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Biomedical subjects

L A Ball

Publications and source records attributed to L A Ball.

At least 73 records · Page 4Linked to original sources
PubMed1982

Coding assignments of the five smaller mRNAs of Newcastle disease virus.

The polypeptide coding assignments for the five messengers of the 18S size class of Newcastle disease virus (NDV) RNA have been determined by cell-free translation of individual RNAs separated by gel electrophoresis. Listed in order of their decreasing electrophoretic mobilities in acid agarose-urea gels, the coding assignments of the RNAs were as follows: RNA 1, M protein; RNA 2, P protein; RNA 3, NP; RNA 4, F glycoprotein; and RNA 5, HN glycoprotein. RNA 2 also directed the synthesis of 33- and 36-kilodalton proteins, which were tentatively identified as being overlapping segments of the P protein. The 33- and 36-kilodalton polypeptides could be detected in infected cells, but not in purified virions of NDV. Since the other unique NDV RNA, a 35S species, has been shown previously to encode the viral L protein, these results complete the coding assignments of the six known NDV mRNAs.

Animals↗
PubMed1982

Induction and maintenance of 2',5'-oligoadenylate synthetase in interferon-treated chicken embryo cells.

Treatment of primary cultures of chicken embryo cells with homologous interferon results in a substantial increase in the level of 2',5'-oligoadenylate synthetase activity that can be detected in cell extracts. This increase can be prevented by inhibitors of RNA or protein synthesis and is thus thought to represent the induction of an interferon-inducible gene, perhaps the 2',5'-oligoadenylate synthetase gene itself. To examine this response in greater detail, we studied its kinetics under the following conditions: (i) cessation of interferon treatment after different lengths of time, (ii) delayed inhibition of RNA or protein synthesis, and (iii) combinations of these treatments. The results showed that in cells treated continuously with interferon, the enzyme level reached a peak after 9 h of treatment and then decreased with a half-life of about 30 h, despite the continued presence of interferon. Removal of interferon during induction reduced the peak level of activity that was attained and somewhat accelerated its decline but did not otherwise affect the time-course of the response. On the other hand, removal of interferon after maximum induction clearly accelerated the decay of enzyme activity. This process could be delayed by inhibitors of protein synthesis, which effectively stabilized the induced enzyme. This behavior is reminiscent of other inducible enzymes, such as the steroid-induced tyrosine aminotransferase, and suggests that the level of 2',5'-oligoadenylate synthetase, which is also inducible by steroid hormones in some cell types, is subject to similar control mechanisms.

2',5'-Oligoadenylate Synthetase↗
PubMed1981

Translation of black beetle virus RNA and heterologous viral RNAs in cell-free lysates derived from Drosophila melanogaster.

A cell-free protein synthesizing system was prepared from cells of Drosophila melanogaster line 1 and made mRNA dependent by treatment with micrococcal nuclease. The system was tested with homologous RNA from black beetle virus propagated in Drosophila cells, with Drosophila heat shock mRNA, and with various heterologous viral mRNA's. Under optimal conditions amino acid incorporation programmed with black beetle virus RNAs was 30-fold higher than endogenous incorporation. RNAs 1 and 2 primarily directed the synthesis of proteins with approximately molecular weights of 120,000 and 46,000, respectively. mRNA's, prepared by transcription from vesicular stomatitis virus or vaccinia virus, were translated efficiently and yielded products that comigrated with authentic viral proteins. Brome mosaic virus RNA and encephalomyocarditis virus RNA were translated poorly. The system retained full activity after freezing.

Animals↗
PubMed1981

Control of expression of the vaccinia virus thymidine kinase gene.

mRNA extracted from vaccinia virus-infected cells early after infection directs cell-free synthesis of enzymatically active viral thymidine kinase (Hruby and Ball, Virology, in press). We used this assay for a specific vaccinia virus mRNA to study the induction and repression of the viral thymidine kinase gene during infection of thymidine kinase-deficient L-cells. As observed previously by other workers, the synthesis of thymidine kinase occurred immediately after infection but was switched off after 4 h later. We observed similar kinetics of accumulation and shutoff under conditions where viral DNA synthesis and late gene expression were inhibited. Cell-free translation of mRNA from infected cells showed that the concentration of functional message for viral thymidine kinase reached a peak 3 to 4 h after infection and then decreased with a half-life of about 1 h. These kinetics indicated that significant levels of thymidine kinase mRNA persisted in cells which had stopped synthesizing the enzyme. Under conditions where late gene expression was inhibited, high concentrations of functional mRNA could be isolated from cells at late times after infection. On the basis of these results, we conclude that the repression of thymidine kinase expression is mediated at the translational level by one or more early or delayed early viral genes. Repression is accompanied by, but does not depend on, the inactivation or degradation of thymidine kinase mRNA, which is a late gene function.

Animals↗
PubMed1980

Induction, purification, and properties of 2'5' oligoadenylate synthetase.

The results presented above relate to several aspects of the 2-5A system. A simple but insensitive radiochemical assay fro 2-5A and its synthetase is described. Progress towards the molecular characterization of the synthetase suggested that it is composed of a single 56,000 dalton polypeptide chain that is synthesized in response to IF treatment. Degradation of 2-5A occurs at the same rapid rate in extracts of IF-treated and untreated chick cells. However, this breakdown can be inhibited by its end-product, 5'AMP, or by the activated synthetase which can further elongate 2-5A and thereby protect it from degradation. The direction of elongation is from the 5' to the 2' terminus. Molecules other than 2-5A can act as substrates fro 2'-adenylation by the activated synthetase. These include some dinucleoside monophosphates, ADP-ribose and NAD+, and methylene-bridged analogues of ATP. The methylene-bridged analogues of 2-5A that are synthesized in the latter case retain some of the biological activity of authentic 2-5A, indicating that cleavage of the 5'-terminal phosphate group(s) is not involved in the mechanism of nuclease activation by 2-5A.

2',5'-Oligoadenylate Synthetase↗
PubMed1980

Transcriptional map for Newcastle disease virus.

A transcriptional map of Newcastle disease virus was determined by measuring the kinetics of UV inactivation of the transcription of individual genes and of viral infectivity. The inactivation of single genes was monitored by measuring the reduction in the accumulation of viral gene products in vivo and in vitro. In vivo, the accumulation of viral polypeptides in infected cells was measured after reversal of a cycloheximide treatment designed to inhibit secondary transcription. Actinomycin D and a hypertonic medium were used to decrease selectively the synthesis of host cell polypeptides in infected cells. In vitro, mRNA's synthesized by irradiated viruses were analyzed by translation in cell-free systems under conditions in which the amount of each polypeptide synthesized reflected the relative abundance of the corresponding mRNA. UV target sizes were obtained for the genes coding for the HN, F0, NP, M, L, and P polypeptides; the 47,000-dalton protein was not detected. A comparison of the UV target sizes with the corresponding gene sizes suggested that transcription of these genes initiated at a single promotor and proceeded in the order NP, P, (F0, M), HN, L. These experiments were performed with Newcastle disease virus strains Australia-Victoria and B1-Hitchner; for both strains, two forms of the P polypeptide which differed in electrophoretic mobility were detected. Proof that the P protein is virus specific was obtained. In addition, infection of chicken embryo cells with avirulent strain B1-Hitchner enhanced the accumulation of at least four polypeptides that appeared to be specified by the host cell rather than by the infecting virus.

Genes, Viral↗
PubMed1978

Oligonucleotide inhibitor of protein synthesis made in extracts of interferon-treated chick embryo cells: comparison with the mouse low molecular weight inhibitor.

Cytoplasmic extracts of interferon-treated primary chick embryo cells contain an enzyme activity that synthesized an inhibitor of chick cell-free protein synthesis. The same activity was detected in extracts of cells treated with mock preparations of interferon, but at <0.3% of the level found in interferon-treated cell extracts. The enzyme was activated by double-stranded RNA and could be isolated by binding to columns of poly(I)-poly(C)-agarose. In the column-bound state, the enzyme reacted with ATP to synthesize the inhibitor, which could then be continuously eluted from the column. The inhibitor was purified and its structure and function were compared with those of the low molecular weight inhibitor of protein synthesis made by an enzyme from interferon-treated mouse L cells. The avian and mammalian inhibitors comigrated on thin layers of polyethyleneimine-cellulose during chromatography in three different solvent systems, and they coeluted as a series of peaks from columns of DEAE-cellulose during sodium chloride gradient elution. Digestion with bacterial alkaline phosphatase or snake venom phosphodiesterase yielded products that similarly comigrated. Functionally, the two inhibitors were interchangeable: both inhibited protein synthesis in extracts of mammalian and avian cells, producing 50% inhibition at a concentration of about 0.3 nM (AMP equivalents). We conclude that the chick cell-derived oligonucleotide inhibitor has a structure that is closely related or identical to that of the inhibitor made in the mouse system, and that both preparations inhibit cell-free protein synthesis in a non-species-specific manner.

Adenosine Triphosphate↗
PubMed1978

Synthesis and breakdown of pppA2'p5'A2'p5'A and transient inhibiton of protein synthesis in extracts from interferon-treated and control cells.

Incubation of the mouse L-cell-free system with a concentration of pppA2'p5'A2'p5'A [(2'-5')An] just sufficient to inhibit protein synthesis results in formation of a high-molecular-weight, heatlabile inhibitor and enhanced ribonuclease activity and in the rapid breakdown of (2'-5')An to ATP. The (2'-5')An-enhanced ribonuclease activity is also unstable and in the absence of a (2'-5')-An-regenerating system inhibiton of protein synthesis is transient. Although interferon treatment enhances the synthesis of (2'-5')An, the rates of degradation of (2'-5')An and levels of activatible nuclease are similar in extracts prepared from control or interferon-treated cells. Interestingly, the sensitivity of different cell-free systems to (2'-5')An, varies with the source of the cell-free systems and with the methods used in their preparation. There is, however, no obvious correlation between the sensitivities of the system and the rate of breakdown of (2'-5')An. The significance of these results is discussed in relation to a possible control function for the (2'-5')An system in both interferon-treated and control cells.

Adenine Nucleotides↗
PubMed1978

Transcription and translation of Newcastle disease virus mRNA's in vitro.

Transcription directed in vitro by Triton-activated Newcastle disease virus (NDV) was stimulated and prolonged by the presence of cytoplasmic extracts of animal cells. The RNA products closely resembled those of NDV transcription in vivo by several criteria: binding to oligodeoxythymidylic acid-cellulose, the mobility and relative abundance of each major band resolved by polyacrylamide gel electrophoresis, and the ability to direct the accurate cell-free synthesis of polypeptides corresponding to the NDV proteins HN, F0/F1, NP, and M. Synthesis of a novel polypeptide related to NP but of higher apparent molecular weight was also detected. These results indicated that cell-free transcription under these conditions was a close facsimile of NDV transcription in vivo. In addition, both in vitro and in vivo, NDV polypeptides were synthesized in nonequimolar amounts which reflected the order of the genes in the transcriptional map: NP, F0, M, (47K, HN), L. Strains AV and HP, virulent strains which have differences in biological activities, exhibited differences in the polypeptides synthesized in infected cells and in cell-free systems.

Cell-Free System↗