Evolutionary implications of multiple SINE insertions in an intronic region from diverse mammals

An analysis of the nuclear beta-fibrinogen intron 7 locus from 30 taxa representing 12 placental orders of mammals reveals the enriched occurrences of short interspersed clement (SINE) insertion events. Mammalian-wide interspersed repeats (MIRs) are present at orthologous sites of all examined species except those in the order Rodentia. The higher substitution rate in mouse and a rare MIR deletion from rat account for the absence of MIR in the rodents. A minimum of five lineage-specific SINE sequences are also found to have independently inserted into this intron in Carnivora, Artiodactyla and Lagomorpha. In the case of Carnivora, the unique amplification pattern of order-specific CAN SINE provides important evidence for the "pan-carnivore" hypothesis of this repeat element and reveals that the CAN SINE family may still be active today. Particularly interesting is the finding that all identified lineage-specific SINE elements show a strong tendency to insert within or in very close proximity to the preexisting MIRs for their efficient integrations, suggesting that the MIR clement is a hot spot for successive insertions of other SINEs. The unexpected MIR excision as a result of a random deletion in the rat intron locus and the non-random site targeting detected by this study indicate that SINEs actually have a greater insertional flexibility and regional specificity than had previously been recognized. Implications for SINE sequence evolution upon and following integration, as well as the fascinating interactions between retroposons and the host genomes are discussed.

Triplophysa lixianensis, a new nemacheiline loach species (Pisces : Balitoridae) from the upper Yangtze River drainage in Sichuan Province, South China

Triplophysa lixianensis, a new nemacheiline loach species, is described from the Min Jiang of the upper Yangtze River drainage in Sichuan Province, South China. It can be separated from all other species of Triplophysa by having a unique combination of the following characters: posterior chamber of gas bladder greatly reduced or absent; caudal peduncle columnar with a roughly round cross- section at its beginning; anterior edge of lower jaw completely exposed or uncovered by lower lip; intestine short, forming a zigzag loop below stomach; dorsal- fin origin closer to caudal- fin base than to snout tip; pelvic fin inserted anterior to dorsal- fin origin; snout length 50.6 - 57.5 % of head length; eye diameter 12.3 15.4 % of head length; caudal peduncle length 25.1 - 27.1 % of standard length; anal fin with five branched rays; lower lip greatly furrowed with two thick lateral lobes; and body smooth or scaleless.

Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites

With current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10(-4) to 10(-5)) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering.

A miniature insertion element transposable in Microcystis sp FACHB 854

A 186-bp sequence with imperfect terminal inverted repeats and target direct repeats but without any transposase-encoding capacity was found to be transposable in an isolate derived from Microcystis sp. FACHB 854. This miniature insertion element, designated as ISM854-1, and with its homologues present at least 10 copies in the genome of Microcystis FACHB 854, is inserted into the 8-bp long and AT-rich target sequences, but none or few in other Microcystis strains. A variant of ISM854-1, denoted ISM854-1A, has perfect inverted repeat sequences and may transpose in pairs in a structure like a composite transposon. This is the first report of non-autonomous transposition of a mini-IS in a cyanobacterium.

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp beta-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.

Cloning of rainbow trout (Oncorhynchus mykiss) histone H3 promoter and the activity analysis in rare minnow (Gobiocypris rarus)

Rainbow trout historic H3 (RH3) promoter was cloned via high fidelity PCR. The cloned RH3 promoter was inserted into a promoter-lacked vector pEGFP-1, resulting in an expression vector pRH3FGFP-1. The linearized pRH3EGFP-1 was microinjected into fertilized eggs of rare minnows and the sequential embryogenetic processes were monitored under a fluorescent microscope. Strong green fluorescence was ubiquitously observed at as early as the gastrula stage and then in various tissues at the fry stage. The results indicate that RH3 promoter, as a piscine promoter, could serve in producing transgenic Cyprinoid such as rare minnow. Promoter activity of RH3, CMV and common carp beta-actin (CA) were compared in rare minnow by the expression of respective recombinant EGFP vectors. The expression of pCMVEGFP occurred earlier than the following one, pRH3EGFP-1, and then pCAEGFP during the embryogenesis of the transgenics. Their expression activities demonstrated that the CMV promoter is the strongest one, followed by the CA and then the RH3.

Retroposon analysis of major cetacean lineages: The monophyly of toothed whales and the paraphyly of river dolphins

SINE (short interspersed element) insertion analysis elucidates contentious aspects in the phylogeny of toothed whales and dolphins (Odontoceti), especially river dolphins. Here, we characterize 25 informative SINEs inserted into unique genomic loci during evolution of odontocetes to construct a cladogram. and determine a total of 2.8 kb per taxon of the flanking sequences of these SINE loci to estimate divergence times among lineages. We demonstrate that: (i) Odontocetes are monophyletic; (ii) Ganges River dolphins, beaked whales, and ocean dolphins diverged (in this order) after sperm whales; (iii) three other river dolphin taxa, namely the Amazon, La Plata, and Yangtze river dolphins, form a monophyletic group with Yangtze River dolphins being the most basal; and (iv) the rapid radiation of extant cetacean lineages occurred some 28-33 million years B.P., in strong accord with the fossil record. The combination of SINE and flanking sequence analysis suggests a topology and set of divergence times for odontocete relationships, offering alternative explanations for several long-standing problems in cetacean evolution.

Construction and analysis of a high-CO2-requiring mutant of the cyanobacterium Anabaena sp strain PCC7120

A mutant of Anabaena sp. strain PCC7120 requiring high CO2 was generated using Tn5 mutagenesis. This is the first data for a filamentous cyanobacterium. The mutant was capable of growing at 5% CO2, but incapable of growing at air levels of CO2. Southern hybridization analysis indicated that the Anabaena genome was inserted by the transposon at one site. The apparent photosynthetic affinity of the mutant to external dissolved inorganic carbon (DIC) was about 300 times lower that of the wild type (WT), and the medium alkalization rate as well as the carboxysomal carbonic anhydrase activity of the mutant was also lower than those of the WT. When the mutant was transferred from the culture medium bubbled with 5% CO2 to higher DIC (8.4% CO2) or 1% CO2, it showed similar responses to the WT. However, aberrant carboxysomes were found in the mutant cells through ultrastructural analysis, indicating it was most probably the wrong organization of the carboxysomes that eventually led to the inefficient operation of carboxysomal carbonic anhydrase and the subsequent defectiveness of the mutant in utilizing DIC.

Quantitative strain characterization of SiGe heterostructures by high-resolution transmission electron microscopy

We report the quantitative strain characterization in semiconductor heterostructures of silicon-germaniums (Si(0.76)Geo(0.24)) grown on Si substrate by an ultra-high vacuum chemical vapor deposition system. The relaxed SiGe virtual substrate has been achieved by thermal annealing of the SiGe film with an inserted Ge layer. Strain analysis was performed using a combination of high-resolution transmission electron microscopy and geometric phase analysis.

Growth and fabrication of AlGaN/GaN HEMT based on Si(111) substrates by MOCVD

AlGaN/GaN high electron mobility transistor (HEMT) hetero-structures were grown on the 2-in Si (1 1 1) substrate using metal-organic chemical vapor deposition (MOCVD). Low-temperature (LT) AlN layers were inserted to relieve the tension stress during the growth of GaN epilayers. The grown AlGaN/GaN HEMT samples exhibited a maximum crack-free area of 8 mm x 5 mm, XRD GaN (0 0 0 2) full-width at half-maximum (FWHM) of 661 arcsec and surface roughness of 0.377 nm. The device with a gate length of 1.4 mu m and a gate width of 60 mu m demonstrated maximum drain current density of 304 mA/mm, transconductance of 124 mS/mm and reverse gate leakage current of 0.76 mu A/mm at the gate voltage of -10 V. (C) 2008 Published by Elsevier Ltd.

The influence of low-temperature Ge seed layer on growth of high-quality Ge epilayer on Si(100) by ultrahigh vacuum chemical vapor deposition

High-quality Ge epilayer on Si(1 0 0) substrate with an inserted low-temperature Ge seed layer and a thin Si0.77Ge0.23 layer was grown by ultrahigh vacuum chemical vapor deposition. The epitaxial Ge layer with surface root-mean-square roughness of 0.7 nm and threading dislocation density of 5 x 10(5) cm(-2) was obtained. The influence of low temperature Ge seed layer on the quality of Ge epilayer was investigated. We demonstrated that the relatively higher temperature (350 degrees C) for the growth of Ge seed layer significantly improved the crystal quality and the Hall hole mobility of the Ge epilayer. (C) 2008 Elsevier B.V. All rights reserved.

High responsivity resonant-cavity-enhanced InGaAs/GaAs quantum-dot photodetector for wavelength of similar to 1 mu m at room temperature

The characteristics of a resonant cavity-enhanced InGaAs/GaAs quantum-dot n-i-n photodiode with only a bottom distributed Bragg reflector used as the cavity mirror, are reported. To suppress the dark current, an AlAs layer is inserted into the device structure as the blocking layer. It turns out that the structure still possesses the resonant coupling nature, and makes Rabi splitting discernible in the photoluminescence spectra. The measured responsivity spectrum of the photocurrent shows a peak at lambda = 1030 nm, and increases rapidly as the bias voltage increases. A peak responsivity of 0.75 A/W, or equivalently an external quantum efficiency of 90.3%, is obtained at V-bias = -1.4 V.

Polarization effects simulation of AlGaN/GaN heterojunction by using a symbolistic delta-doping layer

Polarization effects in AlGaN/GaN heterojunction are simulated based on a traditional semiconductor device simulator. A delta doping layer is purposely inserted at the interface of the heterojunction in the simulation, so the ionized donors or acceptors can represent polarization-induced positive or negative fixed charges. The free electron distribution of single AlGaN/GaN heterostructures with Ga-face and N-face growth is compared, and the results of the simulation show that carrier confinement takes place only in the former structure. The dependence of sheet density of free electrons at the interface of Ga-face growth AlGaN/GaN on Al composition and the thickness of AlGaN is also investigated. The consistency of simulation results with the experiments and calculations reported by other researchers shows that this method can be effectively used to deal with the polarization effects in the simulation of GaN-based heterojunction devices. (C) 2004 Elsevier Ltd. All rights reserved.

Investigation of GaAs/AlGaAs interfaces by reflectance-difference spectroscopy

The in-plane optical anisotropy of several GaAs/AlGaAs quantum well samples with different well widths has been measured at room temperature by reflectance-difference spectroscopy (RDS). The RDS line shapes are found to be similar in all the samples examined here, which dominantly consist of two peak-like signals corresponding to 1HH-->1E and 1LH-->1E transition. As the well width is decreased, or the 1 ML InAs layer is inserted at one interface, the intensity of the anisotropy increases quickly. Our detail analysis shows that the anisotropy mainly arises from the anisotropic interface roughness. The results demonstrate that the RDS technique is sensitive to the interface structures.

Structural and optical properties of GaAsSb/GaAs heterostructure quantum wells

The structural and optical properties of MBE-grown GaAsSb/GaAs multiple quantum wells (MQWs) as well as strain-compensated GaAsSb/GaAs/GaAsP MQWs are investigated. The results of double crystal X-ray diffraction and reciprocal space mapping show that when strain-compensated layers are introduced, the interface quality of QW structure is remarkably improved, and the MQW structure containing GaAsSb layers with a high Sb composition can be coherently grown. Due to the influence of inserted GaAsP layers on the energy band and carrier distribution of QWs, the optical properties of GaAsSb/GaAs/GaAsP MQWs display a lot of features mainly characteristic of type-I QWs despite the type-II GaAsSb/GaAs interfaces exist in the structure. (C) 2004 Elsevier B.V. All rights reserved.