Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: “waiting for Godot”

Genome-wide sequence analysis in the invertebrate chordate, Ciona intestinalis, has provided a comprehensive picture of immune-related genes in an organism that occupies a key phylogenetic position in vertebrate evolution. The pivotal genes for adaptive immunity, such as the major histocompatibility complex (MHC) class I and II genes, T-cell receptors, or dimeric immunoglobulin molecules, have not been identified in the Ciona genome. Many genes involved in innate immunity have been identified, including complement components, Toll-like receptors, and the genes involved in intracellular signal transduction of immune responses, and show both expansion and unexpected diversity in comparison with the vertebrates. In addition, a number of genes were identified which predicted integral membrane proteins with extracellular C-type lectin or immunoglobulin domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motifs (ITAMs) (plus their associated signal transduction molecules), suggesting that activating and inhibitory receptors have an MHC-independent function and an early evolutionary origin. A crucial component of vertebrate adaptive immunity is somatic diversification, and the recombination activating genes (RAG) and activation-induced cytidine deaminase (AID) genes responsible for the Generation of diversity are not present in Ciona. However, there are key V regions, the essential feature of an immunoglobulin superfamily VC1-like core, and possible proto-MHC regions scattered throughout the genome waiting for Godot.     

EST analysis of the immune-relevant genes in Chinese amphioxus challenged with lipopolysaccharide

It is generally accepted that the adaptive immune system is only present in vertebrates but not in invertebrates. Amphioxus is the most basal chordate and hence is an important reference to the evolution of the adaptive immune system. Here, a cDNA library of lipopolysaccharide-challenged amphioxus was constructed in order to identify immune genes. A total of 3024 expressed sequence tags (ESTs) were examined and 63 out of 398 annotated genes (16.3%) appeared related to immunity. Most of them encode cell adhesion molecules or signal proteins that are involved in immune responses. Although the key molecules such as TCR, MHC, Ig or VLR involved in the adaptive immune system were not identified in our database, we demonstrated the presence of histocompatibility-relevant genes and lymphocyte immune signaling-relevant genes. These findings support the statement that amphioxus presents some components that may be recruited by adaptive immune processes.     

In vivo analysis of compound activity and mechanism of action using epistasis in Drosophila

The recent establishment of high-throughput methods for culturing Drosophila provided a unique ability to screen compound libraries against complex disease phenotypes in the context of whole animals. However, as compound studies in Drosophila have been limited so far, the degree of conservation of compound activity between Drosophila and vertebrates or the effectiveness of feeding as a compound delivery system is not well known. Our comprehensive in vivo analysis of 27 small molecules targeting seven signaling pathways in Drosophila revealed a high degree of conservation of compound activity between Drosophila and vertebrates. We also investigated the mechanism of action of AY9944, one of the Hh pathway antagonists that we identified in our compound feeding experiments. Our epistasis analysis of AY9944 provided novel insights into AY9944’s mechanism of action and revealed a novel role for cholesterol transport in Hh signal transduction.     

Presequence Acquisition During Secondary Endocytobiosis and thePossible Role of Introns

Targeting of nucleus-encoded proteins into chloroplasts is mediated by N-terminal presequences. During evolution of plastids from formerly free-living cyanobacteria by endocytobiosis, genes for most plastid proteins have been transferred from the plastid genome to the nucleus and subsequently had to be equipped with such plastid targeting sequences. So far it is unclear how the gene domains coding for presequences and the respective mature proteins may have been assembled. While land plant plastids are supposed to originate from a primary endocytobiosis event (a prokaryotic cyanobacterium was taken up by a eukaryotic cell), organisms with secondary plastids like diatoms experienced a second endocytobiosis step involving a eukaryotic alga taken up by a eukaryotic host cell. In this group of algae, apparently most genes encoding chloroplast proteins have been transferred a second time (from the nucleus of the endosymbiont to the nucleus of the secondary host) and thus must have been equipped with additional targeting signals. We have analyzed cDNAs and the respective genomic DNA fragments of seven plastid preproteins from the diatom Phaeodactylum tricornutum. In all of these genes we found single spliceosomal introns, generally located within the region coding for the N-terminal plastid targeting sequences or shortly downstream of it. The positions of the introns can be related to the putative phylogenetic histories of the respective genes, indicating that the bipartite targeting sequences in these secondary algae might have evolved by recombination events via introns.The nucleotide sequences have been deposited at Genbank under accession numbers AY191862, AY191863, AY191864, AY191865, AY191866, AY191867, and AY191868.     

A putative helical cytokine functioning in innate immune signalling in Drosophila melanogaster

In invertebrates and vertebrates, innate immunity is considered the first line of defense mechanism against non-self material. In vertebrates, cytokines play a critical role in innate immune signalling. To date, however, the existence of genes encoding for invertebrate helical cytokines has been anticipated, but never demonstrated. Here, we report the first structural and functional evidence of a gene encoding for a putative helical cytokine in Drosophila melanogaster. Functional experiments demonstrate that its expression, as well as that of the antimicrobial factors defensin and cecropin A1, is significantly increased after immune stimulation. These observations suggest the involvement of helical cytokines in the innate immune response of invertebrates.     

Immune Related Genes Underpin the Evolution of Adaptive Immunity in Jawless Vertebrates

The study of immune related genes in lampreys and hagfish provides a unique perspective on the evolutionary genetic underpinnings of adaptive immunity and the evolution of vertebrate genomes. Separated from their jawed cousins at the stem of the vertebrate lineage, these jawless vertebrates have many of the gene families and gene regulatory networks associated with the defining morphological and physiological features of vertebrates. These include genes vital for innate immunity, inflammation, wound healing, protein degradation, and the development, signaling and trafficking of lymphocytes. Jawless vertebrates recognize antigen by using leucine-rich repeat (LRR) based variable lymphocyte receptors (VLRs), which are very different from the immunoglobulin (Ig) based T cell receptor (TCR) and B cell receptor (BCR) used for antigen recognition by jawed vertebrates. The somatically constructed VLR genes are expressed in monoallelic fashion by T-like and B-like lymphocytes. Jawless and jawed vertebrates thus share many of the genes that provide the molecular infrastructure and physiological context for adaptive immune responses, yet use entirely different genes and mechanisms of combinatorial assembly to generate diverse repertoires of antigen recognition receptors.     

文昌鱼抗金葡菌感染差减文库的构建及免疫相关基因分析

适应性免疫的起源一直是免疫学研究的关键问题.文昌鱼被认为是最接近于脊椎动物的祖先 自从被发现以来一直是研究脊椎动物起源与进化机制的经典模式动物.为了在文昌鱼中寻找适应性免疫系统的分子证据,采用金黄色葡萄球菌感染文昌鱼以调查免疫的起源.应用抑制性差减杂交(SSH)技术,通过对差减文库克隆序列的测定,共获得588个表达序列标签(EST).对这些EST进行生物信息学分析和进一步功能分类,发现了一些免疫上调基因,如免疫调控基因、凋亡相关基因、细胞黏附相关基因、转录相关基因、信号传导相关基因等,以及一些非免疫相关基因;这些基因在文昌鱼中绝大多数为首次报道.金黄色葡萄球菌差减文库的成功构建,为调查文昌鱼抗细菌感染的分子事件提供了重要线索,对于这些新发现基因的进一步研究将有助于深入了解免疫系统起源与进化的机制.     

Molecular characterisation and expression of two venom allergen-like protein genes in Heterodera glycines

Secretory proteins encoded by genes expressed in the oesophageal gland cells of plant-parasitic nematodes have key roles in nematode parasitism of plants. Two venom allergen-like protein cDNAs (designated hg-vap-1 and hg-vap-2)were isolated from Heterodera glycines gland cell cDNA libraries. Both cDNAs hybridised to genomic DNA of H. glycines in Southern blots. The hg-vap-1 cDNA contained an open reading frame encoding 215 amino acids with the first 25 amino acids being a putative secretion signal. The hg-vap-2 cDNA contained an open reading frame encoding 212 amino acids with the first 19 amino acids being a putative secretion signal. Genes of hg-vap-1 and hg-vap-2 contained four introns, which ranged in size from 44 to 574 bp, and five exons ranging in size from 43 to 279 bp. In situ hybridisation analyses showed that mRNAs of both vap genes accumulated specifically in the subventral gland cells of H. glycines during parasitism. The gland cell-specific expression and presence of predicted secretion signal peptides in both VAPs suggest that these proteins are secreted from the nematode and may play a role in the infection of host plants by this parasite.     

东亚钳蝎毒素BmKT四个同源基因的克隆及基因组织分析

BmKT是从本室构建的cDNA文库中筛选到的1个α钠通道毒素,根据其全长cDNA序列设计引物,采用PCR法以蝎总基因组DNA为模板,获得4个BmKT的同源基因,分别命名为BmKT′和BmKTa、BmKTb、BmKTb′.序列分析表明：BmKT′和BmKTa基因含有大小分别为509 bp和506 bp的内含子,位于信号肽编码区内,插入信号肽-4位残基Gly密码子的第一个碱基G之后;而BmKTb和BmKTb′ 的内含子大小均为418 bp．这4个BmKT的同源基因内含子符合GT/AG拼接规律,其中BmKT′和BmKTa的内含子A+T含量分别为61.7%和61.9%,低于目前已报导的大多数蝎毒素基因A+T含量,大大低于它们第一外显子A+T含量（71.7%）,略高于第二外显子A+T含量（55.5%）;而BmKTb,BmKTb′的内含子A+T含量分别为75.8%和76.1%,与目前已报导的大多数蝎毒素基因A+T含量相似．BmKT′基因的外显子与BmKT基因cDNA所对应氨基酸序列仅在信号肽中-7位有一个氨基残基的差异（BmKT: Leu→BmKTa: Val）;而BmKTa基因外显子所推断的氨基酸序列与BmKT前体比较,则在成熟肽的+54位发生了突变（BmKT: Lys→BmKTa: Asn）,是与BmKT同源的一个新基因．BmKTb基因和BmKTb′基因所编码的前体肽与BmKT基因对应的前体肽同源性约为65%,显然BmKTb和BmKTb′是不同于BmKT的2个新基因（GenBank登录号: BmKT′, AY786186; BmKTa, AY676142; BmKTb, AY676140; BmKTb′, AY676141）     

A putative helical cytokine functioning in innate immune signalling in Drosophila melanogaster

In invertebrates and vertebrates, innate immunity is considered the first line of defense mechanism against non-self material. In vertebrates, cytokines play a critical role in innate immune signalling. To date, however, the existence of genes encoding for invertebrate helical cytokines has been anticipated, but never demonstrated. Here, we report the first structural and functional evidence of a gene encoding for a putative helical cytokine in Drosophila melanogaster. Functional experiments demonstrate that its expression, as well as that of the antimicrobial factors defensin and cecropin A1, is significantly increased after immune stimulation. These observations suggest the involvement of helical cytokines in the innate immune response of invertebrates.     

Evidence for Hox Gene Duplication in Rainbow Trout (Oncorhynchus mykiss): A Tetraploid Model Species

We examined the genomic organization of Hox genes in rainbow trout (Oncorhynchus mykiss), a tetraploid teleost derivative species, in order to test models of presumptive genomic duplications during vertebrate evolution. Thirteen putative clusters were localized in the current rainbow trout genetic map; however, analysis of the sequence data suggests the presence of at least 14 Hox clusters. Many duplicated genes appear to have been retained in the genome and share a high percentage of amino acid similarity with one another. We characterized two Hox genes located within the HoxCb cluster that may have been lost independently in other teleost species studied to date. Finally, we identified conserved syntenic blocks between salmonids and human, and provide data supporting two new linkage group homeologies (i.e., RT-3/16, RT-12/29) and three previously described homeologies (RT-2/9, RT-17/22, and RT-27/31) in rainbow trout. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. The sequence data for this study have been submitted to GenBank under the following accession numbers: AY567792, AY567793, AY567794, AY567795, AY567796, AY567797, AY567798, AY567799, AY567800, AY567801, AY567802, AY567803, AY567804, AY567805, AY567806, AY567807, AY567808, AY567809, AY567810, AY567812, AY567813, AY567814, AY567815, AY567816, and AY567817. [Reviewing Editor : Dr. Axel Meyer]     

Evolution of Hox Clusters in Salmonidae: A Comparative Analysis Between Atlantic Salmon (Salmo salar) and Rainbow Trout (Oncorhynchus mykiss)

We studied the genomic organization of Hox genes in Atlantic salmon (Salmo salar) and made comparisons to that in rainbow trout (Oncorhynchus mykiss), another member of the family Salmonidae. We used these two species to test the hypothesis that the Hox genes would provide evidence for a fourth round of duplication (4R) of this gene family given the recent polyploid ancestry of the salmonid fish. Thirteen putative Hox clusters were identified and 10 of these complexes were localized to the current Atlantic salmon genetic map. Syntenic regions with the rainbow trout linkage map were detected and further homologies and homeologies are suggested. We propose that the common ancestor of Atlantic salmon and rainbow trout possessed at least 14 clusters of Hox genes, and additional clusters cannot be ruled out. Salmonid Hox cluster complements seem to be more similar to those of zebrafish (Danio rerio) than medaka (Oryzias latipes) or pufferfish (Sphoeroides nephelus and Takifugu rubripes), as both Atlantic salmon and rainbow trout have retained HoxCb ortholog, which has been lost in medaka and pufferfish but not in zebrafish. However, our data suggest that phylogenetically, the homologous genes within each cluster express mosaic relationships among the teleosts tested and, thus, leave unresolved the interfamilial relationships among these taxa. Sequence data from this article have been deposited within the EMBL/GenBank Data Libraries under the following accession numbers: AY677341, AY677342, AY677343, AY677344, AY677345, AY677346, AY677347, AY677348, AY677349, AY677350, AY677351, AY677352, AY677353, AY677354, AY677355, AY677356, AY677357, AY677358, AY677359, AY677360, AY677361, AY677362, AY677363, AY677364 and AY677365. [Reviewing Editor: Dr. Axel Meyer]     

Antigen-specific T-cell responses to a recombinant fowlpox virus are dependent on MyD88 and interleukin-18 and independent of Toll-like receptor 7 (TLR7)- and TLR9-mediated innate immune recognition

Fowlpox virus (FWPV) is a double-stranded DNA virus long used as a live-attenuated vaccine against poultry diseases, but more recent interest has focused on its use as a mammalian vaccine vector. Here, in a mouse model system using FWPV encoding the nominal target antigen chicken ovalbumin (OVA) (FWPV(OVA)), we describe for the first time some of the fundamental processes by which FWPV engages both the innate and adaptive immune systems. We show that Toll-like receptor 7 (TLR7) and TLR9 are important for type I interferon secretion by dendritic cells, while TLR9 is solely required for proinflammatory cytokine secretion. Despite this functional role for TLR7 and TLR9 in vitro, only the adapter protein myeloid differentiation primary response gene 88 (MyD88) was shown to be essential for the formation of adaptive immunity to FWPV(OVA) in vivo. The dependence on MyD88 was confined only to the T-cell compartment and was not related to its contribution to TLR signaling, dendritic cell maturation, or the capture and presentation of FWPV-derived OVA antigen. We demonstrate that this is not by means of mediating T-cell-dependent interleukin-1 (IL-1) signaling, but rather, we suggest that MyD88 functions to support T-cell-specific IL-18 receptor signaling, which in turn is essential for the formation of adaptive immunity to FWPV-encoded OVA.     

Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface

The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and α-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated. The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the α-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast α factor, respectively, were fused with the gene encoding the C-terminal half of the yeast α-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and α-amylase activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying glucoamylase and α-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase. Received: 16 June 1998 / Received last revision: 21 August 1998 / Accepted: 3 September 1998     

Molecular cloning,characterization and expression analysis of MHCI and chemokines CXCR3 and CXCR4 gene from freshwater carp,Catla catla

The immune system with large number of molecules protects the host against a plethora of continuously evolving microbes. Major histocompatibility complex (MHC) molecules serve as cardinal elements of the adaptive immune system responsible for the activation of the adaptive immunity in the host. The present study reports MHCI molecule in freshwater carp, Catla catla, and its differential expression in immunologically relevant tissues post‐infection with Gram‐negative and Gram‐positive bacteria. The MHCI sequence of C. catla had 502 bp nucleotides encoding putative 146 amino acids. The phylogenetic analysis exhibited its evolutionary conservation within the Cyprinidae family and formed a different clade with the higher vertebrates. Simultaneously, CXCR3 and CXCR4 chemokines were cloned and characterized for their expression in infected tissues. Analysis of immunologically relevant tissues of the infected fish exhibited an increase of MHCI gene expression and the down‐regulation of CXCR3 and CXCR4 chemokines, indicating a tricky interaction between the innate and adaptive immune system. It was found that intestine, skin and spleen played a crucial role in the contribution of the defense activity which instigated the self‐immunity. These immune activities can provide useful information to understand the interaction of self and non‐self‐ immune system in freshwater fish, Catla catla.     

无颌类脊椎动物适应性免疫系统的进化

适应性免疫系统的起源与进化问题一直是人们研究的热点, 以七鳃鳗为代表的无颌类脊椎动物, 被普遍认为处在进化出适应性免疫系统的边缘。因此, 研究无颌类脊椎动物适应性免疫的机制, 对揭示适应性免疫系统的起源与进化具有重要意义。研究表明, 无颌类在一定范围内具有高等脊椎动物特有的适应性免疫特征, 并发现了一些在结构或功能上与高等脊椎动物免疫相关基因同源的免疫因子。文章就近年来对无颌类脊椎动物适应性免疫系统机制的研究进展作一概述, 为进一步深入研究脊椎动物适应性免疫系统的起源与进化提供有益的参考。     

Normal and neoplastic lymphocyte maturation

Lymphocytes arc cells that are responsible for processes of specific antigen recognition and for those aspects of the immune response that characterize adaptive immunity. In this respect adaptive immunity can be characterized as antigen-induced immune memory and effector functions as compared to native immunity – the nonspecific phagocytic and humoral protective elements in lower vertebrates. In vertebrates both B and T lymphocytes apparently express self-synthesized receptors that (1) are involved in the recognition of antigens, and (2) mediate the interactions between various important cells in the hematolymphoid system. There are three major subclasses of T lymphocytes – those involved with helper/inducer functions, those involved with suppressor functions, and those involved in direct cytotoxicity of antigenic target cells [1,2].     

The immune gene repertoire encoded in the purple sea urchin genome

Echinoderms occupy a critical and largely unexplored phylogenetic vantage point from which to infer both the early evolution of bilaterian immunity and the underpinnings of the vertebrate adaptive immune system. Here we present an initial survey of the purple sea urchin genome for genes associated with immunity. An elaborate repertoire of potential immune receptors, regulators and effectors is present, including unprecedented expansions of innate pathogen recognition genes. These include a diverse array of 222 Toll-like receptor (TLR) genes and a coordinate expansion of directly associated signaling adaptors. Notably, a subset of sea urchin TLR genes encodes receptors with structural characteristics previously identified only in protostomes. A similarly expanded set of 203 NOD/NALP-like cytoplasmic recognition proteins is present. These genes have previously been identified only in vertebrates where they are represented in much lower numbers. Genes that mediate the alternative and lectin complement pathways are described, while gene homologues of the terminal pathway are not present. We have also identified several homologues of genes that function in jawed vertebrate adaptive immunity. The most striking of these is a gene cluster with similarity to the jawed vertebrate Recombination Activating Genes 1 and 2 (RAG1/2). Sea urchins are long-lived, complex organisms and these findings reveal an innate immune system of unprecedented complexity. Whether the presumably intense selective processes that molded these gene families also gave rise to novel immune mechanisms akin to adaptive systems remains to be seen. The genome sequence provides immediate opportunities to apply the advantages of the sea urchin model toward problems in developmental and evolutionary immunobiology.