Targeted disruption of genes for gp138, a cell-fusion-related protein in Dictyostelium discoideum, revealed the existence of a third gene

The sexual cycle of the cellular slime mold, Dictyostelium discoideum , offers a suitable experimental system to analyze sexual cell interactions. We have been analyzing molecular mechanisms involved in sexual cell fusion using complementary heterothallic strains in D. discoideum and have identified several cell surface proteins involved in the process. One of them, gp138 is present in strains of both mating types and considered to be responsible for membrane fusion itself. Two genes with high mutual homology, GP 138 A and GP 138 B , have been identified so far as encoding this protein. Expression of antisense RNA for GP 138 B has been shown to suppress sexual cell fusion, confirming the critical importance of these genes in sexual cell fusion. However, neither the functional relationship of the two gp138 genes nor the possibility of the existence of more genes that encode gp138 has been determined yet. In the present study, GP 138 A and GP 138 B were disrupted by homologous recombination in an effort to clarify these points. Analysis of the double knock-out mutants suggested the presence of a third gene for gp138.     

Activin A-induced differentiation in K562 cells is associated with a transient hypophosphorylation of RB protein and the concomitant block of cell cycle at G1 phase.

The human erythroleukemic cell line, K562, can be induced to differentiate by the addition of activin A, a newly purified protein belonging to the TGF-beta 1 family. The present studies used flow cytometric cell cycle analysis, indirect immunofluorescence staining of the proliferating cell nuclear antigen (PCNA), and thymidine incorporation assay of cell proliferation to study the effects of activin A on the cell cycle during differentiation in K562 cells. Activin A-treated K562 cells were found to undergo a transient block in cell cycle, temporarily halting progression from G1 to S phase. The latter can be observed after approximately 24 hr of incubation with activin A and then disappears after this early stage of induction of differentiation. Cell cycle kinetics analysis using synchronized K562 cells also confirms that in the presence of activin A, K562 cells progress normally through various phases of cell cycle, except that there is prolongation of the G1 phase between 10 to 24 hr of culture. Furthermore, this transient arrest in G1 is correlated with dephosphorylation of a nucleoprotein, the RB gene product, which occurs within 9-24 hr of incubation with activin A; and phosphorylation of RB protein then develops afterward. In addition, these cell cycle-related events are observed to occur earlier than the accumulation of hemoglobins in K562 cells. It is concluded that transient dephosphorylation of RB protein and prolongation of G1 phase of cell cycle precede and accompany erythroid differentiation caused by activin A and chemical inducers, thus constituting part of the mechanism for induction of differentiation in the erythroleukemia cells.     

The expression of nr0b1, the earliest gene in zebrafish tooth development,is a marker for human tooth and ameloblastoma formation

Zebrafish teeth develop on pharyngeal jaws in the 5th branchial arch, but early tooth development is remarkably similar to mammals (Borday-Birraux et al., Evol Dev 8:130, 2006). Recently, eve1 has been shown to be associated with the primary tooth (4V¹) and early ameloblast development, the enamel organ precursor (Laurenti et al., Dev Dyn 230:727, 2004). dax1 is initially expressed in the 5th branchial arch in zebrafish at approximately 26 h postfertilization (hpf) and colocalizes with eve1 expression at ~48 hpf. Embryos injected with dax1 morpholino show downregulation of eve1 expression. Based on the zebrafish observations, we demonstrated novel DAX1 expression in normal human dental, benign ameloblastoma, and malignant ameloblastoma tissues. The association of NR0B1 and its protein product DAX1 with primary tooth development and ameloblastoma tumorigenesis is an association not previously described.     

Suppression and restoration of primordial germ cell marker gene expression in channel catfish,Ictalurus punctatus,using knockdown constructs regulated by copper transport protein gene promoters: Potential for reversible transgenic sterilization

Complementary DNA overexpression and short hairpin RNA interference approaches were evaluated for decreasing expression of primordial germ cell (PGC) marker genes and thereby sterilizing channel catfish, Ictalurus punctatus, by delivering knockdown constructs driven by a constitutive promoter from yeast and a copper transport protein gene into fish embryos by electroporation. Two PGC marker genes, nanos and dead end, were the target knockdown genes, and their expressions, along with that of an off-target gene, vasa, were evaluated temporally using real-time polymerase chain reaction. Copper sulfate was evaluated as a repressor compound. Some of the constructs knocked down PGC marker gene expression, and some of the constructs were partially repressed by application of 0.1-ppm copper sulfate. When the rate of sexual maturity was compared for three-year-old broodfish that had been exposed to the sterilizing constructs during embryologic development and controls that had not been exposed, several treatments had reduced sexual maturity for the exposed fish. Of two promoter systems evaluated, the one which had been designed to be less sensitive to copper generally was more effective at achieving sterilization and more responsive to repression. Knockdown constructs based on 3′ nanos short hairpin RNA interference appeared to result in the best repression and restoration of normal sexual maturity. We conclude that these copper-based systems exhibited good potential for repressible transgenic sterilization. Optimization of this system could allow environmentally safe application of transgenic technology and might be applicable to other applications for aquatic organisms.     

Involvement of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) in the differentiation of primordial germ cells

In order to understand the role of the protein of Xenopus vasa homolog ( Xenopus vasa -like gene 1, XVLG1 ) in germ line cells, an attempt was made to perturb the function of the protein with the anti-vasa antibody 2L-13. The 2L-13 or the control antibody was microinjected with a lineage tracer (FITC-dextran-lysine, FDL) into single vegetal blastomeres containing the germ plasm of Xenopus 32-cell embryos, the descendants of which were destined to differentiate into a small number of primordial germ cells (PGC) and a large number of somatic cells, mostly of endodermal tissues at the tadpole stage. No significant effect of the injection of the antibodies on FDL-labeled, presumptive PGC (pPGC) was observed in embryos until stage 37/38. However, FDL-labeled PGC were not observed in almost all the 2L-13 antibody-injected tadpoles, although a similar number of labeled somatic cells were always present. As 2L-13 antibody specifically reacts with XVLG1 protein in the embryos by immunoblotting, the present results suggest that the antibody perturbed the function of XVLG1 protein in the pPGC, resulting in failure of PGC differentiation at the tadpole stage.     

Structure and expression of a hybrid proline-rich protein gene in the solanaceous species,Solanum brevidens, Solanum tuberosum, andLycopersicum esculentum

The full-length cDNA of a previously identified Solanum brevidens gene was isolated and characterised. DNA sequence analysis revealed an open reading frame that encodes a hybrid proline-rich cell wall protein of 407 amino acids. The putative protein was designated SbrPRP. The SbrPRP harbours three parts, an N-terminal signal peptide followed by a repetitive proline-rich domain and a cysteine-rich C-terminus resembling non-specific lipid-transfer proteins. The repetitive proline-rich domain contains two repeated motifs, PPHVKPPSTPK and PTPPIVSPP extended with TPKYP and TPKPPS motifs, respectively, at their N- or C-terminal. The SbrPRP gene of the non-tuberising Solanum species, Solanum brevidens, possesses highly homologous counterparts in the tuberising species, Solanum tuberosum (StPRP) and in the related species, Lycopersicum esculentum (TFM7). All three genes are present in single- or low copy number in the corresponding genome. Organ-specific expression of the genes, however, is different in the three solanaceous species.     

Identification of transposons,retroelements, and a gene family predominantly expressed in floral tissues in chromosome 3DS of the hexaploid wheat progenitor <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

A multigene family expressed during early floral development was identified on the short arm of wheat chromosome 3D in the region of the Ph2 locus, a locus controlling homoeologous chromosome pairing in allohexaploid wheat. Physical, genetic and molecular characterisation of the Wheat Meiosis 1 (WM1) gene family identified seven members that localised within a region of 173-kb. WM1 gene family members were sequenced and they encode mainly type Ia plasma membrane-anchored leucine rich repeat-like receptor proteins. In situ expression profiling suggests the gene family is predominantly expressed in floral tissue. In addition to the WM1 gene family, a number of other genes, gene fragments and pseudogenes were identified. It has been predicted that there is approximately one gene every 19-kb and that this region of the wheat genome contains 23 repetitive elements including BARE-1 and Wis2-1 like sequences. Nearly 50% of the repetitive elements identified were similar to known transposons from the CACTA superfamily. Ty1-copia, Ty3-gypsy and Athila LTR retroelements were also prevalent within the region. The WM1 gene cluster is present on 3DS and on barley 3HS but missing from the A and B genomes of hexaploid wheat. This suggests either recent generation of the cluster or specific deletion of the cluster during wheat polyploidisation. The evolutionary significance of the cluster, its possible roles in disease response or floral and early meiotic development and its location at or near the Ph2 locus are discussed.     

Fxna, a novel gene differentially expressed in the rat ovary at the time of folliculogenesis, is required for normal ovarian histogenesis

In rodents, the formation of ovarian follicles occurs after birth. In recent years, several factors required for follicular assembly and the growth of the newly formed follicles have been identified. We now describe a novel gene, Fxna, identified by differential display in the neonatal rat ovary. Fxna encodes an mRNA of 5.4 kb, and a protein of 898 amino acids. Fxna is a transmembrane metallopeptidase from family M28, localized to the endoplasmic reticulum. In the ovary, Fxna mRNA is expressed in granulosa cells; its abundance is maximal 48 hours after birth, i.e. during the initiation of follicular assembly. Reducing Fxna mRNA levels via lentiviral-mediated delivery of short hairpin RNAs to neonatal ovaries resulted in substantial loss of primordial, primary and secondary follicles, and structural disorganization of the ovary, with many abnormal follicles containing more than one oocyte and clusters of somatic cells not associated with any oocytes. These abnormalities were not attributable to either increased apoptosis or decreased proliferation of granulosa cells. The results indicate that Fxna is required for the organization of somatic cells and oocytes into discrete follicular structures. As an endoplasmic reticulum-bound peptidase, Fxna may facilitate follicular organization by processing precursor proteins required for intraovarian cell-to-cell communication.     

Quantitation of cell proliferation,colony formation,and carcinogen induced cytotoxicity of rat tracheal epithelial cells grown in culture on 3T3 feeder layers

Summary A cell culture system is described for the growth of rat tracheal epithelial (RTE) cells at clonal density. The system uses normal, early passage RTE cells grown on feeder layers of lethally irradiated 3T3 cells. The RTE cells have a high colony forming efficiency (5 to 10%) in culture, can be passaged up to 5 times, and are capable of more than 20 cumulative doublings per colony forming cell. The epithelial nature of the cells was confirmed by cell and colony morphology, immunoperoxidase staining of intracellular keratin, and cellular ultrastructural studies. The cytotoxic response of RTE cells to a variety of carcinogens, including a direct acting chemical carcinogen, a physical carcinogen, and a series of polycyclic aromatic hydrocarbons, was quantitated. A linear decrease in the logarithm of survival was observed with increasing doses ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), γ-irradiation, 7,12-dimethylbenz(a)anthracene, and a diol-epoxide of benzo(a)pyrene. No toxicity was observed after treatment with benzo(a)pyrene or 3-methylcholanthrene over the concentration range examined. In contrast, phorbol ester tumor promoters stimulated cell growth markedly. Based on these and other studies, the RTE cell culture system represents a model system that will be useful for quantitative studies of epithelial cell growth, differentiation, and carcinogenesis.     

Rat testicular myotubularin, a protein tyrosine phosphatase expressed by Sertoli and germ cells, is a potential marker for studying cell-cell interactions in the rat testis

The full-length cDNA encoding the entire open reading frame (ORF) of rat myotubularin (rMTM) was isolated from a rat testis expression library by PCR. Among the three approximately 2.9-kb cDNAs that were sequenced, one clone was different from the other two clones. It contained seven extra amino acids of FVVLNLQ; this short stretch of extra sequence was found between Gln(421) and Phe(422) within the SET (Suvar3-9, Enhancer-of-zeste, Trithorax) interacting domain (SID) of rMTM. The rMTM ORF had 1,713 bp encoding for a 571 amino acid polypeptide and a calculated molecular weight of 65.8 kDa. A comparison between its deduced amino acid sequence and the GenBank database using BLAST revealed a 53.1% identity with human myotubularin protein (hMTM1), which is a member of the protein tyrosine phosphatase (PTP) family associated with X-linked myotubular myopathy. A 22 amino acid peptide NH(2)-TKVNERYELCDTYPALLAVPAN was synthesized based on the deduced amino acid sequence of rMTM and used for antibody production. By using immunoblot analysis, a 66-kDa protein was indeed detected in both Sertoli and germ-cell cytosols. rMTM mRNA was found in various tissues but was predominantly expressed in the testis, ovary, and skeletal muscle. Sertoli cell rMTM expression was stimulated by germ cells and enhanced when inter-Sertoli junctions were being assembled in vitro. A drastic reduction in testicular rMTM steady-state mRNA level correlated with the depletion of germ cells from the testis in vivo following either glycerol or lonidamine treatment. These results indicate that rMTM is a rat homologue of hMTM1 that may be a useful marker in monitoring the events of cell-cell interactions in the testis.     

Application of quantitative immunoprecipitation combined with knockdown and cross‐linking to Chlamydomonas reveals the presence of vesicle‐inducing protein in plastids 1 in a common complex with chloroplast HSP90C

Knowledge of the interaction partners of a protein of interest may provide important information on its function. Common to currently available tools for the identification of protein–protein interactions, however, is their high rates of false positives. Only recently an assay was reported that allowed for the unequivocal identification of protein–protein interactions in mammalian cells in a single experiment. This assay, termed quantitative immunoprecipitation combined with knockdown (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture, immunoprecipitation, and quantitative MS. We are using the unicellular green alga Chlamydomonas reinhardtii to understand the roles of chaperones in chloroplast biogenesis. The goal of this work was to apply QUICK to Chlamydomonas for the identification of novel interaction partners of vesicle‐inducing protein in plastids 1 (VIPP1), a protein required for the biosynthesis/maintenance of thylakoid membranes and known substrate of chloroplast HSP70B. We report here a robust QUICK protocol for Chlamydomonas that has been improved (i) by introducing a cross‐linking step (‐X) to improve protein complex stability and (ii) by including a control for the correction of unequal immunoprecipitation and/or labeling efficiencies. Using QUICK and cross‐linking we could verify that HSP70B and CGE1 form a complex with VIPP1 and could also demonstrate that chloroplast HSP90C is part of this complex. Moreover, we could show that the chaperones interact with VIPP1 also in membrane fractions.     

Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present.     

Intercellular communications within the rat anterior pituitary. XVI: Postnatal changes of distribution of S-100 protein positive cells,connexin 43 and LH-RH positive sites in the pars tuberalis of the rat pituitary gland. An immunohistochemical and electron microscopic study

The architecture of luteinizing hormone-releasing hormone (LH-RH) nerve ends and the S-100 protein containing folliculo-stellate cells forming gap junctions in the pars tuberalis is basically important in understanding the regulation of the hormone producing mechanism of anterior pituitary glands. In this study, intact male rats 5–60 days old were prepared for immunohistochemistry and electron microscopy. From immunostained sections, the S-100 containing cells in pars tuberalis were first detected on day 30 and increased in number to day 60; this was parallel to the immunohistochemical staining of gap junction protein, connexin 43. LH-RH positive sites were clearly observed on just behind the optic chiasm and on the root of pituitary stalk on day 30. On day 60, the width of layer increased, while follicles and gap junctions were frequently observed between agranular cells in 10 or more layers of pars tuberalis.     

Significance of calcium binding, tyrosine phosphorylation, and lysine trimethylation for the essential function of calmodulin in vertebrate cells analyzed in a novel gene replacement system

Calmodulin (CaM) was shown to be essential for survival of lower eukaryotes by gene deletion experiments. So far, no CaM gene deletion was reported in higher eukaryotes. In vertebrates, CaM is expressed from several genes, which encode an identical protein, making it difficult to generate a model system to study the effect of CaM gene deletion. Here, we present a novel genetic system based on the chicken DT40 cell line, in which the two functional CaM genes were deleted and one allele replaced with a CaM transgene that can be artificially regulated. We show that CaM is essential for survival of vertebrate cells as they die in the absence of CaM expression. Reversal of CaM repression or ectopic expression of HA-tagged CaM rescued the cells. Cells exclusively expressing HA-CaM with impaired individual calcium binding domains as well as HA-CaM lacking the ability to be phosphorylated at residues Tyr(99)/Tyr(138) or trimethylated at Lys(115) survived and grew well. CaM mutated at both Ca(2+) binding sites 3 and 4 as well as at both sites 1 and 2, but to a lesser degree, showed decreased ability to support cell growth. Cells expressing CaM with all calcium binding sites impaired died with kinetics similar to that of cells expressing no CaM. This system offers a unique opportunity to analyze CaM structure-function relationships in vivo without the use of pharmacological inhibitors and to analyze the function of wild type and mutated CaM in modulating the activity of different target systems without interference of endogenous CaM.     

Multipotent nestin-expressing stem cells capable of forming neurons are located in the upper,middle and lower part of the vibrissa hair follicle

We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair follicle stem cells. Nestin-expressing cells were initially identified in the hair follicle bulge area (BA) using a transgenic mouse model in which the nestin promoter drives the green fluorescent protein (ND-GFP). The hair-follicle ND-GFP-expressing cells are keratin 15-negative and CD34-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells and melanocytes in vitro. Subsequently, we showed that the nestin-expressing stem cells could affect nerve and spinal cord regeneration after injection in mouse models. In the present study, we separated the mouse vibrissa hair follicle into three parts (upper, middle and lower). Each part of the follicle was cultured separately in DMEM-F12 containing B-27 and 1% methylcellulose supplemented with basic FGF. After 2 mo, the nestin-expressing cells from each of the separated parts of the hair follicle proliferated and formed spheres. Upon transfer of the spheres to RPMI 1640 medium containing 10% FBS, the nestin-expressing cells in the spheres differentiated to neurons, as well as glia, keratinocytes, smooth muscle cells and melanocytes. The differentiated cells were produced by spheres which formed from nestin-expressing cells from all segments of the hair follicle. However, the differentiation potential is greatest in the upper part of the follicle. This result is consistent with trafficking of nestin-expressing cells throughout the hair follicle from the bulge area to the dermal papilla that we previously observed. The nestin-expressing cells from the upper part of the follicle produced spheres in very large amounts, which in turn differentiated to neurons and other cell types. The results of the present study demonstrate that multipotent, nestin-expressing stem cells are present throughout the hair follicle and that the upper part of the follicle can produce the stem cells in large amounts that could be used for nerve and spinal cord repair.