Identification of a New Gene Family Expressed during the Onset of Sexual Reproduction in the Centric Diatom Thalassiosira weissflogii

An intriguing feature of the diatom life cycle is that sexual reproduction and the generation of genetic diversity are coupled to the control of cell size. A PCR-based cDNA subtraction technique was used to identify genes that are expressed as small cells of the centric diatom Thalassiosira weissflogii initiate gametogenesis. Ten genes that are up-regulated during the early stages of sexual reproduction have been identified thus far. Three of the sexually induced genes, Sig1, Sig2, and Sig3, were sequenced to completion and are members of a novel gene family. The three polypeptides encoded by these genes possess different molecular masses and charges but display many features in common: they share five highly conserved domains; they each contain three or more cysteine-rich epithelial growth factor (EGF)-like repeats; and they each display homology to the EGF-like region of the vertebrate extracellular matrix glycoprotein tenascin X. Interestingly, the five conserved domains appear in the same order in each polypeptide but are separated by variable numbers of nonconserved amino acids. SIG1 and SIG2 display putative regulatory domains within the nonconserved regions. A calcium-binding, EF-hand motif is found in SIG1, and an ATP/GTP binding motif is present in SIG2. The striking similarity between the SIG polypeptides and extracellular matrix components commonly involved in cell-cell interactions suggests that the SIG polypeptides may play a role in sperm-egg recognition. The SIG polypeptides are thus important molecular targets for determining when and where sexual reproduction occurs in the field.     

Rapid evolution of a sexual reproduction gene in centric diatoms of the genus Thalassiosira

Sexual reproduction is commonly assumed to occur in the vast majority of diatoms due to the intimate association of this process with cell size control. Surprisingly, however, little is known about the impact of sexual events on diatom population dynamics. The Sig1 gene is strongly upregulated during sexual reproduction in the centric diatom Thalassiosira weissflogii and has been hypothesized to encode a protein involved in gamete recognition. In the present study, degenerate PCR primers were designed and used to amplify a portion of Sig1 from three closely related species in the cosmopolitan genus Thalassiosira, Thalassiosira oceanica, Thalassiosira guillardii, and Thalassiosira pseudonana. Identification of Sig1 in these three additional species facilitated development of this gene as a molecular marker for diatom sexual events. Examination of the new sequences indicated that multiple copies of Sig1 are probably present in the genome. Moreover, compared to the housekeeping gene beta-tubulin, the Sig1 genes of isolates of T. weissflogii collected from different regions of the Atlantic and Pacific oceans displayed high levels of divergence. The Sig1 genes of the four closely related Thalassiosira species also displayed high levels of sequence divergence compared to the levels observed with a second gene, Fcp, probably explaining why Sig1 could not be amplified from more distantly related species. The high levels of sequence divergence both within and between species suggest that Sig1 is rapidly evolving in a manner reminiscent of the manner observed in other genes that encode gamete recognition proteins. A simple model is presented for Sig1 evolution and the implications of such a rapidly evolving sexual reproduction gene for diatom speciation and population dynamics.     

Rapid Evolution of a Sexual Reproduction Gene in Centric Diatoms of the Genus Thalassiosira

Sexual reproduction is commonly assumed to occur in the vast majority of diatoms due to the intimate association of this process with cell size control. Surprisingly, however, little is known about the impact of sexual events on diatom population dynamics. The Sig1 gene is strongly upregulated during sexual reproduction in the centric diatom Thalassiosira weissflogii and has been hypothesized to encode a protein involved in gamete recognition. In the present study, degenerate PCR primers were designed and used to amplify a portion of Sig1 from three closely related species in the cosmopolitan genus Thalassiosira, Thalassiosira oceanica, Thalassiosira guillardii, and Thalassiosira pseudonana. Identification of Sig1 in these three additional species facilitated development of this gene as a molecular marker for diatom sexual events. Examination of the new sequences indicated that multiple copies of Sig1 are probably present in the genome. Moreover, compared to the housekeeping gene β-tubulin, the Sig1 genes of isolates of T. weissflogii collected from different regions of the Atlantic and Pacific oceans displayed high levels of divergence. The Sig1 genes of the four closely related Thalassiosira species also displayed high levels of sequence divergence compared to the levels observed with a second gene, Fcp, probably explaining why Sig1 could not be amplified from more distantly related species. The high levels of sequence divergence both within and between species suggest that Sig1 is rapidly evolving in a manner reminiscent of the manner observed in other genes that encode gamete recognition proteins. A simple model is presented for Sig1 evolution and the implications of such a rapidly evolving sexual reproduction gene for diatom speciation and population dynamics.     

Platelet-derived growth factor-inducible gene JE is a member of a family of small inducible genes related to platelet factor 4

The platelet-derived growth factor-inducible gene JE was found to encode a 148-residue basic (pI = 10.4) secretory protein which shows striking similarity to the gene products of a family of small inducible genes (SIG), LD78, TCA3, IP10, 3-10C, 9E3/pCEF4, and gro/MGSA, and to several of the proteins secreted from platelet alpha-granules. Members of the SIG family have spatially conserved cysteine residues that vary in distance by only one amino acid residue as well as conserved proline residues at analogous sites. Hydrophilicity plots show alternating hydrophobic and hydrophilic domains which are similar for all members of the SIG family except IP10 and platelet factor 4, which show similarities to each other. The genomic organization of SIG family members is similar in the location of the splice junctions and the number of introns and exons, suggesting that they were derived from a common ancestor. The collective evidence suggests that a family of inducible cytokines, which are mitogenic or chemotactic, may act as intercellular coordinators of diverse responses designed to combat infection and promote the healing and regeneration of injured tissue.     

A Cluster of Vitellogenin Genes in the Mediterranean Fruit Fly Ceratitis Capitata: Sequence and Structural Conservation in Dipteran Yolk Proteins and Their Genes

Four genes encoding the major egg yolk polypeptides of the Mediterranean fruit fly Ceratitis capitata, vitellogenins 1 and 2 (VG1 and VG2), were cloned, characterized and partially sequenced. The genes are located on the same region of chromosome 5 and are organized in pairs, each encoding the two polypeptides on opposite DNA strands. Restriction and nucleotide sequence analysis indicate that the gene pairs have arisen from an ancestral pair by a relatively recent duplication event. The transcribed part is very similar to that of the Drosophila melanogaster yolk protein genes Yp1, Yp2 and Yp3. The Vg1 genes have two introns at the same positions as those in D. melanogaster Yp3; the Vg2 genes have only one of the introns, as do D. melanogaster Yp1 and Yp2. Comparison of the five polypeptide sequences shows extensive homology, with 27% of the residues being invariable. The sequence similarity of the processed proteins extends in two regions separated by a nonconserved region of varying size. Secondary structure predictions suggest a highly conserved secondary structure pattern in the two regions, which probably correspond to structural and functional domains. The carboxy-end domain of the C. capitata proteins shows the same sequence similarities with triacyglycerol lipases that have been reported previously for the D. melanogaster yolk proteins. Analysis of codon usage shows significant differences between D. melanogaster and C. capitata vitellogenins with the latter exhibiting a less biased representation of synonymous codons.     

NOVEL PROTEINS COMPRISING THE STRAMENOPILE TRIPARTITE MASTIGONEME IN OCHROMONAS DANICA (CHRYSOPHYCEAE)1

Two‐dimensional (2‐D) protein analysis of the mastigoneme fraction of the chromophyte alga Ochromonas danica E. G. Pringsh. showed the presence of several component proteins of the tubular mastigoneme. Adding to the reported gene Ocm1, three new genes (Ocm2, Ocm3, and Ocm4) belonging to the Ocm gene family were isolated using degenerate primers designed from predicted Ocm1 amino acid sequences. The predicted polypeptides encoded by Ocm2, Ocm3, and Ocm4 were smaller in size than Ocm1. However, they shared four highly conserved, cysteine‐rich, epithelial growth factor (EGF)‐like motifs, potentially involved in protein–protein interaction. In addition, Ocm2, Ocm3, and Ocm4 showed homology to the SIG protein family in the centric diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle, which is up‐regulated during early stages of sexual reproduction. Immunofluorescence analysis with a polyclonal antibody against the partial amino acid sequences of Ocm2, Ocm3, and Ocm4 showed that Ocm2 and Ocm3 were located in the basal segment region of mastigonemes attached on the surface of the anterior flagellum, and that Ocm4 was located within the tubular shaft portion similar to Ocm1.     

Identification and characterization of three members of a novel subclass of protocadherins

Protocadherins are members of a nonclassic subfamily of calcium-dependent cell-cell adhesion molecules in the cadherin superfamily. Although the extracellular domains have several common structural features, there is no extensive homology between the cytoplasmic domains of protocadherin subfamily members. We have identified a new subclass of protocadherins based on a shared and highly conserved 17-amino-acid cytoplasmic motif. The subclass currently consists of 18 protocadherin members. Two of these, PCDH18 and PCDH19, are novel protocadherins and a third is the human orthologue of mouse Pcdh10. All three genes encode six ectodomain repeats with cadherin-like attributes and, consistent with the structural characteristics of protocadherins, a large first exon encodes the extracellular domain of each gene.     

The six conserved helicase motifs of the UL5 gene product, a component of the herpes simplex virus type 1 helicase-primase, are essential for its function.

The UL5 protein of herpes simplex virus type 1, one component of the viral helicase-primase complex, contains six sequence motifs found in all members of a superfamily of DNA and RNA helicases. Although this superfamily contains more than 20 members ranging from bacteria to mammalian cells and their viruses, the importance of these motifs has not been addressed experimentally for any one of them. In this study, we have examined the functional significance of these six motifs for the UL5 protein through the introduction of site-specific mutations resulting in single amino acid substitutions of the most highly conserved residues within each motif. A transient replication complementation assay was used to test the effect of each mutation on the function of the UL5 protein in viral DNA replication. In this assay, a mutant UL5 protein expressed from an expression clone is used to complement a replication-deficient null mutant with a mutation in the UL5 gene for the amplification of herpes simplex virus origin-containing plasmids. Eight mutations in conserved regions and three similar mutations in nonconserved regions of the UL5 gene were analyzed, and the results indicate that all six conserved motifs are essential to the function of UL5 protein in viral DNA replication; on the other hand, mutations in nonconserved regions are tolerated. These data provide the first direct evidence for the importance of these conserved regions in any member of the superfamily of DNA and RNA helicases. In addition, three motif mutations were introduced into the viral genome, and the phenotypic analyses of these mutants are consistent with results from the transient replication complementation assay. The ability of these three mutant UL5 proteins to form specific interactions with other members of the helicase-primase complex, UL8 and UL52, indicates that the functional domains required for replication activity of UL5 are separable from domains responsible for protein-protein interactions. It is anticipated that this type of structure-function analysis will lead to the identification of protein domains that contribute not only to the enzymatic activities of helicase or primase but also to protein-protein interactions within members of the complex.     

A maize-specifically expressed gene cluster in Ustilago maydis

The corn pathogen Ustilago maydis requires its host plant maize for development and completion of its sexual cycle. We have identified the fungal mig2-1 gene as being specifically expressed during this biotrophic stage. Intriguingly, mig2-1 is part of a gene cluster comprising five highly homologous and similarly regulated genes designated mig2-1 to mig2-5. Deletion analysis of the mig2-1 promoter provides evidence for negative and positive regulation. The predicted polypeptides of all five genes lack significant homologies to known genes but have characteristic N-terminal secretion sequences. The secretion signals of mig2-1 and mig2-5 were shown to be functional, and secretion of a full length Mig2-1-eGFP fusion protein to the extracellular space was demonstrated. The central domains of the Mig2 proteins are highly variable whereas the C-termini are strongly conserved and share a characteristic pattern of eight cysteine residues. The mig2 gene cluster was conserved in a wide collection of U. maydis strains. Interestingly, some U. maydis isolates from South America had lost the mig2-4 gene as a result of a homologous recombination event. Furthermore, the related Ustilago scitaminea strain, which is pathogenic on sugar cane, appears to lack the mig2 cluster. We describe a model of how the mig2 cluster might have evolved and discuss its possible role in governing host interaction.     

Putative Chitin Synthases from Branchiostoma floridae Show Extracellular Matrix-related Domains and Mosaic Structures

The transition from unicellular to multicellular life forms requires the development of a specialized structural component,the extracellular matrix(ECM).In Metazoans,there are two main supportive systems,which are based on chitin and collagen/hyaluronan,respectively.Chitin is the major constituent of fungal cell walls and arthropod exoskeleton.However,presence of chitin/chitooligosaccharides has been reported in lower chordates and during specific stages of vertebrate development.In this study,the occurrence of chitin synthases(CHSs) was investigated with a bioinformatics approach in the cephalochordate Branchiostoma floridae,in which the presence of chitin was initially reported in the skeletal rods of the pharyngeal gill basket.Twelve genes coding for proteins containing conserved amino acid residues of processive glycosyltransferases from GT2 family were found and 10 of them display mosaic structures with novel domains never reported previously in a chitin synthase.In particular,the presence of a discoidin(DS) and a sterile alpha motif(SAM) domain was found in nine identified proteins.Sequence analyses and homology modelling suggest that these domains might interact with the extracellular matrix and mediate protein-protein interaction.The multi-domain putative chitin synthases from B.floridae constitute an emblematic example of the explosion of domain innovation and shuffling which predate Metazoans.     

桃WRKY基因家族全基因组鉴定和表达分析

WRKY转录因子是植物中最大的转录调控家族之一,在生物和非生物胁迫以及植物生长和发育过程中起着重要调控作用.本文利用HMMER 3.0软件,使用WRKY保守域全蛋白序列(Pfam数据库编号:PF03106)鉴定桃(Prunus persica L.)基因组中的WRKY基因;利用DNAMAN 5.0,WebLogo 3,MEGA5.1,MapInspect和MEME等软件对其蛋白序列进行生物信息学分析.本文共鉴定得到61个桃WRKY基因.进化树分析结果显示,桃WRKY蛋白分为Ⅰ,Ⅱ和Ⅲ类型,类型Ⅰ分为Ⅰ-C亚组和Ⅰ-N亚组,类型Ⅱ分为Ⅱ-a,II-b,II-c,II-d和II-e亚组.WRKY结构域分析显示,WRKY结构域高度保守,绝大多数都含有WRKYGQK七肽和锌指结构.染色体定位分析显示,桃WRKY基因分布于8条染色体中,呈不均匀分布.内含子和外显子结构分析表明,WRKY基因结构进化高度保守.保守元件分析表明,桃WRKY基因家族包含5个保守元件,元件1,2和3为WRKY盒,元件4,5为未知盒.桃WRKY基因家族都包含有WRKY盒,类型Ⅰ中含有2个WRKY盒;II-d亚组中含有未知元件5.半定量和荧光定量PCR结果显示,16个WRKY基因均在桃的根,茎,叶,花和果中表达,但其相对表达水平不同.     

Tryptophan residue of Trp-Ser-X-Trp-Ser motif in extracellular domains of erythropoietin receptor is essential for signal transduction.

The Trp-Ser-X-Trp-Ser motif commonly exists just outside the transmembrane domains of all cytokine receptors so far isolated. The role of this conserved motif in erythropoietin receptor was examined by assessing a series of mutant receptors on erythropoietin-induced signal transduction. Replacement of one of the two conserved Trp residues in the motif to Gly was found to completely abolish the binding of erythropoietin to the receptor and also to lose the ability to transduce the factor-dependent growth signal. While the mutants with one Ser residue converted to Gly or Ala retained full biological activities, the replacement of both conserved Ser residues diminished the functions of the receptor. Furthermore, the receptors lacking a part or all of the Trp-Ser-X-Trp-Ser motif did not respond to erythropoietin. The Trp-Ser-X-Trp-Ser motif, especially Trp residue, located in extracellular domains of the erythropoietin receptor thus appears to play a critical role in receptor-mediated signal transduction.     

Polypeptides during early conjugation in Tetrahymena thermophila

As Tetrahymena thermophila cells differentiate from their vegetative life cycle to sexual reproduction, their polypeptide pattern undergoes a series of changes. These changes have been traced in extracellular, cellular, and subcellular compartments. The first alteration is induced by the nutritional shift-down and results in stimulation of at least one ciliary polypeptide and affects a series of polypeptides from other compartments. The second alteration is induced by mixing starved cells of complementary mating types and this stimulates the synthesis of nine ciliary polypeptides before pairs have formed and eight afterwards. At least five of these early and one of the late conjugation-related ciliary polypeptides are removed by low concentrations of EDTA, indicating that they are located on the external side of the plasma membrane. No differences were observed between polypeptides excreted during starvation and after mixing of complementary mating types. At Tris concentrations restrictive for conjugation, cilia lack the conjugation-related polypeptides. Some of these are instead found among the excreted polypeptides. Using O'Farrell gels and silver staining on isogenic cells of all possible mating types, we have been unable to correlate changes in polypeptide patterns to specific mating types.     

Molecular cloning and sexually dimorphic expression of DM-domain genes in Daphnia magna

Daphnia magna is known to switch between sexual and asexual reproduction depending on the environment. It reproduces asexually when in an optimal environment for food, photoperiod, and population density. Once the environment declines, it changes reproductive strategy from asexual to sexual reproduction. However, the molecular bases of environmental sex determination are largely unknown. To understand the molecular mechanisms of environmental sex determination in Daphnia, it is essential to isolate the genes related to sex determination. As DM-domain genes are well known as sex-related genes, we aimed to identify DM-domain genes from Daphnia. Based on degenerate PCR of conserved DM domains using Daphnia cDNA, we identified three DM-domain genes that corresponded to DMRT11E, DMRT93B, and DMRT99B of Drosophila melanogaster. Quantitative gene expression analysis in gonads revealed that DMRT93B was expressed only in the testis. This finding contributes to an improved understanding of the switching mechanism from an asexual to a sexual life cycle depending on the environment.     

Segmented Helical Structure of the Neck Region of the Glycan-Binding Receptor DC-SIGNR

Carbohydrate-recognition domains (CRDs) in the glycan-binding receptors DC-SIGN (dendritic-cell-specific intercellular adhesion molecule 1-grabbing nonintegrin; CD209) and DC-SIGNR (DC-SIGN-related receptor, also known as L-SIGN and variously designated CD209L and CD299) are projected from the membrane surface by extended neck domains containing multiple repeats of a largely conserved 23-amino-acid sequence motif. Crystals of a fragment of the neck domain of DC-SIGNR containing multiple repeats in which each molecule extends through multiple unit cells, such that the observed crystallographic asymmetric unit represents one repeat averaged over six repeats of the protein, have been obtained. The repeats are largely α-helical. Based on the structure and arrangement of the repeats in the crystal, the neck region can be described as a series of four-helix bundles connected by short, non-helical linkers. Combining the structure of the isolated neck domain with a previously determined overlapping structure of the distal end of the neck region with the CRDs attached provides a model of the almost-complete extracellular portion of the receptor. The results are consistent with previous characterization of the extended structure for the isolated neck region and the extracellular domain. The organization of the neck suggests how CRDs may be disposed differently in DC-SIGN compared with DC-SIGNR and in variant forms of DC-SIGNR assembled from polypeptides with different numbers of repeats in the neck domain.     

Cloning and modeling of the first nonmammalian CD4

We have cloned and sequenced the first nonmammalian CD4 cDNA from the chicken using the COS cell expression method. Chicken CD4 contains four extracellular Ig domains that, in analogy to mammalian CD4, are in the order V, C2, V, and C2. The molecule is 24% identical with both human and mouse sequences. The extracellular domains were modeled using human and rat CD4 crystal structures as templates. In the first domain there are two extra Cys residues that are at suitable distance to form an intra-beta-sheet disulfide bridge in addition to the canonical one in the V domain. The region responsible for the interaction with MHC class II is relatively nonconserved in chicken. However, there are positively charged amino acids in the C" region of the N-terminal domain that may mediate the association to the negatively charged residues of the MHC class II beta-chain. Molecular modeling also implies that the membrane-proximal domain mediates dimerization of chicken CD4 in a similar way as it does for human CD4. Furthermore, the cytoplasmic tail is highly conserved, containing the protein tyrosine kinase p56lck recognition site that is preceded by an adjacent di-leucine motif for the internalization of the molecule. Interestingly, there are no Ser residues in the cytoplasmic part, which may explain the slow down-regulation of chicken CD4 after phorbol ester stimulation.