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R Wu

Publications and source records attributed to R Wu.

At least 397 records · Page 22Linked to original sources
PubMed1986

Expression of a foreign gene linked to either a plant-virus or a Drosophila promoter, after electroporation of protoplasts of rice, wheat, and sorghum.

The bacterial chloramphenicol acetyltransferase (CAT) gene was expressed in protoplasts of three important graminaceous plant species after introduction of the gene by electroporation. Gene transfer occurred when high-voltage electric pulses were applied either directly or indirectly (without anode contact) to a solution containing plasmid DNA and protoplasts of rice, wheat, or sorghum. The indirect method was more rapid, resulted in higher protoplast viability, and was less subject to contamination than the direct-contact method. Gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the 35S promoter of cauliflower mosaic virus or the copia long terminal repeat promoter of Drosophila. Together with recent advances in regeneration of callus and whole plants from protoplasts, this system makes it possible to study inheritance and expression of genes introduced into graminaceous monocotyledonous plants.

Journal Article↗
PubMed1986

Biosynthesis of prostaglandins by isolated and cultured airway epithelial cells.

The metabolism of arachidonic acid to prostaglandins and thromboxane by freshly isolated and cultured respiratory tract epithelial cells was examined by HPLC methods. Homogenates prepared from freshly isolated rat and rabbit tracheal epithelial cells did not convert arachidonic acid to prostaglandins. Rat tracheal epithelial cells however, did convert arachidonic acid to uncharacterized metabolites possibly hydroxyfatty acids. In contrast, rat tracheal epithelial cells grown in culture for 9 days acquired the capacity to convert arachidonic acid to PGE2 and related products while cultured rabbit tracheal epithelial cells converted arachidonic acid to TXB2. The conversion of arachidonic acid to PGE2 by rat tracheal cells was studied at various times in culture. The formation of PGE2 appeared to parallel the growth of the cultures. In contrast to freshly isolated rat tracheal cells, enriched rat Clara cell fractions were able to convert 14C-arachidonic acid to prostacyclin (PGI2) ans measured by HPLC analysis of its stable end product 6-keto PGF1 alpha. PGI2 was also the major metabolite of arachidonic acid produced by enriched rat alveolar type II cell fractions. PGF2 alpha, and hydroxyfatty acids were also formed. These results suggest that arachidonic acid metabolism differs in various types of respiratory tract cells and that maintenance of such cells in culture alters the pattern of arachidonic acid metabolism.

Animals↗
PubMed1985

Structure of two unlinked Drosophila melanogaster glyceraldehyde-3-phosphate dehydrogenase genes.

Two Drosophila genes that code for the enzyme glyceraldehyde-3-phosphate dehydrogenase (Gapdh) have been isolated and their structures determined by DNA sequence analysis. The two genes, Gadph-1 and Gapdh-2, are homologous to each other in their coding regions but differ entirely in the 5' and 3' flanking regions. Both genes are functionally expressed in adult flies as determined by Northern blot analysis using gene-specific probes. Gapdh-1 is mapped by in situ hybridization at position 43E-F on the right arm of the second chromosome and Gapdh-2 at position 13F on the left arm of the X chromosome. Transcription initiation sites as well as polyadenylation sites for both Gapdh transcripts have also been determined. Gapdh-1 lacks a sequence homologous to the TATA box in its -30-base pair region that is characteristic of many RNA polymerase II transcribed promoters. In contrast, Gapdh-2 contains a consensus TATA box sequence as well as a CAAT box in its promoter region. Furthermore, a sequence element ATTTGCAT (dc) and nontandem multiple direct repeats have been found in the -35 to -155-base pair 5' flanking region. Other than the intron located in the 5' noncoding region of Gapdh-2, both genes lack intervening sequences.

Amino Acid Sequence↗
PubMed1985

Pea cytochrome oxidase subunit II gene has no intron and generates two mRNA transcripts with different 5'-termini.

We have isolated and sequenced the cytochrome oxidase subunit II (COII) gene from pea mitochondria. The coding sequence (777 bp) shows over 90% homology to the COII genes from three monocotyledonous plants (rice, maize and wheat) and one dicotyledonous plant (Oenothera berteriana). Several codons are deleted, however, in the pea COII gene. Of interest is the deletion in pea of the last three codons, including the stop codon, found at the 3' end of the other four COII genes. Instead, a new stop codon has been created due to a single-base substitution at the 13th bp downstream from the position of the original stop codon. This pea gene does not contain an intron which is found in all three monocots. Two distinct 5' termini of the pea COII transcripts have been identified by S1 nuclease mapping, one at 285 bp (site I) and the other at 302 bp (site II) upstream from the ATG codon. They are located at two identical sites within nearly perfect direct repeats. Transcripts with the 5' end corresponding to site I occur five time more frequently than those with the 5' end corresponding to site II. Both transcripts have the same 3' terminus which has been mapped to be at 193 to 195 bp downstream from the stop codon.

Base Sequence↗
PubMed1985

Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene.

Full length cDNAs encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rat and man have been isolated and sequenced. Many GAPDH gene-related sequences have been found in both genomes based on genomic blot hybridization analysis. Only one functional gene product is known. Results from genomic library screenings suggest that there are 300-400 copies of these sequences in the rat genome and approximately 100 in the human genome. Some of these related sequences have been shown to be processed pseudogenes. We have isolated several rat cDNA clones corresponding to these pseudogenes indicating that some pseudogenes are transcribed. Rat and human cDNAs are 89% homologous in the coding region, and 76% homologous in the first 100 base pairs of the 3'-noncoding region. Comparison of these two cDNA sequences with those of the chicken, Drosophila and yeast genes allows the analysis of the evolution of the GAPDH genes in detail.

Amino Acid Sequence↗
PubMed1985

Characterization of a mouse somatic cytochrome c gene and three cytochrome c pseudogenes.

Mouse contains two functional, but differentially expressed, cytochrome c genes. One of these genes is expressed in all somatic tissues so far examined. The other gene is expressed only in testis and is assumed to be spermatogenesis-specific. The nucleotide sequence of four mouse cytochrome c-like genes has been determined. One of these genes (MC1) contains an intron and encodes a polypeptide sequence identical to the published mouse somatic cytochrome c amino acid sequence. The other three genes can not properly encode a mouse cytochrome c protein and appear to be pseudogenes which have arisen via an insertion into the mouse genome of a cDNA copy of a cytochrome c mRNA molecule.

Amino Acid Sequence↗
PubMed1985

Characterization of two Drosophila melanogaster cytochrome c genes and their transcripts.

Analysis of total Drosophila melanogaster DNA by genomic blot hybridization indicates that two cytochrome c-like sequences exist in the Drosophila genome. These two sequences, DC3 and DC4, have been isolated from a Charon 4A-D. melanogaster genomic library. DC3 and DC4 are located within a 4 kb region of DNA, at position 36A 10-11, on the left arm of chromosome 2. The nucleotide sequence of these two clones has been determined. Both DC3 and DC4 can encode functional cytochrome c proteins. The polypeptide sequences predicted by these two genes, however, differ at 32 amino acid residues. DC4 is expressed at varying, but relatively high levels throughout Drosophila development. In contrast, DC3 is expressed at constant, but relatively low levels throughout development.

Amino Acid Sequence↗
PubMed1985

Expression of tracheal differentiated functions in serum-free hormone-supplemented medium.

Most dissociated airway epithelial cells in culture express few of their in vivo functions and only to a limited degree. In this report, we demonstrate that hamster tracheal epithelial (HTE) cells cultured on a collagen gel substratum in a serum-free hormone-supplemented medium differentiate to cilia-beating and mucus-secreting cell types. The medium is Ham's F-12 supplemented with insulin, epidermal growth factor, transferrin, hydrocortisone, cholera toxin, bovine hypothalamus extract, and vitamin A. Under these culture conditions, HTE cells exhibit a growth rate of 24 h/population doubling and reach confluency, at a density of 2-5 X 10(4) cells/cm2, within 2 weeks. Both the collagen gel substratum and vitamin A of this culture system are important to the growth and differentiation of HTE cells in vitro. Evidence of HTE cell differentiation has been obtained at both the ultrastructural and the histochemical levels. In addition, a variety of biochemical studies (gel filtration, ion exchange column chromatography, enzyme digestion, nitrous acid treatment, and composition analysis) indicate the production of mucin-like glycoprotein in the HTE cultures. The levels of mucin-like glycoprotein were found to closely correlate with the histochemically quantitated levels of the mucous cell type. Kinetic studies demonstrate that HTE cells rapidly lose their differentiated features during the attachment stage of primary culture but redifferentiation occurs after the cultures reach confluency. The ability of HTE cells to grow and differentiate in this serum-free culture system in the absence of other cell types should greatly facilitate the study of mucociliary functions in vitro.

Animals↗
PubMed1985

Induced reversion of a Chinese hamster ovary triple auxotroph. Validation of the system with several mutagens.

A Chinese hamster ovary triple auxotroph (CHO AUXB1) requires glycine, adenosine, and thymidine (GAT) for growth and survival due to a defect in the structural gene for folylpolyglutamate synthetase (FPGS). This auxotroph and others like it contain less than 3% of the parental amounts of FPGS activity. In order to develop a reverse mutation assay with CHO AUXB1, we determined the optimal conditions for measuring reversion and characterized some of the revertants. We also obtained quantitative mutagenicity data for several direct-acting mutagens for comparison to the parental CHO-S/HGPRT locus. Induced revertants appear in the culture immediately following 20-22 h exposures in +GAT complete medium, indicative of dominant genetic changes. They are maximally expressed after 2 population doublings and can be conveniently selected after 44-48 h of expression growth by plating 1 X 10(6) cells/100-mm dish into -GAT-deficient medium and incubating 12-13 days. Plating reconstruction experiments show that the cloning efficiencies of revertants in -GAT medium are not influenced by the presence of up to 1 X 10(6) CHO AUXB1 cells. Dose-dependent increases above the spontaneous revertant frequency (average = 5 X 10(7)) are induced with cis-Pt(NH3)2Cl2 (14-fold) (but not trans-Pt(NH3)2Cl2), PtCl4(10-fold), Pt(SO4)2 (14-fold), K2CrO4 (8-fold), EMS (10-fold), 4-NQO (53-fold), ICR-191 (60-fold), and ICR-170 (30-fold). All of the revertants that have been isolated are stable to repeated subculturing in -GAT medium; 40 out of 42 that have been analyzed are characterized by an increased 72-h growth incorporation of labeled folate and their extracts contain 5-94% as much FPGS as the original, parental CHO-S line. Spontaneous and induced reversion to the GAT+ phenotype primarily reflects mutations involving the FPGS gene locus. But the re-acquisition by most of the revertants of much less than normal amounts of FPGS activity suggests that they arise from compensatory second-site mutations within this gene. Comparison of the mutagenicity patterns of the foregoing compounds as a function of the applied concentration and the relative percent survival reveals some interesting similarities, as well as differences, between the CHO AUXB1/FPGS and CHO-S/HGPRT loci. In particular, the FPGS locus is rather insensitive to EMS (or other simple alkylating agents). However, it seems to be quite susceptible to reversion by other chemicals that are known to react selectively with guanine bases in DNA. CHO AUXBI is a useful supplemental mammalian assay system for assessing quantitatively the generally weak mutagenic activities of metal compounds.

Animals↗
PubMed1985

Growth and differentiation of human nasal epithelial cells in culture. Serum-free, hormone-supplemented medium and proteoglycan synthesis.

Ham's F12 medium supplemented with insulin (Ins), transferrin (Tf), epidermal growth factor (EGF), hydrocortisone (HC), T3, cholera toxin (CT), and bovine hypothalamus extract (BHE) was developed for in vitro growth of human nasal epithelial (HNE) cells. The HNE cells were dissociated from freshly excised nasal polyps or turbinates with protease. Colony-forming efficiency of primary HNE cells was approximately 5%. Growth studies showed Ins, BHE, and CT were essential for growth; HC, EGF, Tf, and T3 were also stimulatory for growth. The growth rate in this serum-free, hormone-supplemented medium was 24 h per population doubling. Up to 20 population doublings and 3 passages of dissociated HNE cells could be achieved. Addition of serum to this culture medium inhibited epithelial cell growth. Vitamin A had no apparent effect on cell growth but induced an alteration in the morphologic characteristics of the cell. The epithelial nature of cultured cells was confirmed by positive staining with antihuman keratin antibody, ultrastructural studies, and by formation of a columnar, ciliated epithelium in denuded tracheal grafts repopulated by these cultured HNE cells. Biochemical analyses of glycoproteins (labeled with 3H-glucosamine and/or 35S-sulfate) secreted by cultured HNE cells were unable to demonstrate the secretion of mucinlike glycoproteins in culture. Instead, major secretory products of cultured cells were hyaluronate and heparan sulfate. These results were in agreement with morphologic observations that showed no mucus-secreting granules in cultured cells. Dome formation was observed in high cell density cultures. We conclude that HNE cells can be cultured in well-defined culture media. As indicated by formation of domes, these cells may be useful for in vitro ion transport studies. Further differentiation, however, may be required for studies of mucin synthesis.

Adolescent↗
PubMed1985

Morphological changes in rat tracheal cells during the adaptive and early growth phase in primary cell culture.

The purpose of our studies was to determine the fate of different cell types present in early primary cultures of tracheal epithelial cells and, if possible, to elucidate the role they play in the establishment of the cultures. Epithelial cells were isolated from rat tracheas with 0.5% Pronase and were cultured on collagen-coated dishes as described previously. Light and transmission electron microscopic studies showed that the cell population harvested from rat trachea was composed of approximately 30% ciliated cells, 50% granule-containing cells and 20% undifferentiated cells (presumably basal cells). Upon seeding the tracheal cell suspensions into culture, approximately 40% of the cells attached. Cell attachment was virtually complete after 16 h. Roughly 60% of the cells attaching during the first 12 h were neither ciliated nor granulated, suggesting that undifferentiated cells played a major role in establishment of the early cultures. Between 20 and 35% of the cells attaching during this time were identified as granulated cells (mucous cells). Ciliated cells did not start to attach in significant numbers until 8 h after seeding. They never amounted to more than 8-12% of the attached cell population. After 12 h of culture, the cell population underwent a progressive loss of differentiation. The number of poorly differentiated cells (i.e. those showing neither cilia nor mucous granules) increased correspondingly. This loss of differentiation preceded the onset of DNA synthesis and cell growth which began at about 24 and 40 h, respectively. Continuous [3H]thymidine-labelling studies showed that at 48 h after the start of culture about 90% of all attached cells had entered DNA synthesis at least once. This finding is consistent with the interpretation that the ciliated cells are terminally differentiated cells and are probably the only part of the tracheal cell inoculum not participating in the growth of the cultures. At 72 h, the cultures (now in mid-log growth phase) were composed of uniformly undifferentiated cells lacking cilia and mucous granules. The cells nevertheless showed unequivocal epithelial characteristics such as tight junctions and desmosomes. The studies suggest that both basal and mucous cells are responsible for the establishment and growth of the rat tracheal epithelial cell cultures.

Animals↗
PubMed1984

Cytochrome oxidase subunit II gene of rice has an insertion sequence within the intron.

We have isolated and sequenced the cytochrome oxidase subunit II gene from rice (Oryza sativa L. var Labelle). The overall structural organization of this gene is very similar to that of the maize gene. This gene contains an intron in a position identical to the intron in the maize gene. However, the intron in the rice gene is longer than that of the maize gene largely due to a 461 bp insertion sequence, which has inverted repeats at its termini and is flanked by direct repeats, characteristic of transposable elements. Apart from this insertion sequence, the remainder of the intron sequence is strikingly homologous to that of maize (98.6% homology), suggesting a possible functional or structural role. The coding regions of the two genes exhibit 99.5% nucleotide sequence homology and their deduced amino acid sequences are identical. Similarly, the 3'-noncoding regions, except for several small insertions and deletions, show complete sequence homology. On the contrary, no sequence homology is detected in the 5'-noncoding regions.

Base Sequence↗
PubMed1984

A potential stem-loop structure and the sequence CAAUCAA in the transcript are insufficient to signal rho-dependent transcription termination at lambda tR1.

It has been suggested that a sequence in the RNA transcript that can form a stem and loop structure, followed by the sequence CAAUCAA, is the signal for rho-dependent transcription termination. We tested this hypothesis by synthesizing a DNA duplex whose sequence corresponds to a region of the lambda tR1 terminator that contains these structural features. We cloned this synthetic DNA fragment under the control of the lacUV5 promoter, and showed that it does not cause rho-dependent termination in vitro. RNA polymerase pauses during in vitro transcription across the synthetic sequence, although less efficiently than at the corresponding sequence on the lambda template. No rho-mediated termination was detected even under conditions that prolonged transcriptional pausing at the synthetic site, indicating that the synthetic sequence is defective as a transcript release site. We suggest that unlike rho-independent terminators, rho-dependent terminators require sequences in addition to those immediately before the sites of termination.

Base Sequence↗
PubMed1984

Synthesis of a human insulin gene. VII. Synthesis of preproinsulin-like human DNA, its cloning and expression in M13 bacteriophage.

A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M 13mp8 vector. A clone pNB82 -121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25-bp affinity leader sequence coding for (glu)7 was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of beta-galactosidase. Preproinsulin was expressed under lac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide.

Base Sequence↗
PubMed1984

In vitro mouse cytomegalovirus infection of mouse tracheal epithelial cells requires the presence of other cell types.

Recently, methods have been developed to culture dissociated tracheal epithelial (TE) cells. Attempts were made to infect these epithelial cells with mouse cytomegalovirus (MCMV) to see if the dissociated epithelial cells share characteristics of infection with MCMV-infected tracheal organ cultures. When isolated TE cells were incubated with MCMV at multiplicities between 0.10 and 2.0, no infection or minimal infection resulted. Centrifugation of virus onto the TE cell sheets also resulted in only minimal infection. If MCMV-infected mouse embryo fibroblasts were added to cultures of TE cells, however, the TE cells subsequently became infected. TE cells could also be infected by incubating MCMV with mixed cultures of uninfected fibroblasts and TE cells. The epithelial nature of infected cells was confirmed by electron microscopy. Reconstruction experiments demonstrated that fibroblast-mediated infection of mouse TE cells with MCMV was not simply due to the large amounts of virus provided by the infected fibroblasts. It is suggested that cell-cell contact, or fusion with infected cells is required for productive infection of TE cells with MCMV.

Animals↗