A new allele of the human estrogen receptor (ER) gene has been identified; it was found in >10% of the samples studied. Sequence analysis revealed two point mutations; an Ala-to-Val change in codon 86 and a silent base change in codon 87. The Ala 86 is within a region of the protein (amino acids 80-89 in human) whose sequence is identical in human, rat and mouse, suggesting a conserved function in this region. Previous studies showed that, in women with ER positive breast tumors, individuals carrying the variant gene had a much higher incidence of spontaneous abortion than did women who were homozygous for the more common allele. Additional correlations with the variant genotype in the breast tumor samples include a lower estrogen receptor/progesterone receptor ratio and a higher progesterone receptor affinity for its steroid. Proposed studies will extend the information about the association of the variant gene and spontaneous abortion independent of breast cancer. Also, the association with other gynecological pathologies will be evaluated. Then, after determining whether there are other mutations in the B-variant allele, tests will be carried out in vitro to compare the function of the B-variant and wild type ER proteins. Here, transfection studies will assess the relative potencies of the B-variant and wild type ER as transcriptional activators of an ER-regulated gene known to be sensitive to deletions of surrounding the B region. Also, transfection of the B-variant and wild type ER will be used to measure the effect of the two alleles on progesterone receptor production and affinity for its steroid. Finally, B-variant and wild type ERs will be compared in assays testing the interaction of the receptor with other nuclear proteins. This combination of clinical and biochemical studies should provide significant new information linking a particular altered ER genotype with one or more gynecological abnormalities. NS25616 Clinical evidence strongly suggests that the thalamus contains critical substrates involved in the central representation of pain. The research proposed in this continuation application is based on the concept that the lamina I spinothalamic (STT) projection is an integral component of the system conveying pain and temperature sensation, and thus, that the identification and the analysis of its termination sites will provide deeper insight into the thalamic substrates involved in pain. Four projects are proposed that test the hypotheses stated under Specific Aims. (1) In the principal project the anatomical identification of lamina I STT terminations performed in the preceding grant period with the anterograde tracer PHA-L in the cat will be extended by identifying lamina I STT and nigemiriothalamic (TrT) terminations with PHA-L in the cynomolgus monkey. This will provide fumdamental information necessary to make reliable inferences relevant to the human thalamus. (2) The anatomical findings will be complemented by antidromically mapping the projections of single, functionally identified lamina I STT and TrT neurons in the cat and the monkey in order to differentiate the projections of nociceptive-specific, thermoreceptive-specific, multireceptive, and wide dynamic range lamina I cells (a collaborative project with J. O. Dostrovsky). (3) The PHA-L method will be used to obtain material that will enable ultrastructural examination (by A. Blomqvist, collaborator) of lamina I STT and TrT tenninations in the medial thalamic n. submedius (Sm) of the cat, to determine whether differences between nociceptive and thermoreceptive lamina I terminals that underly their physiological actions could be reflected in heterogeneity in the synaptology of lamina I terminals in Sm. (4) The endogenous immunoreactivity of PHA-L-labeled lamina I tertninations will (continue to) be examined with co-localization methods in order to determine if chemical markers can be found that reflect the functional selectivity of lamina I neurons and that would serve to selectively identify their termination sites.
