In preliminary experiments with Chironomus eggs, a bioassay for anterior determinant activity has been established. The activity is necessary for head and thorax formation. It is localized anteriorly in the egg and is associated with a relatively small (250-500nt) cytoplasmic poly(A)+RNA. More narrowly defined size fractions containing the activity will be isolated by gel electrophoresis and used to prepare a size-restricted cDNA library. If sense RNA transcribed in vitro from this entire library is active in the bioassay, the library will be subdivided progressively until individual sequences with anterior determinant activity are identified. Alternatively, the bioassay will be combined with a hybrid-depletion procedure. The principle of this procedure is to selectively remove certain RNA sequences from a biologically active complex mixture of RNA molecules. This is accomplished by solution hybridization with cloned cDNA and subsequent digestion with RNase H. These steps have been tested in preliminary experiments and can be carried out with batches of several hundred cDNA clones at a time. If the depleted RNA mixture is no longer active, then the previous activity must have resided in one or more of the removed RNA sequences. These can then be identified by progressive subdivision of the batch of cDNA clones used for depletion. The Chironomus RNA sequences identified as anterior determinants will be compared to similar sequences in other organisms, in particular to known Drosophila transcripts involved in defining the anteroposterior polarity. %%% It appears that the earliest information about the polarity of an organism in laid down in the egg cytoplasm. By the time an egg is fertilized, it is already determined which will be the head and tail end of the organism. Dr. Kalthoff is exploring how this positional information is laid out in the egg and what molecules contain this information. He is using insects for the model system in which to carry out this investigation due to the fact that there are mutants available in insects which affect this aspect of pattern formation.
