Expression and localization of a novel phosducin-like protein from amphioxus Branchiostoma belcheri

A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-N1/Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri.

Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-pax genes

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.

Amphi-Eomes/Tbr1: An amphioxus cognate of vertebrate Eomesodermin and T-Brain1 genes whose expression reveals evolutionarily distinct domain in amphioxus development

A cDNA for a novel T-box containing gene was isolated from the amphioxus Branchiostoma belcheri. A molecular phylogenetic tree constructed from the deduced amino acid sequence of the isolated cDNA indicates that this gene belongs to the T-Brain subfamily. In situ hybridization reveals that the expression is first detected in the invaginating archenteron at the early gastrula stage and this expression is down-regulated at the neurula stage. In early larvae, the expression appears again and transcripts are detected exclusively in the pre-oral pit (wheel organ-Hatschek's pit of the adult). In contrast to the vertebrate counterparts, no transcripts are detected in the brain vesicle or nerve cord throughout the development. These results are interpreted to mean that a role of T-Brain products in vertebrate forebrain development was acquired after the amphioxus was split from the lineage leading to the vertebrates. On the other hand, comparison of the tissue-specific expression domain of T-Brain genes and other genes between amphioxus and vertebrates revealed that the pre-oral pit of amphioxus has several molecular features which are comparable to those of the vertebrate olfactory and hypophyseal placode. (C) 2002 Wiley-Liss, Inc.

Expression of muscle-related genes and two MyoD genes during amphioxus notochord development

The notochord is one of the diagnostic features of the phylum Chordata. Despite the similarities in the early morphogenetic patterns of the notochords of various chordates, they are strikingly distinct from one another at the histological level. The amphioxus notochord is one example of an evolutionary novelty because it is made up of muscle cells. Our previous expressed sequence tag analysis, targeting messenger RNAs expressed in the adult amphioxus notochord, demonstrated that many muscle-related genes are expressed there. To characterize amphioxus notochord cells and to gain insights into the myogenic program in the notochord, we determined the spatial and temporal expression patterns of these muscle-related genes during amphioxus development. We found that BbNA1 (notochord actin), Amphi-Trop I (troponin I), Amphi-TPmyosin (tropomyosin), Amphi-MHC2 (myosin heavy chain), Amphi-nMRLC (notochord-specific myosin regulatory light chain), AmphinTitin/MLCK (notochord-specific titin/myosin light chain kinase), Amphi-MLP/CRP3 (muscle LIM protein), and Amphi-nCalponin (notochord-specific calponin) are expressed with characteristic patterns in notochord cells, including the central cells, dorsally located cells, and ventrally located cells, suggesting that each notochord cell has a unique molecular architecture that may reflect its function. In addition, we characterized two MyoD genes (Amphi-MyoD1 and Amphi-MyoD2) to gain insight into the genetic circuitry governing the formation of the notochord muscle. One of the MyoD genes (Amphi-MyoD2) is expressed in the central notochord cells, and the coexistence of Amphi-MyoD2 transcripts along with the Amphi-MLP/CRP3 transcripts implies the participation of Amphi-MyoD2 in the myogenic program in the notochord muscle.

利用叶绿体DNA trn L-F序列初步探讨菊科风毛菊属的系统发育

风毛菊属Saussurea DC．是菊科物种分化十分剧烈和分类处理十分困难的一个属。该属的单系起源性质、属下分类系统以及一些独特形态物种的系统位置尚不清楚，有待进一步验证。本文测定了代表该属5个亚属37种植物43个样品和川木香属Dolomiaea DC．的1种样品的叶绿体DNA trnL-F序列，并调取菜蓟族Cardueae Cass．与风毛菊属具有一定亲缘关系的13属的该序列，一起进行了分支分析，重点验证该属的属下形态分类系统以及形态特殊、青藏高原地区特有的雪兔子亚属subgen．Eriocoryne中假雪兔子组sect．Pseudoeriocoryne的单系性质。研究结果表明：(1)尽管风毛菊属5个亚属形态变异极大，但种间的trnL-F碱基变异却非常小；(2)根据形态学划分的5个亚属在分支图上没有得到分辨；(3)根据无明显茎、叶呈莲座状包被头状花序等特征建立的假雪兔子组是一多系群，与其他雪兔子亚属的种类也没有密切的亲缘关系，其共同拥有的形态学特征分别起源了4次；(4)雪莲亚属假雪莲组subgen．Amphi- laena sect．Pseudoamphilaena中的多鞘雪莲s．polycolea变种尖苞雪莲S．polycolea var．acuisauama与风毛菊亚属subgen．Saussurea的长毛风毛菊S．hieraciodies、美丽风毛菊s．superba、打箭风毛菊s．tatsienensis和重齿风毛菊s．katochaete具有一段长18个碱基的插入，但多鞘雪莲原变种和重齿风毛菊另一居群无此插入；这一插入在整个菜蓟族和风毛菊属中都十分特殊，是一次进化事件的结果，这一复合体的形态分化应该是在这一进化事件发生之后进行的；而尖苞雪莲和重齿风毛菊拥有这一特殊序列可能是由于杂交而导致种内存在不同的单倍性。研究结果证实青藏高原极端环境下即使在较低的分类等级——属内、种问的形态特征也存在相同选择压力下的生态趋同进化。这一点在利用形态特征研究该地区物种的亲缘关系和命名自然分类等级时应予以特别重视。风毛菊属提供了研究极端环境下物种快速分化、网状进化和趋同进化的典型范例。