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J P Dougherty

Publications and source records attributed to J P Dougherty.

At least 55 records · Page 3Linked to original sources
PubMed1988

Determination of the rate of base-pair substitution and insertion mutations in retrovirus replication.

We recently described a protocol for determination of retrovirus mutation rates, that is, the mutation frequency in a single cycle of retrovirus replication (J.P. Dougherty and H.M. Temin, Mol. Cell. Biol. 6:4378-4395, 1987; J.P. Dougherty and H.M. Temin, p. 18-23, in J. H. Miller and M. P. Calos, ed., Gene Transfer Vectors for Mammalian Cells, 1987). We used this protocol to determine the mutation rates for defined mutations in a replicating retrovirus by using a spleen necrosis virus-based vector. We determined that the mutation rate for a single base pair substitution during replication of this avian retrovirus is 2 x 10(-5) per base pair per replication cycle and the insertion rate is 10(-7) per base pair per replication cycle. It will be possible to use this protocol to determine mutation rates for other retroviruses.

Base Sequence↗
PubMed1987

Isolation of a cDNA clone encoding a high molecular weight precursor to a 6-kDa pulmonary surfactant-associated protein.

Mammalian surfactant is an incompletely defined mixture of lipids and associated proteins of molecular mass 35,000 Da and approximately 6,000 Da. Surfactant preparations which are highly effective in treating respiratory distress syndrome in premature infants lack the 35-kDa proteins, but contain the 6-kDa proteins. We isolated and partially sequenced one of these low molecular weight proteins from the lung lavage material of an alveolar proteinosis patient. Oligonucleotides deduced from the sequence were used as probes to isolate a human cDNA clone. The clone codes for a 42-kDa protein which contains the sequence of the 6-kDa protein. Messenger RNA coding for the 42-kDa protein was identified in human lung RNA by in vitro translation and immunoprecipitation of the translation products with an antiserum against purified bovine surfactant 6-kDa proteins. Immunoprecipitation of the 42-kDa primary translation product is inhibited by the presence of the bovine 6-kDa protein. These observations suggest a precursor-product relationship of the 42-kDa protein to one of the 6-kDa proteins.

Amino Acid Sequence↗
PubMed1987

Immobilization of DNA via oligonucleotides containing an aldehyde or carboxylic acid group at the 5' terminus.

A general method for the immobilization of DNA through its 5'-end has been developed. A synthetic oligonucleotide, modified at its 5'-end with an aldehyde or carboxylic acid, was attached to latex microspheres containing hydrazide residues. Using T4 polynucleotide ligase and an oligonucleotide splint, a single stranded 98mer was efficiently joined to the immobilized synthetic fragment. After impregnation of the latex microspheres with the fluorescent dye, Nile Red and attachment of an aldehyde 16mer, 5 X 10(5) bead-DNA conjugates could be detected with a conventional fluorimeter.

Aldehydes↗
PubMed1987

Rapid hybridization kinetics of DNA attached to submicron latex particles.

We describe a novel method for attaching any DNA molecule to submicron latex beads and characterize the hybridization kinetic properties of these bead-DNA conjugates. The conjugates hybridize to DNA in solution with rates comparable to homogeneous hybridization reactions, are compatible with common hybridization conditions and are conveniently manipulated. They should thus serve as useful reagents for the fractionation and characterization of DNA and RNA.

Bacteriophage lambda↗
PubMed1987

A promoterless retroviral vector indicates that there are sequences in U3 required for 3' RNA processing.

We constructed a retrovirus vector in which all viral transcriptional signals are deleted during provirus formation. This vector would be safer and more useful in gene-transfer experiments. Construction of this vector involved the deletion of all of the U3 sequence except for 10 base pairs, required for integration, from the right-side long terminal repeat. We found that this deletion resulted in an inability to propagate virus efficiently. When we inserted sequences containing all of the signals for polyadenylylation of simian virus 40 late mRNA at the end of our vector, we were able to propagate virus efficiently. This result indicates that there are sequences in U3 upstream from AAUAAA that are essential for 3' processing of viral RNA.

Animals↗
PubMed1986

The mouse immunoglobulin heavy-chain gene enhancer contains sequences that inhibit transcription in vitro in HeLa cell extracts.

The effect of the cell-specific mouse immunoglobulin heavy-chain gene (IgH) enhancer on transcription from heterologous promoter elements was studied in vitro with a HeLa whole-cell extract. No stimulation of transcription could be seen under conditions in which an activation was observed with the simian virus 40 enhancer. We found, however, that a specific segment of the IgH enhancer region contains sequences which inhibit transcription in vitro.

Animals↗
PubMed1986

High mutation rate of a spleen necrosis virus-based retrovirus vector.

Spleen necrosis virus (SNV) is an avian retrovirus that efficiently infects some mammalian cells (e.g., dog and rat cells). We constructed an SNV-based vector, which contains less than 1 kilobase (kb) of the retrovirus sequence, and a number of derivatives containing selectable markers. We obtained high-titer virus stocks, over 10(6) transforming units per ml, with a vector whose genomic RNA consists of 1,850 bases (full-length SNV RNA is 7.7 kb). We also studied two vectors that both carry two genes which should be expressed from a single promoter, one gene from unspliced mRNA and the other gene from spliced mRNA. In one vector, both genes were efficiently expressed as expected. However, in the other vector, expression of the gene 3' to the splice acceptor was inhibited. When we selected for expression of the 3' gene is this latter case, we found that the resistant cells contained mutant proviruses in which the 3' gene could be expressed. Furthermore, we found that mutations were generated during a single round of virus replication (provirus to provirus) at a rate of approximately 0.5% mutations per cycle.

Animals↗
PubMed1984

Stimulation of in vitro transcription from heterologous promoters by the simian virus 40 enhancer.

Insertion of the simian virus 40 enhancer upstream from the +33 to -34 adenovirus major late promoter element or the +62 to -102 conalbumin promoter region causes a 10-fold stimulation of specific transcription using a whole cell extract but not an S100 extract. Many of the in vivo effects of the enhancer were mimicked in vitro. This stimulation occurred only in cis, with either orientation of the 72-base-pair repeat, and was markedly decreased by deletions known to diminish the enhancer activity in vivo. However, in vitro, the 72-base-pair repeat did not stimulate at a distance or in the presence of a strong upstream promoter element.

Adenoviruses, Human↗
PubMed1984

The effect of febrile temperatures on biologic actions of interferons: abrogation of suppression of delayed-type hypersensitivity and antibody production.

Interferons (IFN) have a complex immunoregulatory effect on all cells of the immune system. In most cases in which IFN had an enhancing effect, the suggested mechanism was inhibition of the generation or activity of suppressor cells. In the present study, we examined the effect of IFN on suppression of the delayed-type hypersensitivity (DTH) response. Suppression was induced with a low antigen dose of sheep erythrocytes (SRBC), and IFN was found to abrogate both the suppressed state and the transferability of this state. Cyclophosphamide had the same effect. However, the in vitro generation of suppressor cells was not altered by the addition of IFN to the culture medium at a normal temperature (37 degrees C). To reconcile the disparity between the successful anti-suppressive action of IFN in vivo compared with its failure in vitro, we considered the possibility that the pyrogenic action of IFN in vivo might create the optimal thermal environment for its anti-suppressive action. Indeed, when IFN was then tested in vitro at a febrile temperature (39.3 degrees C), it completely blocked the generation of suppressor cells. On the other hand, once suppressor cells were generated at 37 degrees C, IFN had no effect on their ability to suppress a fresh culture either at 37 degrees C or at 39.3 degrees C. IFN also had no effect on the generation of helper cells at either temperature, but help was greatly enhanced by high temperature alone. In vivo, we found our IFN preparation to be pyrogenic and observed that an anti-pyretic drug given before and during antigen stimulation abrogated the anti-suppressive effect of IFN. We suggest, therefore, that the febrile state induced by IFN promotes its action on suppressor cells.

Animals↗
PubMed1981

Studies with pure mouse Ehrlich ascites tumor interferons alpha and beta: patterns of induction of (2'-5') (A)n synthetase and of a double-stranded RNA-dependent protein kinase in mouse cells and human cells.

The N-terminal sequences of mouse Ehrlich ascites tumor cell IFN beta (35,000-40,000 daltons) and IFN alpha (20,000 daltons) differ in 18 out of 20 positions. Furthermore, these two IFN species show little immunological cross reactivity. We treated mouse L929 cells and human HeLa S3 cells with essentially pure mouse IFN alpha or IFN beta or both at various concentrations and for various lengths of time. From the treated cells we prepared extracts and compared in these the activities of (2'-5')(A)n synthetase, an enzyme that was earlier shown to be induced by partially purified IFN preparations. The effects of treatment of mouse L929 cells with pure IFN alpha or IFN beta on (2'-5') (A)n accumulation in the cell extracts were very similar both in respect to the dependence on the length of exposure of the cells to the IFNs and on IFN concentration. Treatment with both IFN alpha and IFN beta at concentrations resulting in only partial induction of the enzyme led to an additive rather than to a synergistic effect. The maximal level of enzyme induced was the same in cells treated with high concentrations of IFN alpha or IFN beta or both. Mouse IFN alpha was as active as IFN beta in inducing a double-stranded RNA-dependent protein kinase in mouse L cells. The treatment of HeLa S3 cells with IFN beta did not affect the accumulation of (2'-5') (A)n in their extracts whereas treatment with IFN alpha boosted the accumulation though to a lesser extent than in the case of mouse L cells. These results are in line with the finding that mouse IFN alpha can, but mouse IFN beta cannot convert HeLa S3 cells into the antiviral state and also with the more pronounced homology in N-terminal sequence between mouse IFN alpha and a human (lymphoblastoid) IFN chi than between mouse IFN beta and a human IFN beta.

2',5'-Oligoadenylate Synthetase↗
PubMed1980

Synthesis of (2'-5')(A)n from ATP. Characteristics of the reaction catalyzed by (2'-5')(A)n synthetase purified from mouse Ehrlich ascites tumor cells treated with interferon.

The treatment of Ehrlich ascites tumor cells with mouse interferon increases the level of the latent enzyme (2'-5')(A)n synthetase. If activated by double-stranded RNA, this catalyzes the synthesis from ATP of a series of 2'-5'-oligoadenylates: (2'-5')(A)n where n extends from 2 to about 15. We isolated (2'-5')(A)n synthetase in a homogeneous state. In the presence of double-stranded RNA, the purified enzyme can convert the large majority (about 97%) of the ATP into (2'-5')(A)n and pyrophosphate, although it does not cleave the pyrophosphate. The stoichiometry of the reaction can be formulated as: (n + I) ATP leads to (2'-5') pppA(pA)n + n pyrophosphate. Added pyrophosphate does not inhibit the synthesis of (2'-5')(A)n. The extent of the reverse reaction, i.e. the pyrophosphorolysis of (2'-5')(A)n, was below the level of detection under our conditions. The affinity of the enzyme for ATP is low: the rate of the reaction increases by about 10% when the concentration of ATP is increased from 5 mM to 10 mM. The optimal concentration of double-stranded RNA increases with the concentration of the enzyme. As tested at 0.4, 2, and 10 micrograms/ml of enzyme concentrations, close to maximal (2'-5')(A)n synthesis can be obtained if reovirus double-stranded RNA or poly(I) . poly(C) are used at about half the concentration (in w/v) of the enzyme. The plot of the reaction rate versus enzyme concentration is sigmoidal. It remains to be seen if this reflects on a cooperative behavior of the enzyme.

2',5'-Oligoadenylate Synthetase↗
PubMed1980

Interferon, double-stranded RNA, and RNA degradation. Isolation of homogeneous pppA(2'p5'A)n-1 synthetase from Ehrlich ascites tumor cells.

pppA(2'p5'A)n-1 ((2'-5')(A)n) synthetase is one of the mediators of interferon action. On activation by double-stranded RNA, it converts ATP into (2'-5')(A)n; in turn, (2'-5')(A)n activates an endonuclease (RNase L) which cleaves single-stranded RNA. We report a simple procedure for the isolation of pure (2'-5')(A)n synthetase from interferon-treated Ehrlich ascites tumor cells. The procedure involves differential precipitation of the ribosomal salt wash fraction with ammonium sulfate and chromatography on DEAE-cellulose and CM-cellulose. The apparent molecular weight of the enzyme is 105,000 as determined by gel electrophoresis in sodium dodecyl sulfate and about 85,000 when determined by centrifugation through a glycerol gradient. The size range of the (2'-5')(A)n produced by the enzyme extends from the dimer to at least the pentadecamer.

2',5'-Oligoadenylate Synthetase↗