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Biomedical subjects

W F Anderson

Publications and source records attributed to W F Anderson.

At least 253 records · Page 14Linked to original sources
PubMed1985

Gene expression in mice after high efficiency retroviral-mediated gene transfer.

A retroviral expression vector (N2) containing the selectable gene, neoR, has been used to determine the optimal conditions for infecting murine hematopoietic progenitor cells at high efficiency. After infected bone marrow cells were introduced into lethally irradiated mice, the presence, stability, and expression of the vector DNA sequences were analyzed either in individual spleen foci 10 days later or in the blood, bone marrow, and spleens of mice 4 months later. When bone marrow cells were cultured in medium containing virus with titers of more than 10(6) colony-forming units per milliliter in the presence of purified murine interleukin-3, more than 85 percent of the resulting foci contained vector DNA. This proviral vector DNA was intact. Efficient expression of the neoR gene was demonstrated in most of the DNA-positive foci examined. The spleens of reconstituted animals (over a long term) contained intact "vector DNA" and the blood and bone marrow expressed the neoR gene in some animals. Thus, a retroviral vector can be used to introduce intact exogenous DNA sequences into hematopoietic stem cells with high efficiency and with substantial expression.

Animals↗
PubMed1985

delta-Aminolevulinate dehydratase: induced expression and regional assignment of the human gene to chromosome 9q13----qter.

The structural gene encoding human delta-aminolevulinate dehydratase has been assigned to the long arm of chromosome 9 by somatic cell hybridization techniques using murine erythroleukemia-human fibroblast somatic cell hybrids. Dimethyl sulfoxide induction of erythroid differentiation in these hybrid cells resulted in a 3 to 12-fold increase in the levels of total delta-aminolevulinate dehydratase. Human delta-aminolevulinate dehydratase was detected by an immunodiscrimination assay using polyclonal mouse anti-human delta-aminolevulinate dehydratase antibodies. Of four primary hybrid clones, each from an independent fusion, one hybrid line, XX-8, was positive for human delta-aminolevulinate dehydratase. Examination of 23 secondary, tertiary, and quaternary XX-8 subclones revealed that the expression of the human isozyme segregated with human chromosome 9q, confirming the provisional regional assignment made by classical linkage studies. One positive quaternary clone, XX-8-H21-H7-2, expressed human delta-aminolevulinate dehydratase activity and contained only human 9q13----qter. In addition, studies of tertiary and quaternary subclones from two series, XX-8-A31 and XX-8-H21-H7, indicated that murine regulatory factors increased the human as well as the murine enzymatic activity following induction of erythroid differentiation.

Animals↗
PubMed1985

Computer modeling studies of the structure of a repressor.

Due to advances in molecular biology the DNA sequences of structural genes coding for proteins are often known before a protein is characterized or even isolated. The function of a protein whose amino acid sequence has been deduced from a DNA sequence may not even be known. This has created greater interest in the development of methods to predict the tertiary structures of proteins. The a priori prediction of a protein's structure from its amino acid sequence is not yet possible. However, since proteins with similar amino acid sequences are observed to have similar three-dimensional structures, it is possible to use an analogy with a protein of known structure to draw some conclusions about the structure and properties of an uncharacterized protein. The process of predicting the tertiary structure of a protein relies very much upon computer modeling and analysis of the structure. The prediction of the structure of the bacteriophage 434 cro repressor is used as an example illustrating current procedures.

Amino Acid Sequence↗
PubMed1985

Isolation of stable preinitiation, initiation, and elongation complexes from RNA polymerase II-directed transcription.

Distinct RNA polymerase II transcription preinitiation, initiation, and elongation complexes can be formed in vitro on cloned adenovirus 2 DNA sequences containing the major late promoter. These transcription complexes are stable and can be rapidly isolated by gel filtration of HeLa whole cell extracts. In the absence of exogenous nucleotides and under appropriate salt conditions, a stable but transcriptionally incomplete preinitiation complex is formed. When this complex is incubated in the presence of adenosine or deoxyadenosine triphosphates, the beta-gamma phosphodiester bond is hydrolyzed, and RNA polymerase II joins the complex, thereby converting it into a stable initiation complex capable of forming (but prior to the formation of) the first phosphodiester bond. When this complex is isolated and incubated in the presence of all four nucleoside triphosphates, it is converted into an elongation complex that then permits the synthesis of phosphodiester bonds and the correct run-off transcript. A limiting transcription component is sequestered in the preinitiation complex. This factor is released upon elongation and can reassociate with new DNA templates during subsequent rounds of initiation. Therefore, class II genes do not appear to form activated transcription units stable for multiple rounds of transcription; rather, their transcriptional activity may be controlled in part by regulating the association of transcription factors at each initiation event.

Adenoviridae↗
PubMed1985

Human gene therapy: scientific and ethical considerations.

The term 'gene therapy' encompasses at least four types of application of genetic engineering for the insertion of genes into humans. The scientific requirements and the ethical issues associated with each type are discussed. Somatic cell gene therapy is technically the simplest and ethically the least controversial. The first clinical trials will probably be undertaken within the next year. Germ line gene therapy will require major advances in our present knowledge and it raises ethical issues that are now being debated. In order to provide guidelines for determining when germ line gene therapy would be ethical, the author presents three criteria which should be satisfied prior to the time that a clinical protocol is attempted in humans. Enhancement genetic engineering presents significant, and troubling, ethical concerns. Except where this type of therapy can be justified on the grounds of preventive medicine, enhancement engineering should not be performed. The fourth type, eugenic genetic engineering, is impossible at present and will probably remain so for the foreseeable future, despite the widespread media attention it has received.

Bone Marrow Transplantation↗
PubMed1985

Transfer of human and murine globin-gene sequences into transgenic mice.

We have studied the transfer of human and murine globin gene sequences into fertilized mouse oocytes by microinjection. Germline transmission was demonstrated for the human delta- and beta-globin genes contained in the bacteriophage lambda H beta G1. Expression of these human globin-gene sequences was not detectable in either erythroid or nonerythroid tissues. A recombinant plasmid containing the murine beta maj promoter region coupled to the prokaryotic coding sequence for galactokinase was also successfully transferred to two mice, and stable germline transmission of integrated DNA was demonstrated for at least 3 generations. Despite the presence of a murine globin-promoter sequence, expression of the mouse beta maj galactokinase fusion gene was not observed in primary or secondary animals in erythroid or nonerythroid tissues. Analysis of primary and secondary animals from both series of injections revealed extensive de novo methylation in the integrated microinjected DNA. Administration of 5-azacytidine to mice containing the mouse beta maj-promoted galactokinase gene resulted in partial hypomethylation was associated with an apparent two- to threefold increase in galactokinase (gal K) gene expression.

Animals↗
PubMed1985

Human gene therapy: scientific and ethical considerations.

The term "gene therapy" encompasses at least four types of application of genetic engineering for the insertion of genes into humans. The scientific requirements and the ethical issues associated with each type are discussed. Somatic cell gene therapy is technically the simplest and ethically the least controversial. The first clinical trials will probably be undertaken within the next year. Germ line gene therapy will require major advances in our present knowledge and it raises ethical issues that are now being debated. In order to provide guidelines for determining when germ line gene therapy would be ethical, the author presents three criteria which should be satisfied prior to the time that a clinical protocol is attempted in humans. Enhancement genetic engineering presents significant, and troubling, ethical concerns. Except where this type of therapy can be justified on the grounds of preventive medicine, enhancement engineering should not be performed. The fourth type, eugenic genetic engineering, is impossible at present and will probably remain so for the foreseeable future, despite the widespread media attention it has received. There are four potential levels of the application of genetic engineering for the insertion of gene into a human being. Somatic cell therapy: this would result in correcting a genetic defect in the somatic (i.e.,body) cells of a patient. Germ line gene therapy: this would require the insertion of the gene into the reproductive tissue of the patient in such a way that the disorder in his or her offspring would also be corrected. Enhancement genetic engineering: this would involve the insertion of a gene to try to "enhance" a known characteristic; for example, the placing of an additional growth hormone gene into a normal child. Eugenic genetic engineering: this is defined as the attempt to alter or "improve" complex human traits each of which is coded by a large number of genes; for example, personality, intelligence, character, formation of body organs, and so on.

Adenosine Deaminase↗
PubMed1984

Prospects for human gene therapy.

Procedures have now been developed for inserting functional genes into the bone marrow of mice. The most effective delivery system at present uses retroviral-based vectors to transfer a gene into murine bone marrow cells in culture. The genetically altered bone marrow is then implanted into recipient animals. These somatic cell gene therapy techniques are becoming increasingly efficient. Their future application in humans should result in at least partial correction of a number of genetic disorders. However, the safety of the procedures must still be established by further animal studies before human clinical trials would be ethical.

Animal Experimentation↗
PubMed1984

A model for the structure of fumarate reductase in the cytoplasmic membrane of Escherichia coli.

By a recombinant DNA approach we have prepared Escherichia coli cytoplasmic membranes that are highly enriched in the terminal electron transfer enzyme fumarate reductase. This enzyme is composed of four nonidentical subunits in equal molar ratio. A 69,000-dalton covalent flavin-containing subunit and a 27,000-dalton nonheme iron-containing subunit make up a membrane extrinsic catalytic domain. Two very hydrophobic subunits of 15,000 and 13,000 daltons make up the hydrophobic membrane anchor domain. Electron microscopy of negatively stained membranes shows a characteristic knob-and-stalk-type structure composed of the catalytic domain. The anchor polypeptides have been analyzed for hydrophobic segments and alpha-helical content and a model for their organization within the lipid bilayer is presented. The results reviewed in this paper suggest a model for the fumarate reductase complex in the cytoplasmic membrane.

Bacterial Proteins↗
PubMed1984

Human globin gene expression in hybrid 2S MEL X human fibroblast cells.

A somatic cell hybrid line, called M11-X, was developed in order to study the expression and regulation of the human beta-like globin genes in a mouse erythroid environment. M11-X cells were obtained by fusing the human fibroblast cell line GM3552 (which contains the translocation chromosome t(11;X) that carries the human beta-like globin genes) with hypoxanthine phosphoribosyltransferase (HPRT) -negative tetraploid (2S) mouse erythroleukemia (MEL) cells. After induction with 5 mM hexamethylene bisacetamide (HMBA), these cells contain approximately 300-600 copies per cell of correctly initiated, processed, and terminated human beta-globin mRNA; however, neither human epsilon- nor gamma-globin mRNAs were detected. Carboxymethylcellulose chromatography followed by SDS-polyacrylamide gel electrophoresis and Western blotting revealed that normal human beta-globin protein was also present. These results suggest that the human beta-globin gene, when present in mouse erythroid cells, can be transcribed and its mRNA translated into normal products, but at a much lower level than the mouse beta-globin genes. Analysis of the frequency of cytosine methylation near the human gamma-globin genes indicated that these genes are heavily methylated in M11-X cells. The inability to express the human gamma-globin genes of these cells might be accounted for, at least in part, by DNA methylation.

Animals↗
PubMed1984

Fate and structure of DNA microinjected into mouse TK-L cells.

Co-microinjection of single linearized molecules of plasmids containing the human beta-globin gene (pRK1) and the herpes simplex virus (HSV) type I thymidine kinase gene (pX1) into the mouse TK-L cell nucleus results in covalent linkage between these (or derived) molecules within the nucleus as revealed by Southern blotting, plasmid rescue, and recovery of plasmid-derived DNA from a Charon 4A phage library of cellular DNA. The microinjected DNA is predominantly found as high molecular weight DNA as determined by Hirt fractionation. Southern blotting data and recombinants from the Charon 4A library suggest that the plasmid DNA is in the form of a head-to-tail linear concatamer of up to 80 copies. Passage of these microinjected cells in selective medium (HAT) results in coordinate amplification of both plasmids, which are maintained in an approx. 3:1 molar ratio of pRK1 to pX1-derived molecules. Hybridization in situ shows the DNA to be integrated on a translocation chromosome, t(4;4). Integration does not appear to be site-specific, since plasmid DNA from another microinjected cell line, C2B, appears on a different translocation chromosome, t(8?;14). Plasmid rescue experiments confirm a previous finding that passage of pBR322 DNA through eukaryotic cells may result in deletions of normally stable plasmid DNA upon subsequent transformation of E. coli. These deletions appear to occur in the bacteria, and originate in a 128 bp region between the Sal I and Hae II sites of pBR322.

Animals↗
PubMed1984

Isolation of an active transcription initiation complex from HeLa cell-free extract.

A two-step procedure has been developed for the formation of RNA polymerase II transcription initiation and elongation complexes. Initiation complexes are rapidly formed in HeLa cell-free extract supplemented with a DNA template containing the adenovirus 2 major late promoter and ATP. Assembly of transcription components required for correct initiation is absolutely dependent on specific eukaryotic promoter sequences. Sarkosyl-sensitive transcription initiation complexes are rapidly converted to Sarkosyl-resistant elongation complexes when supplemented with the remaining nucleoside triphosphates. The 60S initiation complex can be extensively purified by glycerol gradient centrifugation and is easily separated from free RNA polymerase II and free DNA template. Recovery of this stable complex is greater than 90%. Specific transcription cannot be detected if the DNA template is subsequently added to gradient fractions containing HeLa cell-free extract components alone. This suggests that the DNA templates promote the specific assembly of RNA polymerase II and transcription factors required for accurate initiation. Since conversion of purified initiation complexes to elongation complexes can occur without additional HeLa cell components, the presence of transcription components required for initiation and elongation in a single complex is indicated.

Cell-Free System↗
PubMed1984

DNA methylation and regulation of the human beta-globin-like genes in mouse erythroleukemia cells containing human chromosome 11.

The human beta-globin gene is expressed--but the human fetal (gamma) and embryonic (epsilon) globin genes are not--in an induced mouse erythroleukemia cell line (M11-X) that contains most of human chromosome 11. A 24-hr exposure of M11-X cells to 5-azacytidine before induction causes "global" DNA hypomethylation but selective activation of the human gamma-globin genes. Genomic DNA is remethylated 2-3 days after exposure to 5-azacytidine, but sequences near the human and mouse globin genes remain hypomethylated, suggesting that the remethylation process is inhibited in these regions.

Acetamides↗
PubMed1984

Homologous globin cell-free transcription system with comparison of heterologous factors.

Mouse erythroleukemia (MEL) cells provide a useful model system to examine the regulation of globin gene expression. MEL cells ordinarily do not express globin genes, but in the presence of inducers, such as dimethyl sulfoxide or hexamethylene bisacetamide, they mimic erythroid differentiation. We have developed a cell-free transcription system from uninduced MEL cells to determine the requirements for mRNA synthesis. The MEL system directs accurate transcription of adenovirus type 2 major late DNA and mouse betamaj-globin with an efficiency comparable to those of HeLa and KB cell extracts. Using the procedure of Matsui et al. (T. Matsui, J. Segall, P.A. Weil, and R.G. Roeder, J. Biol. Chem. 255:11992-11996, 1980), we have isolated three active fractions from both MEL and HeLa cell extracts which are required for accurate transcription and have shown that equivalent fractions from MEL and HeLa cell extracts are interchangeable. Our findings suggest that the components required for initiation of transcription are similar in different cell types, at least to the extent that they can be assayed in these in vitro systems.

Animals↗
PubMed1984

Correlation of gene expression and transformation frequency with the presence of an enhancing sequence in the transforming DNA.

The transformation frequency of cultured mammalian cells is increased 10- to 100-fold when certain DNA sequences are present in the transforming DNA. We wanted to determine whether enhancers, which stimulate gene expression, can cause this phenomenon. Three plasmids, each containing a galactokinase K (galK) gene, were used to transform galK- Chinese hamster cells. One plasmid has no enhancer, another has the simian virus 40 (72-base-pair repeat) enhancer, and the third has the Harvey sarcoma virus (73-base-pair repeat) enhancer. The presence of either enhancer significantly increased the appearance of GalK+ colonies. Galactokinase transient assays in this Chinese hamster strain in the presence of the same plasmids demonstrated an increase in GalK enzyme levels when either enhancer was present. These data indicate that there is a strong correlation between galK expression and transformation frequency that is dependent on the presence of an enhancer in the transforming DNA.

Animals↗
PubMed1984

Promoter dependence of enhancer activity.

The interaction of enhancers with different promoters was studied by measuring the influence of two enhancers (from simian virus 40 and from Harvey sarcoma virus) on the activity of expression vectors that are identical except for their promoter region. The promoters examined were from the simian virus 40 early region, with or without its own 72-base-pair repeat, and the mouse beta major-globin gene. It is clear that the promoter acted upon strongly influences the level of activity of an enhancer.

Base Sequence↗