Mutagenesis of the conserved 51-nucleotide region of Sindbis virus.

We have constructed 25 site-specific mutations in a domain of 51 nucleotides in Sindbis virus that is highly conserved among all alphaviruses sequenced to date. These 51 nucleotides are capable of forming two hairpin structures and are found from nucleotides 155 to 205 in Sindbis virus within the region encoding nsP1. Of the mutations, 21 were silent and did not lead to a change in the amino acid sequence encoded. These silent mutations changed not only the linear sequence but also the stability of the hairpins in most cases. Two double mutants that were constructed led to the replacement of one base pair by another so that the linear sequence was altered but the nature of the hairpins was not. All of the mutants with silent mutations were viable, but 19 of the 21 mutants were severely impaired for growth in both chicken and mosquito cells. Compared with the parental virus, they grew slowly and produced virus at rates of 10(-1) to 10(-4) times the parental rate. Surprisingly, however, the plaques produced by these mutants were indistinguishable from those produced by the parental virus. Two of the silent mutations, found within the first hairpin structure, produced virus at a faster rate than the parental virus. It is clear that the exact sequence of this region is important for some aspect of virus replication. We suggest that one or more proteins, either virus encoded or cellular, bind to the hairpin structures in a sequence-specific fashion in a step that promotes replication of the viral RNA. Of the mutations that resulted in a change of coding, only one of four was viable, suggesting that the amino acid sequence encoded in this domain is essential for virus replication.     

The control of O6-methylguanine-DNA methyltransferase (MGMT) activity in mammalian cells: a pre-molecular view

Human peripheral blood lymphocytes (PBLs) can have a range of O⁶-methylguanine-DNA methyltransferase (MGMT) activities. PBLs from some individuals may have almost no MGMT activity. Such individuals have most often been subject to malignancy or to immunodeficiency disease. Long-term lymphoblastoid lines (LCLs) prepared from PBLs of normal subjects by Epstein-Barr virus (EBV) transformation have MGMT activities which are in general somewhat higher than the PBLs from which they derive. Such cultures are therefore generally MGMT-positive. Only in rare cases, and generally from patients with low MGMT activity, are freshly obtained lines with very low activity obtained. There is however a 4-fold range of MGMT activity over which multiple lines derived from the same PBL sample can be found. Long-term cultivation can lead to LCLs with low activity as well as to lines of high activity. On rare occasions an MGMT-positive line may, within a few divisions, give a negative line. Some (but not all) MGMT-negative (or very low) lines have been known to gain (some) activity. Chinese hamster ovary (CHO) cell lines are in general very low in MGMT activity. Lines of higher activity can be selected by treatment with mutagenic crosslinking alkylating agents. Chinese hamster lines with high MGMT activity can be obtained by transfection with human DNA from MGMT-positive cells. Lines with significant activity can also be obtained by transfection of CHO cells with human DNA from MGMT-negative (or very low) cells. Resistance to MNNG treatment can be acquired without the acquisition of significant MGMT activity. Crosses of lines of high and low MGMT activity give equivocal results. Hybrids of low × low activity have no activity. Crosses of positive × positive strains give varied results. It has not been possible to identify MGMT-positive hybrids as including one particular chromosome by this type of experiment. There is no evidence for a general adaptive effect on MGMT synthesis greater than the variation within the cell cycle.     

The development of the U.S. EPA health effects research laboratory frozen blood cell repository program

We have suggested that proper blood-cell freezing and storage is necessary in longitudinal studies with reduced between tests error, for specimen sharing between laboratories and for convenient scheduling of assays. Our present purpose is to describe the design and use of a prototype computer program, Cryovial, for data-based management of frozen cell repositories. We are pleased to provide, free of charge upon request, a 5.25" (specify 0.36 or 1.2 MB) disk containing all files required to run Cryovial.     

Hypochlorite-Modified Adenine Nucleotides: Structure, Spin-Trapping, and Formation by Activated Guinea Pig Polymorphonuclear Leukocytes

Adenosine and its nucleotides react with hypochlorite to form unstable products that have been identified as the N⁶ chloramine derivatives. These chloramines spontaneously oligomerize. form stable adducts with proteins and nucleic acids, and are converted with loss of chlorine to the original nucleoside or nucleotide by reducing agents. The chloramines are associated with a free radical. and the spin-trapping of adenosine chloramine with 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) yielded a mixture of unstable nitroxyl adducts that corresponded to nitrogen-centered radicals from the parent nucleoside. When activated guinea pig polymorphonuclear leukocytes were stimulated with phorbol myristate acetate to produce hypochlorite, they actively incorporated [¹⁴C] adenosine into acid-insoluble products by a process that was dependent on oxygen and inhibited by azide and thiols. These findings suggest that adenine nucleotide chloramines are generated by activated phagocytic cells and form ligands with proteins and nucleic acids as observed in model systems. The results imply that nucleotide chloramines are among the cytotoxic and possibly mutagenic factors that are associated with the icflammatory process.     

The Action Of Sulphur As A Fungicide And As An Acaricide

The conclusion arrived at by chemical methods, that the volatile agent produced when sulphur is applied to a heated surface is gaseous sulphur, has been subjected to biological tests in which the fungi Ery-siphe graminis and Sphaerotheca Humulind the gall mite Eriophyes ribis were employed.
The fungi were found not to be sufficiently sensitive to yield satisfactory and concordant results and strong support is given to the view that actual contact of the sulphur particle with the fungus is necessary before fungicidal action can occur.
It was shown that the agent present in air passed over heated sulphur and responsible for the death of the gall mite was not removed by filtration through a heated glass-wool plug, this observation being contrary to the view that the toxic agent is produced initially in solid form.
Filtration through a cooled glass-wool plug only removed part of the volatile agent and it was shown that the gall mite is affected by the traces of sulphur volatilised at ordinary temperatures.
The results of the experiments with the gall mite were in complete accord with those obtained in the previous chemical work.
At relatively large concentrations sulphur dioxide and hydrogen sulphide are without permanent effect upon the gall mite and these gases are therefore not responsible for the acaricidal action of sulphur.