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1.
LIM homeobox genes are characterized by encoding proteins in which two cysteine-rich LIM domains are associated with a homeodomain. We report the isolation of a gene, named Xlim-5 in Xenopus and lim5 in the zebrafish, that is highly similar in sequence but quite distinct in expression pattern from the previously described Xlim-1/lim1 gene. In both species studied the lim5 gene is expressed in the entire ectoderm in the early gastrula embryo. The Xlim-5 gene is activated in a cell autonomous manner in ectodermal cells, and this activation is suppressed by the mesoderm inducer activin. During neurulation, expression of the lim5 gene in both the frog and fish embryo is rapidly restricted to an anterior region in the developing neural plate/keel. In the 2-day Xenopus and 24-hr zebrafish embryo, this region becomes more sharply defined, forming a strongly lim5-expressing domain in the diencephalon anterior to the midbrain-forebrain boundary. In addition, regions of less intense lim5 expression are seen in the zebrafish embryo in parts of the telencephalon, in the anterior diencephalon coincident with the postoptic commissure, and in restricted regions of the midbrain, hindbrain, and spinal cord. Expression in ventral forebrain is abolished from the 5-somite stage onward in cyclops mutant fish. These results imply a role for lim5 in the patterning of the nervous system, in particular in the early specification of the diencephalon. 相似文献
2.
We have isolated a previously unknown human homeobox-containing cDNA, VENT-like homeobox-2 ( VENTX2), using PCR with a bone marrow cDNA library and primers designed from the VENTX1 (alias HPX42) homeobox sequence. Here we describe the molecular cloning, chromosomal localization to 10q26.3, and functional analysis of this gene. The 2.4-kb human VENTX2 cDNA encoded a protein with a predicted molecular weight of 28 kDa containing a homeodomain with 65% identity to the Xenopus laevis ventralizing gene Xvent2B. VENTX2 antisera detected a 28-kDa protein in cells transfected with a VENTX2 expression construct, in a human erythroleukemic cell line and in bone marrow samples obtained from patients in recovery phase after chemotherapy. The similarity of the homeodomains from VENTX2 and the X. laevis Vent gene family places them in the same homeodomain class. Consistent with this structural classification, overexpression of VENTX2 in zebrafish embryos led to anterior truncations and failure to form a notochord, which are characteristics of ventralization. 相似文献
3.
CNOT2,a newly identified homeobox gene, is physically linked to the CNOT1gene in the chicken genome. The two chicken genes represent two different subgroups of the Notgene family, the first including CNOT1and the Xenopusgenes XNot1and XNot2,and the second CNOT2and the zebrafish floating headgene. The overall expression pattern of CNOT2in Hensen's node, notochord, neural plate, tailbud, and epiphysis resembled the CNOT1pattern. However, several significant differences occurred: CNOT2expression was much stronger and more widespread in the pregastrulation embryo, it showed an additional, transient domain on the anterior intestinal portal, and lacked expression on the early anterior neural folds and the anterodistal limb bud. We studied CNOT expression by transplanting parts of the primitive streak into growing embryos or by explanting them into tissue culture. CNOTgene expression from young nodes was maintained in vivo,but required in vitrothe addition of retinoic acid. The generation of differentiated notochord structures could only be obtained, if either older node grafts were used in vitroor young node grafts were transplanted close to the primary axis in vivo.We conclude that CNOTexpression in the anterior streak is not enough for notochord differentiation, but further influences are necessary. A Not-related gene has previously been isolated from Drosophila melanogasterand its expression was detected in the posterior brain and the neuroblasts (Dessain and McGinnis, 1993. Adv. Dev. Biochem.2, 1–55). The correspondence between Notgene-expressing cells in the nervous system of Drosophilaand the early neuroectoderm in the chick and its implication for a phylogenetic relationship between neuroectoderm and the notochord is discussed. 相似文献
4.
To explore the role of homeobox genes in the intestine, the human colon adenocarcinoma cell line Caco2-TC7 has been stably transfected with plasmids synthesizing Cdx1 and Cdx2 sense and antisense RNAs. Cdx1 overexpression or inhibition by antisense RNA does not markedly modify the cell differentiation markers analyzed in this study. In contrast, Cdx2 overexpression stimulates two typical markers of enterocytic differentiation: sucrase-isomaltase and lactase. Cells in which the endogenous expression of Cdx2 is reduced by antisense RNA attach poorly to the substratum. Conversely, Cdx2 overexpression modifies the expression of molecules involved in cell–cell and cell–substratum interactions and in transduction process: indeed, E-cadherin, integrin-β4 subunit, laminin-γ2 chain, hemidesmosomal protein, APC, and α-actinin are upregulated. Interestingly, most of these molecules are preferentially expressed in vivo in the differentiated villi enterocytes rather than in crypt cells. Cdx2 overexpression also results in the stimulation of HoxA-9 mRNA expression, an homeobox gene selectively expressed in the colon. In contrast, Cdx2-overexpressing cells display a decline of Cdx1 mRNA, which is mostly found in vivo in crypt cells. When implanted in nude mice, Cdx2-overexpressing cells produce larger tumors than control cells, and form glandular and villus-like structures. Laminin-1 is known to stimulate intestinal cell differentiation in vitro. In the present study, we demonstrate that the differentiating effect of laminin-1 coatings on Caco2-TC7 cells is accompanied by an upregulation of Cdx2. To further document this observation, we analyzed a series of Caco2 clones in which the production of laminin-α1 chain is differentially inhibited by antisense RNA. We found a positive correlation between the level of Cdx2 expression, that of endogenous laminin-α1 chain mRNA and that of sucrase-isomaltase expression in these cell lines. Taken together, these results suggest (a) that Cdx1 and Cdx2 homeobox genes play distinct roles in the intestinal epithelium, (b) that Cdx2 provokes pleiotropic effects triggering cells towards the phenotype of differentiated villus enterocytes, and (c) that Cdx2 expression is modulated by basement membrane components. Hence, we conclude that Cdx2 plays a key role in the extracellular matrix–mediated intestinal cell differentiation. 相似文献
5.
The monoclonal antibody, TS19, (Heimfeld et al., 1985), labels the apical surface of ectodermal epithelial cells of tentacles and lower peduncles in Hydra. To investigate the patterning process in a tissue whose original pattern was completely destroyed, the TS19 staining pattern was examined in developing aggregates of Hydra cells. Two types of aggregates were prepared. G-aggregates were made from tissue of the gastric portion of animals and RG-aggregates from gastric tissue allowed to regenerate for 24 hr before making aggregates. G-aggregates were initially TS19-negative, and later dim and uniformly TS19-positive. Thereafter, TS19 staining broke up into brightly stained and unstained regions. The brightly staining regions developed into head or foot structures. The TS19 pattern in RG-aggregates developed differently. Since the initial aggregates contained cells of regenerating tips, they started with TS19-positive cells as well as TS19-negative cells. The numbers of brightly staining TS19-positive cells increased with time. Some patches of these cells developed into head or foot structures, while others did not. These results and a simulation using a reaction-diffusion model suggest that the changes in activation levels affected the temporal changes in the pattern of TS19 staining, and that the de novo pattern formation in hydra can be explained in terms of a process involving activation and inhibition properties. 相似文献
6.
The reeler Albany2mutation ( Relnrl-Alb2) in the mouse is an allele of reelerisolated during a chlorambucil mutagenesis screen. Homozygous animals had drastically reduced concentrations of reelinmRNA, in which an 85-nt exon was absent. At the genomic level, the mutation was shown to be due to an intracisternal A-particle insertion leading to exon skipping. This appears to be the first observation of retrotransposon insertion during chlorambucil mutagenesis. 相似文献
7.
Progression through the G1 phase of the cell cycle is dependent on the activity of holoenzymes formed between D-type cyclins and their catalytic partners, the cyclin-dependent kinases cdk4 and cdk6. p16 INK4a,p15 INK4b, and p18 INK4c, a group of structurally related proteins, function as specific inhibitors of the cyclin D-dependent kinases and are likely to play physiologic roles as specific regulators of these kinases in vivo.A new member of the INK4 gene family, murine INK4d,has recently been identified. Here we report the isolation of human INK4d(gene symbol CDKN2D), which is 86% identical at the amino acid level to the murine clone and 44% identical to each of the other human INK4 family members. The INK4dgene is ubiquitously expressed as a single 1.4-kb mRNA with the highest levels detected in thymus, spleen, peripheral blood leukocytes, fetal liver, brain, and testes. The abundance of INK4dmRNA oscillates in a cell-cycle-dependent manner with expression lowest at mid G1 and maximal during S phase. Using a P1-phage genomic clone of INK4dfor fluorescence in situhybridization analysis, the location of this gene was mapped to chromosome 19p13. No rearrangements or deletions of the INK4dgene were observed in Southern blot analysis of selected cases of pediatric acute lymphoblastic leukemia (ALL) containing a variant (1;19)(q23′3) translocation that lacks rearrangement of either E2Aor PBX1, or in ALL cases containing homozygous or hemizygous deletions of the related genes, INK4aand INK4b. 相似文献
9.
Orosomucoid 1-like 3 (ORMDL3) gene was strongly linked with the development of asthma in genetic association studies, and its expression could be significantly induced by allergen in airway epithelial cells of mice. However, the expression mechanism of ORMDL3 was still unclear. Here we have identified and characterized the mouse ORMDL3 gene promoter. Deletion constructs of the 5′ flanking region were fused to a luciferase reporter gene. After transient transfection in mouse fibroblast cell line NIH3T3, a CRE (−27/−20) binding CREB was identified in the core promoter region. Deletion or mutation of the CRE consensus sequence resulted in a significant loss of the promoter activity. EMSA and ChIP assays demonstrated the binding of CREB to the core promoter. Knocking down endogenous CREB led to a reduction in ORMDL3 expression. Conversely, overexpression of CREB up-regulated ORMDL3 expression. Moreover, forskolin, a PKA activator, could facilitate the phosphorylation of CREB, which in turn heightens ORMDL3 expression. H-89, a PKA-specific inhibitor, could significantly inhibit ORMDL3 expression. This study delineates the characterization of mouse ORMDL3 gene promoter and shows signaling pathway cAMP/PKA/CREB plays an important role in regulating ORMDL3 expression, which will be helpful for future animal model studies regarding the regulation or function of ORMDL3 gene. 相似文献
10.
Axin is a critical component of the β-catenin destruction complex and is also necessary for Wnt signaling initiation at the level of co-receptor activation. Axin contains an RGS domain, which is similar to that of proteins that accelerate the GTPase activity of heterotrimeric Gα/Gna proteins and thereby limit the duration of active G-protein signaling. Although G-proteins are increasingly recognized as essential components of Wnt signaling, it has been unclear whether this domain of Axin might function in G-protein regulation. This study was performed to test the hypothesis that Axin RGS-Gna interactions would be required to attenuate Wnt signaling. We tested these ideas using an axin1 genetic mutant (masterblind) and antisense oligo knockdowns in developing zebrafish and Xenopus embryos. We generated a point mutation that is predicted to reduce Axin-Gna interaction and tested for the ability of the mutant forms to rescue Axin loss-of-function function. This Axin point mutation was deficient in binding to Gna proteins in vitro, and was unable to relocalize to the plasma membrane upon Gna overexpression. We found that the Axin point mutant construct failed to rescue normal anteroposterior neural patterning in masterblind mutant zebrafish, suggesting a requirement for G-protein interactions in this context. We also found that the same mutant was able to rescue deficiencies in maternal axin1 loss-of-function in Xenopus. These data suggest that maternal and zygotic Wnt signaling may differ in the extent of Axin regulation of G-protein signaling. We further report that expression of a membrane-localized Axin construct is sufficient to inhibit Wnt/β-catenin signaling and to promote Axin protein turnover. 相似文献
11.
The role of glial cells for the inactivation and synthesis of precursors for amino acid transmitters was studied in the brains of anesthetized rats in vivo using the microdialysis technique. The dialysis probes were inserted stereotactically into each neostriatum. One neostriatum was treated with the gliotoxin fluorocitrate, whereas the contralateral side served as a control. The basal efflux of amino acids, reflecting the extracellular level, was measured as well as the efflux during depolarization with 100 mM K+ in the dialysis stream. The potassium-evoked efflux of transmitter amino acids was calcium dependent and thus considered to reflect release from the transmitter pool. gamma-Aminobutyric acid (GABA) and glutamate release from the treated side was higher than the control value during the first 2-3 h, a result indicating an important role of glial cells in the inactivation of released transmitter. After 6-7 h with fluorocitrate, the release of glutamate was lower than the control value, a result indicating an important role of glial cells in the synthesis of precursors for the releasable pool of glutamate. The role of glutamine for the production of transmitter glutamate and GABA in vivo was further investigated by inhibiting glutamine synthetase with intrastriatally administered methionine sulfoximine. The release of gluatamate into the dialysis probe decreased to 54% of the control value, whereas the release of GABA decreased to 22% of the control value, a result indicating that glutamine may be more important for transmitter GABA than for transmitter glutamate. 相似文献
12.
The intermediate filament keratin, K15, is present in variable abundance in stratified epithelia. In this study we have isolated and characterized the sheep K15gene, focusing on its expression in the follicles of sheep and mice. We show that K15is expressed throughout the hair cycle in the basal layer of the outer root sheath that envelops the follicle. Strikingly, however, in large medullated wool follicles, a small group of basal outer root sheath cells located in the region thought to contain hair follicle stem cells are K15-negative. In the follicle bulb K15is expressed in cells situated next to the dermal papilla but not in the inner bulb cells. Elsewhere, K15is expressed at a low, variable level in the basal layer of the epidermis and sebaceous gland, often in a punctate pattern. In the esophagus of the sheep K15expression is restricted to the basal layer, in contrast to human esophagus where it is expressed throughout the epithelium. Transgenic mouse lines established with a 15-kb sheep K15gene construct exhibited faithful expression and showed no phenotypic consequences of K15overexpression. An investigation of transgene expression showed that K15is continuously expressed in outer root sheath cells during the hair cycle. Given its expression in the mitotically active basal cell layers of diverse epithelia and the follicle, K15expression appears to signal an early stage in the pathway of keratinocyte differentiation that precedes the decision of a cell to become epidermal or hair-like. 相似文献
13.
A solid-phase enzyme immunoassay for prostaglandin D 2 (PGD 2) was developed in which PGD 2 was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labelled and free PGD 2, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)- propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3 – 100 pg for PGD 2. The IC 50 value for PGD 2 in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enyzme immunoassay was applied to the measurement of PGD 2 content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD 2. PGD 2 contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay ( r = 0.966) after raising its contents by intravenous administration of PGD 2. The
level of PGD 2 in rat brain was extremely low but determined to be 0.11 ± 0.03 ng/g tissue (mean ± S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change. 相似文献
14.
Abstract: We synthesized a potent and selective antagonist radioligand for the neurokinin (NK)-1 receptor and characterized its binding to guinea pig striatal membranes. ( R ) - N - [2 - [Acetyl[ 3H 3][(2 - methoxyphenyl) - methyl]amino] - 1 - (1 H - indol - 3 - ylmethyl)ethyl][1,4' - bipiperidine]-1'-acetamide ([ 3H]LY303870) binds to a single class of sites with an equilibrium K D of 0.22 n M and a B max of 723 fmol/mg of protein. Unlabeled LY303870 potently inhibited the binding with an IC 50 of 0.56 n M , whereas the less active ( S )-enantiomer (LY306155) was substantially less potent. The nonpeptide NK-1 antagonists (±)-CP96,345 and (±)-RP 67580 had IC 50 values of 0.74 and 49 n M , respectively. Substance P (SP) was also a potent inhibitor with with an IC 50 of 3.1 n M . The inhibition by SP could be separated into two components: a high-affinity component with a K i of 0.53 n M and a lower-affinity component with a K i of 155 n M . Addition of 100 µ M guanylyl 5'-imidodiphosphate [Gpp(NH)p] in the incubation increased the relative amount of the low-affinity agonist state of the receptor. Consistent with the antagonist properties of LY303870, the dissociation rate of [ 3H]LY303870 was not changed by the presence of 100 µ M Gpp(NH)p. The distribution of [ 3H]LY303870 binding sites in the guinea pig brain closely matched the distribution of NK-1 receptors labeled by [ 3H]SP. Therefore, [ 3H]LY303870 is a potent and selective antagonist radioligand for NK-1 receptors in guinea pig brain. In addition, regulation of NK-1 agonist affinity by guanine nucleotides is similar to that seen for monoaminergic receptors. 相似文献
15.
Opitz G/BBB syndrome (OS) is a genetically heterogeneous disorder with an X-linked locus and an autosomal locus linked to 22q11.2. OS affects multiple organ systems with often variable severity even between siblings. The clinical features, which include hypertelorism, cleft lip and palate, defects of cardiac septation, hypospadias, and anorectal anomalies, indicate an underlying disturbance of the developing ventral midline of the embryo. The gene responsible for X-linked OS, FXY/MID1, is located on the short arm of the human X chromosome within Xp22.3 and encodes a protein with both an RBCC (RING finger, B-box, coiled coil) and a B30.2 domain. The Fxy gene in mice is also located on the X chromosome but spans the pseudoautosomal boundary in this species. Here we describe a gene closely related to FXY/MID1, called FXY2, which also maps to the X chromosome within Xq22. The mouse Fxy2 gene is located on the distal part of the mouse X chromosome within a region syntenic to Xq22. Analysis of genes flanking both FXY/MID1 and FXY2 (as well as their counterparts in mouse) suggests that these regions may have arisen as a result of an intrachromosomal duplication on an ancestral X chromosome. We have also identified in both FXY2 and FXY/MID1 proteins a conserved fibronectin type III domain located between the RBCC and B30.2 domains that has implications for understanding protein function. The FXY/MID1 protein has previously been shown to colocalize with microtubules, and here we show that the FXY2 protein similarly associates with microtubules in a manner that is dependent on the carboxy-terminal B30.2 domain. 相似文献
16.
Gram-negative bacteria use the sophisticated type II secretion system (T2SS) to secrete a large number of exoproteins into the extracellular environment. Five proteins of the T2SS, the pseudopilins GspG-H-I-J-K, are proposed to assemble into a pseudopilus involved in the extrusion of the substrate through the outer membrane channel. Recent structural data have suggested that the three pseudopilins GspI-J-K are organized in a trimeric complex located at the tip of the GspG-containing pseudopilus. In the present work we combined two biochemical techniques to investigate the protein-protein interaction network between the five Pseudomonas aeruginosa Xcp pseudopilins. The soluble domains of XcpT-U-V-W-X (respectively homologous to GspG-H-I-J-K) were purified, and the interactions were tested by surface plasmon resonance and affinity co-purification in all possible combinations. We found an XcpV I-W J-X K complex, which demonstrates that the crystallized trimeric complex also exists in the P. aeruginosa T2SS. Interestingly, our systematic approach revealed an additional and yet uncharacterized interaction between XcpU H and XcpW J. This observation suggested the existence of a quaternary, rather than ternary, complex (XcpU H-V I-W J-X K) at the tip of the pseudopilus. The assembly of this quaternary complex was further demonstrated by co-purification using affinity chromatography. Moreover, by testing various combinations of pseudopilins by surface plasmon resonance and affinity chromatography, we were able to dissect the different possible successive steps occurring during the formation of the quaternary complex. We propose a model in which XcpV I is the nucleator that first binds XcpX K and XcpW J at different sites. Then the ternary complex recruits XcpU H through a direct interaction with XcpW J. 相似文献
19.
MTG8(HGMW-approved symbol CBFA2T1) was originally identified as one of the loci involved in the t(8;21)(q22;q22) of acute myeloid leukemia. We characterize two human MTG8-related genes, MTGR1and MTGR2(HGMW-approved symbols CBFA2T2and CBFA2T3). The former is duplicated in mouse, one locus possibly being a retroposon. Multiple MTG8-related sequences are found in several vertebrate species, from fish to mammals, albeit not in a urodele. MTGR2maps to 16q24 and, like MTG8and MTGR1,is close to one of three loci encoding a syntrophin (dystrophin-associated proteins). Moreover, an alternative MTGR1promoter/5′ exon is contained within the α1-syntrophin locus. Thus, the two classes of genes may define novel paralogous groups. MTGR1is expressed mainly in brain, while MTGR2is expressed in the thymus and possibly in monocytes. Like MTG8, MTGR1is transcribed into a number of isoforms due to alternative splicing of different 5′ exons onto a common splice acceptor site. Comparison of the three predicted human MTG8-related polypeptides to their Drosophilacounterpart ( nervy) highlights four separate regions of sequence conservation that may correspond to distinct domains. The most NH 2-terminal of these is proportionately more conserved among the human polypeptides, presumably due to specific structural/functional constraints. 相似文献
20.
Mutations in the X-linked gene FMR1 cause fragile X syndrome, the leading cause of inherited mental retardation. Two autosomal paralogs of FMR1 have been identified, and are known as FXR1 and FXR2. Here we describe and compare the genomic structures of the mouse and human genes FMR1, FXR1, and FXR2. All three genes are very well conserved from mouse to human, with identical exon sizes for all but two FXR2 exons. In addition, the three genes share a conserved gene structure, suggesting they are derived from a common ancestral gene. As a first step towards exploring this hypothesis, we reexamined the Drosophila melanogaster gene Fmr1, and found it to have several of the same intron/exon junctions as the mammalian FXRs. Finally, we noted several regions of mouse/human homology in the noncoding portions of FMR1 and FXR1. Knowledge of the genomic structure and sequence of the FXR family of genes will facilitate further studies into the function of these proteins. 相似文献
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