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The amphioxus is a promising animal model for evolutionary-developmental studies due to its key position on the animal phylogenetic tree. In the present study, we reported a genetically modified amphioxus strain on the Hedgehog (Hh) gene locus using the TALEN method. The result showed that our TALEN pair injection could bring about 34% mutations in the amphioxus Hh coding region. Further analysis on the F(0) gametic DNA revealed that the mutations had entered into gametes. So, we paired one F(0) male carrying an 8 bp deletion with a wild-type (WT) female, and carefully nursed the F(1) embryos up to adulthood. We then screened F(1) individually via analyzing their genomic DNA from a tiny tail tip, and obtained eight heterozygous mutants from the F(1) offspring. Moreover, our observation on the F(2) embryos generated by mating F(1) mutants also revealed that about 25% of early larvae developed aberrantly with head and tail curving ventrally, agenesis of the mesoblastic tissue under their anterior notochord, and no mouth opening. With the larva growth, deformities (such as twist of head and tail, mouth absent, ventrally localized endostyle and gill slits) became more severe, and eventually those malformed larvae died due to no food intake. Genetic analysis showed that all these deformed embryos were homozygous mutants and the ratio of Hh hetorozygotes vs WT agreed with Mondel's law. WT amphioxus larvae are asymmetric with the mouth on the left and gill slits on the right side. However, the homozygous mutant larvae became left-right symmetric with the gill slits on the ventral side, indicating a conserved role of Hedgehog signaling in establishing the left-right embryonic axis.

In addition to protein coding sequence, the organism genome contains a significant amount of regulatory DNA. Comparative genomics reveals that the organism genomes of vertebrates, tunicate, cephalochordate, flies, and nematodes contain cis-regulatory elements with highly conserved non-coding elements (CNEs). CNEs that cluster around trans-dev genes are part of core gene regulatory networks (GRNs), and usually, they can act as transcriptional enhancers. In this review, we described the identification of CNEs and summarized their key properties across the metazoans, and then discussed the evolution of CNEs after large-scale genome duplication events and the role of CNEs in the evolution of ani-mal body plan.

A series of signal transduction pathways have been found to regulate the polarity establishment and formation of animal primary body axis. Among them, Wnt signaling pathway is extremely conserved and several key components in the pathway have been identified in the demosponge lineage. This implies that it is one of the earliest pathways involved in the ancestral metazoan axis development and might play an important role in specification and development of posterior and ventral fate of animal axis. Recently, with the establishment of functional experiments in vitro, the body plan formation has been found to be affected, in varying degrees, by many genes in the Wnt signaling pathway, such as members of wnt gene family, maternal gene beta-catenin and some transcription factor encoding genes. In this review, we analyzed the evolutionary origin of the wnt gene family involved in development of metazoan body plans, and then made a brief review on the roles of canonical Wnt/beta-catenin signaling in the polarity establishment and formation of primary body axis in diverse deuterostomes including sea urchin, amphioxus, zebrafish, frog, and mouse.

As one of the closest living invertebrate relatives of vertebrates, amphioxus (subphylum Cephalochordata) occupies a key position in animal evolution and is becoming the best available proxy and model animal for studying the last common ancestor of all chordates, especially vertebrates. As long-term continuous culturing of amphioxus in laboratory became reliable, for pushing this animal to be a more successful model system, whole-genome sequencing of one or more species derived from this branch will be another urgent issue needed to address. In the present study, we described the construction and characterization of a bacterial artificial chromosome (BAC) library, using a single individual of Chinese amphioxus (Branchiostoma belcheri). High quality genomic DNA extracted from the spermary was partially digested with EcoRand EcoRmethylase. Desirable DNA fragments were isolated by pulsed field gel electrophoresis (PFGE), ligated to linearized and phosphorylased carrier pCC1BAC, and then transformed to Escherichia coli EPI300. The constructed library consists of 44 706 clones with the average insert fragment size around 80 kb as estimated by PFGE. The representation of the library is about 9 equivalents to the amhioxus genome. These results indicate that the BAC library will be useful for functional genomic studies and facilitate the whole-genome sequencing of Chinese amphioxus.

The ATP-binding cassette (ABC) superfamily of genes encodes membrane proteins that transport many kinds of substrates across membranes. Based on amino acid sequence similarities and phylogeny, 48 ABC genes in the human genome were divided into seven subfamilies: ABCA to ABCG. Among them, ABCA transporters have a unique topology and the members of this subfamily had expanded twice by gene duplication after the divergences between amphibian and mammal, respectively. The new duplicated genes are well retained in rodent and carnivore, while half of them became pseudogenes or were absent in primate genome. Transporters of the ABCA subclass were responsible for critical physiological functions in the transmembrane transport of endogenous lipid and cholesterol substrates. Mutations of members of ABCA are associated with human genetic diseases.

Alternative splicing, a fundamental and important regulatory mechanism in eukaryotes, allows one pre-mRNA to be processed into many different mature forms within a cell, each of which can have distinct functions. As alternative splicing is associated with human diseases, the study of alternative splicing becomes quite important. Bioinformatics is a new subject for the study of alternative splicing, especially for its regulatory mechanism, prediction and origin. Of course, bioinformatics must be combined with experimental research so as to clarify these aspects of alternative splicing. This paper reviewed the recent research progress in this field in the hope to gain a deeper understanding of eukaryotic gene expression regulation.

The venom of elapid snakes contains a number of small proteins that display a broad spectrum of pharmacological activities. In study on the neurotoxin from the venom gland of Bungarus multicinctus, five cDNA sequences (MNCTL1-a,MNCTL1-b,MNCTL1-c,MNCTL1-d and MNCTL2) encoding two novel proteins were obtained from the total RNA by reverse transcription-polymerase chain reaction. Among them, four sequences (MNCTL1-b,MNCTL1-c,MNCTL1-d and MNCTL2) are at the length of 505 bp, composing of a 32 bp 5'-untranslational region,a 212 bp 3'-untranslational region, and a 261 bp open reading frame which encodes a 20 amino acids of signal peptide and a 65 amino acids of mature peptide. But the sequence of MNCTL1-a has only 481 bp because of a 24 bp deletion in 3'-untranslational region. Comparison between cDNA sequences obtained here and that of cardiotoxin-like protein reported by Chang previously shows a homological value above 95.8%. Computer simulation based on deduced amino acid sequences reveals that both proteins encoded by these cDNAs and cardiotoxin-like protein share similar molecular characters, suggesting their functional similarity. This result implies that multicopy genes existing in the genome of Bungarus multicinctus encode cardiotoxin-like proteins.