The evolutionary foundation of genomic imprinting in lower vertebrates

In mammals,genomic imprinting confers developmental asymmetry and complementation on the parental genomes and makes both parental genomes essential for complete development.Genomic imprinting is,therefore,the first regulatory step of genome-wide gene expression of embryogenesis and thought to be the epigenetic foundation of bisexual reproduction.However,how the genomic imprinting is originated,established and maintained during vertebrate evolution remains unknown.Because no endogenous imprinting gene has be...     

Evolution of the serum resistance-associated SRA gene in African trypanosomes

Serum resistance-associated (SRA) protein, a protein unique for Trypanosoma brucei rhodesiense, is responsible for resistance of this parasite to the lysis by normal human serum (NHS) and is a vital molecular marker to distinguish this species from other African trypanosomes. We cloned and sequenced the SRA basic copy (SRAbc) gene from T. b. rhodesiense and related species and found that this gene is confined to the subgenus Trypanozoon. The average 82% identity among the sequenced SRAbc genes indicates that they may have a common origin and are highly conserved. Since SRAbc coexists in the T. b. rhodesiense genome with SRA, we propose that SRAbc might be the ‘donor VSG’, which after duplication became inserted into the expression site by recombination. Under natural selection, SRAbc could reform into SRA following mosaic formation. Supported by National Natural Science Foundation of China (Grant Nos. 30570245, 30670275), Changjiang Scholars and Innovative Research Team in University (Grant No. DPCKSCU/IRT0447), International Foundation for Science of Sweden (Grant No. B/4318-1), Grant Agency of the Czech Republic (Grant No. Z60220518) and Education Foundation of the Czech Republic (Grant No. 2B06129)     

Expression of chromatin modification genes in organs of cloned cattle that died within hours after birth

Several mammalian species including sheep[1,2], bo- vine[3], goats[4], mice[5], pigs[6], cat[7] and rabbits[8], have been successfully cloned by somatic nuclear transfer (NT), but the efficiency of this process is poor, with only a small proportion of the…     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize （Zea mays L.）

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutinl gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors （pUUOAH）. The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of CrylAh protein in the construct containing the ubil intron （pUUOAH） was 20% higher than that of the intronless construct （pUOAH）. Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubil intron was higher than that of the intronless construct. These results indicated that the maize ubil intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

Epigenetic reprogramming in mammalian nuclear transfer

Somatic cloning has been succeeded in some species, but the cloning efficiency is very low, which limits the application of the technique in many areas of research and biotechnology. The cloning of mammals by somatic cell nuclear transfer (NT) requires epigenetic reprogramming of the differentiated state of donor cell to a totipotent, embryonic ground state. Accumulating evidence indicates that incomplete or inappropriate epigenetic reprogramming of donor nuclei is likely to be the primary cause of failures in nuclear transfer. This review summarizes the roles of various epigenetic mechanisms, including DNA methylation, histone acetylation, imprinting, X-chromosome inactivation, telomere maintenance and expressions of development-related genes on somatic nuclear transfer.     

Evolutionary analysis of allotetraploid hybrids of red crucian carp × common carp, based on ISSR, AFLP molecular markers and cloning of cyclins genes

The allotetraploid hybrids of red crucian carp x common carp are the first reported artificially cultured polyploid fish with bisexual fertility and stable inheritance in vertebrate. Using ISSR and AFLP markers and the cyclins genes, the genomes and cyclin gene sequence changes were analyzed between the allotetraploid hybrids and their parents. The results indicated that the allotetraploids inherited many genetic characteristics from their parents and the genetic characteristics were stable after 15 generations. However, the allotetraploids had a closer genetic relationship with their original female parents and represented a bias toward the maternal progenitor. DNA fingerprinting analysis showed that the allotetraploids had undergone sequences deletion from their original parents and that the deleted sequences were mostly from the male parent＇s genome. Some non-parental bands were found in the allotetraploid hybrids. Sequences analysis of the cyclin A1 and B1 genes showed nonsynonymous substitutions of single nucleotides in codons that were different from their original parents, leading to non-parental amino acid loci. We speculate that the non-additivity in the allotetraploids, compared with their progenitors, could be an adjustment to the genomic shock from heterozygosity and polyploidy, allowing maintenance of genetic stability.     

Cloning and sequence analysis of Sox genes in a tetraploid cyprinid fish, Tor douronensis

A PCR survey for Sox genes in a young tetraploid fish Tor douronensis （Teleostei： Cyprinidae） was performed to access the evolutionary fates of important functional genes after genome duplication caused by polyploldizatlon event. Totally 13 Soxgenes were obtained in Tordouronensis, which represent SoxB, SoxC and SoxE groups. PhylogeneUc analysis of Sox genes in Tor douronensis provided evidence for fish-specific genome duplication, and suggested that Sox19 might be a teleost specific Sox gene member. Sequence analysis revealed most of the nucleoUde substitutions between duplicated copies of Soxgenes caused by tetreploldlzatlon event or their orthologues in other species are silent substitutions. It would appear that the sequences are under purifying selective pressure, strongly suggesting that they represent functional genes and supporting selection against all null allele at either of two duplicated loci of Sox4a, Sox9a and Sox9b. Surprising variations of the intron length and similarities of two duplicated copies of Soxga and Sox9b, suggest that Tor douronensis might be an allotetreploidy.     

Phylogenetic relationships and divergence times of the family Araucariaceae based on the DNA sequences of eight genes

Araucariaceae is one of the most primitive families of the living conifers, and its phylogenetic relationships and divergence times are critically important issues. The DNA sequences of 8 genes, i.e., nuclear ribosomal 18S and 26S rRNA, chloroplast 16S rRNA, rbcL, matK and rps4, and mitochondrial coxl and atpl, obtained from this study and GenBank were used for constructing the molecular phylogenetic trees of Araucariaceae, indicating that the phyiogenetic relationships among the three genera of this family should be （（Wollemia, Agathis）, Araucaria）. On the basis of the fossil calibrations of Wollemia and the two tribes Araucaria and Eutacta of the genus Araucaria, the divergence time of Araucariaceae was estimated to be （308± 53） million years ago, that is, the origin of the family was in the Late Carboniferous rather than Triassic as a traditional view. With the same gene combination, the diver- gence times of the genera Araucaria and Agathis were （246 ± 47） and （61 ±15） Ma, respectively. Statis- tical analyses on the phylogenetic trees generated by using different genes and comparisons of the divergence times estimated by using those genes suggested that the chloroplast matK and rps4 genes are most suitable for investigating the phylogenetic relationships and divergence times of the family Araucariaceae.     

Modified fiber qualities of the transgenic cotton expressing a silkworm fibroin gene

A silkworm gene for fibroin was introduced into the upland cotton WC line by Agrobacterium-mediated transformation. PCR detection for fibroin, nptII and gus genes, Kanamycin (Km)-resistance analysis and GUS-histochemical assay were conducted on 30 regenerated plants from 9 callus lines, and 17 positive plants were obtained by these 5 screening methods. By Km-resistance analysis and PCR for fibroin, 6 homozygous lines in T₃ were obtained. Southern blot and Northern bolt demonstrated that the fibroin gene was inserted into the genome of these 6 lines, stably inherited and expressed. Compared to the control, the surface structure of mature fiber in the 6 lines was significantly distorted and an increased number of convolution was observed by scanning electron microscopy (SEM). Fiber quality traits analysis indicated that fiber elongation of the 6 homozygous lines was all increased and fiber strength of 3 lines was enhanced. These results indicated that fibroin expression influenced cotton fiber structure and quality, suggesting that fibroin has great potential for improving cotton fiber quality by genetic engineering. Supported by National High Technology Research and Development Program of China (Grant No. 2006AA100105), Science & Technology Pillar Program of Jiangsu Province (Grant No. BE2008310) and Programme of Introducing Talents of Discipline to Universities (Grant No. B08025) Contributed equally to this work     

Production of transgenic calves by somatic cellnuclear transfer

Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector(pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant(Neo^r) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was cartied out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves,and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves.PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.     

Cloning and functional analysis of the sequences flanking mini-Tn5 in the magnetosome-deleted mutant NM21 of Magnetospirillum gryphiswaldense MSR-1

A magnetosome-deleted mutant NM21 of Magnetospirillum gryphiswaldense MSR-1 was generated by mini-Tn5 lacZ2 transposon mutagenesis, and a 3073-bp fragment flanking mini-Tn5 lacZ2 in NM21 was cloned by Anchored PCR. Sequencing analysis showed that this fragment involved three putative ORFs; the mini-Tn5 lacZ2 was inserted into ORF1. Functional complementary test indicated that the 3073-bp fragment was required for biosynthesis of magnetosomes in M. gryphiswaldense MSR-1. The majority of proteins, which had homology with the protein encoded by ORF1, were the cation transporter. Transmembrane domain analysis showed that the protein encoded by ORF1 contained four transmembrane domains. It may be a transmembrane protein. The protein encoded by ORF1 contained two putative conserved domains: COG0053 and PRK09509. The MMT1 and FieF, containing conserved domains COG0053 and PRK09509 too, were Fe²⁺ transporter (cation diffusion facilitator superfamily). It was suggested that the protein encoded by ORF1 might take part in the magnetosomes biosynthesis as Fe²⁺ transporter. Supported by National Natural Science Foundation of China (Grant No. 30570023) and Scientific Research Project of Huaibei City, Anhui Province (Grant No. 070114)     

Clone of Chinese Jinan red-cross yellow cattle and evaluation of reproductive characteristics of cloned calf

Somatic cell clone technology is a viable approach to preserving endangered livestock and wildlife genetic resources. In the present research, somatic cell nuclear transfer （SCNT） was performed using granulose cells from the critical endangered Chinese red-cross yellow cattle as donor cells. A total of 211 oocytes were manipulated and 166 （79%） of them were successfully enucleated. 112 （67.4%） SCNT embryos were reconstructed, 94 （83%） of them cleaved, and 48 （43 %） of them developed to blastocyst stage. SCNT blastocysts were transferred to 6 Holstein recipients, and 2 （33%） of them were found to be pregnant. One of them maintained to term and delivered a calf, whereas another aborted. Effect of different fusion buffer （mannitol vs. Zimmerman fusion buffer） and different activation methods （calcium ionophore＋6-DMAP vs. cycloheximide＋CB） on fusion rate and development of SCNT embryos were investigated. The results indicated that： （i） on condition of two DC pulses of 2.5 kV/cm for 10 μs each, fusion rates were higher in mannitol solution than in Zimmerman fusion buffer （71% vs. 61%, respectively, p 〈 0.05）, but the blastocysts rates did not differ between two treatments （36 % vs. 39 %, p〉0.05 ）; （ii） There was no significant difference in development rates to the blastocyst stage for SCNT embryos activated by calcium ionophore＋6-DMAP or by cycloheximide＋CB （42% vs. 46%, respectively, p〉0.05）. Microsatellite DNA analysis examining 28 loci confirmed that the cloned calf was genetically identical to the donor Jinan red-cross yellow cattle and different from the recipient females. Growth and reproductive performance of cloned cow were evaluated, and there were no difference i cross-red n it between cloned and normal control Jinan yellow cattle. Furthermore, the cloned yellow cow has delivered a healthy yellow calf.     

Reporter-based screen for <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants compromised in nonhost resistance

Plants are exposed to many potentially pathogenic microbes in the environment, but each species is only susceptible to a limited number of pathogens. The broad resistance is referred to as nonhost resistance. To date, little is known about the underlying mechanism of nonhost resistance and the signaling transduction process. Here we describe a simple method for isolating Arabidopsis nonhost resistance mutants against a nonadapted bacterial pathogen. A RAP2.6 promoter-driven LUC reporter system was developed to replace the tedious bacterial growth assay during the primary screening. The RAP2.6-LUC reporter gene is normally induced by the virulent bacterium Pseudomonas syringae pv tomato but not the nonadapted bacterium P. syringae pv phaseolicola. By using this method we iso- lated 4 mutants displaying strong reporter activity in response to P. syringae pv phaseolicola, which were characterized in some details, ebsl, ebs2, ebs3, and ebs4 （enhanced bacterial susceptibility） were compromised in resistance against P. syringae pv phaseolicola and/or P. syringae pv tomato. In addition, ebs4 showed enhanced hypersensitive response to the incompatible bacterium P. syringae pv tomato （avrB）. These results demonstrated that the method is suited for large scale screening for nonhost resistance mutants.     

Functional characterization and mapping of two MADS box genes from peach （Prunus persica）

Previously an AGAMOUS gene homologue PpMADS4 and a FRUITFULL gene homologue PpMADS6 were isolated from peach （Prunus persica）, and both genes were shown to express in the developing floral and fruits. To gain insight into their function, the two genes were constitutively expressed in Arabidopsis thaliana and their effects on plant growth and floral organ development were studied in this work. The transgenic plants all displayed early flowering and conversion of inflorescence to floral meristem. However, the two genes had different effects on the floral organ structures in A. thaliana. The transgenic plants overexpressing PpMADS4 displayed homeotic conversion of floral organs, and particularly the perianth abscission was inhibited. The plants overexpressing PpMADS6 showed early flowering, produced higher number of carpels, petals, and stamens than nontransgenic plants, and pod shatter was prevented; significantly, the transgenic plants yielded more than one siliques from a single flower. A SSR molecular marker was developed for PpMADS4, and it was then assigned into the G5 linkage group of Prunus sp. Both PpMADS4 and PpMADS6 genes were located at the same region in the G5 linkage group. Our results showed the potential application of these two MADS box genes for crop and fruit tree improvement.     

Genome-wide distribution of histone H3 acetylation in all- trans retinoic acid induced neuronal differentiation of SH-SY5Y cells

With chromatin immunoprecipitation (ChIP) and promoter DNA microarray analyses (ChIP-on-chip), we analyzed the variations of acetylation on histone H3 in all-trans retinoic acid (RA) induced neuronal cell differentiation. Neuroblastoma SH-SY5Y cells were treated with RA for 24 h and the acetylation on histone H3 in the promoter region of the genes was detected. Results showed that, after treatment, the level of acetylation on histone H3 elevated in 597 genes in the genome, and reduced in the other 647 genes compared with those of the control. In summary, we have successfully adopted a high throughput technique to detect and analyze variations of acetylation of histone H3 in human genome at the early phage of RA induced neuronal differentiation of the SH-SY5Y cells. Supported by National Natural Science Foundation of China (Grant Nos. 90408007 and 30721063) and National Key Basic Research and Development Program of China (Grant No. 2004CB518605)     

The Escherichia coli O157:H7 DNA detection on a gold nanoparticle-enhanced piezoelectric biosensor

This paper presents development of a quartz crystal microbalance （QCM） biosensor for real-time detection of E. coil O157：H7 DNA based on nanogold particles amplification. Many inner Au nanoparticles were immobilized onto the thioled surface of the Au electrode, then more specific thiolated sin- gle-stranded DNA （ssDNA） probes could be fixed through Au-SH bonding. The hybridization was induced by exposing the ssDNA probe to the complementary target DNA of E. coli O157：H7 gene eaeA, then resulted in a mass change and corresponding frequency shifts （ △f ） of the QCM. The outer avidin-coated Au nanoparticles could combine with the target DNA to increase the mass. The electrochemical techniques, cyclic voltammetry （CV） and electrochemical impedance spectroscopy （EIS） were adopted to manifest and character each step. The target DNA corresponding to 2.0×10^3 colony forming unit （CFU）/mL E. coil O157：H7 cells can be detected by this biosensor, so it is practical to develop a sensitive and effective QCM biosensor for pathogenic bacteria detection based on specific DNA analysis. The piezoelectric biosensing system has potential for further applications, such as food safety and environment monitoring, and this approach lays the groundwork for incorporating the method into an integrated system for in-field bacteria detection.     

Transformation of japonica rice with<Emphasis Type="Italic">RHL</Emphasis> gene and salt tolerance of the transgenic rice plant

Overexpression of the yeastHAL2 gene increases salt tolerance of yeast and plant. RiceHAL2-like (RHL) gene was introduced into ajaponica rice cultivar HJ19 withAgrobacterium tumefaciens-mediated transformation. Transgenic plants in R₀ generation were selected on the principle of GUS-positive,RHL gene PCR-positive and normal growth. Hygromycin-resistant plants of some transgenic lines in R₁ generation increased salt tolerance during the seedling and booting stage, being less damaged in the cytomembrane and stronger in leaf tissue viability under salt stress during booting period. Southern analysis of transgenic lines tolerant to salt in R₁ generation showed that theRHL gene expression cassette had been successfully integrated into rice genome. Moreover, gene engineering breeding methodology and really salt-tolerant rice cultivar were discussed.