Differential influence of nucleoside analog-resistance mutations K65R and L74V on the overall mutation rate and error specificity of human immunodeficiency virus type 1 reverse transcriptase.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) variants with the K65R or L74V substitution display resistance to several nucleoside analogs. An in vitro dNTP exclusion assay revealed an increased fidelity for K65R RT compared with wild-type RT, but little change for L74V RT. When the forward mutation rates were measured via a gap-filling assay, the K65R variant displayed an 8-fold decrease in the overall mutation rate (1.0 x 10(-3) versus 8.6 x 10(-3) for wild-type HIV-1 RT), whereas the rate for the L74V variant was closer to that for wild-type RT (5.0 x 10(-3)). The increase in overall fidelity observed for K65R RT is the largest reported for any drug-resistant HIV-1 RT variant. Nucleotide sequence analysis of lacZalpha mutants generated by variant RTs indicated that K65R RT displays uniform reduction in most types of errors, whereas L74V RT does not. Modeling the substitutions into the x-ray structure of the ternary complex revealed that the major influence of Leu(74) in stabilizing the templating base is unaffected by Val substitution, whereas the K65R substitution appears to increase the stringency of dNTP binding. It is speculated that the increased fidelity of K65R RT is due to an altered interaction with the dNTP substrate.

The influence of 3TC-resistance mutations E89G and M184V in the human immunodeficiency virus reverse transcriptase on mispair extension efficiency.

Two nucleoside analog resistance mutations in HIV-1 reverse transcriptase (RT), E89G and M184V, were previously shown to increase the dNTP insertion fidelity of HIV-1 RT. However, forward mutation assays using a lacZ alpha reporter gene have revealed a lack of impact on the overall error rate of these variants. In an effort to investigate the basis for this discrepancy, we have examined whether the increases in misinsertion fidelity observed for E89G and M184V RTs are accompanied by an increase in mispair extension fidelity. The relative efficiencies with which the wild type, E89G, M184V and M184V/E89G HIV-1 RTs extend model template-primer duplexes containing 3'-OH terminal mismatches were measured. The calculated efficiencies of mispair extension ( f ext) were, in general, not significantly decreased from the wild type HIV-1 RT. In fact, the efficiency of extension from one of the mispaired primer-template duplexes was significantly increased for two of the mutants tested. These results suggest that amino acid substitutions that increase the fidelity of dNTP insertion do not necessarily increase misextension fidelity, and that the decreased misextension fidelity may counterbalance the increases in misinsertion fidelity observed for E89G and M184V RTs.

Evidence for repair in trypan blue-treated mouse egg cylinders: an electron microscopic study.

The question of whether mammalian fetuses can correct damage induced by teratogens during early stages of embryogenesis was reinvestigated. Toward this purpose female mice were injected on day 6 of gestation with the teratogenic dye trypan blue. Within 6, 24, and 48 hr of exposure to the teratogen, egg cylinders were removed, sectioned, and prepared for electron microscopic analysis. The following organelles were examined: 1) mitochondria, 2) Golgi apparatus, 3) endoplasmic reticulum, and 4) free polysomes. On the ultrastructural level, exposure to trypan blue lead within 6 hr to fragmentation of the endoplasmic reticulum and a depletion of free polysomes in the endoderm of the egg cylinders. In ectodermal cells only the distribution of polysomes was disturbed following exposure to trypan blue. Egg cylinders harvested 24 hr after injection of trypan blue had partially recovered. Their endoplasmic reticulum and polysomes appeared closer to controls. The cells of both germ layers of most egg cylinders obtained 48 hr after injection were indistinguishable from controls when viewed with the electron microscope. No consistent changes were found in mitochondria or Golgi apparatus following trypan blue treatment. It is concluded that mouse embryos appear to be able to correct damage sustained during the egg cylinder stage, and that in spite of earlier injury affecting both germ layers such egg cylinders can develop normally as revealed by microscopic examination.

Development of home orientation in hypothyroid and hyperthyroid rat pups.

In order to assess the effects of perinatal hypothyroidism or hyperthyroidism on the development of an integrated behavioral response, we tested hypothyroid, hyperthyroid, and control pups, as well as pups receiving thyroxine replacement therapy, for the development of the home orientation response. Hypothyroidism was induced in the pups by feeding the pregnant or lactating female a diet of .2% propylthiouracil from Day 15 of gestation to Day 22 postpartum. Pups receiving replacement therapy and pups made hyperthyroid were injected daily with thyroxine, starting at birth. The ability of the pups to initiate and maintain locomotion toward the nest was assessed between Days 4 and 22. Hyperthyroid, control, and replacement therapy pups behaved very similarly on the task, showing a peak in the percentage of pups homing between Days 12 and 16. Hypothyroid pups showed a delay in the peak percentage until Day 20, although the percentage of pups was similar to that found in other treatments. An integrated behavioral response can be delayed by hypothyroidism and still emerge apparently intact at a later age.

Is the holocaust relevant to sociobiology?

Sociobiologists have emphasized that altruism and benevolent behavior are part of the genetic repertoire of most animals and certainly of man. They have constructed a theory of "ethics" as a biological phenomenon without reference to the concept of evil. It is concluded here however, that holocaust behavior is not equivalent to the "natural manifestation of an incompletely tamed animal flashing its teeth." Biologists have been too rigid in trying to equate "ethical behavior" with "social behavior." The added dimension of ethical behavior is a special kind of sensitivity to the needs of others, just as evil is the total lack of it. The evolution of this moral sense may itself have important selective value for the human species, whose survival depends on creating maximal diversity in its gene pool.

Some additional observations relating to the mechanism of trypan blue induced teratogenesis.

The mechanism and site of teratogenic action of trypan blue on mammalian embryos was reinvestigated. The experiments to be presented include (1) an analysis of the effect of trypan blue treatment on the morphology of the early mouse egg cylinder, (2) a demonstration of the effect of dye treatment on the enzyme acid phosphatase of yolk sac epithelium using histochemical procedures. Results obtained from these experiments indicate that trypan blue injected into mothers on day 7 of gestational age leads, within 12 to 24 hours after treatment, to dramatic abnormalities in 90-95% of egg cylinders examined. The frequency of gross malformations obtained by this treatment is considerably less when litters are examined at later stages of gestation. Acid phosphatase activity in yolk sac epithelium is depressed by the dye treatment, but there is no difference between enzymatically depressed yolk sacs of malformed embryos and yolk sacs surrounding normally appearing litter mates both obtained from trypan blue treated mothers. The hypothesis that trypan blue may exert its teratogenic effect by the direct exposure of egg cylinder stages to the dye, and that some of the egg cylinders affected may subsequently repair, is recommended for further testing.