Coactivators and corepressors regulate transcription by controlling interactions between sequence-specific transcription factors, the basal transcriptional machinery and the chromatin environment. This review consider the access of nuclear and steroid receptors to chromatin, their use of corepressors and coactivators to modify chromatin structure and the implications for transcriptional control. The assembly of specific nucleoprotein architectures and targeted histone modification emerge as central controlling elements for gene expression.
The complex transformation of a tadpole to a frog during amphibian development is under the control of thyroid hormone (T3). T3 is known to regulate gene transcription through its nuclear receptors. We have previously isolated many genes which are up-regulated by T3 in the intestine of Xenopus laevis tadpoles. We have now cloned a full-length cDNA for one such gene (IU12). Sequence analysis shows that the IU12 cDNA encodes a plasma membrane protein with 12 transmembrane domains and homologous to a mammalian gene associated with cell activation and organ development. Similarly, we have found that IU12 is activated during intestinal remodeling when both cell death and proliferation take place. Furthermore, IU12 is an early T3-response gene and its expression in the intestine during T3-induced metamorphosis mimics that during normal development. These results argue for a role of IU12 in the signal transduction pathways leading to intestinal metamorphosis.
To make further investigation of the IgE antibody repertoire in Trichosanthin (TCS) allergic responses, a murine IgE phage surface display library was constructed (3.0 x 10(5) independent clones). We first constructed the V epsilon cDNA library (4.6 x 10(5) independent clones) and V kappa cDNA library (3.0 x 10(5) independent clones). Then, the V epsilon and V kappa gene segments were amplified from both libraries by PCR respectively, and assembled into Fab fragment by SOE PCR. The phage library containing Fabs was thus constructed. The diversity of V epsilon from this library was analyzed and proved. Fab clones with high specificity to TCS have been screened out.
Hydroxyurea has been used to synchronize cultured cells to S-phase and used to treat patients with sickle-cell anemia. Recently, we found that hydroxyurea can induce the apoptosis of HEL (human erythroleukemia) cells. The induced HEL cells showed ultrastructurally chromatin condensation with regular crescents at the nuclear edges and apoptotic bodies. However, the cells of K562, another human erythroleukemia cell line, did not show such morphological changes. Under fluoroscope, the HEL cells after induction often displayed a clear reduction in nuclear diameter and nuclear chromatin cleavage and condensation and the presence of nuclear ring and apoptotic bodies. Analysis with flow cytometry showed that the percentage of apoptotic cells is about 30-40% after HEL cells were induced by hydroxyurea for 3 days. DNA ladder can be observed by electrophoretic analysis.
This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous intron 1 sequence. hFIX minigene was obtained with middle sequence truncated intron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5i'IX, retroviral vector GINaCi'IX were constructed. These vectors were transduced into target cells of PA317, C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/pKGoi'IX, 151 ng/10(6) cells/24h; PA317/G1NaCi'IX, 308 ng/10(6) cells/24 h; C2C12/G1 NaCi'IX, 188 ng/10(5) cells/24 h; RSF/G1NaCi'IX, 1929 ng/10(5) cells/24 h; HSF/G1NaCi'IX, 1646 ng/10(6) cells/24 h. These results indicated that hFIX minigene with intron 1 is able to increase the expression level to about 3 times of that of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from intron splicing during viral production, a retroviral vector G1NaCi'IXR with reversely inserted hFIX minigene expression cassette was constructed. The expression level of reverse constructor in PA317 cells was 390 ng/10(6) cells/24 h with 79% of bioactivity. PCR detection of HT/G1NaCi'IXR cells infected with PA317/G1NaCi'IXR supernatant confirmed the existence of intron 1 sequence. These results suggested that expression vector with forward-inserted intron1-carrying hFIX expression cassette can be used in directed gene transfer, but when using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying hFIX expression cassette should be considered.
The organization of the higher order structure of chromatin in chicken erythrocytes has been examined with tapping-mode scanning force microscopy under conditions close to their native environment. Reproducible high-resolution AFM images of chromatin compaction at several levels can be demonstrated. An extended beads-on-astring (width of approximately 15-20 nm, height of approximately 2-3 nm for each individual nucleosome) can be consistently observed. Furthermore, superbeads (width of approximately 40 nm, height of approximately 7 nm) are demonstrated. Visualization of the solenoid conformation at the level of 30 nm chromatin fiber is attained either by using AFM or by using electron microscopy. In addition, tightly coiled chromatin fibers (approximately 50-60 nm and approximately 90-110 nm) can be revealed. Our data suggest that the chromatin in the interphase nucleus of chicken erythrocyte represents a high-order conformation and AFM provides useful high-resolution structural information concerning the folding pattern of interphase chromatin fibers.
Epidermal growth factor (EGF) is produced primarily by Leydig cells of human testis. Expression of the EGF gene was assessed in mouse testis during the course of sexual maturation by the application of the RT-PCR method and the use of specific oligonucleotide primers. Testis EGF mRNA content increased with the developmental age of the mice, i.e., day 15 < day 30 < day 45 postnatal. The expression of the EGF gene appears to correlate with maturation of the testis and proliferation of Leydig-cells.
Random Amplified Polymorphic DNA (RAPD) analysis is a new technology of molecular marking which has proved very powerful in detecting genetic diversity at the level of population. The genomic DNAs used in our experiment were extracted from fresh leaves taken from 59 individuals sampled from three natural populations in Yan An, Shanxi Province. Through more than 2,000 PCRs, deep-going RAPD analysis was carried out on DNA samples from 49 individuals. The percentage of polymorphic RAPD loci found in these three populations were respectively 27.2%, 18.6% and 5.4%; the average genetic distances within population, 0.055, 0.036 and 0.008; the average genetic distances between populations (I-II), (I-III) and (II-III), 0.105, 0.096 and 0.060. The genetic diversity of A. brachypus within and between populations was found, for the first time, to be rather poor, thus revealing innate factors as the cause contributing to its endangered status. In addition, our work also provides basic materials for elucidating the underlying cause of its endangerment and for its protection biology.
Expression of opioid receptor-like receptor (ORL1) and its endogenous peptide agonist nociceptin/orphanin FQ (N/OFQ) during mouse embryogenesis have been investigated. Transcripts of ORL1 and N/OFQ were detected by RT-PCR in mouse brain of day 8 embryo (E8) and the expression continued afterwards. Northern blot analysis revealed abundant expression of ORL1 at postnatal day 1 (P1) and N/OFQ at E17 and P1 in the brain but none was detected in other embryonic tissues. The presence of functional ORL1 in mouse embryonic brain was also confirmed by specific binding of [3H] N/OFQ (kd = 1.3 +/- 0.5 nM and Bmax = 72 +/- 9 fmol/mg protein) as well as by N/OFQ-stimulated G protein activation.
The developmental stage-specific silencing of the human epsilon-globin gene during embryonic life is controlled, in part, by the silencer (-392 bp approximately -177 bp) upstream of this gene. In order to elucidate its role, the nuclear extract from the human fetal liver has been prepared and the interactions between trans-acting factors and this silencer element have been examined. By using DNaseI footprinting assay, a major protected region from -278 bp to -235 bp within this silencer element was identified. Furthermore, we found in gel mobility shift assay and Southwestern blotting assay that there were at least four trans-acting factors (MW approximately 32, 28, 26 and 22 kD) in the nuclear extract isolated from the human fetal liver, which could specifically bind to this region. Our results suggested that these trans-acting factors might play an important role in silencing the human embryonic epsilon-globin gene expression at the fetal stage through the interactions with this silencer.
Human gastric cancer MKN-45 cells were transfected with pULB 3238, a plasmid carrying MVMp NS-1 gene with its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR. After the integration and expression of NS-1 gene, some of the transfectants died, while others remained alive, but the growth features of survived cells were changed. For further study on the antineoplastic function of parvoviral NS-1 protein in vivo, transgenic mice carrying NS-1 genes were established by conventional method. Among 4 founders, one of them was found to be able to transmit the transgene to around 50% of their offsprings. RT-PCR was performed to indicate the expression of NS-1 gene in transgenic mice and its mRNA appeared in a variety of tissues. The expression of integrated NS-1 gene may correlate with the decreased incidence of tumor induced in vivo by chemical carcinogens.
Recently we constructed a murine IgE phage surface display library and screened out two IgE (Fab) clones with specific binding activity to Trichosanthin (TCS). In this work, the V epsilon and V kappa genes of the two clones were sequenced and their putative germline gene usages were studied. On the basis of the known 3D structure of Trichosanthin and antibody, molecular modeling was carried out to study the antigen-antibody interaction. The possible antigenic determinant sites on the surface of TCS recognized by both the clones were analyzed, and the reaction forces between TCS and two Fab fragments were also analyzed respectively.
Recent studies have revealed that the gamma-chain of the IL-2 receptor is shared by the receptors for IL-4, IL-7, IL-9, IL-13, and IL-15, and it is therefore also referred to as the common gamma-chain (gamma c). Mutations of gamma c result in X-linked severe combined immunodeficiency syndrome in humans, indicating that gamma c is essential for normal development and function of the immune system. We demonstrate that human hematopoietic cells express two gamma c transcripts differing in their carboxyl terminal coding region. One transcript is the previously reported sequence (gamma c-long), whereas the newly identified sequence exhibits a deletion of 72 nucleotides close to the 3'-end of the open reading frame (gamma c-short). This alteration predicts a loss of 24 amino acids including a conserved tyrosine residue which is shared by several members of the cytokine receptor family. The presence of these two distinct forms of gamma c transcripts was demonstrated by sequencing of reversely transcribed and polymerase chain reaction (RT-PCR) amplified mRNA, restriction digestion of the RT-PCR products, RNAse protection, and Northern blotting from human cell lines and human peripheral blood lymphocytes. Furthermore, the two variants were present in peripheral blood lymphocytes from both female and male donors, which rules out allelic variants since gamma c is a single copy gene located on the X chromosome. A truncation mutant at a site near the observed changes in gamma c-short has been reported by others to alter biochemical events activated by cytokines. This combined with the loss of a potential SH2 "docking" site in gamma c-short suggests that gamma c-long and gamma c-short may link to different signaling pathways and may play an important role in determining the cellular response to IL-2, IL-4, IL-7, IL-9, IL-13, IL-15.
Previous investigation on the mutagenic effects of 3, N4-Ethenocytosine (epsilon C), a nonpairing DNA lesion, revealed the existence of a novel SOS-independent inducible mutagenic mechanism in E. coli termed UVM for UV modulation of mutagenesis. To investigate whether UVM is mediated by an alteration of DNA replication, we have set up an in vitro replication system in which phage M13 viral single-stranded DNA bearing a single site-specific (epsilon C) residue is replicated by soluble protein extracts from E. coli cells. Replication products were analyzed by agarose gel electrophoresis and the frequency of translesion synthesis was determined by restriction endonuclease analyses. Our data indicate that DNA replication is strongly inhibited by epsilon C, but that translesion DNA synthesis does occur in about 14% of the replicated DNA molecules. These results are very similar to those observed previously in vivo, and suggest that this experimental system may be suitable for evaluating alterations in DNA replication in UVM-induced cells.
The production of transgenic swine for xenotransplantation has been proposed as an optimal option to overcome the chronic shortage of human organ donors. Generation of genetically engineered swine has been elusive due to the difficulties in gene transfer. In order to achieve effective gene delivery, a key step for the genetic modification, we applied electronic pulse delivery (EPD) technology to introduce H2Kb-DC DNA construct into swine eggs. Using the developed EPD Protocols, we have achieved good viability of the EPD treated oocytes, satisfactory embryonic development of the EPD treated embryos, and stable DNA transfer into the swine embryos with high efficiency. Thus, application of the EPD technology promises to effectively facilitate the generation of large transgenic mammals.