Cloning and Expression of the vp39 Gene of Bombyx mori Nuclear Polyhedrosis Virus in E.coli

The nuclear capsid protein gene (vp39) ofBombyx mori nuclear polyhedrosis virus (BmNPV) was amplified successfully by PCR technique and inserted into pGEM 3zf(+). The 5′ and 3′ terminal area of the amplified vp39 gene were sequenced with silver-staining dideoxy method. Bmvp39 gene was sub-cloned into the expression vector pRSET-A, and transformed intoE. coli BL21. This gene was highly expressed by IPTG induction. SDS-PAGE analysis showed that the expressed protein is about 38 kd, and the expressed amount reached maxium in 4 h with IPTG induction.

The Clam Cyclina sinensis (Gmelin) Phylogeography Study with 28S rRNA Gene and Potential of Nuclear rRNA Genes in Genetic Assessments of Molluscs

Intraspecific diversity of molluscan species is usually studied based on maternally inherited mitochondrial DNA,from which only part of the evolutionary history can be reflected.Some nuclear ribosomal RNA genes such as 28S rRNA represent poten-tial candidates that can be easily applied in phylogeography because of lacking intraindividual variation.However,considering their low polymorphism,genetic appraisals on whether and how they can be used in population studies are necessary.Here,we applied a short 28S rRNA to assess genetic patterns of the clam Cyclina sinensis along the coast of China and compared the results with a for-mer study based on COI and ITS-1 analyses.The results revealed the 28S rRNA data set was characterized by an extremely low level of variation,with only seven haplotypes defined for 93 individuals.Haplotype and nucleotide diversity for each population was al-most the lowest when compared with the other two markers.However,the distribution of two dominant haplotypes showed clear geo-graphic patterns,and significant population differentiation was revealed between the East China Sea and the South China Sea.These patterns were highly concordant with findings of the former study that populations of C.sinensis were historically separated by land bridges among sea basins.Our study suggested that although the nuclear rRNAs have shortcomings such as low variation,they have advantages including lack of intraindividual variation and high amplification rates.Applying rRNA genes can enrich the toolbox of nuclear markers in molluscan phylogeographic studies.

Phylogenetic relationship of Podocopida (Ostracoda: Podocopa) based on 18S ribosomal DNA sequences

Nucleotide sequences from 18S rDNA of 11 ostracodes, which represent four suborders and six superfamilies of podocopidan, were determined. The phylogenetic relationships were analyzed based on three kinds of methods (maximum-likelihood, maximum-parsimony, and neighbor-joining), and the three topologies gained were basically similar. The results have showed that (1) a monophyletic Podocopida was supported strongly; (2) the phylogenetic relationships of four suborders were (Darwinulocopina plus (Bairdiocopina plus (Cytherocopina plus Cypridocopina))), which indicated that a close relationship between Cytherocopina and Cypridocopina, and Darwinulocopina had separated early from the main podocopinan; (3) Cypridocopinan formed a monophyletic group, among which the phylogenetic relationship of three superfamilies was (Cypridoidea plus (Macrocypridoidea plus Pontocypridoidea)).

Nuclear regulation of mitochondrial functions during oocyte development

Mitochondria are important in eukaryotic cells due to their functions in energy production and regulation over other cellular activities.Oocytes are produced by a long and precisely controlled process,the dysfunction of which leads to impaired female fertility.As oocytes mature,mitochondria are constantly under the regulation of nuclear genes,the process of which can be modulated by extracellular signals.Understanding how nuclear genes regulate mitochondrial functions is important for studying animal reproduction and human fertility.As more and more genes regulating mitochondrial functions in oocytes are being revealed,new approaches for improving female fertility in both human and animals through mitochondria can be developed.

A Novel NR0B1 Gene Mutation Causes Different Phenotypes in Two Male Patients with Congenital Adrenal Hypoplasia

X-linked congenital adrenal hypoplasia is characterised by the acute onset of primary adrenal insufficiency in infancy or early childhood and hypogonadotropic hypogonadism(HH)at puberty,arising from mutations of the nuclear receptor subfamily 0 group B member 1(NR0B1)gene.This study investigated an extended family with two affected males(patient A:23 years and patient B:2 months old)and three carrier females.Sequencing analysis of the NR0B1 gene coding region from the family revealed a novel hemizygous deletion[c.604delT;p.(C202Afs*62)]in the two male patients.Furthermore,the patients'respective mothers and their common grandmother had this heterozygous mutation,but it was not present in the Human Gene Mutation Database.The two male patients showed inconsistent clinical features at onset,particularly in early childhood;however,it is possible that the younger patient will eventually show a delay of puberty,feminisation,and nonspermatogenesis in adulthood,similar to that in the older patient.Identification of a novel NR0BI mutation in this family is important for the diagnosis and genetic counselling of children with primary adrenal insufficiency and HH,and will be helpful for predicting long-term clinical symptoms.

Phylogeny,character evolution,and classification of Selaginellaceae(lycophytes)

Selaginella is the largest and most taxonomically complex genus in lycophytes.The fact that over 750 species are currently treated in a single genus makes Selaginellales/Selaginellaceae unique in pteridophytes.Here we assembled a dataset of six existing and newly sampled plastid and nuclear loci with a total of 684 accessions(74%increase of the earlier largest sampling)representing ca.300 species to infer a new phylogeny.The evolution of 10 morphological characters is studied in the new phylogenetic context.Our major results include:(1)the nuclear and plastid phylogenies are congruent with each other and combined analysis well resolved and strongly supported the relationships of all but two major clades;(2)the Sinensis group is resolved as sister to S.subg.Pulviniella with strong support in two of the three analyses;(3)most morphological characters are highly homoplasious but some characters alone or combinations of characters well define the major clades in the family;and(4)an infrafamilial classification of Selaginellaceae is proposed and the currently defined Selaginella s.l.is split into seven subfamilies(corresponding to the current six subgenera t the Sinensis group)and 19 genera(the major diagnosable clades)with nine new species-poor genera.We support the conservation of Selaginella with a new type,S.flabellata,to minimize nomenclatural instability.We provide a key to subfamilies and genera,images illustrating their morphology,their morphological and geographical synopses,a list of constituent species,and necessary new combinations.This new classification will hopefully facilitate communication,promote further studies,and help conservation.

Nuclear phylogenomics of angiosperms and insights into their relationships and evolution

Angiosperms(flowering plants) are by far the most diverse land plant group with over 300,000 species. The sudden appearance of diverse angiosperms in the fossil record was referred to by Darwin as the “abominable mystery,”hence contributing to the heightened interest in angiosperm evolution. Angiosperms display wide ranges of morphological, physiological,and ecological characters, some of which have probably influenced their species richness. The evolutionary analyses of these characteristics help to address questions of angiosperm diversification and require well resolved phylogeny. Following the great successes of phylogenetic analyses using plastid sequences,dozens to thousands of nuclear genes from next-generation sequencing have been used in angiosperm phylogenomic analyses, providing well resolved phylogenies and new insights into the evolution of angiosperms. In this review we focus on recent nuclear phylogenomic analyses of large angiosperm clades, orders, families,and subdivisions of some families and provide a summarized Nuclear Phylogenetic Tree of Angiosperm Families. The newly established nuclear phylogenetic relationships are highlighted and compared with previous phylogenetic results. The sequenced genomes of Amborella,Nymphaea, Chloranthus, Ceratophyllum, and species of monocots, Magnoliids, and basal eudicots, have facilitated the phylogenomics of relationships among five major angiosperms clades. All but one of the 64 angiosperm orders were included in nuclear phylogenomics with well resolved relationships except the placements of several orders. Most families have been included with robust and highly supported placements, especially for relationships within several large and important orders and families.Additionally, we examine the divergence time estimation and biogeographic analyses of angiosperm on the basis of the nuclear phylogenomic frameworks and discuss the differences compared with previous analyses. Furthermore,we discuss the implications of nuclear phylogenomic analyses on ancestral reconstruction of morphological, physiological, and ecological characters of angiosperm groups, limitations of current nuclear phylogenomic studies, and the taxa that require future attention.

Comparative Analysis of Codon Usage Patterns Among Mitochondrion, Chloroplast and Nuclear Genes

In many organisms, the difference in codon usage patterns among genes reflects variation in local base compositional biases and the intensity of natural selection. In this study, a comparative analysis was performed to investigate the characteristics of codon bias and factors in shaping the codon usage patterns among mitochondrion, chloroplast and nuclear genes in common wheat （Triticum aestivum L.）. GC contents in nuclear genes were higher than that in mitochondrion and chloroplast genes. The neutrality and correspondence analyses indicated that the codon usage in nuclear genes would be a result of relative strong mutational bias, while the codon usage patterns of mitochondrion and chloroplast genes were more conserved in GC content and influenced by translation level. The Parity Rule 2 （PR2） plot analysis showed that pyrimidines were used more frequently than purines at the third codon position in the three genomes. In addition, using a new alterative strategy, 11, 12, and 24 triplets were defined as preferred codons in the mitochondrion, chloroplast and nuclear genes, respectively. These findings suggested that the mitochondrion, chloroplast and nuclear genes shared particularly different features of codon usage and evolutionary constraints.

Summary of Laurasiatheria （Mammalia） Phylogeny

Laurasiatheria is one of the richest and most diverse superorders of placental mammals. Because this group had a rapid evolutionary radiation, the phylogenetic relationships among the six orders of Laurasiatheria remain a subject of heated debate and several issues related to its phylogeny remain open. Reconstructing the true phylogenetic relationships of Laurasiatheria is a significant case study in evolutionary biology due to the diversity of this suborder and such research will have significant implications for biodiversity conservation. We review the higher-level （inter-ordinal） phylogenies of Laurasiatheria based on previous cytogenetic, morphological and molecular data, and discuss the controversies of its phylogenetic relationship. This review aims to outline future researches on Laurasiatheria phylogeny and adaptive evolution.

A peep into mitochondrial disorder： multifaceted from mitochondrial DNA mutations to nuclear gene modulation

Mitochondrial genome is responsible for multiple human diseases in a maternal inherited pattern, yet phenotypes of patients in a same pedigree frequently vary largely. Genes involving in epigenetic modification, RNA processing, and other biological pathways, rather than ＂threshold effect＂ and environmental factors, provide more specific explanation to the aberrant phenotype. Thus, the double hit theory, mutations both in mito- chondrial DNA and modifying genes aggravating the symptom, throws new light on mitochondrial dysfunc- tion processes. In addition, mitochondrial retrograde signaling pathway that leads to reconfiguration of cell metabolism to adapt defects in mitochondria may as well play an active role. Here we review selected examples of modifier genes and mitochondrial retrograde signaling in mitochondrial disorders, which refine our understanding and will guide the rational design of clinical therapies.

Summary of Laurasiatheria(Mammalia)Phylogeny

Laurasiatheria is one of the richest and most diverse superorders of placental mammals.Because this group had a rapid evolutionary radiation,the phylogenetic relationships among the six orders of Laurasiatheria remain a subject of heated debate and several issues related to its phylogeny remain open.Reconstructing the true phylogenetic relationships of Laurasiatheria is a significant case study in evolutionary biology due to the diversity of this suborder and such research will have significant implications for biodiversity conservation.We review the higher-level(inter-ordinal)phylogenies of Laurasiatheria based on previous cytogenetic,morphological and molecular data,and discuss the controversies of its phylogenetic relationship.This review aims to outline future researches on Laurasiatheria phylogeny and adaptive evolution.

A New Species of the Asian Leaf Litter Toad Genus Leptobrachella(Amphibia,Anura,Megophryidae)from Chongqing City,Southwest China

Herein we describe a new species,Leptobrachella yunyangensis sp.nov.,from Yunyang County,Chongqing City,China,based on a combination of molecular and morphological data.Phylogenetic analysis based on the mitochondrial 16 S rRNA gene and six nuclear genes indicated that this new species represented an independent evolutionary lineage.The uncorrected genetic distance between the new species and its closest congener species,L.oshanensis,was 5.4 % for 16 S rRNA.The new species can be distinguished from its congeners by a combination of the following characters:(1) moderate body size(SVL28.3-30.6 mm in males);(2) rough dorsal skin,with sparse large granules and tubercles and short longitudinal ridges on the shoulder;(3) distinctly discernible tympanum with a diameter smaller than that of the eye(TMP/EYE ratio:0.51) and a distinct black supra tympanic line present;(4)an internasal distance almost equal to interorbital distance(IND/IOD ratio:1.05);(5) flanks with several dark spots arranged longitudinally in two rows;(6) distinctly visible supra-axilla ry,femoral,pectoral,a nd ventrola teral glands;(7) bicolored iris,with the upper 1/3 of the iris being copper orange and the lower 2/3 a light silvery grey;(8) relative finger lengths of Ⅰ <Ⅱ=Ⅳ <Ⅲ and relative toe lengths of Ⅰ <Ⅱ <Ⅴ <Ⅲ <Ⅳ;(9) absence of webbing and lateral fringes on fingers,and toes with rudimentary webbing and narrow lateral fringes;(10) heels overlapping when the thighs are positioned at right angles to the body;(11) tibiotarsal articulation reaching to the anterior corner of the eye when the leg is stretched forward;(12) ventral surfaces of the throat,chest,and belly greyish white with purple-brown speckling;(13) lacking distinct blackish dorsolateral markings;and(14) transverse dark brown bars on the surfaces of limbs and digits.

Overexpression of the nuclear protein gene AtDUF4 increases organ size in Arabidopsis thaliana and Brassica napus

Organ size is an important trait of many crops that is influenced by internal and environmental signals and controlled by a combina- tion of factors during organogenesis （I（rizek, 2009）. The final size of plant organs is determined by two successive but overlapping pro- cesses： cell division, which increases cell number, and cell expan- sion, which determines final cell size （Anastasiou and Lenhard, 2007）. Some genes have been identified to control organ size by regulating cell division and for cell expansion in plants.

Growth inhibiting effects of antisense eukaryotic expression vector of proliferating cell nuclear antigen gene on human bladder cancer cells

Objective To explore the growth inhibiting effects on human bladder cancer by antisense RNA targeting the proliferating cell nuclear antigen (PCNA) gene. Methods The eukaryotic expression vector for antisense PCNA cDNA was constructed and transferred into a bladder cancer EJ cell line. The PCNA expression in the cancer cells was detected by RT-PCR and Western blotting assays. The in vitro proliferation activities of the transferred cells were observed by growth curve,tetrazolium bromide (MTT) colorimetry,tritiated thymidine ( 3H-TdR)incorporation, flow cytometry and clone formation testing,while its in vivo anti-tumor effects were detected on nude mice allograft models.Results After the antisense vector,pLAPSN,was transferred,cellular PCNA expression was inhibited at both protein and mRNA levels. The growth rates of EJ cells were reduced from 27.91% to 62.07% ( P <0.01),with an inhibition of DNA synthesis rate by 52.31% ( P <0.01). Transferred cells were blocked at G 0/G 1 phases in cell-cycle assay,with the clone formation ability decreased by 50.81% ( P <0.01). The in vivo carcinogenic abilities of the transferred cancer cells were decreased by 54.23% ( P <0.05). Conclusions Antisense PCNA gene transfer could inhibit the growth of bladder cancer cells in vitro and in vivo,which provided an ideal strategy for gene therapy of human cancers.

Chloroplast DNA Underwent Independent Selection from Nuclear Genes during Soybean Domestication and Improvement

The chloroplast is one of the most important organs in plants because of its essential role in photosynthesis.Studies have shown that the chloroplast was once a free-living cyanobacteria and was integrated into the host species through endosymbiosis（Goksoyr.1967）.after which a large number of its genes had been donated to the host nuclear genome（Heins and Soll, 1998）.

Rapid Evolution of the Mammalian HILS1 Gene and the Nuclear Condensation Process during Mammalian Spermiogenesis

Many genes associated with reproduction show rapid evolution across diverse animal groups, a result commonly due to adaptive evolution driven by positive selection （Swanson and Vacquier, 2002）. Different theories have been proposed to explain the elevated rates of evolution （Swanson and Vacquier, 2002）, including sperm competition, where sperm compete to fertilize eggs leading to the proteins in the sper- matozoa adaptively evolving to increase their ability to fertilize eggs; sexual conflict, where the egg experiences a loss of fitness when sperm are too abundant; sexual selection, where eggs bind sperm carrying adaptive alleles （Palumbi, 1999）; and cryptic female choice （reviewed in Swanson and Vacquier, 2002）.

Vampyroteuthis pseudoinfernalis sp.nov.:the second extant widespread deep sea squid species of Vampyromorpha(Cephalopoda:Coleoidea)

The vampire squid,Vampyroteuthis infernalis Chun,1903,is currently the only extant species in the family Vampyroteuthidae Chun,1903.However,specimens from the Gulf of Guinea,Africa,and California suggested the possibility of additional taxa.Here,we report the second species of Vampyroteuthis,collected from the South China Sea,China,which differs from V.infernalis by its tail shape,lower beak,photophores position,and the phylogenetic analysis of mitochondrial COI and nuclear large subunit ribosomal DNA(LSU)sequences:V.infernalis present by the lacking of the tail,photophores located approximately one-third of the points between the fins and end,and the lower beak with a broad,short wing;V.pseudoinfernalis Qiu,Liu&Huang,sp.nov.present by an acuate tail,a pair of photophores located at the midpoints between the fins and tail,and a lower beak with a broad,elongate wing.

Identification of four novel mutations in the HNF-1A gene in Chinese early-onset and/or multiplex diabetes pedigrees

Background Mutations in the hepatocyte nuclear factor-lA gene cause the type 3 form of maturity-onset diabetes of the young （MODY3）. This study was undertaken to determine mutations and sequence variations of the HNF-1A gene in Chinese with familial early-onset and/or multiplex diabetes mellitus.

Emerging roles of NudC family： from molecular regulation to clinical implications

Nuclear distribution gene C （NudC） was first found in Aspergillus nidulans as an upstream regulator of NudF, whose mamma- lian homolog is Lissencephaly 1 （Lisl）. NudC is conserved from fungi to mammals. Vertebrate NudC has three homologs： NudC, NudC-like protein （NudCL）, and NudC-like protein 2 （NudCL2）. All members of the NudC family share a conserved p23 domain, which possesses chaperone activity both in conjunction with and independently of heat shock protein 90 （Hsp90）. Our group and the others found that NudC homologs were involved in cell cycle regulation by stabilizing the components of the LIS l/dynein complex. Additionally, NudC plays important roles in cell migration, ciliogenesis, thrombopoiesis, and the in- flammatory response. It has been reported that NudCL is essential for the stability of the dynein intermediate chain and cilio- genesis via its interaction with the dynein 2 complex. Our data showed that NudCL2 regulates the LISl/dynein pathway by stabilizing LIS 1 with Hsp90 chaperone. The fourth distantly related member of the NudC family, CML66, a tumor-associated antigen in human leukemia, contains a p23 domain and appears to promote oncogenesis by regulating the IGF-1R-MAPK sig- naling pathway. In this review, we summarize our current knowledge of the NudC family and highlight its potential clinical relevance.

Phylogenomic conflict analyses in the apple genus Malus s.l.reveal widespread hybridization and allopolyploidy driving diversification,with insights into the complex biogeographic history in the Northern Hemisphere

Phylogenomic evidence from an increasing number of studies has demonstrated that different data sets and analytical approaches often reconstruct strongly supported but conflicting relationships.In this study,785 single-copy nuclear genes and 75 complete plastomes were used to infer the phylogenetic relationships and estimate the historical biogeography of the apple genus Malus sensu lato,an economically important lineage disjunctly distributed in the Northern Hemisphere and involved in known and suspected hybridization and allopolyploidy events.The nuclear phylogeny recovered the monophyly of Malus s.l.(including Docynia);however,the genus was supported to be biphyletic in the plastid phylogeny.An ancient chloroplast capture event in the Eocene in western North America best explains the cytonuclear discordance.Our conflict analysis demonstrated that ILS,hybridization,and allopolyploidy could explain the widespread nuclear gene tree discordance.One deep hybridization event(Malus doumeri)and one recent event(Malus coronaria)were detected in Malus s.l.Furthermore,our historical biogeographic analysis integrating living and fossil data supported a widespread East Asianwestern North American origin of Malus s.l.in the Eocene,followed by several extinction and dispersal events in the Northern Hemisphere.We also propose a general workflow for assessing phylogenomic discordance and biogeographic analysis using deep genome skimming data sets.