Gene Expression and Regulation of Blastocyst Formation

Blastocyst formation is a crucial stage of early embryo development.Cell junction proteins and cell adhesion associated proteins are involved in the establishment of cell junction,and subsequently induce cell compaction,blastocyst formation,differentiation of trophectoderm and maintenance of blastocyst expansion.Genes regulating development and differentiation participate in embryo development and differentiation of inner cell mass and trophectoderm,which controls the transition from the undifferentiation to differentiation state.Furthermore,cytokine and growth factor have influence on the proliferation of cells of inner cell mass.In a word,many proteins and factors are involved in the gene expression and regulation of blastocyst formation.

Epigenetic changes in the regulation of carotenoid metabolism during honeysuckle flower development

Flower development is one of the most vital pathways in plant development, during which the epigenetic regulation of gene expression is essential. DNA methylation, the most conserved epigenetic modification, participates in gene expression regulation and transposable element silencing. Honeysuckle(Lonicera japonica) is an important medicinal plant renowned for its colorful and fragrant flowers. Honeysuckle flowers change color from white to gold as a result of carotenoid accumulation during development. However, the role of DNA methylation in flower color changes is not well understood in L. japonica. Here, we performed whole-genome bisulfite sequencing and transcriptome sequencing during flowering development in honeysuckle. The results showed that a decrease in the levels of genome-wide average DNA methylation during flower development and changes in DNA methylation were associated with the expression of demethylase genes. Moreover, many genes involved in carotenoid biosynthesis and degradation, such as Lj PSY1, LjPDS1, LjLCYE, and LjCCD4, have altered expression levels because of hypomethylation, indicating that DNA methylation plays an important role in flower color changes in honeysuckle. Taken together, our data provide epigenetic insights into flower development and color change in honeysuckles.

Polyploid Gene Expression and Regulation in Polysomic Polyploids

Polyploidization is one of the most crucial pathways in introducing speciation and broadening biodiversity, especially in the Plant Kingdom. Although the majority of studies have focused only on allopolyploid or disomic polyploids, polysomic polyploid species have occurred frequently in higher plants. Due to the occurrence of the capabilities of more copies of alleles in a locus which can have additive dosage effects and/or allelic interactions, polysomic polyploids can lead to unique gene regulations to silence or adjust the expression level to create variations in organ size, metabolic products, and abiotic stress tolerance and biotic stress resistance, etc. This review aims to comprehensively summarize the contemporary understanding and findings concerning the molecular mechanisms of gene expression as well as gene regulation in natural typed and resynthesized polysomic polyploid plants. The review investigates the molecular level of phenomena in polysomic polyploid plants such as 1) typically enlarging organ size and stabilizing meiosis, 2) increasing phytochemical content and metabolic products, 3) enhancing the ability to adapt with biotic and abiotic stress, and 4) changing in gene regulation to silence or adjust the expression levels involve in sequence elimination, methylation, gene suppression, subfunctionalization, neo-functionalization, and transposon activation.

Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.

Differential expression of a novel colorectal cancer differentiation-related gene in colorectal cancer

AIM: To investigate SBA2 expression in CRC cell lines and surgical specimens of CRC and autologous healthy mucosa. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used for relative quantification of SBA2 mRNA levels in 4 human CRC cell lines with different grades of differentiation and 30 clinical samples. Normalization of the results was achieved by simultaneous amplification of beta-actin as an internal control. RESULTS: In the exponential range of amplification, fairly good linearity demonstrated identical amplification efficiency for SBA2 and beta-actin (82%). Markedly lower levels of SBA2 mRNA were detectable in tumors, as compared with the coupled normal counterparts P【0.01). SBA2 expression was significantly (0.01】P 【 0.05) correlated with the grade of differentiation in CRC, with relatively higher levels in well-differentiated samples and lower in poorly-differentiated cases. Of the 9 cases with lymph nodes affected, 78% (7/9) had reduced SBA2 mRNA expression in contrast to 24% (5/21) in non-metastasis samples 0.01】P【0.05). CONCLUSION: SBA2 gene might be a promising novel biomarker of cell differentiation in colorectal cancer and its biological features need further studies.

Expression Regulation of Starch and Storage Protein Synthesis Related Genes in Rice Grains

Starch and the storage proteins are the main nutritious substances in crop grains,and their composition and content in grains play a decisive role in the grain quality of rice and other staple food crops.This review has mainly summarized the new advances in the expression regulation of starch and storage protein synthesis related genes in rice grains.Moreover,the challenges of the starch and storage protein synthesis substances in rice genetic improvement were also discussed.This review will provide important information for genetic improvement of grain quality in rice and,potentially,other staple cereals.

DIFFERENCES IN EXPRESSION AND REGULATION BETWEEN TRANSFORMED CELLS OF THE HUMAN GASTRIC CARCINOMA ONCOGENE Ha-ras AND THE UNTRANSFORMED PARENT CELLS

An Ha-ras oncogene was isolated from a cell line of gastric carcinoma called BGE-823 in order to elucidate genetic control and the influence of DNA sequences. The oncogene was cloned and identified as a single nucleotide substitution of thymine for guanine in the 12th codon through the sequencing of its first axon. We compared the differences of expression and regulation between the transformed Ha-ras cells and untransformed parent cells. Data indicated that the expression of Ha-ras in the transformed cells was five-fold higher than in the untransformed cells and that the Ha-ras gene in the former was hypersensitive toward DNase I. In addition, a nuclear protein of 35 kilodaltons bound strongly to the 2.5 Kb fragment located upstream of the 6.6 Kb Ha-ras gene and contained a CC rich region. These results suggest that there might be another mechanism of activation for the ras gene besides point mutation.

Transcriptional Regulation of 10 Mitochondrial Genes in Different Tissues of NCa CMS System in Brassica napus L. and Their Relationship with Sterility

Northern blot analysis was conducted with mitochondrial RNA from seedling leaves, floral buds, and developing seeds of NCa CMS, maintainer line and fertile F1 using ten mitochondrial genes as probes. The results revealed that 9 out of the 10 mitochondrial genes, except for atp6, showed no difference in different tissues of the corresponding materials of NCα CMS system and that they might be constitutively expressed genes. Eight genes, such as orf139, orf222, atpl, cox1, cox2, cob, rm5S, and rm26S, showed no difference among the three tissues of all the materials detected. So the expression of these eight genes was not regulated by nuclear genes and was not tissue-specific. The transcripts of atp9 were identical among different tissues, but diverse among different materials, indicating that transcription of atp9 was neither controlled by nuclear gene nor tissue-specific. Gene atp6 displayed similar transcripts with the same size among different tissues of all the materials but differed in abundance among tissues of corresponding materials and its expression might be tissue-specific under regulation of nuclear gene. Moreover, three transcripts of orf222 were detected in the floral buds of NCa cms and fertile F1, but no transcript was detected in floral buds of the maintainer line.The transcription of orf139 was similar to that of orf222 but only two transcripts of 0.8 kb and 0.6 kb were produced. The atp9 probe detected a single transcript of 0.6 kb in NCa cms and in maintainer line and an additional transcript of 1.2 kb in fertile F1. The relationship of expression of orf222, orf139, and atp9 with NCa sterility was discussed.

Advances in the Regulation of RpoS Protein Expression and Its Function in Bacteria

RpoS protein is a σ factor of RNA polymerase that can control the expression of a group-specific gene, thus playing a vital role in bacteria. In bacteria, RpoS expression is under strict control and is mainly regulated at three levels： transcription level, translation level and post-translational level. Environmental stress enters bacterial cells through signal transduction and leads to a series of variations in microenvironment, thereby causing changes of regulator and controlling its levels based on the direct and indirect interaction between regulator and RpoS protein. In addition, RpoS protein has played special roles in bacteria, therefore the changes of RpoS protein levels will lead to variations in expression levels of a large number of genes, thereby causing variations of bacterial response to different environmental stress and changes of certain characteristics of bacteria, which provides a new strategy for the control of bacterial diseases in the future. This paper reviewed the recent progress on the regulation of RpoS protein expression and its function in several common bacteria. Due to the functional complexity of RpoS protein, there are still a lot of unknown functions to be further identified.

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.

Effect on Survivin Regulation of Transcription Level by p21^(waf1) Overexpression in HepG2 Hepatocellular Carcinoma Cells

The effect of cyclin-dependent kinase inhibitors Cip1/Wafl （p21） on regulatory expression of survivin transcription in human hepatocellular carcinoma cell HepG2 was observed and the related mechanisms explored. Doxorubicin （DOX） was used to treat HepG2. Eukaryotic vector pEGFP-C2-p21 was transfected into HepG2 by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21 and survivin was detected by real-time fluorescent quantitative polymerase chain reaction （RQ-PCR）. Flow cytometry was used to examine the cell cycle, and reverse transcription polymerase chain reaction （RT-PCR） was used to measure the levels of E2F-1 and p300. The results showed that： （1） After treatment with DOX, the expression of p21 was increased, whereas that of survivin was reduced during 24 h of treatment; （2） After transfection of pEGFP-C2-p21 into HepG2, p21 level was significantly enhanced to 2100.11-folds or 980.89-folds in comparison to HepG2 or HepG2-C2 group, and survivin level was markedly down-regulated to 0.54% or 0.59% relative to the control groups; （3） Overexpressed p21 resulted in GI/G0 phase arrest （F=31.59, P〈0.01）, meanwhile E2F-1 mRNA and p300 mRNA were reduced as compared with those of controls （FE2F-1=I25.28, P〈0.05; Fp300= 46.01, P〈0.01）. It was suggested that p21 could be a potential mediator of survivin suppression at transcription level in HepG2 cell, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.

Effects of hypoxia,hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells

AIM: To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells (HSC). METHODS: The expressions of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1 (MT1-MMP) in cultured rat HSC were detected by immunocytochemistry (ICC) and in situ hybridization (ISH). The contents of MMP-2 and TIMP-2 in culture supernatant were detected with ELISA and the activity of MMP-2 in supernatant was revealed by zymography. RESULTS: In the situation of hypoxia for 12h, the expression of MMP-2 protein was enhanced (hypoxia group positive indexes: 5.7 +/- 2.0, n=10; control: 3.2 +/- 1.0, n = 7; P【0.05), while TIMP-2 protein was decreased in HSC (hypoxia group positive indexes: 2.5 +/- 0.7, n = 10; control: 3.6 +/- 1.0, n = 7; P 【 0.05), and the activity (total A) of MMP-2 in supernatant declined obviously (hypoxia group: 7.334 +/- 1.922, n = 9; control: 17.277 +/- 7.424, n = 11; P 【 0.01). Compared the varied duration of hypoxia, the changes of expressions including mRNA and protein level as well as activity of MMP-2 were most notable in 6h group. The highest value(A(hypoxia)-A(control)) of the protein and the most intense signal of mRNA were in the period of hypoxia for 6h, along with the lowest activity of MMP-2. In the situation of hyperoxia for 12h, the contents (A(450)) of MMP-2 and TIMP-2 in supernatant were both higher than those in the control, especially the TIMP-2 (hyperoxia group: 0.0499 +/- 0.0144, n = 16; control: 0.0219 +/- 0.0098, n = 14; P 【 0.01), and so was the activity of MMP-2 (hyperoxia group: 5.252 +/- 0.771, n = 14; control: 4.304 +/- 1.083, n = 12; P 【 0.05), and the expression of MT1-MMP was increased. CONCLUSION: HSC is sensitive to the oxygen, hypoxia enhances the expression of MMP-2 and the effect is more marked at the early stage; hyperoxia mainly raises the activity of MMP-2.

Rice bicoid-related cDNA sequence and its expression during early embryogenesis

Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation. To clone and characterize the rice bicoid-related genes, one cDNA clone, Rb24 (EMBL accession number: AJ2771380), was isolated by screening of rice unmature seed cDNA library. Sequence analysis indicates that Rb24 contains a putative amino acid sequence, which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity, 75% similarity) and involves a lys-9 in putative helix 3. Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner. The transcript was detected strongly in young panicles, but less in young leaves and roots. This results are further confirmed with paraffin section in situ hybridization. The signal is intensive in rice globular embryo and located at the apical tip of the embryo, then, along with the development of embryo, the signal is getting reduced and transfers into both sides of embryo. The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

Genetic engineering and lignin biosynthetic regulation in forest tree species

Genetic engineering of forest tree species is regarded as a strategy to reduce worldwide pressure on natural forests, to conserve genetic resources and ameliorate stress on global climate, and to meet growing demand for forest wood and timber products. Genetic engineering approaches toward the control or management of fungal pathogens, arthropod herbivores, bacterial and viral diseases, the use of pest resistance genes, and weed competitors are being studied. Although the production of transgenic trees is relatively recent and only a few species have been successfully genetically engineered in forest tree species, very useful and valuable information is available on the application of transgenic trees. Genes involved in important agricultural traits such as herbicide resistance, insect resistance, and wood quality have been isolated and have been used to genetically engineer trees. New technologies of plant molecular biology and genomics now make it possible high-efficient genetic improvement of forest trees. Genetic engineering promises to expand greatly the potential for genetic manipulation as new genes of commercial interest are discovered and utilized. Lignification is a process essential to the nature and evolution of vascular plants that is still poorly understood, even though it has been studied for more than a century. Recent studies on mutant and transgenic plants indicate that lignification may be far more flexible than previously realized. Rines with a mutation affecting the biosynthesis of the major lignin precursor, coniferyl alcohol, show a high level of an unusual subunit, dihydroconiferyl alcohol. It is also unusual as a plant polymer in that there are no plant enzymes for its degradation. These results have significant implications regarding the tradiational definition of lignin, and highlight the need for a better understanding of the lignin precursor biosynthetic pathway. In this review, we describe the progress made recently in genetic engineering of forest tree species.

Roles and regulation of bone morphogenetic protein-7 in kidney development and diseases

The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the final number of nephrons in and proper size of the organ. The secreted BMP7 acts on the nephron progenitor cells to exert its dual functions: To maintain and expand the progenitor population and to provide them with competence to respond to differentiation cues, each relying on distinct signaling pathways. Intriguingly, in the adult organ, BMP7 has been implicated in protection against and regeneration from injury. Exogenous administration of recombinant BMP7 to animal models of kidney diseases has shown promising effects in counteracting inflammation, apoptosis and fibrosis evoked upon injury. Although the expression pattern of BMP7 has been well described, the mechanisms by which it is regulated have remained elusive and the processes by which the secretion sites of BMP7 impinge upon its functions in kidney development and diseases have not yet been assessed. Understanding the regulatory mechanisms will pave the way towards gaining better insight into the roles of BMP7, and to achieving desired control of the gene expression as a therapeutic strategy for kidney diseases.

Identification and Expression Analysis of the Phosphoenolpyruvate Carboxylase Gene Family in Peanut (Arachis hypogaea L.)

Phosphoenolpyruvate carboxylase （PEPC） is widely distributed in plants and bacteria, and catalyzes the carboxylation of phosphoenolpyruvate to form oxaloacetate and inorganic phosphate. To investigate the molecular mechanisms of the regulation and control of peanut oil, with the degenerated primers and RACE-PCR approach, five PEPC genes were cloned from peanut, and designated as AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, and AhPEPC5, respectively. The structure and phylogenetic analysis of PEPC protein indicated that AhPEPC1-4 genes encoded a typical plant-type PEPC-enzyme, and AhPEPC5 a bacterial-type. By real-time quantitative RT-PCR approach the expression pattern of each gene was detected in various tissues of normal and high oil-content peanut varieties. It was found that there was a lower expression level of AhPEPCs genes except for the AhPEPC2 in high-oil peanut than normal-oil peanut line. The results provide some fundamental information for the further investigation of plant PEPC proteins and their role in regulation of oil-content in peanut seeds.

Regulated Gene Expression with Promoters Responding to Inducers

Genetically engineered transgenic animals and plants have proven to be extremely useful for analyzing biochemical and developmental processes.Promoters responding to chemical inducers will be powerful tools for basic research in molecular biology and biotechnological applications.Various chemical inducible systems based on activation and inactivation of the target gene had been described.The transfer of regulatory elements from prokaryotes,insects,and mammals has opened new avenues to construct chemically inducible promoters that differ in their ability to regulate the temporal and spatial expression patterns,and this will dramatically increase the application of transgenic technology.This review provides an overview on regulation of gene expression,promoter activating systems,promoter inactivation systems,inducible gene over expression,and inducible anti suppression.

Molecular characterization and expression analysis of three homoeologous Ta14S genes encoding 14-3-3 proteins in wheat(Triticum aestivum L.)

The purpose of this study was to characterize Ta14 S homoeologs and assess their functions in wheat seed development.The genomic and c DNA sequences of three Ta14 S homoeologous genes encoding 14-3-3 proteins were isolated.Sequence analysis revealed that the three homoeologs consisted of five exons and four introns and were very highly conserved in the coding regions and in exon/intron structure,whereas the c DNA sequences were variable in the 5′ and 3′-UTR.The three genes,designated as Ta14S-2A,Ta14S-2B and Ta14S-2D,were located in homoeologous group 2 chromosomes.The polypeptide chains of the three Ta14 S genes were highly similar.These genes were most homologous to Hv14 A from barley.Real-time quantitative PCR indicated that the three Ta14 S genes were differentially expressed in different organs at different developmental stages and all exhibited greater expression in primary roots of 1-day-old germlings than in other tissues.Comparison of the expression patterns of the three homoeologous genes at different times after pollination also revealed that their expression was developmentally regulated.The transcription of Ta14S-2B was clearly higher during seed germination,whereas expressions of Ta14S-2A and Ta14S-2D were up-regulated at the beginning of seed imbibition(0–12 h),but declined thereafter.The results suggested that the three Ta14 S homoeologous genes have regulatory roles in seed development and germination.

Differentially expressed genes in hepatocellular carcinoma induced by woodchuck hepatitis B virus in mice

INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.

Epigenetic regulation of stemness maintenance in the neurogenic niches

In the adult mouse brain, the subventricular zone lining the lateral ventricles and the subgranular zone in the dentate gyrus of the hippocampus are two zones that contain neural stem cells(NSCs) with the capacity to give rise to neurons and glia during the entire life of the animal. Spatial and temporal regulation of gene expression in the NSCs populationis established and maintained by the coordinated interaction between transcription factors and epigenetic regulators which control stem cell fate. Epigenetic mechanisms are heritable alterations in genome function that do not involve changes in DNA sequence itself but that modulate gene expression, acting as mediators between the environment and the genome. At the molecular level, those epigenetic mechanisms comprise chemical modifications of DNA such as methylation, hydroxymethylation and histone modifications needed for the maintenance of NSC identity. Genomic imprinting is another normal epigenetic process leading to parentalspecific expression of a gene, known to be implicated in the control of gene dosage in the neurogenic niches. The generation of induced pluripotent stem cells from NSCs by expression of defined transcription factors, provide key insights into fundamental principles of stem cell biology. Epigenetic modifications can also occur during reprogramming of NSCs to pluripotency and a better understanding of this process will help to elucidate the mechanisms required for stem cell maintenance. This review takes advantage of recent studies from the epigenetic field to report knowledge regarding the mechanisms of stemness maintenance of neural stem cells in the neurogenic niches.