Molecular and morphological analysis of endangered species Gymnodiptychus integrigymnatus (Teleostei: Cyprinidae)

Gymnodiptychus integrigymnatus is a critically endangered species endemic to the Gaoligongshan Mountains. It was thought to be only distributed in several headwater-streams of the Longchuanjiang River (west slope of the Gaoligongshan Mountains, belonging to the Irrawaddy River drainage). In recent years, dozens of G. integrigymnatus specimens have been collected in some streams on the east slope of the Gaoligongshan Mountains (the Salween drainage). We performed a morphological and genetic analyses (based on cytochrome b and D-loop) of the newly discovered populations of G. integrigymnatus to determine whether the degree of separation of these populations warrants species status. Our analysis from the cytochrome b gene revealed that nine individuals from the Irrawaddy drainage area and seven individuals from the Salween drainage area each have only one unique haplotype. The genetic distance between the two haplotypes is 1.97%. Our phylogenetic analysis revealed that G. integrigymnatus is closely related to highly specialized schizothoracine fishes. Analysis from the mitochondrial control region revealed that G. integrigymnatus has relatively high genetic diversity (pi was 0.00891 and h was 0.8714), and individuals from different river drainages do not share the same haplotypes. The AMOVA results indicated 87.27% genetic variability between the Salween and Irrawaddy populations. Phylogenetic trees show two major geographic groups corresponding to the river systems. We recommend that G. integrigymnatus should be considered as a high priority for protected species status in the Gaoligongshan Mountains National Nature Reserve, and that the area of the Gaoligongshan Mountains National Nature Reserve should be expanded to cover the entire distribution of G. integrigymnatus. Populations of G. integrigymnatus from different river systems should be treated as evolutionarily significant units.

A call for mtDNA data quality control in forensic science

There is increasing evidence that many of the mitochondrial DNA (mtDNA) databases published in the fields of forensic science and molecular anthropology are flawed. An a posteriori phylogenetic analysis of the sequences could help to eliminate most of the errors and thus greatly improve data quality. However, previously published caveats and recommendations along these lines were not yet picked up by all researchers. Here we call for stringent quality control of mtDNA data by haplogroup-directed database comparisons. We take some problematic databases of East Asian mtDNAs, published in the Journal of Forensic Sciences and Forensic Science International, as examples to demonstrate the process of pinpointing obvious errors. Our results show that data sets are not only notoriously plagued by base shifts and artificial recombination but also by lab-specific phantom mutations, especially in the second hypervariable region (HVR-II). (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Molecular characterization and expression profiles in response to bacterial infection of Chinese soft-shelled turtle interleukin-8 (IL-8), the first reptilian chemokine gene

In this study, an IL-8 homologue has been cloned and identified from a reptile, Chinese soft-shelled turtle for the first time. The full-length cDNA of turtle IL-8 was 1188 bp and contained a 312 bp open reading frame (ORF) coding for a protein of 104 amino acids. The chemokine CXC domain, which contained Glu-Leu-Arg (ELR) motif and four cysteine residues, was well conserved in turtle IL-8. The 4924 bp genomic DNA of turtle IL-8 contained four exons and three introns. Phylogenetic analysis showed that the amino acid sequence of turtle IL-8 clustered together with birds. RT-PCR analysis showed that turtle IL-8 mRNA was constitutively expressed liver, spleen, kidney, heart, blood and intestine tissues of control turtles. Real-time quantitative PCR analysis further indicated that the turtle IL-8 mRNA expression was apparent in various tissues at 8 h and up-regulated significantly during 8 h-7 d after Aeromonas hydrophila infection. The present studies will help us to understand the evolution of IL-8 molecule and the inflammatory response mechanism in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.

Molecular cloning and expression characterization of ApoC-I in the orange-spotted grouper

Endogenous yolk nutrients are crucial for embryo and larval development in fish, but developmental behavior of the genes that control yolk utilization remains unknown. Apolipoproteins have been shown to play important roles in lipid transport and uptake through the circulation system. In this study, EcApoC-I, the first cloned ApoC-I in teleosts, has been screened from pituitary cDNA library of female orange-spotted grouper (Epinephelus coioides), and the deduced amino acid sequence shows 43.5% identity to one zebrafish (Danio rerio) hypothetical protein similar to ApoC-I, and 21.2%, 21.7%, 22.5%, 20%, and 22.5% identities to Apo C-I of human (Homo sapiens), house mouse (Mus musculus), common tree shrew (Tupaia glis), dog (Canis lupus familiaris) and hamadryas baboon (Papio hamadryas), respectively. Although the sequence identity is low, amphipathic alpha-helices with the potential to bind to lipid were predicted to exist in the EcApoC-I. RT-PCR analysis revealed that it was first transcribed in gastrula embryos and maintained a relatively stable expression level during the following embryogenesis. During embryonic and early larval development, a very high level of EcApoC-I expression was in the yolk syncytial layer, indicating that it plays a significant role in yolk degradation and transfers nutrition to the embryo and early larva. By the day 7 after hatching, EcApoC-I transcripts were observed in brain. In adult, EcApoC-I mRNA was detected abundantly in brain and gonad. In transitional gonads, the EcApoC-I expression is restricted to the germ cells. The data suggested that EcApoC-I might play an important role in brain and gonad morphogenesis and growth.

Molecular cloning, characterization and expression analysis of the PKZ gene in rare minnow Gobiocypris rarus

Double-stranded RNA-activated protein kinase (PKR) plays an important rote in interferon-induced antiviral responses, and is also involved in intracellular signaling pathways, including the apoptosis, proliferation, and transcription pathways. In the present study, a PKR-like gene was cloned and characterized from rare minnow Gobiocypris rarus. The full length of the rare minnow PKR-like (GrPKZ) cDNA is 1946 bp in Length and encodes a polypeptide of 503 amino acids with an estimated molecular mass of 57,355 Da and a predicted isoelectric point of 5.83. Analysis of the deduced amino acid sequence indicated that the mature peptide contains two Zalpha domains and one S_TKc domain, and is most similar to the crucian carp (Carassius auratus) PKR-like amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed that GrPKZ mRNA expression is at low levels in gill, heart, intestine, kidney, liver, muscle and spleen tissues in healthy animals and up-regulated by viruses and bacteria. After being infected by grass carp reovirus, GrPKZ expression was up-regulated from 24 h post-injection and lasted until the fish became moribund (P < 0.05). Following infection with Aeromonas hydrophila, GrPKZ transcripts were induced at 24 h post-injection (P < 0.05) and returned to control levels at 120 h post-injection. These data imply that GrPKZ is involved in antiviral defense and Toll-like receptor 4 signaling pathway in bacterial infection. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning and stress-induced expression of paralichthys olivaceus heme-regulated initiation factor 2 alpha kinase

The heme-regulated initiation factor 2 alpha kinase (HRI) is acknowledged to play an important role in translational shutoff in reticulocytes in response to various cellular stresses. In this study, we report its homologous cDNA cloning and characterization from cultured flounder embryonic cells (FEC) after treatment with UV-inactivated grass carp haemorrhagic virus (GCHV). The full-length cDNA of Paralichthys olivaceus HRI homologue (PoHRI) has 2391 bp and encodes a protein of 651 amino acids. The putative PoHRI protein exhibits high identity with all members of eIF2 alpha kinase family. It contains 12 catalytic subdomains located within the C-terminus of all Ser/Thr protein kinases, a unique kinase insertion of 136 amino acids between subdomains IV and V, and a relatively conserved N-terminal domain (NTD). Upon heat shock, virus infection or Poly PC treatment, PoHRI mRNA and protein are significantly upregulated in FEC cells but show different expression patterns in response to different stresses. In healthy flounders, PoHRI displays a wide tissue distribution at both the mRNA and protein levels. These results indicate that PoHRI is a ubiquitous eIF2a kinase and might play an important role in translational control over nonheme producing FEC cells under different stresses. (c) 2006 Elsevier Ltd. All rights reserved.

Comparison of evolutionary rates in the mitochondrial DNA cytochrome b gene and control region and their implications for phylogeny of the Cobitoidea (Teleostei : Cypriniformes)

It is widely accepted that mitochondrial DNA (mtDNA) control region evolves faster than protein encoding genes with few exceptions. In the present study, we sequenced the mitochondrial cytochrome b gene (cyt b) and control region (CR) and compared their rates in 93 specimens representing 67 species of loaches and some related taxa in the Cobitoidea (Order Cypriniformes). The results showed that sequence divergences of the CR were broadly higher than those of the cyt b (about 1.83 times). However, in considering only closely related species, CR sequence evolution was slower than that of cyt b gene (ratio of CR/cyt b is 0.78), a pattern that is found to be very common in Cypriniformes. Combined data of the cyt b and CR were used to estimate the phylogenetic relationship of the Cobitoidea by maximum parsimony, neighbor-joining, and Bayesian methods. With Cyprinus carpio and Danio rerio as outgroups, three analyses identified the same four lineages representing four subfamilies of loaches, with Botiinae on the basal-most clade. The phylogenctic relationship of the Cobitoidea was ((Catostomidae + Gyrinocheilidae) + (Botiinae + (Balitorinae + (Cobitinae + Nemacheilinae)))), which indicated that Sawada's Cobitidae (including Cobitinae and Botiinae) was not monophyletic. Our molecular phylogenetic analyses are in very close agreement with the phylogenetic results based on the morphological data proposed by Nalbant and Bianco, wherein these four subfamilies were elevated to the family level as Botiidae, Balitoridae, Cobitidae, and Nemacheilidae. (c) 2005 Elsevier Inc. All rights reserved.

Parentage determination of an isolated Yangtze finless porpoise population Neophocaena phocaenoides asiaeorientalis in the Yangtze Tian-e-Zhou Baiji National Natural Reserve based on molecular data

Reproductive behaviors are poorly known for the Yangtze finless porpoise Neophocaena phocaenoides asiaeorientalis. In this study, the parentage of an isolated Yangtze finless porpoise population inhabiting the Yangtze Tian-e-Zhou Baiji National Natural Reserve is determined by analysis of microsatellite loci and mitochondrial DNA (mtDNA) control region sequences, and the porpoise's reproductive behaviors are studied. Overall 4 full parentage assignments and additional 3 single parentage assignments were determined for the population of 23 individuals. The analysis shows that their estimated reproductive cycle is shorter than that reported previously and there probably exists an overlapping between gestation and lactation period. The Study also shows that the female does not show fidelity to a particular male for breeding and vice versa, the oldest males did not monopolize mating and the dominance rank could not be so strict for the porpoise society. Moreover, the porpoise's mating pattern and relatedness among candidate parents are discussed here. These results provide important information for making guidelines of management and conservation for this protected population.

Phylogeny of the East Asian cyprinids inferred from sequences of the mitochondrial DNA control region

With 210 genera and 2010 species, Cyprinidae is the largest freshwater fish family in the world. Several papers, based on morphological and molecular data, have been published and have led to some solid conclusions, such as the close relationships between North American phoxinins and European leuciscins. However, the relationships among major subgroups of this family are still not well resolved, especially for those East Asian groups. In the present paper, the mitochondrial DNA (mtDNA) control region, 896-956 base pairs, of 17 representative species of East Asian cyprinids was sequenced and compared with those of 21 other cyprinids to study their phylogenetic relationships. After alignment, there were 1051 sites. The comparison between pairwise substitutions and HKY distances showed that the mtDNA control region was suitable for phylogenetic study. Phylogenetic analysis indicated that there are two principal lineages in Cyprinidae: Cyprinine and Leuciscine. In Cyprinine, the relationships could be a basal Labeoinae, an intermediate Cyprininae, and a diversified Barbinae (including Schizothroaxinae). In Leuciscine, Rasborinae is at the basal position; Gobioninae and Leuciscinae are sister groups; the East Asian cultrin-xenocyprinin taxa form a large monophyletic group with some small affiliated groups; and the positions of Acheilognathinae and Tincinae are still uncertain.

Molecular cloning and characterization of crucian carp (Carassius auratus L.) interferon regulatory factor 7

Interferon (IFN) can induce an antiviral state via interferon-regulatory transcription factors (IRFs), which bind to and control genes directed by the interferon-stimulated response element (ISRE). Here we describe a fish IRF, termed CaIRF7, cloned from a subtractive cDNA library which is constructed with mRNAs obtained from crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. CaIRF7 cDNA was found to be 1816 bp in length, with a 42 bp 5' UTR and a 508 bp 3' UTR. The open reading frame translates into 421 amino acids in which a DNA-binding domain (DBD) containing the repeated tryptophan motif and IRFs association domain have been identified. Like chicken GgIRF3, CaIRF7 was most similar to mammalian IRF7 with 27 to 30% identity overall and some 37% identity in their DBDs. A single transcript of 1.9 kb was detected in virally induced CAB cells by virtual Northern blotting. RT-PCR analysis revealed a wide tissue distribution of CaIRF7 constitutive expression, with detectable transcript in non-infected CAB cells and various tissues of healthy crucian carp. In addition, CaIRF7 expression was differentially increased by stimulation of the CAB cells with active GCHV, UV-inactivated GCHV or CAB IFN, indicating that the activation of CaIRF7 was directly regulated by IFN. (C) 2003 Published by Elsevier Ltd.

Surface morphology control of strained InAs/GaAs(331)A films: From nanowires to island-pit pairs

We have studied the effect of molecular beam epitaxy growth conditions on the surface morphology of strained InAs/GaAs(331)A films. Our results reveal that InAs nanowires aligned along the [1 (1) over bar0] direction are formed under As-rich conditions, which is explained by the effect of anisotropic buffer layer surface roughing. Under In-rich conditions, however, the surface morphology of the InAs layers is characterized by a feature of island-pit pairs. In this case, cooperative nucleation of islands and pits can lower the activation barrier for domain growth. These results suggest that the surface morphology of strained InAs layers is highly controllable. (C) 2005 American Institute of Physics.

Shape and spatial correlation control of InAs-InAlAs-InP(001) nanostructure superlattices

The control of shape and spatial correlation of InAs-InAlAs-InP(001) nanostructure superlattices has been realized by changing the As overpressure during the molecular-beam epitaxy (MBE) growth of InAs layers. InAs quantum wires (QWRs) are obtained under higher As overpressure (1x10(-5) Torr), while elongated InAs quantum dots (QDs) are formed under lower As overpressure (5x10(-6) or 2.5x10(-6) Torr). Correspondingly, spatial correlation changes from vertical anti-correlation in QWR superlattices to vertical correlation in QD superlattices, which is well explained by the different alloy phase separation in InAlAs spacer layers triggered by the InAs nanostrcutures. It was observed that the alloy phase separation in QD superlattices could extend a long distance along the growth direction, indicating the vertical correlation of QD superlattices can be kept in a wide range of spacer layer thickness.

A method to obtain ground state electroluminescence from 1.3 mu m emitting InAs/GaAs quantum dots grown by molecular beam epitaxy

1.3 mum emitting InAs/GaAs quantum dots (QDs) have been grown by molecular beam epitaxy and QD light emitting diodes (LEDs) have been fabricated. In the electroluminescence spectra of QD LEDs, two clear peaks corresponding to the ground state emission and the excited state emission are observed. It was found that the ground state emission could be achieved by increasing the number of QDs contained in the active region because of the state filling effect. This work demonstrates a way to control and tune the emitting wavelength of QD LEDs and lasers.

Study of GaN thin films grown on intermediate-temperature buffer layers by molecular beam epitaxy

A detailed characterisation study of GaN thin films grown by rf-plasma molecular beam epitaxy on intermediate-temperature buffer layers (ITBL) was carried out with Hall, photoluminescence (PL) and deep-level transient Fourier spectroscopy (DLTFS) techniques. The unique feature of our GaN thin films is that the GaN epitaxial layers are grown on top of a double layer that consists of an ITBL, which is grown at 690 degreesC, and a conventional low-temperature buffer layer deposited at 500 degreesC. It is observed that the electron mobility increases steadily with the thickness of the ITBL, which peaks at 377 cm(2)V(-1)S(-1) for an ITBL thickness of 800 nm. The PL also demonstrated systematic improvements with the thickness of the ITBL. The DLTFS results suggest a three-order-of-magnitude reduction in the deep level at E-c-0.40 eV in the device fabricated with the GaN films grown on an ITBL thickness of 1.25 mum in comparison with the control device without an ITBL. Our analyses indicate that the utilization of an ITBL in addition to the conventional low-temperature buffer layer leads to the relaxation of residual strain within the material, resulting in an improvement in the optoelectronic properties of the films. (C) 2002 Elsevier Science BN. All rights reserved.

Molecular beam epitaxy growth of Iny2Al1-y2As/In0.73Ga0.27As/Iny1Al1-y1As/InP P-HEMTs with enhancement conductivity using an intentional nonlattice-matched buffer layer

Pseudomorphic Iny2Al1-y2As/In0.73Ga0.27As/Iny1Al1-y1As (y1 greater than or equal to 0.52) modulation-doped heterostructures with an intentional nonlattice-matched buffer layer were successfully grown by molecular beam epitaxy on (100)InP substrates. Fourier transform photoluminescence and double crystal x-ray diffraction measurements show a superior crystalline quality in the high In content channel, when In mole fraction increases from y1=0.52 to 0.55 in the Iny1Al1-y1As buffer layer. In this case, an increasing of 16.3% and 23.5% for conductivity (mu xn(s)) and mobility, related to the strain compensation in the In0.73Ga0.27As channel, was achieved, respectively, comparing to the structure containing a well-lattice matched buffer layer. With increasing the mismatch further (y1=0.58), a morphology with cross-hatched pattern was observed due to the onset of a large amount of misfit dislocations, and the electronic characterization is not able to be improved continuously. Because we can realize high quality strained P-HEMTs in a relative wide range of equivalent beam flux (EBF) ratios, the stringent control over the constant EBF is not indispensable on this In-based material system. (C) 1997 American Vacuum Society.