MOLECULAR GENETICS OF THE XG BLOOD GROUP SYSTEM

Gene activity is regulated at multiple levels. Thus, tissue-specific
gene regulations is controlled at a level above the gene promoter by
interactions between cis-acting DNA elements (e.g., enhancers) with
factors (e.g., transcription factors) that activate or repress gene
expression. In contrast, what may be called domain gene regulation acts
at a higher level, to control the expression of multiple contiguous
genes. X-chromosome inactivation is perhaps the best known and most
dramatic example of this level of regulation. The XG blood group is chosen here as a model system for the study of
domain gene regulation: two tightly linked genes on the X chromosome are
known to be co-regulated, XG and MIC2; both genes escape X-chromosome
inactivation; they are coordinately expressed in red blood cells. The
experiments proposed are designed to characterize a recently isolated
gene, BM22, that we consider to be an excellent candidate for being the
XG structural locus. We propose further to clone and to characterize the
cis-acting regulatory element XGR that is postulated to control the
coordinate expression of XG and MIC2. Our specific experimental aims are
the following: In one set of experiments, we propose to analyze the RNA expression of
BM22 in adult venous and umbilical cord bloods from Xg(a+) and Vg(a-)
individuals employing a quantitative RT-PCR method; to raise antibodies
to BM22 and determine whether they react differentially to Xg(a+) and
Xg(a-) red cells; and to transfect BM22 into cultured cells and determine
whether Xg antibodies react differentially to transfected untransfected
cells. In the second set, we propose to localize the postulated regulatory locus
XGR by analysis of DNA polymorphisms segregating in several unusual
families in which recombination between XGR and MIC2 is known to have
occurred; to employ S1 nuclease protection assays on RNA from transfected
cells to detect differential transcriptional activation of the BM22 and
MIC2 promoters in reporter gene constructs into which the XGR elements
from either homozygous Xga or homozygous Xga individuals have been
subcloned; and to sequence the XGR elements from homozygous Xga and
homozygous Xg individuals and determine by transfection studies whether
mutations that convert each element into the alternate allelic form can
cause a switch in the differential transcriptional activation of the BM22
and MIC2 promoters.