Molecular data suggests the ciliate Mesodinium (Protista: Ciliophora) might represent an undescribed taxon at class level

The well-known ciliate, Mesodinium Stein, 1863, is of great importance to marine microbial food webs and is related to the "red tides". However, it is possibly one of the most confusing ciliate taxa in terms of its systematic position: either the morphological or the molecular data excluded it from all the other known assemblages or groups. In the current work, the sequences of small subunit ribosomal RNA（SSU rR NA） genes for all isolates available are analysed and an examination of the secondary structure patterns of related groups is carried out. The results indicate that（1） Mesodinium invariably represents a completely separated and isolated clade positioned between two subphyla of ciliates with very deep branching, which indicates that they should be a primitive or ancestral group for the subphylum Intramacronucleata;（2） the secondary structure of the SSU r RNA of Mesodinium species is unusual in that, while the secondary structure of V4 in Mesodinium sp. has the deletions common to all litostome ciliates, it has more extensive deletions in helix E23₈ and a longer helix E23₁;（3） combining the phylogenetic and morphological information, we suggest establishing Mesodiniea cl. nov., including the order Mesodiniida Grain, 1994, belonging to the subphylum Intramacronucleata.     

Community Characteristics of Early Recovery Vegetation on Abandoned Lands of Shifting Cultivation in Bawangling of Hainan Island, South China

Shifting cultivation is a major form of agricultural practice in most parts of tropical regions worldwide. In places where the bush fallow period is excessively shortened or the period of cultivation is extended for too long, the rate of vegetation recovery and biodiversity on abandoned lands of shifting cultivation would decline. The recovery of the secondary plant communities could even be inhibited for a prolonged period because of grass occupancy. Because of the vital significance of the early recovery communities to secondary succession, we studied the community characteristics of early recovery vegetation on abandoned lands of shifting cultivation in Bawangling of Hainan Island. Measurements were made of the community composition and structure of early recovery vegetation. The sprouting abilities of different functional groups and different species in the same functional group, and the effect of the grass functional group on the composition and quantitative characteristics of tree and shrub functional groups were analyzed. Results indicated that only a few families, genera, or species apparently dominated in the early recovery vegetation on the abandoned lands of shifting cultivation and that deciduous species occurred with a rather high percentage in this early recovery community compared with the natural secondary or old growth forests. Smallsized individuals dominated the woody community. The abundance and basal area of sprouting stems for species in the tree functional group were greater than those of seeder stems, whereas the abundance and basal area of resprouters and seeders for species in the shrub functional group did not differ. The total abundance of stems for the community, stem abundances for species in tree or shrub functional groups, and for seeder or resprouter stems were all negatively correlated with coverage of the grass functional group. The mean sprouting ability in the tree functional group was greater than in the shrub functional group. The sprouting ability for different species in the same functional group was also significantly different.     

cDNA sequence analysis of ribosomal protein S13 gene in Plutella xylostella （Lepidoptera： Plutellidae）

Ribosomal protein S 13 gene has been cloned and analyzed in many organisms,but there are few documents relating to insects. In this communication, the full-length cDNA sequence of ribosomal protein S 13 gene in the diamondback moth, Plutella xylostella(Lepidoptera: Plutellidae), was determined by using PCR amplification technique. The features of the ribosomal protein S 13 gene sequence were analyzed and the deduced amino acids sequence was compared with those from other insects. The results of multi-alignment of the amino acid sequences between the diamondback moth and other insect species revealed that this gene sequence is highly conserved in insects. Based on maximum likelihood method, a phylogenetic tree was constructed from 10 different species using PHYLIP software. It showed that nematode is one separate lineage and the five insect speciesbe long to another lineage, whereas those species higher than insects form the third one. The pattern of this phylogenetic tree evidently represented the evolution of different species.     

A novel gene——samfR involved in early stage of Streptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library of Streptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_(TH4) as probe. The experiments revealed that this DNA fragment consists of saw D gene and a 1.4 kb Pvu Ⅱ fragment which can accelerate mycelium formation of S. ansochromogerms. The nucleofide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was desiguated as samfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded by hppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) in Rhodococcus globerulus. The function of samfR gene was studied using strategy of gene disruption, and the resulting samfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped     

Molecular Characterization and Expression Analysis of Matrix Metalloproteinase 3 in the Asian Yellow Pond Turtle Mauremys mutica

Matrix metallopeptidase 3 is a zinc-containing proteinase that participates in tissue remodeling and immune responses. In this study, a cDNA encoding matrix metallopeptidase 3 was isolated and characterized from the Asian yellow pond turtle Mauremys mutica（designated as MaMMP3）. The MaMMP3 cDNA is 1805 bp and consists of a 5＇-untranslated region（UTR） of 56 bp, a 3＇-UTR of 243 bp, and an open reading frame（ORF） of 1506 bp encoding 481 amino acids. Homology analysis of MaMMP3 revealed that the MaMMP3 shared 25%–63% similarity to other known MMP3 sequences. The genomic sequence covers 6007 bp. Comparative analysis of the cDNA sequence revealed that the Asian yellow pond turtle MMP3 has eight exons and seven introns. The phylogenetic tree showed that the MaMMP3 is closely related to Gallus gallus MMP3 and Taeniopygia guttata MMP3. The mRNA expression of the MaMMP3 in normal group without any bacterial challenge could be detected in all studied tissues including kidney, heart, live and spleen, with the highest level in the spleen. The results of immune challenge showed that the expression level of MaMMP3 was up-regulated in the spleen and liver. These results provided an important information for studying the roles of Asian yellow pond turtle MMP3 in immunity further.     

Phylogeography and Cryptic Species Diversity of Paramesotriton caudopunctatus Species Group(Salamandridae: Paramesotriton) in Guizhou,China

The Paramesotriton caudopunctatus species group is mainly distributed in the karst mountain ecosystems of Guizhou, China. Although some species have been included in previous phylogenetic studies, the evolutionary relationships and divergence-time of members of this species group as a whole remain unexplored. In this study, we report the sequencing of one protein coding mitochondrial gene fragment(ND2) and one nuclear gene(POMC), and use a combination of phylogenetic analyses and coalescent simulations to explore the cryptic diversity and evolutionary history of the P. caudopunctatus species group. Phylogenetic relationships revealed that the P. caudopunctatus species group is composed of two major groups, i. e., East Clade and Western-South Clade. The divergence-time and ancestral area estimation suggested that the P. caudopunctatus species group likely originated in the Doupeng Mountains in Guizhou, China at 12.34 Ma(95% HPD: 8.30–14.73), and intraspecific divergence began at about 2.17 Ma(95% HPD: 1.39–2.97). This timing coincides with the orogenesis of the Miaoling Mountains during the Late Miocene to early Pleistocene. The delimitation of species in the P. caudopunctatus species group supports the existence of the currently identified species, and consensus was confirmed across methods for the existence of least to two cryptic species within what has been traditionally considered to be P. caudopunctatus species group. This study is of significance for understanding the species formation, dispersal, and diversity of the tailed amphibians in the karst mountains ecosystem of Guizhou and the role of the Miaoling Mountains as a geographical barrier to species dispersal.     

番茄烟粉虱传双生病毒PCR检测

From the conserved regions of the reported nucleotide sequences of whitefly-transmitted geminiviruses (WTGV), a pair of degenerate primers was designed to anneal to the conserved sequence.The tomato samples infected geminivirus-like from Guangdong were detected by PCR. The results showed that a 356bp specific fragment was amplified from the samples. The specific fragment was cloned and sequenced, and the sequence was compared with all nucleotide sequences in GenBank by Blast of NCBI. The result showed that the fragment belonged to Geminiviridae DNA. So the degenerate primers may be used to detect the WTGV from tomato in Guangdong. Moreover, both of the homology of the fragment between WTGV from tomato in Guangdong and the reported WTGV in the world and WTGV from tomato in Guangxi were under 82%. These results implied that the WTGV from tomato in Guangdong differed from the above-mentioned WTGV.     

Sequence Divergence of Microsatellites and Phylogeny Analysis in Tetraploid Cotton Species and Their Putative Diploid Ancestors

To determine the level of microsatellite sequence differences and to use the information to construct a phylogenetic relationship for cultivated tetraploid cotton （Gossypium spp.） species and their putative diploid ancestors, 10 genome-derived microsatellite primer pairs were used to amplify eight species, including two tetraploid and six diploid species, in Gossypium. A total of 92 unique amplicons were resolved using polyacrylamide gel electrophoresis. Each amplicon was cloned, sequenced, and analyzed using standard phylogenetic software. Allelic diversities were caused mostly by changes in the number of simple sequence repeat （SSR） motif repeats and only a small proportion resulted from interruption of the SSR motif within the locus for the same genome. The frequency of base substitutions was 0.5%-1.0% in different genomes, with only few indels found. Based on the combined 10 SSR flanking sequence data, the homology of A-genome diploid species averaged 98.9%, even though most of the amplicons were of the same size, and the sequence homology between G. gossypioides （Ulbr.） Standl. and three other D-genome species （G. raimondii Ulbr., G. davidsonii Kell., and G. thurberi Tod.） was 98.5%, 98.6%, and 98.5%, respectively. Phylogenetic trees of the two allotetraploid species and their putative diploid progenitors showed that homoelogous sequences from the A- and D-subgenome were still present in the polyploid subgenomes and they evolved independently. Meanwhile, homoelogous sequence interaction that duplicated loci in the polyploid subgenomes became phylogenetic sisters was also found in the evolutionary history of tetraploid cotton species. The results of the present study suggest that evaluation of SSR variation at the sequence level can be effective in exploring the evolutionary relationships among Gossypuim species.     

Treatment of chronical myocardial ischemia by adenovirus-mediated hepatocyte growth factor gene transfer in minipigs

Growth factor gene transfer-induced therapeutic angiogenesis has become a novel approach for the treatment of myocardial ischemia. In order to provide a basis for the clinical application of an adeno- virus with hepatocyte growth factor gene (Ad-HGF) in the treatment of myocardial ischemia, we estab- lished a minipig model of chronically ischemic myocardium in which an Ameroid constrictor was placed around the left circumflex branch of the coronary artery (LCX). A total of 18 minipigs were ran- domly divided into 3 groups: a surgery control group, a model group and an Ad-HGF treatment group implanted with Ameroid constrictor. Ad-HGF or the control agent was injected directly into the ischemic myocardium, and an improvement in heart function and blood supply were evaluated. The results showed that myocardial perfusion remarkably improved in the Ad-HGF group compared with that in both the control and model groups. Four weeks after the treatment, the density of newly formed blood vessels was higher and the number of collateral blood vessels was greater in the Ad-HGF group than in the model group. The area of myocardial ischemia reduced evidently and the left ventricular ejection fraction improved significantly in the Ad-HGF group. These results suggest that HGF gene therapy may become a novel approach in the treatment of chronically ischemic myocardium.     

An Endoreticulatus species from Ocinara lida (Lepidoptera: Bombycidae) in Taiwan

A microsporidium from the Ficus pest, Ocinara lida, in Taiwan is characterized. The taxonomic position of this species was preliminarily determined by sequencing small subunit rRNA gene (SSUrRNA). Analysis of the SSUrRNA sequence indicated that this isolate from O. lida is a member of the genus Endoreticulatus and belongs to the genetic grouping containing other lepidopteran Endoreticulatus species we have analyzed phylogenetically. The taxonomic position of this isolate was also confirmed by the ultrastructural characteristics of this isolate. The congruence between SSUrRNA sequence analysis and ultrastructural characteristics shows that this isolate is more closely related to Endoreticulatus bombycis than to Endoreticulatus schubergi Zw?lfer.     

家蚕微粒子病原虫(Nosema bombycis)小亚基核糖体RNA全基因的克隆及其二级结构的构建

在用PCR技术扩增、克隆、测序了家蚕微粒子病原虫Nosema bombycis (镇江株)小亚基核糖体RNA基因核心序列(5'-端起1 200 bp)的基础上,用SSP-PCR技术克隆了核心序列3'-端下游序列,从而获得了家蚕微粒子病原虫小亚基核糖体RNA基因的全序列共1 233 bp。 用RnaViz 、Forcon、DCSE等生物软件构建了家蚕微粒子病原虫小亚基核糖体RNA的二级结构,与其它微孢子虫及真核生物小亚基核糖体RNA的二级结构相比,该二级结构缺乏螺旋10、E10-1、11、18、E23-n和43。     

The novel organization and complete sequence of the ribosomal RNA gene of Nosema bombycis

We present here for the first time the complete DNA sequence data (4301bp) of the ribosomal RNA (rRNA) gene of the microsporidian type species, Nosema bombycis. Sequences for the large subunit gene (LSUrRNA: 2497bp, GenBank Accession No. ), the internal transcribed spacer (ITS: 179bp, GenBank Accession No. ), the small subunit gene (SSUrRNA: 1232bp), intergenic spacer (IGS: 279bp), and 5S region (114bp) are also given, and the secondary structure of the large subunit is discussed. The organization of the N. bombycis rRNA gene is LSUrRNA-ITS-SSUrRNA-IGS-5S. This novel arrangement, in which the LSU is 5' of the SSU, is the reverse of the organizational sequence (i.e., SSU-ITS-LSU) found in all previously reported microsporidian rRNAs, including Nosema apis. This unique character in the type species may have taxonomic implications for the members of the genus Nosema.     

微孢子虫一新种的描述及分子鉴定

随着集约化、高密度养殖业的发展, 传播性寄生虫病给水产业造成了严重的损失, 微孢子虫是养殖鱼类的主要病原生物之一, 迄今, 已报道的鱼类寄生微孢子虫多达100 余种。       

Arnoldiellina fluorescens gen. et sp. nov. – A new green autofluorescent foraminifer from the Gulf of Eilat (Israel)

A new monothalamous (single-chambered) soft-walled foraminiferal species, Arnoldiellina fluorescens gen. et sp. nov., was isolated from samples collected in the Gulf of Eilat, Israel. The species is characterized by a small elongate organic theca with a single aperture of allogromiids. It is characterized by the emission of green autofluorescence (GAF) that has so far not been reported from foraminifera. Phylogenetic analysis of a fragment of the 18S rDNA indicates that the species is related to a group of monothalamous foraminiferans classified as clade I. Although the morphology of the new species is very different compared to the other members of this clade, a specific helix in 18S rRNA secondary structure strongly supports this position.     

Conformation and molecular dynamics calculations on uteroglobin fragment 18-47

The conformational properties of fragment 18–47 of rabbit uteroglobin in aqueous solution containing SDS micelles were investigated by two-dimensional nmr spectroscopy and molecular dynamics calculations. The fragment comprises helices II and III and the β-turn connecting the two helices. The nmr results and nmr-restrained molecular dynamics calculations showed that in the isolated fragment the elements of secondary structure present in the intact protein are preserved only in part. Specifically, a well-defined α-helix was found in the sequence 33–44, corresponding to helix III of uteroglobin, while the regions of helix II and β-turn are characterized by high flexibility in the fragment. © 1994 John Wiley & Sons, Inc.     

Ultrastructure and development of Pleistophora ronneafiei n. sp., a microsporidium (Protista) in the skeletal muscle of an immune-compromised individual

This report provides a detailed ultrastructural study of the life cycle, including proliferative and sporogonic developmental stages, of the first Pleistophora species (microsporidium) obtained from an immune-incompetent patient. In 1985, the organism obtained from a muscle biopsy was initially identified as belonging to the genus Pleistophora, based on spore morphology and its location in a sporophorous vesicle. Since that initial report, at least two new microsporidial genera, Trachipleistophora and Brachiola, have been reported to infect the muscle tissue of immunologically compromised patients. Because Trachipleistophora development is similar to Pleistophora, and as Pleistophora was only known to occur in cold-blooded hosts, the question of the proper classification of this microsporidium arose. The information acquired in this study makes it possible to compare Pleistophora sp. (Ledford et al. 1985) to the known human infections and properly determine its correct taxonomic position. Our ultrastructural data have revealed the formation of multinucleate sporogonial plasmodia, a developmental characteristic of the genus Pleistophora and not Trachipleistophora. A comparison with other species of the genus supports the establishment of a new species. This parasite is given the name Pleistophora ronneafiei n. sp.     

The gene encoding cytochrome c oxidase subunit II from Rhodobacter sphaeroides; comparison of the deduced amino acid sequence with sequences of corresponding peptides from other species.

The gene (coxII) encoding subunit II of Rhodobacter sphaeroides cytochrome c oxidase (cytochrome aa3) has been isolated by screening a genomic DNA library in phage lambda with a probe derived from coxII of Paracoccus denitrificans. A 2-kb fragment containing coxII DNA was subcloned into the phage M13mp18 and the sequence determined. The 2-kb insert contains the entire coding region for coxII gene, including the ATG start codon and a TGA stop codon. The deduced amino acid (aa) sequence of subunit II of R. sphaeroides shows regions of substantial homology to the corresponding subunit of the bovine mitochondrial oxidase (63% overall) and P. denitrificans oxidase (68% overall). The postulated redox-active copper ion (CuA) binding site involving two Cys and two His residues (as well as an alternative Met residue) is conserved among these species, along with four invariant acidic aa residues (two Asp and two Glu) that may be involved in interactions with cytochrome c, and a region of aromatic residues (Tyr-Gln-Trp-Tyr-Trp-Gly-Tyr-Glu-Tyr) which is postulated to play a role in electron transfer. Hydropathy profile analysis suggests that while the bovine COXII secondary structure contains two transmembrane helices, the R. sphaeroides subunit II has a third such helix that may function as part of a signal sequence, as suggested for P. denitrificans.     

The characterization of microsporidian isolates (Nosematidae: Nosema) from five important lepidopteran pests in Taiwan

Microsporidian isolates from five lepidopteran pests-Spodoptera litura, Spodoptera exigua, Helicoverpa armigera, Plutella xylostella, and Pieris spp.-were compared by spore morphology, infectivity to S. litura, Western-blot banding patterns, the sequences of small subunit rRNA gene (SSUrRNA sequence), and random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). All the isolates could infect experimentally and multiply in the larvae of S. litura. The S. exigua isolate showed the highest virulence to the larvae of S. litura while the P. xylostella isolate showed the lowest. No significant differences either in spore morphology or in SSUrRNA sequences of these isolates were found. The SSUrRNA sequences of these isolates confirmed they are members of the genus Nosema. Based on the result of Western-blot hybridization with the rabbit anti-Nosema spodopterae spore antiserum, three serotypes could be distinguished: N. spodopterae (S. litura isolate) and Pi. spp. isolate; S. exigua and H. armigera isolates; and P. xylostella isolate. The amplicons of RAPD-PCR with 60 primers yielded clear patterns that were selected and used for identification and also for phylogenic analysis of these five isolates. Based on analysis by the computer, isolates could be clearly divided into three groups that were coincident with the serotypes; therefore we suggest that N. spodopterae and isolates of Pi. spp., S. exigua, and H. armigera are more closely related in phylogenesis. In addition, in the amplification with the Nosema bombycis specific primer set, only DNAs from P. xylostella isolate and N. bombycis yielded amplicons. Therefore, we suggest that four isolates, excluding the P. xylostella isolate, are N. spodopterae, and the taxonomic position of P. xylostella isolate needs to be elucidated.     

菜粉蝶微孢子虫核糖体RNA(rRNA)编码基因的研究

用PCR方法扩增、克隆了菜粉蝶微孢子虫核糖体小亚单位RNA(SSUrRNA)编码基因的核心序列 1 2 0 5bp后 ,进一步克隆到菜粉蝶微孢子虫SSUrRNA基因 3′端至LSUrRNA基因 5′端 (580R区 ) 657bp长的序列。与GenBank中对应序列比较后 ,在 657bp这段序列鉴定出菜粉蝶微孢子虫SSUrRNA基因 3′末端、rRNA基因内转录间隔区 (ITS)及LSUrRNA基因 5′端 (580R区 ) ,它们分别位于该序列中 1 45位、1 46 1 86位及 1 87位。与SSUrRNA基因核心序列拼接后SSUrRNA全基因长为 1 2 4 5bp ,rRNA基因内转录间隔区为 41bp及核糖体大亚单位RNA(LSUr RNA)编码基因 580R区为 470bp。同时还构建了菜粉蝶微孢子虫SSUrRNA的完整二级结构。关于微孢子虫rRNA基因的克隆及SSUrRNA的二级结构在国内尚属首次报道 ,它为进一步利用核糖体RNA编码基因及SSUrRNA的二级结构对不同微孢子虫的分类及亲缘关系的确定奠定了基础