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Biomedical subjects

E Linney

Publications and source records attributed to E Linney.

At least 37 records · Page 2Linked to original sources
PubMed1989

The need for enhancers in gene expression first appears during mouse development with formation of the zygotic nucleus.

Microinjection of the firefly luciferase gene coupled to a thymidine kinase (tk) promoter provided a quantitative assay to evaluate the requirements for gene expression in individual mouse oocytes and embryos. Polyoma virus (PyV) enhancers had no effect on the level of gene expression or competition for transcription factors as long as the DNA remained either in the oocyte germinal vesicle or the pronuclei of one-cell embryos. Expression of injected genes could be observed in pronuclei because the signal that normally triggers zygotic gene expression in two-cell embryos still occurred in one-cell embryos arrested in S phase. However, when the tk promoter was injected into zygotic nuclei of two-cell embryos, enhancers increased the number of embryos that expressed luciferase as well as the level of luciferase activity per embryo. PyV enhancer mutation F101, selected for growth in mouse embryonal carcinoma F9 cells, stimulated expression in developing two-cell embryos about seven times better than the wild-type PyV enhancer and competed effectively for factors required for transcription. These results were consistent with the fact that enhancers are required to activate the PyV origin of DNA replication in developing two-cell embryos but not in one-cell embryos. The maximum levels of gene expression in oocytes, one-cell embryos, and developing two-cell embryos (1:67:21) were inversely related to the extent of chromatin assembly, but the need for enhancers was independent of chromatin assembly. Therefore, it appears that the need for enhancers to activate promoters or origins of replication results from some negative regulatory factor that first appears as a component of zygotic nuclear structure.

Animals↗
PubMed1989

Retinoic acid receptor expression vector inhibits differentiation of F9 embryonal carcinoma cells.

Expression vectors have been constructed for a region of the human retinoic acid receptor-alpha (hRAR-alpha) and transferred into F9 embryonal carcinoma (EC) cells. When the vectors are overexpressed in F9 cells, clones can be selected for resistance to retinoic acid-induced differentiation. This effect is obtained even when the hRAR-alpha region is expressed as a beta-galactosidase fusion protein. Using the beta-galactosidase component of the fusion protein as a marker, overexpression of the fusion protein has been correlated with the retinoic acid-resistance effect. The clones resistant to retinoic acid no longer exhibit the normal retinoic acid induction of endo B cytokeratin, laminin B-1, and tissue plasminogen activator mRNAs observed with normal F9 cells. Retinoic acid induction of type IV alpha-1 collagen and Hox-1.3 RNAs is observed with these clones. When transfected with a thyroid receptor DNA-binding sequence (TRE)/thymidine kinase promoter/luciferase construct, the retinoic acid-resistant clones do not yield the same retinoic acid-induced level of luciferase obtained with F9 cells. It is hypothesized that the RAR vectors are interfering with endogenous RAR(s) in a dominant-negative manner to inhibit retinoic acid-induced differentiation of F9 EC cells.

Blotting, Northern↗
PubMed1989

Development and characterization of an Fv-1-sensitive retrovirus-packaging system: single-hit titration kinetics observed in restrictive cells.

We have constructed an RNA-packaging-deficient mutant of N-tropic murine leukemia virus WN1802N by removal of 330 nucleotides located between the upstream long terminal repeat and the start of the gag gene region. Transfection into mink CCL64 cells produced a cell line capable of packaging retrovirus vectors into ecotropic, Fv-1 N-tropic virions. Using retrovirus vectors that confer resistance to the antibiotic G418, we demonstrated that the magnitude of restriction in BALB/3T3 and SIM.R cells (both Fv-1b/b) and in RFM/3T3 cells (Fv-1nr/nr) is approximately 100-fold compared with that in AKR or NIH 3T3 cells (both Fv-1n/n). Furthermore, titration kinetics were single hit in restrictive cells. Colonies of antibiotic-resistant cells recovered after infection of genotypically restrictive cultures were phenotypically restrictive when reinfected, ruling out selection of stably nonrestrictive subpopulations. These results suggest that the ability to infect some fraction of cells in a genotypically restrictive culture does not require specific abrogation and that multihit kinetics may not be an essential feature of Fv-1 restriction.

Animals↗
PubMed1988

Differential expression of the homeobox gene Hox-1.3 in F9 embryonal carcinoma cells.

The Hox-1.3 gene is located on mouse chromosome 6 and has been previously shown to be expressed in mouse embryos and adults. In this study, we have examined the steady-state levels of the Hox-1.3 transcripts in undifferentiated and differentiated F9 embryonal carcinoma cells. We find that there is a rapid increase of Hox-1.3 transcripts after differentiation induction of F9 cells. The level of the major 1.85-kilobase (kb) transcript peaks at 16-24 hr after differentiation induction of F9 cells. By using primer extension techniques the 5' ends of the major 1.85-kb transcript have been mapped to two sites in induced F9 cells. Cellular fractionation of RNA and transfer blot gel analysis has localized one minor transcript to the nucleus, whereas the major transcript and two additional minor transcripts appear in the nucleus and the cytoplasm of induced F9 cells. The results of nuclear run-off experiments with uninduced and induced F9 cell nuclei indicate that there is a substantial increase in the rate of Hox-1.3 transcription upon induction of F9 cells with retinoic acid.

Animals↗
PubMed1987

Suppression of endo B cytokeratin by its antisense RNA inhibits the normal coexpression of endo A cytokeratin.

Antisense endo B cytokeratin RNA encoded by a retrovirus vector was expressed in a derivative of the F9 embryonal carcinoma cell line. Two G418-resistant clones were selected that expressed a colinear transcript containing both neomycin and antisense endo B cytokeratin sequences. Expression of a 5-fold excess of antisense endo B RNA over endogenous, retinoic acid-induced endo B RNA resulted in suppression of endo B cytokeratin protein expression. In addition, the normal induction of endo A protein, the type II cytokeratin that polymerizes with endo B, was suppressed at the RNA and protein levels. Revertant clones, which synthesize little if any neo or antisense endo B RNA, regain the ability to express the affected gene products in response to retinoic acid. These results indicate that the suppression of endo B cytokeratin protein synthesis influences the stable levels of endo A mRNA.

Cell Line↗
PubMed1987

Retroviral vector gene expression in F9 embryonal carcinoma cells.

When F9 embryonal carcinoma (EC) cells are infected with retroviral vectors, the efficiency of expression of selectable genes is considerably lower than that in mouse fibroblasts infected with the same retroviral vectors. In this study, several retroviral vectors with regulatory sequences placed immediately 5' to a selectable gene were constructed, packaged, and used to infect mouse fibroblasts and F9 EC cells. With selection as an assay, there was a hierarchy of relative expression in F9 cells compared with that in mouse fibroblasts. These internally placed regulatory sequences are the source of the mRNAs detected in F9 EC cells, while both retroviral long-terminal-repeat promoters and internal promoters are the source of steady-state mRNAs in mouse fibroblasts. This effect was observable with both the internally placed herpes simplex virus thymidine kinase promoter and the Moloney murine leukemia virus promoter.

Animals↗
PubMed1983

Molecular cloning and nucleotide sequence of a cDNA clone coding for the cell attachment domain in human fibronectin.

A cDNA clone coding for the cell attachment domain in human fibronectin has been isolated using synthetic oligonucleotides. Three sets of mixed tetradecamer oligonucleotides were synthesized based on amino acid sequences in the 108-amino acid cell attachment domain (Pierschbacher, M. D., Ruoslahti, E., Sundelin, J., Lind, P., and Peterson, P. A. (1982) J. Biol. Chem. 257, 9593-9597). One of these sets was made complementary to amino acids located near the COOH terminus of the cell attachment domain and synthesized as a mixture of 24 sequences. This oligonucleotide mixture was used to prime cDNA synthesis with mRNA prepared from a human fibrosarcoma as a template. A cDNA library was constructed with the oligonucleotide-primed sequences in the vector pBR322. Colonies that hybridized with the primer were isolated from the library and further identified by hybridization with oligonucleotides deduced from an amino acid sequence located 45 amino acid residues NH2-terminal of the primer sequence. One clone which hybridized to both probes was characterized in detail. The insert was 380 base pairs long and its nucleotide sequence agreed completely with the corresponding amino acid sequence of human plasma fibronectin, showing that the sequences for this region are identical in plasma fibronectin and fibronectin from a cell line. This clone should be useful for studies on the expression of fibronectins and may also allow for the production of the biologically active cell attachment domain of fibronectin in bacteria.

Amino Acid Sequence↗
PubMed1983

DNA fragments from F9 PyEC mutants increase expression of heterologous genes in transfected F9 cells.

Restrictions fragments encompassing the DNA sequence changes, which allow polyoma mutants to productively infect embryonal carcinoma (EC) cells, have been coupled to the Herpes simplex virus thymidine kinase (HSV TK) gene and to the bacterial chloramphenicol acetyl transferase (CAT) gene. F9 TK(-) EC cells have been transfected with the TK constructions and transformation to TK(+) colonies has been determined. F9 EC cells, retinoic-acid-induced differentiating F9 cultures, mouse fibroblasts, and mouse myoblasts have been transfected with the CAT constructions and CAT enzyme activity has been measured from the transfected cells. The results suggest that the mutant changes function at the level of gene-expression enhancement, the mutant constructions increasing the TK transformation frequency and also yielding a higher level of CAT enzyme activity when compared with constructions having no polyoma fragment or the analogous wild-type polyoma restriction fragment. The sequence specificity for enhancement changes upon differentiation of F9 EC cells, with the CAT enzyme levels from the wild-type construction approaching the values obtained from a PyEC mutant construction having a single base-pair difference.

Animals↗
PubMed1982

Polyoma mutants that productively infect F9 embryonal carcinoma cells do not rescue wild-type polyoma in F9 cells.

Mouse embryonal carcinoma cells are refractory to infection by wild-type polyoma virus, the infection process apparently being blocked at a stage after adsorption and penetration but before early protein synthesis. Polyoma virus mutants capable of productive infection of mouse embryonal carcinoma cells have been isolated and these mutants all have DNA sequence alterations in a noncoding region near the origin of replication of the viral genome. PyF101 and PyF441 are two mutants selected for their ability to infect the embryonal carcinoma cell line F9. Here we show that these PyF mutants do not rescue replication of wild-type polyoma during a mixed infection of F9 cells. The mutant and wild-type DNAs were distinguished on the basis of restriction fragments obtained by digestion with Msp I or BstNI, and no wild-type DNA was detected in F9 cells coinfected with wild-type polyoma and with either PyF101 or PyF441. The mutant viruses do not appear to inhibit wild-type replication during a mixed infection because both mutant and wild-type DNAs can replicate efficiently in coinfected 3T6 cells which are permissive for both mutant and wild-type viruses. A double mutant having the PyF101 mutation and the ts-25E temperature-sensitive mutation in polyoma large tumor antigen was constructed and found to be temperature-sensitive for replication in F9 cells. This double mutant, designated PyFts-1, can be rescued in F9 cells at the restrictive temperature by coinfection with PyF441. These results suggest that the PyF mutations affect two processes in F9 cells, one involving expression of polyoma early genes and a second involving viral DNA replication.

Animals↗
PubMed1981

Mutation near the polyoma DNA replication origin permits productive infection of F9 embryonal carcinoma cells.

F9 mouse embryonal carcinoma cells are resistant to productive infection by wild-type polyoma virus. Continued passage of F9 cells initially infected with wild-type polyoma virus eventually leads to the selection of polyoma virus mutants that are capable of productive infection of undifferentiated F9 cells. Three mutants, PyF101, PyF111 and PyF441, have been plaque-purified and examined. All three PyF mutant DNAs are altered from the wild-type sequence in the Pvu II-4 fragment that spans 67.6 to 70.2 map units on the polyoma genome. PyF441 has a single base change of A to G at 69.6 map units. PyF101 and PyF111 DNAs also contain this point mutation at 69.6 map units. In addition, PyF101 and PyF111 DNAs have exact tandem duplications of 54 and 31 bp, respectively, of sequences encompassing the point mutation, and both copies of the tandem duplication have the point mutation. Other than these changes, no difference exists in the nucleotide sequences of wild-type and PyF mutant DNAs from the BcI I site at 65.6 map units clockwise through the origin of viral DNA replication to the BgI I site at 72.2 map units. DNA infections of F9 cells with wild-type-mutant hybrid DNAs formed by ligation of heterologous combinations of the small and large DNA fragments generated by double digestion with the restriction enzymes BcII and BGI I show that the DNA sequence changes described above are responsible for the ability of the PyF mutants to infect F9 cells.

Animals↗
PubMed1980

H1 histone and nucleosome repeat length alterations associated with the in vitro differentiation of murine embryonal carcinoma cells to extra-embryonic endoderm.

The histone compositions and average distance between nucleosomes have been determined for F9.22 and PSA1 murine embryonal carcinoma cell lines, for primary extra-embryonic endoderm derived from the in vitro differentiation of PSA1 embryonal carcinoma cells, and for two long-term extra-embryonic endodermal cell lines. A change in the relative proportions of two forms of the H1 histones (H1A and H1B) was found to correlate with the extra-embryonic endodermal differentiated phenotype. The embryonal carcinoma cells had a ratio of H1A/H1B of 1.49 or greater. In contrast, extra-embryonic endoderm from either cell lines or freshly isolated from differentiating embryonal carcinoma cell cultures had a ratio of H1A/H1B of less than 0.9. Partial peptide mapping of gel purified H1A and H1B suggest the two proteins differ in primary structure. The nucleosome repeat length of the embryonal carcinoma cell lines was 196 bp of DNA. Primary extra-embryonic endoderm was found to have a value of 205 bp, but the long-term extra-embryonic endodermal cell lines had an average nucleosome repeat length of 187 bp. Since both freshly isolated primary endoderm and the long-term endodermal cell lines express differentiated functions (basement membrane glycoproteins and plasminogen activator activity), there appears to be no simple correlation between the nucleosome repeat length and the expression of these differentiated functions.

Animals↗
PubMed1977

Teratocarcinoma differentiation: plasminogen activator activity associated with embryoid body formation.

Changes in plasminogen activator activity have been examined as a clonal line of mouse embryonal carcinoma cells aggregate and differentiate to form cystic embryoid bodies in vitro. Within the first 10 days of study, the pluripotent embryonal carcinoma cells aggregate; a layer of endodermal cells appears on the outside of the aggregate forming an embryoid body; a basement membrane forms between the outer layer of endodermal cells and the internal cells; a cyst forms within the embryoid body; and the internal cells assume a columnar appearance along the inner portion of the basement membrane. After the formation of the endodermal layer, there is a rise in intracellular plasminogen activator activity. This rise continues for up to 25 days in culture, providing that the three-dimensional integrity of the embryoid bodies is maintained by culturing them on bacterial petri dishes. Selective removal of the outer endodermal layer of cells reduces the plasminogen activatory activity of the resulting embryoid body cores. Intracellular and secreted plasminogen activator activity of simple embryoid bodies composed of only two cell types can be increased by culturing the embryoid bodies in dbcAMP, theophylline, or cholera toxin. These results suggest that the embryoid body endodermal cells are the source of a cAMP-inducible plasminogen activator activity.

Alkaline Phosphatase↗
PubMedDate not supplied

NIEHS/EPA Workshops. Growth and differentiation factors.

The work group identified a number of research areas where they felt there were significant data gaps where additional research was critical to better under standing of the origins of birth defects and to developing ways for their prevention. These included: 1. Studies designed to determine the role of growth and differentiation factors during pre- and postimplantation stages of development. These investigations could include descriptive studies involving the localization and timing of genes expressed and their products, but must also emphasize and include studies involving the function of these molecules and their interactions in normal and abnormal development. 2. Studies designed to develop and utilize models for investigating normal and abnormal development. These approaches could include in vivo and in vitro techniques, such as creation of genetically defined systems and cell, organ, and whole embryo cultures. These technologies should emphasize ways to study the functions of growth and differentiation factors and the effects of environmental factors. 3. Studies designed to identify environmental agents and their targets in embryonic, extraembryonic, and maternal tissues that may play a role in producing developmental abnormalities through peturbations of growth and differentiation factors. 4. Studies designed to determine cellular and molecular mechanisms for protection and recovery from environmental insults.

Animals↗
PubMedDate not supplied

Magnetic resonance microscopy of embryos.

We demonstrate that magnetic resonance (MR) microscopy provides a mechanism to investigate normal and abnormal developmental anatomy in a non-destructive and distortion-free manner. Techniques for the fixation, embedding, perfusion and image acquisition of embryos between 3 and 30 mm crown rump length are described. We describe the perfusion of a contrast agent to enhance images of the developing embryonic vasculature. Data are acquired as three-dimensional isotropic arrays which permit images to be reformatted retrospectively in any plane. The data are available for archiving, distributing and for post-acquisition manipulations. MR microscopy is a fast technique for producing three-dimensional reconstructions and is free from registration and sectioning artifacts.

Anatomy, Artistic↗