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J H Sang

Publications and source records attributed to J H Sang.

At least 19 recordsLinked to original sources
PubMed1986

Functional analysis of the transcriptional control regions of the copia transposable element.

The introduction of copia-based vectors in Drosophila hydei cells results in their high-level transient expression and the subsequent establishment of stably transformed cell lines containing multiple copies of vector integrated into host genomic DNA. Using transformation frequency and transient expression analysis as assays of promoter strength, we have defined the regions of copia essential for expression. We find that the essential sequences reside within the long terminal repeat, but 3' to the site of initiation of copia RNA. Deletion of the consensus enhancer-like sequences from copia appears to have no effect on vector expression.

Journal Article↗
PubMed1985

Efficient expression of an Epstein-Barr nuclear antigen in Drosophila cells transfected with Epstein-Barr virus DNA.

In a search for exogenous promoters which function in cultured Drosophila cells, we have co-transfected a D. melanogaster cell line with an Epstein-Barr virus (EBV) cosmid clone which encodes the Epstein-Barr nuclear antigen (EBNA-1). Here we report that Drosophila cells containing stably integrated copies of EBNA-1 encoding DNA synthesise a polypeptide of mol. wt. identical to that of authentic EBNA-1, which is detectable with EBNA-positive but not EBNA-negative human serum. As in EBV-transformed lymphoblastoid cells, this neo-antigen is associated with the nucleus of transfected cells suggesting that cellular localisation signals which operate in mammalian cells are also recognised in insect cells.

Animals↗
PubMed1985

Regulated expression of a Drosophila melanogaster heat shock locus after stable integration in a Drosophila hydei cell line.

DNA-mediated cotransformation has been used to transfer a Drosophila melanogaster heat shock locus into cultured Drosophila hydei cells by use of the copia-based selectable vector pCV2gpt and of pMH10A, a cloned 87A7 heat shock locus encoding a mutant heat shock protein (hsp). Transformed lines contain between 50 and 200 copies of both plasmids, each separately organized as a head-to-tail concatemer which is stably maintained in the transformed lines. Exposure of the cotransformants to heat shock temperatures induces the regulated expression of the hsp RNA and the mutant hsp in all the lines analyzed.

Animals↗
PubMed1984

An assay for transient gene expression in transfected Drosophila cells, using [3H]guanine incorporation.

We have developed an assay for transient gene expression using a dominant-selectable marker previously employed to transform Drosophila cultured cells. Drosophila hydei cells transfected with a functional Escherichia coli xanthine guanine phosphoribosyl transferase gene (gpt), under the control of the long terminal repeats (LTRs) of the copia transposable element, rapidly incorporate guanine into acid-precipitable counts. Autoradiographic analysis in situ shows that approximately 20% of cells take up, and express, the gpt gene. This transient gpt expression depends on the Drosophila promoter sequences since vectors with the gpt gene in reverse orientation to the copia LTRs fail to incorporate guanine. Deletion analysis confirms that the LTRs are essential for gpt gene expression. Similarly, cells transfected with gpt controlled by the Drosophila 70 000 mol. wt. heat-shock (hsp 70) promoter show regulated guanine incorporation when heat shocked. The efficiency of the copia LTRs varies considerably between the cell lines we tested, whereas that of the hsp 70 promoter does not. The heterologous promoters of the Rous sarcoma virus (RSV) and simian virus 40 (SV40) function poorly in these cells.

Animals↗
PubMed1984

Rescue of a Drosophila temperature-sensitive mutant cell line by DNA transfection.

At the nonpermissive temperature, the shibirets gene of Drosophila causes adult paralysis and cell death during development. We have used DNA-mediated gene transfer with wild-type genomic DNA to rescue the lethal phenotype of shits cultured cells and have isolated cell lines which grow at the nonpermissive temperature of 29 degrees C. By using restriction endonucleases, we confirm that this rescue is DNA sequence dependent. Further, cells cotransfected with wild-type genomic DNA and plasmid DNA contain plasmid sequences when selected for survival at 29 degrees C. This confirms the uptake of DNA by the transformed cells and provides a simple temperature selection technique allowing coselection of genes which cannot themselves be selected for.

Animals↗
PubMed1984

Integration of Drosophila heat-shock genes transfected into cultured Drosophila melanogaster cells.

We have used DNA-mediated gene transfer to introduce into Drosophila melanogaster cells DNA sequences for which no selective criteria exist. We have introduced a Drosophila heat-shock locus into cultured Drosophila cells by calcium phosphate cotransfection with the copia vector pCV31gpt and selection for xanthine utilization. We recovered cell lines containing between three and about 50 copies of both pCV31gpt and pMH10A, a cloned 87 A7 hsp70 heat-shock locus that encodes a mutant 40,000-dalton heat-shock protein (hsp40). The stable inheritance of the transformed DNAs argues that the input DNAs have integrated into the genome. We show that this is indeed the case for one cell line by cloning back the transfected DNA and detecting the flanking chromocentral sequences by in situ hybridization. Surprisingly, the integrated hsp70 genes are not expressed. This report represents the first example of the cointroduction of DNA sequences into Drosophila cells by cotransfection with a dominant selectable marker.

Animals↗
PubMed1983

When does determination occur in Drosophila embryos?

Small groups of blastoderm cells were transplanted from wild-type donor embryos into genetically marked host embryos of the same age. Donor cells were injected either into an homologous or an ectopic region of the recipient, and both donor and recipient embryos were allowed to develop. Donor flies were examined for defects in external structures. Recipients were scored for patches of donor-type marked tissue derived from the injected cells. After ectopic transfer, the donor cells recovered in chimaeric recipients differentiated structures consistent with the donor site of cell removal. No apparent fate change was observed. In the rare cases when both individuals of a donor/host pair survived, a direct correspondence could be made between the deleted region in the donor and the chimaeric patch in the host. The results show that blastoderm cells are stably determined to within a segment.

Animals↗
PubMed1981

Developmental analysis of the homoeotic mutation bithoraxoid of Drosophila melanogaster.

The homoeotic transformations caused by bxd are described in detail. The anterior histoblast nests of the first abdominal segment are missing, and are replaced by one or two leg discs ventrally. Mainly anterior compartment patterns are found in the ectopic, abdominal legs of adult flies. However, cell lineage analyses show that both anterior and posterior polyclones are established early in the development of these ectopic legs, but the posterior polyclone is smaller. Cells of the anterior polyclone may regulate later in development to adjust for this and form pattern elements normally derived from the posterior polyclone. In addition, experiments show that bxd+ is required by the second larval instar stage, and possibly as early as the blastoderm stage.

Animals↗
PubMed1978

Cell culture of individual Drosophila embryos. I. Development of wild-type cultures.

A new procedure is described for the preparation of in vitro cell cultures from individual early gastrulae of Drosophila melanogaster. In these cultures several identifiable cell types differentiate within 24 h (nerve, muscle, fat-body, haemocyte and chitin-secreting); their initial appearance and continuing development over a period of weeks is described. It is proposed that this technique may be used to analyse abnormalities of cellular development in embryonic lethal mutants. Culture in vitro of cells from lethal embryos is seen to have two broad roles: (1) to test the developmental capacity of individual cell types in a situation where they are relatively free from possible deleterious interactions with other cell types and are liberated from the system of the dying embryo, and (2) through the preparation of mixed cultures from normal and mutant embryos, to determine the influence of the presence of wild-type cells on observed abnormalities of a particular cell type.

Animals↗
PubMed1978

Cell culture of individual Drosophila embryos. II. Culture of X-linked embryonic lethals.

Results are reported from the culturing in vitro of cells from individual early gastrulae of the following four groups of X-linked embryonic lethal mutants of Drosophila melanogaster. (1) Notch lethals, Five Notch mutants were studied which have been reported to give similar abnormalities in whole embryos: the nervous system displays a three-fold hypertrophy as part of a shift in the pattern of differentiation within ectodermal derivatives, and mesodermal derivatives do not differentiate. An hypertrophy of nerve was found in cell cultures prepared from embryos of all five mutants. In addition, four of the five alleles consistently gave abnormalities of muscle differentiation: when compared to controls, Notch cultures had a reduced frequency of myotubes, and displayed unusual clusters of myocytes which had either failed to fuse or had fused incompletely. Results from mixed cultures prepared from two embryos were consistent with the autonomous expression of nerve and muscle abnormalities by Notch-8 cells in the presence of wild-type cells. It is argued that the Notch locus has a direct role in the differentiation of both nerve and muscle. (2) white deficiencies. Cells carrying either of two deficiencies gave a clear-cut pattern of abnormalities: initial cellular differentiations were normal, but nerve, muscle and fat-body cells progressively deteriorated during the culture period. Mixed cultures showed that wild-type cells could not 'rescue' mutant muscle and fat-body cells; however, the status of the autonomy of mutant nerve abnormalities in these cultures was unclear. Both white deficiencies remove cytological band 3C1, and this permits a comparison of results with those from cultures of cells from Notch-8 embryos (also deficient for 3C1). Abnormalities displayed in cultures of the two types of mutant show no overlap. Therefore no consistent cellular abnormality can be attributed to absence of band 3C1. (3) lethal(1)myospheroid. In contrast to earlier observations on in vitro cell cultures (Donady & Seecof, 1972) muscle was seen to differentiate, though its morphology was extremely abnormal. Observations indicated that all cell types within the cultures had poor properties of adhesion to a glass substrate. It is argued that the observed abnormalities are not consistent with a mutant lesion which is restricted to the basement membrane (contra Wright, 1960), and that all cell types carry a basic defect which may reside in the cell membrane. (4) shibirets alleles. Cultures of two temperature-sensitive lethal shibire alleles (shits1), shits3) were normal at the permissive temperature of 22 degrees C. At the restrictive temperature (29 degrees C) early cell differentiation was normal but subsequent development was blocked. This blockage could be partially reversed by shifting cultures to the permissive temperature after as much as 10 days exposure to the high temperature. It is suggested that shits cells are mutant in a process which is basic to several cell types.

Animals↗
PubMed1977

The development of a medium supplemented with egg extract for the maintenance of Drosophila cell lines.

A saline extract was prepared from Drosophila eggs. When diluted to a concentration of 1% with Drosophila tissue culture medium, it did not support growth of cells from the Drosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time four Drosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonic Drosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells.

Blood↗
PubMed1977

Active ion transport and beta-acdysone induced differentiation of Drosophila melanogaster imaginal discs cultured in vitro.

The theory that beta-ecdysone initiates developmental changes during insect metamorphosis by causing an increase in intranuclear levels of potassium, together with a concomitant decrease in sodium levels, has been investigated by two methods. First, imaginal discs from late third instar larvae have been cultured with 0-2 microng/ml of beta-ecdysone together with inhibitors of active ion transport. Non-specific inhibitors, which may have general effects on sulphydryl groups, such as iodoacetic acid, N-ethylmaleimide ethacrinic acid and furosemide, inhibit both eversion and differentiation at concentrations of from 10(-3) M to 2 X 10(-3) M. Ouabain, the only specific inhibitor of the active transport of Na+ and K+ across membranes, had no effect on development even at a concentration of 10(-2) M. Second, a medium containing raised levels of K+, and reduced concentrations of Na+, neither initiated disc development in the absence of beta-ecdysone, nor stimulated development induced by suboptimal levels (0-02 microng/ml) of beta-ecdysone, either in the presence or absence of ouabain. These results suggest that beta-ecdysone induced morphogenesis is not dependent upon Na+ and K+ concentrations, or on the activity of an ouabain-sensitive ion pump.

Animals↗
PubMed1975

Differentiation in vitro of larval cell types from early embryonic cells of Drosophila melanogaster.

A variety of cell types develop when cells of 6 1/2-8 1/2 h Drosophila embryos are cultured in an improved medium. Nerve, muscles, fat-body, chitin-secreting, and macrophage-like cells (possibly haemocytes) appear in the first 24 h and mature over the next week. Tracheal, imaginal disc, a second stage of the macrophage-like, and a anumber of unidentified fibroblastic and epithelial cells appear in the 2nd and 3rd week, following a resumption of cell multiplication. There is some organization of some of the cell types into higher structures.

Adipose Tissue↗
PubMed1975

Development and differentiation in vitro of Drosophila imaginal disc cells from dissociated early embryos.

Embryos of Drosophila melanogaster, 6-8 h after oviposition, were dissociated and the cells cultured in vitro. Besides larval cell types, imaginal disc cells, assembled and growing in bloated monolayered vesicles, were obtained. The cells of these vesicles become competent to differentiate adult structures when treated with alpha-ecdysone or ecdysterone in vitro. Recognizable patterns of the adult fly are not formed though. If metamorphosis of imaginal cell vesicles from in vitro-cultures is induced in vivo by transplantation into host larvae of various ages within the third larval instar, recognizable patterns can differentiate provided the host larva does not metamorphose prior to 2 days after transplantation. The frequency of specific patterns in the implants can be increased by providing 9 days of culture in vivo (adult host flies) before metamorphosis. Passage through the third larval instar is not essential for these cells to produce identifiable patterns since culture in adult flies alone can achieve this. The quality of the differentiated pattern is not correlated with the extent of cell proliferation in the cultured tissues. The problem of pattern specification in vitro and in vivo is discussed.

Animals↗