show that Mls(a) incompatibility alone is sufficient for tolerance induction. Data from these experiments also show that the T cells seen responding at high frequency to stimulators from mice expressing Mls(a) determinants, as well as many other non-H-2 encoded incompatibilities, are indeed responding to Mls(a) determinants. In addition, experiments involving neonatal injection of Mls(b) mice with Mls(a) and Mls(c) spleen cells show no cross-reactivity of tolerance between Mls(a) and Mls(c) haplotypes. Our findings also show coexpression of determinants common to both Mls(a) and Mls(c) haplotypes by the Mls(d) haplotype. In all, the described experiments elucidate a pattern of Mls determinant specific hyporesponsiveness, in mice neonatally injected with appropriate allogeneic spleen cells, which bears all the hallmarks of functional, alloantigen specific, clonal deletion type tolerance.     

MHC recognition by clones of Mls specific T-lymphocytes

To test whether M1s determinants, like other non-MHC or nominal antigens, are recognized by T-cells in association with H-2 determinants, the in vitro proliferative responses of T-cell lines and clones were studied. Lines and clones were prepared by soft agar cloning (B10.BR x BALB/c)F₁ (H-2^k/H-2^d, M1s^b/M1s^b) T-cells responding in a primary MLR to AKD2F1 (H-2^k/H-2^d, M1s^a/M1s^a) stimulator cells. All the T-cell clones obtained could respond equally well in a proliferative assay to the Mls^a determinant in association with the H-2 haplotype of either parent, i. e., DBA/2 (H-2^d, M1s^a), and AKR (H-2^k, M1s^a) both stimulated equally well. When the T-cell lines and clones were screened against stimulators from recombinant inbred (RI) strains, it became apparent that strains exhibiting the H-2^b, M1s^a genotype stimulated poorly or not at all. This shows that the T-cell response to M1s^a involves MHC recognition, and raises the possibility that the response to M1s^a can involve recognition of H-2 specificities shared between the H-2 ^k and H-2 ^d haplotypes.Abbreviations used in this paper MHC major histocompatibility complex - MLC mixed lymphocyte culture - IL-2 interleukin 2 - Con A concanavalin A - RI recombinant inbred Howard Hughes Medical Institute     

Allostimulatory analysis of a newly-defined and widely-distributed Mls superantigen

We previously noted that Mls^a,c C58/J responder cells proliferated unexpectedly to H-2^k-compatible Mls^a or Mls^c prototypic stimulator cells in a primary mixed lymphocyte reaction. The present investigation was performed to evaluate whether the response of C58/J T cells to these H-2- and Mls-compatible stimulator cells could functionally identify a newly-defined member of the Mls superantigen family through its allostimulatory ability. We observed that C58/J responder cells also proliferated when cultured with H-2-compatible prototypic Mls^null, Mls^b (nonstimulatory), or Mls^a,c splenic stimulator cells. The widely distributed nature of the non-MHC ligand recognized by C58/J T cells is indicated by the finding that 11 of 12 H-2^k inbred mouse strains clearly expressed this specificity. A gradient of stimulatory capacity from low to high across this newly-defined non-MHC difference was detected with splenocytes from these different inbred mouse strains. The Mls^a,c genetic composition of C58/J was confirmed by the observation that crossing C58/J with parental B10.BR (responsive to both Mls^a and Mls^c determinants) generated F₁ progeny that were unresponsive to H-2^k-compatible Mls^a, Mls^c, or Mls^a,c stimulator cells. Like prototypic Mls^a and Mls^c, the non-MHC specificity recognized by C58/J responder cells, termed Mls^f, was particularly sensitive to radiation (versus mitomycin C) treatment of the stimulator cells, was greatly augmented after anti-IgD activation of splenic stimulator cells, was blocked with anti-MHC class II antibody, and was effectively presented by phenotypically normal female but not B cell-defective xid⁺ male CBA/N F₁ stimulator cells. Address correspondence and offprint requests to: J. J. Ryan     

Specific neonatally induced tolerance to Mls locus determinants

Neonatal injection of CBA/HT6T6 (H-2k, Mlsb) mice with adult, Mls-incompatible (CBA/J [H-2k, Mlsd] X CBA/HT6T6)F1 spleen cells results in the abrogation of cell proliferation and interleukin 2 (IL 2) production in bulk mixed lymphocyte cultures, when spleen cells from the inoculated mice are tested at 6 to 8 wk of age with stimulator cells expressing the Mlsd of the tolerizing inoculum. In limiting dilution assays, this tolerant state was manifested in a 25- to 550-fold (280-fold average) decrease in the frequency of precursors of Mlsd-responsive IL 2-producing T cells. Tolerance was specific in that the frequencies of precursors of IL 2-producing cells responding to Con A, allogeneic H-2d, and self-Ia were not affected. The observed low frequency of Mls-responsive cells was due neither to extensive chimerism resulting in the dilution of Mlsd-responsive cells by the nonresponsive F1 cells of the inoculum, nor to the action of suppressor cells. These findings indicate that neonatal injection of Mls-incompatible spleen cells produces a state of specific tolerance by a clonal deletion or inactivation mechanism. This specific tolerance supports the view that 1) the Mls locus encodes or regulates the expression of defined alloantigenic determinants and 2) Mls-incompatible responder mice have specific receptors for Mls determinants on clonally distributed IL 2-producing responder T cells.     

Multigene families and the evolution of complexity

Summary Higher organisms are complex, and their developmental processes are controlled by the sequential expression of genes that often form multigene families. Facts are surveyed on how functional diversity of genes is related to duplication of genes or segments of genes, by emphasizing that diversity is often enhanced by alternate splicing and proteolytic cleavage involving duplicated genes or gene segments. Analyses of a population genetics model for the origin of gene families suggest that positive Darwinian selection is needed for acquiring gene families with desirable functions. Based on these considerations, examples that show acceleration of amino acid substitution relative to synonymous change during evolutionary processes are surveyed. Some of such examples strongly suggest that positive selection has worked. In other cases it is difficult to judge whether or not acceleration is caused by positive Darwinian selection. As a general pattern, acceleration of amino acid substitution is often found to be related to gene duplication. It is thought that complexity and diversity of gene function have been advantageous in the long evolutionary course of higher organisms.     

链霉菌ACCC40021的多基因系统进化分析

链霉菌ACCC40021是尹莘耘1953年分离筛选,用于农业生产的抗生菌肥。该菌株1979年经鉴定为泾阳链霉菌(Streptomyces jingyangensis),但该菌株名称一直没有在国际上得到有效发表。为了在分子水平上明确该菌株的分类地位,对ACCC40021的菌株进行了16SrRNA和gyrB,recA,rpoB和trpB等基因的进化分析,将ACCC40021鉴定为黄赭色链霉菌(Streptomyces silaceus)。     

植物多基因转化研究进展

通过转基因技术改良植物品质近几年已成为热点研究问题,基因工程不断发展,单基因转化技术已不能满足人们对植物改良的需要。更多的研究者投身于参与某个代谢途径的多个基因在植物体中共同表达的研究,通过多基因调控来获得更好的植物性状。基因的协调表达有四种研究思路,在此基础上多基因转化方法可概述为传统转化法、改进后的共转化法,及新兴的基因融合方法,综合分析每种方法在植物代谢调控中的优缺点与应用,并探讨多基因整合的不稳定及相互作用问题。     

Multigene analysis of lophophorate and chaetognath phylogenetic relationships

Maximum likelihood and Bayesian inference analyses of seven concatenated fragments of nuclear-encoded housekeeping genes indicate that Lophotrochozoa is monophyletic, i.e., the lophophorate groups Bryozoa, Brachiopoda and Phoronida are more closely related to molluscs and annelids than to Deuterostomia or Ecdysozoa. Lophophorates themselves, however, form a polyphyletic assemblage. The hypotheses that they are monophyletic and more closely allied to Deuterostomia than to Protostomia can be ruled out with both the approximately unbiased test and the expected likelihood weights test. The existence of Phoronozoa, a putative clade including Brachiopoda and Phoronida, has also been rejected. According to our analyses, phoronids instead share a more recent common ancestor with bryozoans than with brachiopods. Platyhelminthes is the sister group of Lophotrochozoa. Together these two constitute Spiralia. Although Chaetognatha appears as the sister group of Priapulida within Ecdysozoa in our analyses, alternative hypothesis concerning chaetognath relationships could not be rejected.     

The role of H-2 in T cell recognition of Mls

The role of H-2 was evaluated in T cell recognition of Mls-encoded antigens during primary mixed lymphocyte responses (MLR). Mlsc was used as a stimulating determinant in MLR and its recognition by T cells was assessed by linear regression analysis under culture conditions in which (A x B)F1 responder cell number was the factor limiting total response. Results of such experiments indicated the presence of distinct (A x B)F1 responder T cell subpopulations capable of differentially recognizing the foreign Mls antigen in association with one or the other parental H-2 haplotype. These findings demonstrate that T cells do not recognize Mlsc products in isolation, but rather are restricted to recognition of Mlsc in the context of "self" H-2 determinants.     

Multigene phylogeny of choanozoa and the origin of animals

Animals are evolutionarily related to fungi and to the predominantly unicellular protozoan phylum Choanozoa, together known as opisthokonts. To establish the sequence of events when animals evolved from unicellular ancestors, and understand those key evolutionary transitions, we need to establish which choanozoans are most closely related to animals and also the evolutionary position of each choanozoan group within the opisthokont phylogenetic tree. Here we focus on Ministeria vibrans, a minute bacteria-eating cell with slender radiating tentacles. Single-gene trees suggested that it is either the closest unicellular relative of animals or else sister to choanoflagellates, traditionally considered likely animal ancestors. Sequencing thousands of Ministeria protein genes now reveals about 14 with domains of key significance for animal cell biology, including several previously unknown from deeply diverging Choanozoa, e.g. domains involved in hedgehog, Notch and tyrosine kinase signaling or cell adhesion (cadherin). Phylogenetic trees using 78 proteins show that Ministeria is not sister to animals or choanoflagellates (themselves sisters to animals), but to Capsaspora, another protozoan with thread-like (filose) tentacles. The Ministeria/Capsaspora clade (new class Filasterea) is sister to animals and choanoflagellates, these three groups forming a novel clade (filozoa) whose ancestor presumably evolved filose tentacles well before they aggregated as a periciliary collar in the choanoflagellate/sponge common ancestor. Our trees show ichthyosporean choanozoans as sisters to filozoa; a fusion between ubiquitin and ribosomal small subunit S30 protein genes unifies all holozoa (filozoa plus Ichthyosporea), being absent in earlier branching eukaryotes. Thus, several successive evolutionary innovations occurred among their unicellular closest relatives prior to the origin of the multicellular body-plan of animals.     

Multigene DNA prime-boost vaccines for SHIV89.6P

We assessed four prime-boost vaccine regimens with a Gene Gun component for SHIV89.6P in Macaca nemestrina. A dosing experiment using beta-galactosidase plasmid showed that 30 or 45 shots per dose elicited higher titer antibody than smaller doses. For SHIV89.6P, we administered a six-plasmid vaccine capable of producing non-infectious virions in vivo in combination with either vaccinia recombinants or inactivated virus. DNA prime/vaccinia boost, or the reverse, elicited strong immune responses. The SHIV89.6P challenge virus was grown in M. nemestrina peripheral blood mononuclear cells and titered in vivo intrarectally. As has been observed for SHIV89.6P in M. mulatta, the infected M. nemestrina experienced rapid and severe loss of circulating CD4+ T cells. Vaccinated macaques were challenged three weeks after the last boost. DNA prime/vaccina boost or vaccina prime/DNA boost protected 11/12 animals from acute CD4+ T cell depletion and disease, while other regimens were not effective.     

载脂蛋白多基因家族分子进化的研究

与脂质运输有关的载脂蛋白基因构成一个复杂的多基因家族。为探讨这种演化时间长的基因家族的进化规律,本文首先建立了一种在非均衡进化速率条件下计算系统发生树中任意分支长度的简易方法,并可在此基础上算出无根分支系统树中分歧年代的期望值。进一步对本文科１０个种属共２６种载脂蛋白的系统演作作了实际分析,结果提示：①ＡｐｏＡ－Ｉ＇ＡｐｏＡ－ＩＶ,ＡｐｏＥ及ＡｐｏＡ－ＩＩ的共同祖先可能在奥陶纪水生脊椎动物中就已存