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Biomedical subjects

N S Yang

Publications and source records attributed to N S Yang.

At least 55 records · Page 3Linked to original sources

Inheritance and expression of foreign genes in transgenic soybean plants.

DNA-coated gold particles were introduced into meristems of immature soybean seeds using electric discharge particle acceleration to produce transgenic fertile soybean plants. The lineages of integrated foreign DNA in two independently transformed plants were followed in the first (R(1)) and second (R(2)) generation of self-pollinated progeny. One plant (4615) was transformed with the Escherichia coli genes for beta-glucuronidase and neomycin phosphotransferase II; the other (3993) was transformed only with the gene for beta-glucuronidase. Segregation ratios for the introduced gene(s) were approximately 3:1 for plant 4615 and 1:1 for plant 3993 in the R(1) generation. DNA analysis showed 100% concordance between presence of the foreign gene sequences and enzyme activity. Moreover, all copies of the foreign genes are inherited as a unit in each plant. Plant 3993 segregated in a 1:1 ratio in the R(2) generation. R(1) plants derived from plant 4615, which expressed both genes, gave either 100% or 3:1 expression of both genes in the R(2) generation, demonstrating recovery of both homozygous and heterozygous R(1) plants. Our results show that foreign DNA introduced into soybean plants using electric discharge particle acceleration can be inherited in a Mendelian manner. Results also demonstrate cotransformation of tandem markers and show that both markers are inherited as closely linked genes in subsequent generations. These results indicate that whole plants can be derived from single transformed cells by a de novo organogenic pathway.

Journal Article↗

Dot assay for neomycin phosphotransferase activity in crude cell extracts.

A dot assay for determining neomycin phosphotransferase (NPT II) activity in crude cell extracts has been developed. The assay provides for the rapid screening of large numbers of cell cultures generated in gene transformation experiments using NPT II as a dominant selectable marker. Currently, the commonly used procedure for NPT II assay employs a time-consuming electrophoretic protein separation step to eliminate a positive interference resulting from putative protein kinase activities present in crude cell extracts. The dot method we have developed is based upon the ability of nitrocellulose membrane to eliminate that positive interference without a prior protein separation step. It provides a sensitive, reproducible, and significantly more convenient and rapid means of screening large numbers of cell extracts in order to distinguish cultures producing high levels of NPT II from those that do not.

Collodion↗

Detection of opines by colorimetric assay.

A colorimetric procedure for confirming the presence of arginine-derived opines (nopaline and octopine) in plant tissue extracts is described. Those materials are widely used as markers of plant cell transformation and tumorigenesis mediated by the tumor-inducing plasmids of Agrobacterium tumefaciens. Nopaline and octopine are generally detected, following resolution by paper electrophoresis, by observation of the uv-fluorescent products formed upon reaction with phenanthrenequinone. We found that a further heat treatment step, compatible with paper electrophoresis, results in rapid production of a red-purple pigment. Our colorimetric assay is sensitive to 1.25-micrograms quantities of opine and eliminates problems of background fluorescence encountered with crude plant extract in the usual assay.

Arginine↗

Bacillus thuringiensis section sign-Endotoxin Expressed in Transgenic Nicotiana tabacum Provides Resistance to Lepidopteran Insects.

The crystal proteins, or section sign-endotoxins, of Bacillus thuringiensis are specifically lethal to Lepidopteran insects. We utilized a truncated and modified portion of a cloned crystal protein gene to construct a chimeric gene capable of expression in plant cells. Using an Agrobacterium tumefaciens binary vector system, we then transferred the chimeric toxin gene into tobacco (Nicotiana tabacum cv Havana 425) cells and regenerated recombinant plants. One to several copies per cell of the toxin gene are routinely present in the recombinant plants. Hybridization experiments demonstrated that these plants had a new RNA species of the size expected for the truncated toxin mRNA, and a polypeptide having the mobility expected for the truncated toxin was detected by immunoblotting. Significant variation was found in the levels of toxin-specific RNA expression between different recombinants, but the levels of hybridizing RNA in transformants correlated with the level of toxicity demonstrated against Manduca sexta (tobacco hornworm), and other Lepidopteran insects. The recombinant genes were transmitted to progeny and resistance to insects was maintained, thus demonstrating that the introduction of toxin genes into plants may be a practical method of providing protection against certain insect pests.

Journal Article↗

Effect of the extracellular matrix on plasminogen activator isozyme activities of human mammary epithelial cells in culture.

Multiple molecular forms of plasminogen activator were detected in normal human mammary epithelial cells in culture. Cells derived from (normal) breast mammoplasty specimens and grown on the surface of collagen gels exhibited three major classes of plasminogen activator isozymes (Mr = 100,000 [100K], 75,000 [75K], and 55,000 [55K]). The activity of the 100K and 75K isozymes was greatly reduced when the cells were grown on conventional tissue-culture-grade plastic surfaces. MCF-7, a human mammary carcinoma cell line, exhibited predominantly or exclusively the 55K isozyme, irrespective of the cell growth substratum. The activity of the 55K isozyme was more than twofold higher for MCF-7 cells grown on collagen gels than for cells grown on plastic. Progesterone, diethylstilbestrol, and estrogen stimulated the activity of the 55K isozyme of MCF-7 cells, but only when the cells were grown on a plastic surface. The plasminogen activator activities of the normal human mammary epithelial cells were not stimulated by these hormones, irrespective of the growth substratum. These results show that the expression of plasminogen activator isozymes by human mammary epithelial cells is subject to modulation by the extracellular matrix. Normal and malignant cells may differ in their responsiveness to these effects.

Breast↗

Growth of human mammary epithelial cells on collagen gel surfaces.

We have developed a method for sustained growth of human mammary epithelial cells in monolayer cultures. Epithelial organoids derived from solid breast tissues were grown on the surface of thin (approximately 1 mm) collagen gel layers in an enriched growth medium supplemented with hormones, growth factors, fetal calf serum, and horse serum. To transfer the cultures, the collagen layers were dislodged and digested with collagenase. The monolayers of cells released into suspension were then dissociated into single cells using trypsin-ethylene-diaminetetraacetate. Dissociated single cells were repleted with 75 to 95% efficiency onto collagen layers or tissue culture plastic surfaces. The dissociated cells could also be cryopreserved and reactivated with greater than 80% plating efficiency on collagen layers. Normal human mammary epithelial cells grown under these conditions progressed through 12 to 15 population doublings. The population-doubling times for normal and malignant mammary cells on collagen layers were 34 and 65 hr, respectively. After reaching confluence, cells in some cultures, derived from either normal or malignant tissues, penetrated the gel surface and grew into the collagen. Within the gels, the cells became organized into three-dimensional tubular structures. The use of collagen layers eliminates a major problem in growth of human mammary epithelial cells in culture, difficulty in efficient dissociation, and cell transfer from monolayers.

Breast↗

Absence of fibronectin and presence of plasminogen activator in both normal and malignant human mammary epithelial cells in culture.

Primary monolayer cultures of normal and malignant human mammary epithelial cells were tested for fibronectin by indirect immunofluorescence using antisera specific for fibronectin. This protein was not detectable on either the normal or malignant epithelial cells. Similar results were obtained for normal and malignant mouse mammary epithelial cell cultures. Control normal and transformed fibroblasts exhibited the expected result: the normal cells were positive and the transformed cells were negative. With the use of supernatant fluids from the same cultures or an agar-overlay assay on viable cells, high levels of plasminogen-dependent fibrinolytic activity were detectable in both the normal and malignant mammary cells. Thus, two characteristics that distinguish normal from transformed fibroblasts do not serve as markers of malignancy in mammary epithelial/carcinoma systems.

Animals↗

Growth of normal and malignant human mammary epithelial cells in culture.

Normal and malignant human mammary epithelial cells were placed in culture. Normal cells were recovered from late-lactation milk and breast fluids, and malignant cells were isolated from primary breast tumors by collagenase digestion. The concentration of cells obtained from breast fluid samples was inversely proportional to the volume of fluid secreted. Most of these cells adhered rapidly to the substrate, did not replicate, displayed Fc receptor-dependent phagocytic activity, and were thus identified as macrophages. The remaining cells grew out into large islands comprised of one or two distinct morphologic types of mammary epithelial cells. Optimum growth of these cells was obtained in medium buffered to pH 6.8, and the epidermal growth factor markedly prolonged the exponential growth phase of the cells. Two morphologically distinct populations of epithelial cells were also observed in cultures established from individual breast tumors. Growth of the malignant cells was relatively unaffected by the pH of the culture medium, and the cells were unresponsive to exogenously added hormones. Overgrowth of malignant epithelial cells in primary cultures by stromal fibroblasts was retarded by replacement of standard growth medium with fresh medium containing a serum substitute; growth of the malignant epithelial cells was unaffected. A feeder layer of mitomycin C-treated human fibroblasts increased the plating efficiency of both normal and malignant cells in primary culture and also facilitated passage of these cells to secondary and tertiary cultures.

Breast↗

Genetic control of mitochondrial malate dehydrogenases: evidence for duplicated chromosome segments.

The genetic control of the major mitochondrial isoenzymes of malate dehydrogenase (L-malate:NAD+ oxidoreductase; EC 1.1.1.37) has been investigated in Zea mays. The mitochondrial isozymes are coded at four nuclear gene loci. Two of the loci (mdh1 and mdh2) are diallelic and tightly linked. The other two loci (mdh3 and mdh4) appear to have arisen by duplication of the chromosome segment carrying mdh1 and mdh2, but are not linked to them. The segregation of such a duplicate segment can explain anomalous backcross and F2 segregation ratios.

Alleles↗

Effects of cycloheximide and chloramphenicol on the synthesis of polypeptides found in three subcellular fractions of maize scutellum.

The effects of two protein synthesis inhibitors, cycloheximide and chloramphenicol, on the synthesis of mitochondrial proteins in maize (Zea mays) have been studied. The results of these investigations suggest that while most of the mitochondrial proteins are synthesized in the cytoplasm and are subsequently associated with the mitochondrion, several proteins of the mitochondrial inner membrane are synthesized within the mitochondrion. These results are consistent with those observed in several other systems, but not previously reported for higher plants.

Journal Article↗

The blocked and methylated 5' terminal sequence of a specific cellular messenger: the mRNA for silk fibroin of Bombyx mori.

The messenger RNA for silf fibroin, laveled with 32PO4 and methyl-3H L-methionine, was purified to near homogeneity from the posterior silk gland of the sildworm Bombyx mori, and the sequence of a methylated, RNAase T2-resistant structure was determined. This sequence is similar structurally to 5' terminal blocked and methylated sequences found on the total populations of polyadenylated eucaryotic cellular and certain viral mRNAs. The RNAase T2-resistant oligomer from fibroin mRNA was cleaved by nucleases P1 into three components: a blocked and methylated sequence containing three phosphates; a 2'-0-methyl UMP residue (pUm), and an unmethylated CMP (pC). The blocked and methylated sequence comigrated in three chromatographic systems with the blocked and methylated terminus of silkworm cytoplasmic polyhedrosis virus mRNA, which has the structure m7GpppAm. The fibroin mRNA cap was cleaved by nucleotide pyrophosphatase to yeild 7-methyl GMP Tpm7G) and 2'-0-methyl AMP (pAm). This sequence also appeared to be terminally located, with the m7G joined by a 5'-5' pyrophosphate linkage to the Am. It was concluded that the 5' terminal sequence of fibroin mRNA molecules is m7G(5')ppp(5')AmpUmpCp. The regulation of expression of the highly specialized gene for fibroin is discussed in light of this finding.

Animals↗