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Biomedical subjects

W F Anderson

Publications and source records attributed to W F Anderson.

At least 235 records · Page 13Linked to original sources
PubMed1987

The purification and characterization of subunits alpha, beta, and gamma from the rabbit reticulocyte eukaryotic initiation factor 2.

Eukaryotic initiation factor 2 (eIF-2) contains three nonidentical subunits, alpha, beta, and gamma. The simultaneous purification of all three subunits was achieved by reverse-phase HPLC using a 0.1% trifluoroacetic acid-acetonitrile binary solvent system. The order of the eluted subunits, beta, alpha, and gamma, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After hydrolysis in 6 N HCl, picomole level amino acid composition analysis was achieved by the ninhydrin reaction on a Beckman 6300 system. Using second-derivative spectroscopic analysis, Trp was detected in all three subunits. All three subunits were subjected to amino-terminal sequence analysis. The amino-terminal of eIF-2 alpha from amino acid positions 1 to 23 inclusive was determined. The order of eight amino acids from the amino-terminal of eIF-2 gamma was also determined. This characterization and partial determination of the primary sequence of these subunits permit the utilization of molecular biology techniques in order to elucidate the complete primary structure. Additionally, the partial amino acid sequence data permitted the designation of synthetic gene probes as well as the identification of eIF-2 alpha and gamma cDNA and/or genomic clones.

Amino Acid Sequence↗
PubMed1987

The expression of integrated plasmid DNA depends on copy number.

The effect of copy number, integration site, and enhancers on the expression of stably integrated exogenous DNA was examined in Chinese hamster cells. Three similar plasmids were constructed with the mouse beta maj-globin promoter fused to the galK gene either with no enhancer or with the SV40 or Harvey sarcoma virus (HaSV) enhancer. Eighteen stable cell lines were obtained and characterized with respect to plasmid copy number and galactokinase activity. At copy numbers of four or less, the enhancers showed detectable activity and a DNase I hypersensitive site was present. Above four copies, gene activity decreased as the copy number increased, the enhancer sequences were apparently inactive, and the DNase I hypersensitive site disappeared. These data suggest that, at least in this model system, when exogenous DNA is integrated as multiple head-to-tail copies, the entire multigene unit expresses poorly and inappropriately. When the same exogenous DNA integrates as a single (or low number) copy, expression appears to be relatively normal as judged by enhancer stimulation and DNase I hypersensitivity.

Animals↗
PubMed1987

Production of glycosylated physiologically "normal" human alpha 1-antitrypsin by mouse fibroblasts modified by insertion of a human alpha 1-antitrypsin cDNA using a retroviral vector.

Alpha 1-Antitrypsin (alpha 1AT) deficiency is a hereditary disorder characterized by reduced serum levels of alpha 1AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment alpha 1AT levels in this disorder with physiologically normal human alpha 1AT, we have integrated a full-length normal human alpha 1AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the alpha 1AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi 2 and infected NIH 3T3. The clones produced three mRNA transcripts (5.8, 4.8, and 2.4 kilobases) containing human alpha 1AT sequences, secreted an alpha 1AT molecule recognized by an anti-human alpha 1AT antibody, with the same molecular mass (52 kDa) as normal human alpha 1AT and that complexed with and inhibited human neutrophil elastase. The psi 2 produced alpha 1AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal alpha 1AT purified from human plasma and markedly longer than that of nonglycosylated human alpha 1AT cDNA-directed yeast-produced alpha 1AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human alpha 1AT cDNA into non-alpha 1AT-producing cells, resulting in the synthesis and secretion of physiologically "normal" human alpha 1AT.

Animals↗
PubMed1987

Structural and functional homology of parvovirus and papovavirus polypeptides.

We have compared the sequences of the putative polypeptides of the human pathogenic B19 parvovirus with protein sequences in the National Bethesda Research Foundation Library, and have discovered a significant homology between a B19 parvovirus non-structural (NS) protein and the T antigens of polyomaviruses and simian virus 40 (SV40) and the putative E1 proteins of papillomaviruses. The region of highest homology with the papovavirus proteins corresponds to the region that is most highly conserved in the NS1 proteins of several other parvoviruses. Studies with the T antigen of both polyomaviruses and SV40 have implicated this region as having an ATPase activity and nucleotide-binding function.

Amino Acid Sequence↗
PubMed1987

Effect of internal viral sequences on the utility of retroviral vectors.

Expression of the human ADA cDNA encoded by the Moloney murine leukemia virus spliced RNA form is enhanced by intron-contained sequences. The presence of sequences corresponding to the viral gag gene in a Moloney murine leukemia virus-based vector results in the generation of 10- to 40-fold higher titers of virus.

Adenosine Deaminase↗
PubMed1987

Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) shortens the period of neutropenia after autologous bone marrow transplantation in a primate model.

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on hematopoietic reconstitution after autologous bone marrow transplantation was evaluated in a primate model. Animals were given a continuous intravenous infusion of recombinant human GM-CSF for several days both before and after transplantation or only after the transplant procedure. Marrow ablation was accomplished by total body irradiation. In both groups of animals, the neutrophil count reached 1,000/mm3 by 8-9 d posttransplant compared with an interval of 17 and 24 d for two concurrent controls. After withdrawal of GM-CSF, neutrophil counts fell to values comparable to those observed in untreated controls. Accelerated recovery of platelet production was also observed in four of the five animals. Two additional animals were initially given GM-CSF several weeks posttransplantation because of inadequate engraftment. Prompt and sustained increases in neutrophil and platelet counts were observed. We conclude that GM-CSF may be useful in accelerating bone marrow reconstitution.

Agranulocytosis↗
PubMed1986

Compensatory increase in levels of beta minor globin in murine beta-thalassemia is under translational control.

A 3.7-kilobase pair deletion including the entire beta major globin gene results in beta-thalassemia in a murine model of the disease (Skow, L. C., Burkhart, B. A., Johnson, F. M., Popp, R. A., Popp, D. M., Goldberg, S. Z., Anderson, W. F., Barnett, L. B., and Lewis, S. E. (1983) Cell 34, 1043-1052). There is a compensatory increase in synthesis of beta minor globin, resulting in a beta minor/alpha globin ratio of 0.75 in the homozygous thalassemic mouse, as compared to 0.2 in the normal homozygous diffuse mouse. The results presented here demonstrate that the increase in beta minor globin synthesis occurs at translation rather than at transcription. RNase T1 analysis of reticulocyte mRNA reveals that the beta/alpha ratio of globin mRNA is 0.3, significantly lower than the globin synthetic ratio of 0.7. However, the beta/alpha ratio of mRNA on polysomes is higher than unassociated mRNA, demonstrating that beta minor mRNA is preferentially translated. Elevated synthesis of beta minor globin is maintained during in vitro translation in thalassemic reticulocyte lysate. In this system, partial inhibition of translational elongation by cycloheximide decreases the beta minor/alpha globin synthetic ratio, whereas partial inhibition of initiation by hemin deficiency increases the beta minor/alpha synthetic ratio. This suggests that beta minor mRNA competes with alpha-mRNA for a limiting mRNA binding factor at initiation of translation.

Animals↗
PubMed1986

Multiple sequence alignment.

A method has been developed for aligning segments of several sequences at once. The number of search steps depends only polynomially on the number of sequences, instead of exponentially, because most alignments are rejected without being evaluated explicitly. A data structure herein called the "heap" facilitates this process. For a set of n sequence segments, the overall similarity is taken to be the sum of all the constituent segment pair similarities, which are in turn sums of corresponding residue similarity scores from a Table. The statistical models that test alignments for significance make it possible to group sequences objectively, even when most or all of the interrelationships are weak. These tests are very sensitive, while remaining quite conservative, and discourage the addition of "misfit" sequences to an existing set. The new techniques are applied to a set of five DNA-binding proteins, to a group of three enzymes that employ the coenzyme FAD, and to a control set. The alignment previously proposed for the DNA-binding proteins on the basis of structural comparisons and inspection of sequences is supported quite dramatically, and a highly significant alignment is found for the FAD-binding proteins.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1986

A 66-base pair insert bridges the deletion responsible for a mouse model of beta-thalassemia.

The breakpoints of the deletion responsible for the Hbb(th-1) mouse model of beta-thalassemia have been isolated. A 3709 (+/- 2)-base pair (bp) region, including the entire beta major globin gene and 2 kilobases of 5' flanking region, is deleted. A novel 66 (+/- 2)-bp sequence, ending in a stretch of 25 dA:dT base pairs, was found to bridge the deletion. A region of the normal murine genome, containing the first 43 bp of the deletion-associated insert (DAI), but lacking the 25-bp dA:dT sequence, was isolated. All normal mice tested contain this DAI-like element and several inbred strains contain an additional DAI-like element. The sequence spanning the Hbb(th-1) deletion may be a reverse transcript of this region.

Animals↗
PubMed1986

Different interactions used by Cro repressor in specific and nonspecific DNA binding.

The mode of interaction of Cro repressor with specific and nonspecific sites on DNA was explored by chemical modification and protection of lysine and tyrosine residues. Cro has 8 lysines. In the presence of DNA, lysines 32 and 56 are fully protected and lysines 21, 62, and 63 are partially protected from alkylation. However, the terminal amino group and lysines 8, 18, and 39 are not protected. Location of the protected and unprotected lysines on the three-dimensional Cro structure defines a DNA-binding region. The results provide direct experimental support for a mode of interaction between Cro and DNA, in which Cro buries its 2-fold related alpha-helices in consecutive DNA major grooves (Anderson, W. F., Ohlendorf, D. H., Takeda, Y., and Matthews, B. W. (1981) Nature 290, 754-758; Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., and Matthews, B. W. (1982) Nature 298, 718-723). In the model, the carboxyl-terminal part of Cro was tentatively presumed to interact with the DNA minor groove. Protection of lysines 62 and 63 confirms the involvement of the carboxyl terminus in DNA binding. Although nonspecific and specific DNA protect the same lysine residues, there are differences in the nature of the interaction of Cro with nonspecific and specific DNA. Cro-nonspecific DNA interaction is salt-sensitive, suggesting that the interaction is predominantly electrostatic. On the other hand, Cro-specific DNA interaction is salt-resistant, suggesting that the interaction may include nonelectrostatic components (hydrogen bonds and hydrophobic interactions) as well. Protection experiments of tyrosine residues (against iodination) suggest that the conformation of Cro repressor changes in two stages: first, when Cro binds at nonspecific sites, and, second, when Cro binds to specific sites on DNA.

Amino Acid Sequence↗
PubMed1986

Crystallization of a complex of cro repressor with a 17 base-pair operator.

Crystals of the lambda cro repressor complexed to a 17 base-pair synthetic binding site related to the OR3 operator have been obtained. The complex crystallizes in the hexagonal space group P6(2) (or P6(4)) with unit cell dimensions a = b = 154.8 A, c = 85.6 A. Preliminary photography reveals that the crystals are stable to X-rays and display measurable reflections to a resolution of about 3.7 A. The diffraction patterns suggest that the cro-DNA complexes are arranged in an open hexagonal network with the DNA fragments stacked end-to-end. The DNA is in the B-form but appears to be bent or curved into an approximate superhelix.

Bacteriophage lambda↗
PubMed1986

Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells.

A retrovirus-derived vector called self-inactivating (SIN) vector was designed for the transduction of whole genes into mammalian cells. SIN vectors contain a deletion of 299 base pairs in the 3' long terminal repeat (LTR), which includes sequences encoding the enhancer and promoter functions. When viruses derived from such vectors were used to infect NIH 3T3 cells, the deletion was transferred to the 5' LTR, resulting in the transcriptional inactivation of the provirus in the infected cell. Introduction of a hybrid gene (human metallothionein-promoted c-fos) into cells via a SIN vector was not associated with rearrangements and led to the formation of an authentic mRNA transcript, which in some cases was induced by cadmium. SIN vectors should be particularly useful in gene transfer experiments designed to study the regulated expression of genes in mammalian cells. Absence of enhancer and promoter sequences in both LTRs of the integrated provirus should also minimize the possibility of activating cellular oncogenes and may provide a safer alternative to be used in human gene therapy.

Chromosome Deletion↗
PubMed1986

Prediction of 3-D structure of the Cro protein from phage 434.

A comparative model building process has been utilized to predict the three-dimensional structure of the bacteriophage 434 Cro protein. Amino acid sequence similarities between the 434 Cro protein and other bacteriophage repressor and Cro proteins have been used, in conjunction with secondary structure prediction and the known structures of other base sequence specific DNA binding proteins, to derive the model. From this model the interactions between the 434 Cro protein and its operator DNA have been deduced. These proposed interactions are consistent with the known properties of the bacteriophage 434 Cro protein.

Amino Acid Sequence↗