Identification of low-abundance differentially expressed transcripts using arrayed cDNA clones.

Analysis of comparative gene expression by the use of DNA microarrays has become a widely used tool. However, this technique is only readily applied to organisms where sequence information is known. This paper describes the development of a low-cost method of gene discovery by enrichment of differentially expressed transcripts, which uses cDNA library arrays of bacterial clones on nylon membranes (macroarrays) coupled with a subtractive probe preparation method to discover differentially expressed genes. The method requires no prior knowledge of the organism's genome sequence and overcomes the inherent insensitivity of standard methods of macroarray hybridisation.

Recovery of developmentally defined gene sets from high-density cDNA macroarrays.

New technologies for isolating differentially expressed genes from large arrayed cDNA libraries are reported. These methods can be used to identify genes that lie downstream of developmentally important transcription factors and genes that are expressed in specific tissues, processes, or stages of embryonic development. Though developed for the study of gene expression during the early embryogenesis of the sea urchin Strongylocentrotus purpuratus, these technologies can be applied generally. Hybridization parameters were determined for the reaction of complex cDNA probes to cDNA libraries carried on six nylon filters, each containing duplicate spots from 18,432 bacterial clones (macroarrays). These libraries are of sufficient size to include nearly all genes expressed in the embryo. The screening strategy we have devised is designed to overcome inherent sensitivity limitations of macroarray hybridization and thus to isolate differentially expressed genes that are represented only by low-prevalence mRNAs. To this end, we have developed improved methods for the amplification of cDNA from small amounts of tissue (as little as approximately 300 sea urchin embryos, or 2 x 10(5) cells, or about 10 ng of mRNA) and for the differential enhancement of probe sequence concentration by subtractive hybridization. Quantitative analysis of macroarray hybridization shows that these probes now suffice for detection of differentially expressed mRNAs down to a level below five molecules per average embryo cell.

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.

A sea urchin genome project: sequence scan, virtual map, and additional resources.

Results of a first-stage Sea Urchin Genome Project are summarized here. The species chosen was Strongylocentrotus purpuratus, a research model of major importance in developmental and molecular biology. A virtual map of the genome was constructed by sequencing the ends of 76,020 bacterial artificial chromosome (BAC) recombinants (average length, 125 kb). The BAC-end sequence tag connectors (STCs) occur an average of 10 kb apart, and, together with restriction digest patterns recorded for the same BAC clones, they provide immediate access to contigs of several hundred kilobases surrounding any gene of interest. The STCs survey >5% of the genome and provide the estimate that this genome contains approximately 27,350 protein-coding genes. The frequency distribution and canonical sequences of all middle and highly repetitive sequence families in the genome were obtained from the STCs as well. The 500-kb Hox gene complex of this species is being sequenced in its entirety. In addition, arrayed cDNA libraries of >10(5) clones each were constructed from every major stage of embryogenesis, several individual cell types, and adult tissues and are available to the community. The accumulated STC data and an expanding expressed sequence tag database (at present including >12, 000 sequences) have been reported to GenBank and are accessible on public web sites.

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.

A novel multigene family encodes diversified variable regions.

Antigen recognition in the adaptive immune response by Ig and T-cell antigen receptors (TCRs) is effected through patterned differences in the peptide sequence in the V regions. V-region specificity forms through genetically programmed rearrangement of individual, diversified segmental elements in single somatic cells. Other Ig superfamily members, including natural killer receptors that mediate cell-surface recognition, do not undergo segmental reorganization, and contain type-2 C (C2) domains, which are structurally distinct from the C1 domains found in Ig and TCR. Immunoreceptor tyrosine-based inhibitory motifs that transduce negative regulatory signals through the cell membrane are found in certain natural killer and other cell surface inhibitory receptors, but not in Ig and TCR. In this study, we employ a genomic approach by using the pufferfish (Spheroides nephelus) to characterize a nonrearranging novel immune-type receptor gene family. Twenty-six different nonrearranging genes, which each encode highly diversified V as well as a V-like C2 extracellular domain, a transmembrane region, and in most instances, an immunoreceptor tyrosine-based inhibitory motif-containing cytoplasmic tail, are identified in an approximately 113 kb P1 artificial chromosome insert. The presence in novel immune-type receptor genes of V regions that are related closely to those found in Ig and TCR as well as regulatory motifs that are characteristic of inhibitory receptors implies a heretofore unrecognized link between known receptors that mediate adaptive and innate immune functions.

Origins of immunity: transcription factors and homologues of effector genes of the vertebrate immune system expressed in sea urchin coelomocytes.

Echinoderms share common ancestry with the chordates within the deuterostome clade. Molecular features that are shared between their immune systems and that of mammals thus illuminate the basal genetic framework on which these immune systems have been constructed during evolution. The immune effector cells of sea urchins are the coelomocytes, whose primary function is protection against invasive marine pathogens; here we identify six genes expressed in coelomocytes, homologues of which are also expressed in cells of the mammalian immune system. Three coelomocyte genes reported here encode transcription factors. These are an NFKB homologue (SpNFKB); a GATA-2/3 homologue (SpGATAc); and a runt domain factor (SpRunt-1). All three of these coelomocyte genes respond sharply to bacterial challenge: SpNFKB and SpRunt-1 genes are rapidly up-regulated, while transcripts of SpGATAc factor disappear within hours of injection of bacteria. Sham injection also activates SpNFKB and SpRunt, though with slower kinetics, but does not affect SpGATAc levels. Another gene, SpHS, encodes a protein related to the signal transduction intermediate HS1 of lymphoid cells. Two other newly discovered genes, SpSRCR1 and SpSRCR5, encode proteins featuring SRCR repeats. These genes are members of a complex family of SRCR genes all expressed specifically in coelomocytes. The SRCR repeats most closely resemble those of mammalian macrophage scavenger receptors. Remarkably, each individual sea urchin expresses a specific pattern of SRCR genes. Our results imply some shared immune functions and more generally, a shared regulatory architecture which underlies immune system gene expression in all deuterostomes. We conclude that the vertebrate immune system has evolved by inserting new genes into old gene regulatory networks dedicated to immunity.

Evolution of antigen binding receptors.

This review addresses issues related to the evolution of the complex multigene families of antigen binding receptors that function in adaptive immunity. Advances in molecular genetic technology now permit the study of immunoglobulin (Ig) and T cell receptor (TCR) genes in many species that are not commonly studied yet represent critical branch points in vertebrate phylogeny. Both Ig and TCR genes have been defined in most of the major lineages of jawed vertebrates, including the cartilaginous fishes, which represent the most phylogenetically divergent jawed vertebrate group relative to the mammals. Ig genes in cartilaginous fish are encoded by multiple individual loci that each contain rearranging segmental elements and constant regions. In some loci, segmental elements are joined in the germline, i.e. they do not undergo genetic rearrangement. Other major differences in Ig gene organization and the mechanisms of somatic diversification have occurred throughout vertebrate evolution. However, relating these changes to adaptive immune function in lower vertebrates is challenging. TCR genes exhibit greater sequence diversity in individual segmental elements than is found in Ig genes but have undergone fewer changes in gene organization, isotype diversity, and mechanisms of diversification. As of yet, homologous forms of antigen binding receptors have not been identified in jawless vertebrates; however, acquisition of large amounts of structural data for the antigen binding receptors that are found in a variety of jawed vertebrates has defined shared characteristics that provide unique insight into the distant origins of the rearranging gene systems and their relationships to both adaptive and innate recognition processes.

Organization of an echinoderm Hox gene cluster.

The Strongylocentrotus purpuratus genome contains a single ten-gene Hox complex >0.5 megabase in length. This complex was isolated on overlapping bacterial artificial chromosome and P1 artificial chromosome genomic recombinants by using probes for individual genes and by genomic walking. Echinoderm Hox genes of Paralog Groups (PG) 1 and 2 are reported. The cluster includes genes representing all paralog groups of vertebrate Hox clusters, except that there is a single gene of the PG4-5 types and only three genes of the PG9-12 types. The echinoderm Hox gene cluster is essentially similar to those of the bilaterally organized chordates, despite the radically altered pentameral body plans of these animals.

A long form of the skate IgX gene exhibits a striking resemblance to the new shark IgW and IgNARC genes.

Differential screening has been used to identify cDNAs encoding a long form of IgX in Raja eglanteria (clearnose skate). Comparisons of the IgX long form with the previously described short-form IgX cDNAs and the genomic IgX locus indicate that the V and two 5' C regions of the short and long forms of IgX are >90% identical at the nucleotide level. Differences between the V sequences of the long- and short-form IgX genes are concentrated in complementarity determining regions, suggesting that these forms are derived through alternative splicing of the same genomic loci or transcription of highly related loci. The extreme conservation of nucleotide sequence, including third position codons, among different cDNAs as well as the near identity of nucleotide sequence in the intervening sequences of germline IgX, IgX short-form sterile transcripts and IgX long-form sterile transcripts indicate that the multiple IgX loci are recently diverged from one another and/or are under intense gene correction. Phylogenetic analyses of the known cartilaginous fish immunoglobulin loci demonstrate that the long form of IgX is orthologous to IgW/IgNARC (NARC) and is most consistent with: 1) the divergence of the IgX/IgW/NARC and IgM-like loci from a common ancestral locus prior to the divergence of the cartilaginous/bony fish lineages and 2) the divergence of the NAR locus from the IgX/IgW/NARC gene(s) after the cartilaginous/bony fish split but prior to the shark/skate split, approximately 220 million years ago.

Distinct patterns of IgH structure and organization in a divergent lineage of chrondrichthyan fishes.

Immunoglobulin heavy chain (IgH) genes in representative chondrichthyan fishes (sharks and skates) consist of independently functioning clusters, containing separate variable (VH), diversity (DH), and joining (JH) region elements and constant (CH) region exons. IgH loci have been characterized in Hydrolagus colliei (spotted ratfish), a modern representative of a major independent chondrichthyan lineage. Three distinct families of IgH gene clusters were identified. The most numerous genes consist of unjoined VH-D1-D2-JH segments that correspond to the most abundant Hydrolagus spleen (cDNA) transcripts which apparently arise from a diversified gene family. In the second cluster type, VH, DH, and JH segments are germline-joined, whereas the CH exons exhibit typical organization. This gene type is found in only a few copies per haploid genome and both transmembrane and secretory transcripts have been identified. A third cluster type has been identified that consists of unjoined VH elements but lacks a typical CH1 exon, which is substituted with a second CH2-like exon. Transcripts from this third cluster type also appear to derive from a diversified gene family. Genomic D regions of the two unjoined clone types exhibit structural differences that are consistent with incorporation of recombination machinery-mediated events. Genomic library screening indicates that 90% of VH+ clones are truncated, nearly identical pseudogenes (lacking JH and CH). These studies demonstrate an early phylogenetic origin for the cluster type of gene organization and document extensive organizational diversification within an apparent single class of IgH genes.

Towards understanding the evolutionary origins and early diversification of rearranging antigen receptors.

The rearranging antigen binding receptors, immunoglobulin heavy (IgH) and light (IgL) chains and the four classes of T-cell antigen receptors (TCR) are found in all contemporary species of jawed vertebrates examined thus far. Ig genes have undergone marked changes in organization and mechanisms of diversification during vertebrate phylogeny; whereas TCR genes, which are found in species as phylogenetically removed as man and cartilaginous fishes (e.g. skate), are generally similar in terms of structure, diversification and, presumably, function. The patterns of Ig divergence in cartilaginous fish are informative as to both the potential for genetic variation and the mechanisms that bring about such change. No evidence has been found for homologs of either Ig, TCR, recombination activating gene (RAG)1 or RAG2 in jawless vertebrates or invertebrates. Thus, a phylogenetic demarcation exists in terms of the presence and absence of the rearranging antigen binding receptor genes. It is presumed that the rearranging antigen binding receptors arose from a non-rearranging predecessor. The recent discovery of non-rearranging homologs of antigen binding receptor genes in several species offers insight into alternative forms of recognition, relationships between adaptive and innate mechanisms of immunity, and the origins of antigen recognition.

Green Fluorescent Protein in the sea urchin: new experimental approaches to transcriptional regulatory analysis in embryos and larvae.

The use of Green Fluorescent Protein (GFP) as a reporter for expression transgenes opens the way to several new experimental strategies for the study of gene regulation in sea urchin development. A GFP coding sequence was associated with three different previously studied cis-regulatory systems, viz those of the SM50 gene, expressed in skeletogenic mesenchyme, the CyIIa gene, expressed in archenteron, skeletogenic and secondary mesenchyme, and the Endo16 gene, expressed in vegetal plate, archenteron and midgut. We demonstrate that the sensitivity with which expression can be detected is equal to or greater than that of whole-mount in situ hybridization applied to detection of CAT mRNA synthesized under the control of the same cis-regulatory systems. However, in addition to the important feature that it can be visualized nondestructively in living embryos, GFP has other advantages. First, it freely diffuses even within fine cytoplasmic cables, and thus reveals connections between cells, which in sea urchin embryos is particularly useful for observations on regulatory systems that operate in the syncytial skeletogenic mesenchyme. Second, GFP expression can be dramatically visualized in postembryonic larval tissues. This brings postembryonic larval developmental processes for the first time within the easy range of gene transfer analyses. Third, GFP permits identification and segregation of embryos in which the clonal incorporation of injected DNA has occurred in any particular desired region of the embryo. Thus, we show explicitly that, as expected, GFP transgenes are incorporated in the same nuclei together with other transgenes with which they are co-injected.

alpha, beta, gamma, and delta T cell antigen receptor genes arose early in vertebrate phylogeny.

A series of products were amplified using a PCR strategy based on short minimally degenerate primers and R. eglanteria (clearnose skate) spleen cDNA as template. These products were used as probes to select corresponding cDNAs from a spleen cDNA library. The cDNA sequences exhibit significant identity with prototypic (alpha, beta, gamma, and delta T cell antigen receptor (TCR) genes. Characterization of cDNAs reveals extensive variable region diversity, putative diversity segments, and varying degrees of junctional diversification. This demonstrates expression of both alpha/beta and gamma/delta TCR genes at an early level of vertebrate phylogeny and indicates that the three major known classes of rearranging antigen receptors were present in the common ancestor of the present-day jawed vertebrates.

Characterization of avian T-cell receptor gamma genes.

In birds and mammals T cells develop along two discrete pathways characterized by expression of either the alpha beta or the gamma delta T-cell antigen receptors (TCRs). To gain further insight into the evolutionary significance of the gamma delta T-cell lineage, the present studies sought to define the chicken TCR gamma locus. A splenic cDNA library was screened with two polymerase chain reaction products obtained from genomic DNA using primers for highly conserved regions of TCR and immunoglobulin genes. This strategy yielded cDNA clones with characteristics of mammalian TCR gamma chains, including canonical residues considered important for proper folding and stability. Northern blot analysis with the TCR gamma cDNA probe revealed 1.9-kb transcripts in the thymus, spleen, and a gamma delta T-cell line, but not in B or alpha beta T-cell lines. Three multimember V gamma subfamilies, three J gamma gene segments, and a single constant region C gamma gene were identified in the avian TCR gamma locus. Members of each of the three V gamma subfamilies were found to undergo rearrangement in parallel during the first wave of thymocyte development. TCR gamma repertoire diversification was initiated on embryonic day 10 by an apparently random pattern of V-J gamma recombination, nuclease activity, and P-and N-nucleotide additions to generate a diverse repertoire of avian TCR gamma genes early in ontogeny.

A third Ig light chain gene isotype in Xenopus laevis consists of six distinct VL families and is related to mammalian lambda genes.

Xenopus laevis is a unique model for studying the ontogenetic development of immune functions. A short primer PCR amplification method was employed to amplify fragments from Xenopus genomic DNA that are related to Ig light chains and TCR. One fragment was identified that appeared to represent a novel type of light chain and was used as a probe to recover the corresponding cDNA from a spleen cDNA library. We designate these light chains type III. Using an iterative screening procedure, six families of VL genes, two distinct JL and two distinct CL sequences, were identified. In a comparison of phylogenetically diverse light chains, the type III genes align with higher vertebrate lambda genes. Southern blot hybridization analyses with genomic DNA from different animals showed the VL and CL sequences to be both diverse and polymorphic. Intrafamily sequence comparisons of VL genes revealed additional diversity. Collectively, these studies confirm the existence of a third type of light chain gene in Xenopus, establish a high degree of genetic variation in the sequences encoding the light chain V regions, and provide the most significant evidence to date for the presence of a lambda-like light chain gene at the phylogenetic level of the amphibians.

Extensive diversity of transcribed TCR-beta in phylogenetically primitive vertebrate.

The genetic complexity of the expressed TCR Vbeta repertoire in Heterodontus francisci, the horned shark, a member of the most phylogenetically primitive vertebrate class in which TCR genes have been identified, is addressed. The sequences of 55 spleen cDNA clones encoding TCR-beta genes are compared, and 7 diverse Vbeta families are defined by overall sequence identity and clustering based on phylogenetic distance analyses. At least 18 putative Jbeta sequence types, as well as a consensus diversity (D) element that resembles most closely mammalian TCR Dbeta, can he recognized. Extensive sequence diversity, as well as characteristic TCR-beta length variation in CDR3, is evident. Unlike Ig genes in this species, TCR-beta genes exhibit considerable V family multiplicity and appear to utilize combinatorial mechanisms in the generation of immunologic diversity. in this sense, the TCR-beta genes in this cartilaginous fish and humans are more similar than are the genes encoding Ab proteins in these species.