Relatedness threshold for the production of female sexuals in colonies of a polygynous ant, Myrmica tahoensis, as revealed by microsatellite DNA analysis.

The genetic relationships of colony members in the ant Myrmica tahoensis were determined on the basis of highly polymorphic microsatellite DNA loci. These analyses show that colonies fall into one of two classes. In roughly half of the sampled colonies, workers and female offspring appear to be full sisters. The remaining colonies contain offspring produced by two or more queens. Colonies that produce female sexuals are always composed of highly related females, while colonies that produce males often show low levels of nestmate relatedness. These results support theoretical predictions that workers should skew sex allocation in response to relatedness asymmetries found within colonies. The existence of a relatedness threshold below which female sexuals are not produced suggests a possible mechanism for worker perception of relatedness. Two results indicate that workers use genetic cues, not queen number, in making sex-allocation decisions. (i) The number of queens in a colony was not significantly correlated with either the level of relatedness asymmetry or the sex ratio. (ii) Sex-ratio shifts consistent with a genetically based mechanism of relatedness assessment were seen in an experiment involving transfers of larvae among unrelated nests. Thus workers appear to make sex-allocation decisions on the basis of larval cues and appear to be able to adjust sex ratios long after egg laying.

Mutational analysis of the conserved region 2 site of adenovirus E1A and its effect on binding to the retinoblastoma gene product: use of the "double-tagging" assay.

We have explored the feasibility of using a "double-tagging" assay for assessing which amino acids of a protein are responsible for its binding to another protein. We have chosen the adenovirus E1A-retinoblastoma gene product (pRB) proteins for a model system, and we focused on the high-affinity conserved region 2 of adenovirus E1A (CR2). We used site-specific mutagenesis to generate a mutant E1A gene with a lysine instead of an aspartic acid at position 121 within the CR2 site. We demonstrated that this mutant exhibited little binding to pRB by the double-tagging assay. We also have shown that this lack of binding is not due to any significant decrease in the level of expression of the beta-galactosidase-E1A fusion protein. We then created a "library" of phage expressing beta-galactosidase-E1A fusion proteins with a variety of different mutations within CR2. This library of E1A mutations was used in a double-tagging screening to identify mutant clones that bound to pRB. Three classes of phage were identified: the vast majority of clones were negative and exhibited no binding to pRB. Approximately 1 in 10,000 bound to pRB but not to E1A ("true positives"). A variable number of clones appeared to bind equally well to both pRB and E1A ("false positives"). The DNA sequence of 10 true positive clones yielded the following consensus sequence: DLTCXEX, where X = any amino acid. The recovery of positive clones with only one of several allowed amino acids at each position suggests that most, if not all, of the conserved residues play an important role in binding to pRB. On the other hand, the DNA sequence of the negative clones appeared random. These results are consistent with those obtained from other sources. These data suggest that a double-tagging assay can be employed for determining which amino acids of a protein are important for specifying its interaction with another protein if the complex forms within bacteria. This assay is rapid and up to 1 x 10(6) mutations can be screened at one time.