Evolution of actin gene families of sea urchins

The actin gene family of the sea urchin Lytechinus pictus includes a single muscle actin gene, LpM, and four cytoskeletal actin genes: LpC1, LpC2, LpC3, and LpC4. The origin and relationship of these actin genes to members of the actin gene family of the sea urchin Strongylocentrotus purpuratus were considered. Comparison of deduced amino acid sequences suggested a close relationship between LpC1 and the CyI–CyII subfamily of S. purpuratus actin genes, and between LpC2 and the CyIII subfamily of S. purpuratus actin genes; the muscle actin genes were orthologous. It is proposed that two divergent cytoskeletal actin genes of the common ancestral sea urchin gave rise by duplication to the extant cytoskeletal actin genes of these species, some of which have changed 3 noncoding sequences while others have maintained a terminus highly conserved among sea urchin actin genes.Correspondence to: B.P. Brandhorst     

Dissociation of expression patterns of homeodomain transcription factors in the evolution of developmental mode in the sea urchins Heliocidaris tuberculata and H. erythrogramma

The direct-developing sea urchin species Heliocidaris erythrogramma has a radically modified ontogeny. Along with gains of novel features, its entire ectoderm has been reorganized, resulting in the apparent absence of a differentiated oral ectoderm, a major module present in the pluteus of indirect-developing species, such as H. tuberculata. The restoration of an obvious oral ectoderm in H. erythrogrammaxH. tuberculata hybrids, indicates the action of dominant regulatory factors from the H. tuberculata genome. We sought candidate regulatory genes based on the prediction that they should include genes that govern development of the oral ectoderm in the pluteus, but play different roles in H. erythrogramma. Such genes may have a large effect in the evolution of development. Goosecoid (Gsc), Msx, and the sea urchin Abd-B-like gene (Hox11/13b) are present and expressed in both species and the hybrid embryos. Both Gsc and Msx are oral ectoderm specific in H. tuberculata, and show novel and distinct expression patterns in H. erythrogramma. Gsc assumes a novel ectodermal pattern and Msx shifts to a novel and largely mesodermal pattern. Both Gsc and Msx show a restoration of oral ectoderm expression in hybrids. Hox11/13b is not expressed in oral ectoderm in H. tuberculata, but is conserved in posterior spatial expression among H. tuberculata, H. erythrogramma and hybrids, serving as a control. Competitive RT-PCR shows that Gsc, Msx, and Hox11/13b are under different quantitative and temporal controls in the Heliocidaris species and the hybrids. The implications for the involvement of these genes in the rapid evolution of a direct developing larva are discussed.     

Involvement of Tcf/Lef in establishing cell types along the animal-vegetal axis of sea urchins

Members of the Tcf/Lef family interact with β-catenin to activate programs of gene expression during development. Recently β-catenin was shown to be essential for establishing cell fate along the animal-vegetal axis of the sea urchin embryo. To examine the role of Tcf/Lef in sea urchins we cloned a Strongylocentrotus purpuratus Tcf/Lef homolog. Expression of SpTcf/Lef was maximal when β-catenin became localized to nuclei of vegetal blastomeres, consistent with its acting in combination with β-catenin to specify vegetal cell fates. Expression of a dominant-negative SpTcf/Lef inhibited primary and secondary mesenchyma, endoderm, and aboral ectoderm formation in a manner similar to that observed when nuclear accumulation of β-catenin was prevented. Our results suggest that SpTcf/Lef functions by interacting with β-catenin to specify cell fates along the sea urchin animal-vegetal axis. Received: 6 July 1999 / Accepted: 27 August 1999     

Effect of droplet vitrification on development competence, actin cytoskeletal integrity and gene expression in in vitro cultured mouse embryos

The effect of modified droplet vitrification was assessed on cellular actin filament organization, apoptosis related gene expression and development competence in mouse embryos cultured in vitro. Mouse zygotes, 2-cell embryos and morulae were vitrified in ethylene glycol (VS-1) and ethylene glycol plus DMSO (VS-2) and thawed by directly placing the vitrified drop into 0.3 M sucrose solution at 37 °C. High recovery (93-99%) of morphologically normal embryos was evident following vitrification and thawing. No detectable actin filament disruption was observed in the embryos at any development stage following vitrification and thawing and/or in vitro culture. The expression pattern of Bax, Bcl2 and p53 genes was altered (P < 0.05) in vitrified zygotes and 2-cell embryos, but not in morulae. Although a large proportion of the vitrified zygotes (59.5 ± 4.4% in VS-1 and 57.9 ± 4.5% in VS-2; mean ± S.E.M.) and 2-cell embryos (63.1 ± 4.4% in VS-1 and 59.2 ± 4.3% in VS-2) developed into blastocysts, development of control embryos (70.2 ± 5.0% of zygotes and 75.5 ± 4.4% of 2-cell embryos) into blastocysts was higher (P < 0.05). In contrast, development of the control and vitrified morulae into blastocysts (more than 85%) was similar. We concluded that the modified droplet vitrification procedure supported better survival of morula stage compared to zygotes and 2-cell mouse embryos.     

Maternal factors and the evolution of developmental mode: Evolution of oogenesis in Heliocidaris erythrogramma

 Evolutionary change in developmental mode in sea urchins is closely tied to an increase in maternal provisioning. We examined the oogenic modifications involved in production of a large egg by comparison of oogenesis in congeneric sea urchins with markedly different sized oocytes and divergent modes of development. Heliocidaris tuberculata has small eggs (95 μm diameter) and the ancestral mode of development through feeding larvae, whereas H. erythrogramma has large eggs (430 μm diameter) and highly modified non-feeding lecithotrophic larvae. Production of a large egg in H. erythrogramma involved both conserved and divergent mechanisms. The pattern and level of vitellogenin gene expression is similar in the two species. Vitellogenin processing is also similar with the gonads of both species incorporating yolk protein from coelomic and hemal stores into nutritive cells with subsequent transfer of this protein into yolk granules in the developing vitellogenic oocyte. Immunocytology of the eggs of both Heliocidaris species indicates they incorporate similar levels of yolk protein. However, H. erythrogramma has evolved a highly divergent second phase of oogenesis characterised by massive deposition of non-vitellogenic material including additional maternal protein and lipid. Maternal provisioning in H. erythrogramma exhibits recapitulation of the ancestral vitellogenic program followed by a novel oogenic phase with hypertrophy of the lipogenic program being a major contributor to the increase in egg size. Received: 12 August 1998 / Accepted: 25 November 1998     

Quantitative changes in cytoskeletal beta- and gamma-actin mRNAs and apparent absence of sarcomeric actin gene transcripts in early mouse embryos

Actin is known to be synthesized both during oogenesis and in cleavage-stage embryos in mice. Cytoskeletal beta-actin appears to be the major component, followed by gamma-actin, but the synthesis of alpha-actin has also been inferred from protein electrophoretic patterns. We have studied the expression of cytoskeletal (beta- and gamma-) and sarcomeric (alpha-cardiac and alpha-skeletal) actin genes at the level of the individual mRNAs in blot hybridization experiments using isoform-specific RNA probes. The results show that there are about 2 x 10(4) beta-actin mRNA molecules in the fully grown oocyte; this number drops to about one-half in the egg and less than one-tenth in the late two-cell embryo but increases rapidly during cleavage to about 3 x 10(5) molecules in the late blastocyst. The amount of gamma-actin mRNA is similar to that of beta-actin in oocytes and eggs but only about 40% as much in late blastocysts, indicating a differential accumulation of these mRNAs during cleavage. The developmental pattern of beta- and gamma-actin mRNA provides a striking example of the transition from maternal to embryonic control that occurs at the two-cell stage and involves the elimination of most or all of the maternal actin mRNA. There was no detectable alpha-cardiac or alpha-skeletal mRNA (i.e., less than 1,000 molecules per embryo) at any stage from oocyte to late blastocyst, suggesting that the sarcomeric actin genes are silent during preimplantation development.     

Control of tubulin and actin gene expression in Tetrahymena pyriformis during the cell cycle

Poly(A)-containing mRNA was isolated from division synchronized populations of the ciliated protozoan, Tetrahymena pyriformis. The level of tubulin and actin mRNA at specific cell cycle stages was analyzed by hybridization to tubulin and actin cDNA probes and by gel analysis of their in vitro translation products. The pattern of fluctuation of tubulin mRNA levels was similar to that observed for the in vivo tubulin synthesis previously reported [1]. This suggests that as the cells progress through the cell cycle, tubulin synthesis is controlled at the mRNA level. There was little fluctuation of actin synthesis or actin mRNA levels during the cell cycle, which may be indicative of a different regulatory mechanism for actin than for tubulin.     

葡萄Actin基因家族的鉴定及进化和表达分析

采用生物信息学方法从葡萄(Vitis vinifera Linn.)全基因组中鉴定Actin基因家族,并对各基因的染色体定位和结构特征,编码蛋白质的理化性质、亚细胞定位、二级结构、三级结构和系统进化,以及不同组织的基因表达进行研究.结果表明:葡萄Actin基因家族16个基因分布在12条染色体上.16个基因的结构特征及其编码蛋白质的理化性质差异较大.16个基因的长度及其内含子总长度的变化范围较大,编码序列(CDS)和外显子总长度的变化范围较小.除登录号GSVIVG01008254001和GSVIVG01014035001的基因外,其他14个基因的GC含量均低于其CDS的GC含量.除登录号GSVIVG01008254001的基因外,其他15个基因编码的蛋白质的理论相对分子质量为12534.54~82612.33,理论等电点为pI 4.92~pI 9.13.16个基因编码蛋白质的消光系数为14105~73645,脂肪族氨基酸指数为65.54~92.06,其中9个为稳定蛋白,7个为不稳定蛋白.除登录号GSVIVG01014035001的基因外,其他15个基因编码的蛋白质均为亲水性蛋白.登录号GSVIVG01016517001的基因编码的蛋白质定位于细胞质和细胞核,其他15个基因编码的蛋白质定位于细胞质.二级结构和三级结构显示:葡萄Actin基因家族16个基因编码的蛋白质均由α螺旋、无规则卷曲和延伸链构成,且总体以无规则卷曲为主.系统进化分析和不同组织的基因表达分析结果显示:与拟南芥〔Arabidopsis thaliana(Linn.)Heynh.〕相似,葡萄Actin基因家族16个基因编码的蛋白质分为3个亚家族,ClassⅡ亚家族(营养型)包括登录号GSVIVG01003099001和GSVIVG01026580001的基因编码的蛋白质,这2个基因在所有组织中的表达均较高;ClassⅢ亚家族(生殖型)包括登录号GSVIVG01033494001、GSVIVG01024980001和GSVIVG01016550001的基因编码的蛋白质,这3个基因在花粉、雄蕊和花中的表达均较高;ClassⅠ亚家族包括其他11个基因编码的蛋白质,这11个基因在各组织中的表达总体上较低.研究结果显示:葡萄Actin基因家族的表达具有组织特异性.     

Conservation of the WD-repeat, microtubule-binding protein, EMAP, in sea urchins, humans, and the nematode C. elegans

The echinoderm microtubule-associated protein (EMAP) is the most abundant microtubule-binding protein in the first cleavage mitotic apparatus in sea urchin embryos. The first goal of this study was to determine whether there is sufficient EMAP in the egg and embryo to modify microtubule dynamics during the early cleavages divisions and whether EMAP functions at a specific time or place in the embryo. To accomplish this goal, we examined the relative abundance, tissue distribution, and temporal pattern of EMAP expression during embryonic development. The second goal of this study was to identify important functional domains within the EMAP coding sequence. A conserved sequence might reveal a potential microtubule-binding domain. We cloned, sequenced and compared overlapping EMAP cDNAs from two different sea urchin species that diverged approximately 80 million years ago, and compared these with cDNA sequences from a vertebrate and nematode species. From quantitative immunoblots, we determined the EMAP concentration in eggs to be 4 μM. The steady-state levels of EMAP mRNA and protein accumulated during development, and all three germ layers expressed EMAP. During the early stages of development, EMAP and tubulin were both abundant in the ectoderm, mesoderm and endoderm. However, during late gastrulation and the formation of the early pluteus larvae, EMAP was enriched in the mesoderm, while tubulin staining was most abundant in the archenteron. These results indicate that EMAP may have tissue-specific functions in the late stage embryo. To identify conserved functional domains, we compared the predicted amino acid sequence encoded by Strongylocentrotus purpuratus and Lytechinus variegatus EMAP cDNAs, and determined that these two sea urchin EMAPs were 95% conserved and shared an identical domain organization. A parsimonious analysis of these sea urchin protein sequences, as well as human and C. elegans EMAP sequences was used to construct a gene tree. Together these results suggest that EMAP is an important microtubule protein required at all developmental stages of sea urchins, and whose cellular function may be conserved amongst metazoans. Received: 2 March 1999 / Accepted: 28 June 1999     

Cloning, mapping, and analyses of expression of the Em-like gene family in soybean [Glycine max (L). Merr.]

 The entire Em-like Group-1 late embryogenesis abundant (Lea) gene family from soybean was cloned and characterized. The five Group-1 Lea genes (Sle1-5) were divided into two classes based on sequence identity. Sle1-4 were genetically mapped to four different linkage groups. Nucleotide sequencing indicated that Sle1, Sle2, Sle3, and Sle5 encode polypeptides differing primarily by the presence of a repeated 20-amino acid motif. Sle1 and Sle5 were shown by Northern analysis to be expressed in developing embryos weeks earlier than Sle2 and Sle3. Sle4 was shown to be a pseudogene. Maximal levels of mRNA for all functional Sle genes accumulated in maturation-phase seeds, before significant desiccation had occurred, and declined rapidly upon seed imbibition. Desiccation did not induce Sle expression in seeds or vegetative tissue. Sle expression was confined to embryo tissues and Sle mRNA accumulated at similar levels in both the embryo axis and in the cotyledons. Received: 25 September 1996 / Accepted: 27 September 1996     

Evolutionary Patterns of Gene Families Generated in the Early Stage of Vertebrates

In this paper we have analyzed 49 vertebrate gene families that were generated in the early stage of vertebrates and/or shortly before the origin of vertebrates, each of which consists of three or four member genes. We have dated the first (T₁) and second (T₂) gene duplications of 26 gene families with 3 member genes. The means of T₁ (594 mya) and T₂ (488 mya) are largely consistent to a well-cited version of two-round (2R) genome duplication theory. Moreover, in most cases, the time interval between two successive gene duplications is large enough that the fate of duplicate genes generated by the first gene duplication was likely to be determined before the second one took place. However, the phylogenetic pattern of 23 gene families with 4 members is complicated; only 5 of them are predicted by 2R model, but 11 families require an additional gene (or genome) duplication. For the rest (7 families), at least one gene duplication event had occurred before the divergence between vertebrate and Drosophila, indicating a possible misleading of the 4:1 rule (member gene ratio between vertebrates and invertebrates). Our results show that Ohno's 2R conjecture is valid as a working hypothesis for providing a most parsimonious explanation. Although for some gene families, additional gene duplication is needed, the credibility of the third genome duplication (3R) remains to be investigated. Received: 13 December 1999 / Accepted: 7 April 2000     

RFLP- and physical mapping of resistance gene homologues in rice (O. sativa) and Barley (H. vulgare)

 The deduced peptide sequences of 25 gene fragments of NBS-LRR resistance (R) gene homologues from rice and barley and of characterized R genes were compared, revealing a string of six conserved motifs. Mapping of the R-gene candidates in rice showed linkage to genes conferring race-specific resistance to rice blast (Pi-k, Pi-f and Pi-1) and bacterial blight disease (Xa-1, Xa-3 and Xa-4), in barley to powdery mildew (Mla) and the rust fungus (Rpg1). In rice four mixed clusters were detected, each harboring at least two highly dissimilar NBS-LRR genes. A YAC-contig was established for one of these mixed clusters. YAC fragmentation experiments revealed the presence of at least five NBS-LRR genes within 200 kb in head-to-tail orientation. Received: 24 July 1998 / Accepted: 14 August 1998     

ASW: a gene with conserved avian W-linkage and female specific expression in chick embryonic gonad

Vertebrates exhibit a variety of sex determining mechanisms which fall broadly into two classes: environmental or genetic. In birds and mammals sex is determined by a genetic mechanism. In mammals males are the heterogametic sex (XY) with the Y chromosome acting as a dominant determiner of sex due to the action of the testis-determining factor, SRY. In birds females are the heterogametic sex (ZW); however, it is not known whether the W chromosome carries a dominant ovary-determining gene, or whether Z chromosome dosage determines sex. Using an experimental approach, which assumes only that the sex-determining event in birds is accompanied by sex-specific changes in gene expression, we have identified a novel gene, ASW (Avian Sex-specific W-linked). The putative protein for ASW is related to the HIT (histidine triad) family of proteins. ASW shows female-specific expression in genital ridges and maps to the chicken W chromosome. In addition, we show that, with the exception of ratites, ASW is linked to the W chromosome in each of 17 bird species from nine different families of the class Aves. Received: 18 October 1999 / Accepted: 10 January 2000