雌核发育银鲫原肠期胚胎和尾芽期胚胎差异表达基因的呈现

构建了雌核发育银鲫原肠期胚胎和尾芽期胚胎间的抑制性差减杂交cDNA质粒文库。对原肠期’739个和尾芽期816个PCR阳性克隆进行斑点杂交,得到72个原肠期和98个尾芽期斑点杂交阳性克隆。测序和基因数据库比对结果表明：72个原肠期斑点杂交阳性克隆中,包括19个已知基因的cDNA片段和31个没有同源性的cDNA片段;98个尾芽期斑点杂交阳性克隆中,包括52个已知基因的cDNA片段和37个没有同源性的cDNA片段。采用虚拟Northern杂交和RT-PCR证实了部分基因在银鲫胚胎发育过程中的差异表达。这些差异表达基因的呈现为进一步研究银鲫胚胎发育的分子机制奠定了基础。     

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 d     

Identification and characterization of genes expressed in early embryogenesis from microspores of <Emphasis Type="Italic">Brassica napus</Emphasis>

To understand the mechanism in induction of embryogenesis from microspores of Brassica napus, we isolated exhaustively the genes expressed differentially during the early stage of microspore culture. A subtracted cDNA library composed of up-regulated genes during androgenic initiation was produced by suppression subtractive hybridization followed by differential screening by dot blot hybridization, and a total of 136 non-redundant expressed sequence tags were identified. Analysis of the potential functions of the genes showed that 64% of these genes were homologous to known genes, and the remaining ones have not been previously reported to participate in embryogenesis. Many embryo-specific genes were contained in the isolated genes, for example, genes cording lipid transfer protein, napin, cruciferin, oleosin, and phytosulfokine. Real-time RT-PCR analysis for 15 selected genes, which are understood to not be related with embryogenesis, demonstrated that all genes were expressed highly in the early stage of microspore embryogenesis. A few genes also showed higher expression in microspores cultured in non-embryogenic condition or in later stages of embryos. A principal component analysis based on expression profiles of the 15 genes demonstrated that these genes were classified into 2 groups, one characterized by their high expression in initiation of embryogenesis, and the other characterized by their expression in the early to middle stage of embryogenesis. The expressions of these genes were confirmed in zygotic embryos. The identification and characterization of the genes isolated in the present study provide novel information on microspore embryogenesis in Brassica.     

Application and limitations of differential hybridization in the isolation of T cell-specific cDNA clones

We investigated the limitations and effectiveness of differential hybridization in the cloning of T cell-specific cDNA (complementary DNA) molecular clones. By using the technique with T cell and B cell cDNA probes, together with Northern blot analysis, we successfully isolated cDNA clones exclusively expressed in T cells from 1 X 10(4) plaque-forming units of a T cell hybridoma. These clones represent 0.068% of the mass of the cytoplasmic mRNA. Our result shows that differential hybridization is an effective procedure when used in combination with Northern blot analysis for screening of genes selectively expressed in T cells.     

Isolation and characterization of a diverse set of genes from carrot somatic embryos.

The early events in plant embryogenesis are critical for pattern formation, since it is during this process that the primary apical meristems and the embryo polarity axis are established. However, little is known about the molecular events that are unique to the early stages of embryogenesis. This study of gene expression during plant embryogenesis is focused on identifying molecular markers from carrot (Daucus carota) somatic embryos and characterizing the expression and regulation of these genes through embryo development. A cDNA library, prepared from polysomal mRNA of globular embryos, was screened using a subtracted probe; 49 clones were isolated and preliminarily characterized. Sequence analysis revealed a large set of genes, including many new genes, that are expressed in a variety of patterns during embryogenesis and may be regulated by different molecular mechanisms. To our knowledge, this group of clones represents the largest collection of embryo-enhanced genes isolated thus far, and demonstrates the utility of the subtracted-probe approach to the somatic embryo system. It is anticipated that many of these genes may serve as useful molecular markers for early embryo development.     

Molecular cloning of cDNA corresponding to mRNA species whose steady state levels in the thyroid are enhanced by thyrotropin. Homology of one of these sequences with ferritin H

A lambda gt11 cDNA library was constructed using poly(A)+ mRNA from thyrotropin (TSH)-stimulated Fisher rat thyroid (FRTL5) cells. The library was screened for nonthyroglobulin cDNA sequences by differential plaque filter hybridization using single-stranded cDNA probes synthesized from mRNA prepared from quiescent and TSH-stimulated FRTL5 cells. Thyroglobulin cDNA-containing recombinants in the library were avoided by prehybridizing the TSH probe to excess thyroglobulin cDNA. Of 48,000 clones screened, 60 were chosen as representing mRNA species whose abundance was increased in TSH-stimulated versus quiescent cultures. Southern blot analysis of 9 clones confirmed that the TSH-cDNA probe hybridized to a greater extent to the cDNA inserts than did the control probe. cDNA insert sizes varied between 0.3 kilobase (kb) and 1.0 kb. Northern slot blot analysis using as probes the cDNA of four of these clones (FC4, FC26, FC29, and FC43) demonstrated that TSH stimulation of FRTL5 cells increased the steady state levels of the respective mRNA species by 4-12-fold. For all 4 clones, increases in mRNA levels were apparent within approximately 1 h and were maximal after 14-18 h of TSH stimulation. Determination of the partial nucleotide sequence of these 4 clones confirmed that none was thyroglobulin, thyroid peroxidase, or any other gene previously reported to be stimulated by TSH. Three of the clones bore no homology to any known nucleotide sequence, but FC26 was 85% homologous with human ferritin H. Northern blot analysis using the FC26 cDNA insert as a probe confirmed hybridization to an mRNA species of 1 kb, the known size of ferritin H mRNA. In summary, using the technique of differential plaque filter hybridization, we have identified 4 new genes whose mRNA levels are increased by TSH stimulation of thyroid cells. One of these genes is homologous to human ferritin H.     

Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation.

Mouse embryonic stem cells are pluripotent cells that are derived from the inner cell mass of blastocysts. When induced to synchronously enter a program of differentiation in vitro, they form embryoid bodies that contain cells of the mesodermal, hematopoietic, endothelial, muscle, and neuronal lineages. Here, we used a panel of marker genes with early expression within the germ layers (oct-3, Brachyury T, Fgf-5, nodal, and GATA-4) or a variety of lineages (flk-1, Nkx-2.5, EKLF, and Msx3) to determine how progressive differentiation of embryoid bodies in culture correlated with early postimplantation development of mouse embryos. Using RNA in situ hybridization, we found that the temporal and spatial relationships existing between these marker genes in vivo were maintained also in vitro. Studying the onset of marker gene expression allowed us also to determine the time course of differentiation during the formation of embryoid bodies. Thus, stages equivalent to embryogenesis between implantation and the beginning of gastrulation (4.5-6.5 d.p.c.) occur within the first two days of embryoid body differentiation. Between days 3 and 5, embryoid bodies contain cell lineages found in embryos during gastrulation at 6.5 to 7.0 d.p.c., and after day 6 in culture, embryoid bodies are equivalent to early organogenesis-stage embryos (7.5 d.p.c.). In addition, we demonstrate that the panel of developmental markers can be applied in a screen for stage- or lineage-specific genes. Reporter gene expression from entrapment vector insertions can be co-localized with expression of specific markers within the same cell during embryoid body formation as well as during embryogenesis. Our results thus demonstrate the power of embryoid body formation as an in vitro model system to study early lineage determination and organogenesis in mammals, and indicate that they will prove to be useful tools for identifying developmental genes whose expression is restricted to particular lineages.     

Differential gene expression in the anterior neural plate during gastrulation of Xenopus laevis

We have isolated three cDNA clones that are preferentially expressed in the cement gland of early Xenopus laevis embryos. These clones were used to study processes involved in the induction of this secretory organ. Results obtained show that the induction of this gland coincides with the process of neural induction. Genes specific for the cement gland are expressed very early in the anterior neural plate of stage-12 embryos. This suggests that the anteroposterior polarity of the neural plate is already established during gastrulation. At later stages of development, two of the three genes have secondary sites of expression. The expression of these genes can be induced in isolated animal caps by incubation in 10 mM-NH4Cl, a treatment that is known to induce cement glands.     

DNA array profiling of gene expression changes during maize embryo development

We are using DNA microarray-based gene expression profiling to classify temporal patterns of gene expression during the development of maize embryos, to understand mRNA-level control of embryogenesis and to dissect metabolic pathways and their interactions in the maize embryo. Genes involved in carbohydrate, fatty acid, and amino acid metabolism, the tricarboxylic acid (TCA) cycle, glycolysis, the pentose phosphate pathway, embryogenesis, membrane transport, signal transduction, cofactor biosynthesis, photosynthesis, oxidative phosphorylation and electron transfer, as well as 600 random complementary DNA (cDNA) clones from maize embryos, were arrayed on glass slides. DNA arrays were hybridized with fluorescent dye-labeled cDNA probes synthesized from kernel and embryo poly(A)⁺RNA from different stages of maize seed development. Several characteristic developmental patterns of expression were identified and correlated with gene function. Patterns of coordinated gene expression in the TCA cycle and glycolysis were analyzed in detail. The steady state level of poly(A)⁺ RNA for many genes varies dramatically during maize embryo development. Expression patterns of genes coding for enzymes of fatty acid biosynthesis and glycolysis are coordinately regulated during development. Genes of unknown function may by assigned a hypothetical role based on their patterns of expression resembling well characterized genes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s10142-002-0046-6. Electronic Publication