We have developed a new method which measures the rate of synthesis, processing and degradation of messenger RNAs. Heavy isotopes are used to density-label newly-synthesized RNA. The labelled RNA is then physically separated from pre-existing RNA by equilibrium density-gradient centrifugation. The sequences present in this RNA are then amplified, identified and quantitated using the polymerase chain reaction (PCR) or by in vitro translation followed by 2-D gel electrophoresis and computer-aided quantitation. Using these methods, the rate of accumulation and degradation of specific mRNAs and their precursors can be measured. This approach will be used to answer several questions about the control of mRNA metabolism in the lens. These studies will provide information about the mechanisms responsible for the dramatic accumulation of crystallin mRNAs and protein in lens fibers. Because the accumulation of lens crystallins is responsible for the high refractive index and transparency of the lens, this information will be directly related to the factors responsible for normal lens function and cataract formation. Recent studies have identified DNA sequences that appear to play important roles in targeting gene expression to the lens. However, these studies have not told us why these genes are expressed in such abundance in fibers, but at lower or undetectable levels in lens epithelial cells. We will determine the regulatory mechanism(s) responsible for the differential accumulation of crystallin mRNAs in lens epithelial cells and fibers. We will also determine whether the stability of the mRNAs which accumulate in lens fibers is due to sequences present in the mRNAs themselves, or is simply due to the lack of RNA-degrading enzymes in the fiber cell cytoplasm. If lens-specific mRNAs have an intrinsic stability, it will be possible, in the future, to identify the sequence elements responsible for this stability. Finally, we will determine whether different mRNAs are produced during the process of lens fiber cell maturation. The transcripts produced during normal fiber cell maturation will be compared with those found in the fiber cells of transgenic animals which have congenital cataracts. This will provide information on the signals which control the end stages of fiber formation and the defects that occur when this process in disrupted during cataract formation.
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