The RP14 autosomal recessive Retinitis pigmentosa (arRP) locus has been mapped to a 2cM region of chromosome 6p21.3. TULP1 (the gene encoding tubby-like protein 1) is a candidate target for the disease mutation because it maps to the RP14 minimum genetic region and because a mutation in the highly homologous mouse tub gene leads to obesity, deafness and early progressive retinal degeneration. Here we report a splice-site mutation (IVS14+1, G-->A) that is homozygous in all affected individuals (N=33) and heterozygous in all obligate carriers (N=50) from two RP14-linked kindreds. The mutation was not observed in 210 unrelated controls. The data indicate that impairment of TULP1 protein function is a rare cause of arRP and that the normal protein plays an essential role in the physiology of the retina.

1. To identify the type(s) and properties of inhibitory postsynaptic receptor(s) involved in synaptic transmission in cultured rat embryonic spinal cord and medullary neurons, we have used whole cell patch-clamp techniques to record miniature inhibitory postsynaptic currents (mIPSCs) in the presence of tetrodotoxin, DL-2-amino-5-phosphonovaleric acid, and 6-cyano-7-nitroquinoxaline-2,3-dione. 2. The mIPSCs recorded from both spinal cord and medullary neurons had skewed amplitude distributions. 3. The glycinergic antagonist strychnine and the GABAergic antagonist bicuculline each decreased both the frequency and mean peak amplitudes of mIPSCs. We conclude that both glycine and gamma-aminobutyric acid (GABA) are neurotransmitters at inhibitory synapses in our cultured cells. 4. Most (approximately 96-97%) mIPSCs decay with single-exponential time constants, and decay time distributions were consistently best fitted by the sum of four Gaussians with decay constants as follows: D1 = 5.8 +/- 0.1 (SE) ms (n = 63), D2 = 12.2 +/- 0.2 ms (n = 61), D3 = 23.2 +/- 0.4 ms (n = 54), and D4 = 44.7 +/- 1.0 ms (n = 57). We conclude that the four classes of decay times represent kinetically different inhibitory postsynaptic receptor populations. 5. Strychnine and bicuculline usually had one of two different effects on the mIPSC decay time constant distributions; either selective decreases in the frequency of mIPSCs with decay times in certain classes (i.e., the D1 class was reduced by bicuculline, the D2 class by strychnine, and the D3 and D4 classes by both antagonists) or a nonselective depression in the frequency of mIPSCs with decay times in all four classes. The particular effect observed in a given neuron was correlated with the presence or absence of ATP and guanosine 5'-triphosphate (GTP) in the patch pipette. Namely, in 71% of the antagonist applications where the pipette contained ATP and GTP, the result was a nonselective decrease in mIPSCs in all decay time constant classes. Conversely, in 54% of the antagonist applications in their absence, the result was a selective decrease in the frequency of mIPSCs in specific decay time constant classes. 6. In some experiments, mIPSCs reappeared in antagonist solution after an essentially complete block. Recovery from block in the continued presence of antagonist was never observed in the absence of ATP and GTP (8 neurons), and, at the same time, 5 of 9 neurons patched with ATP and GTP in the pipette did show recovery (56%).

Numerous biochemical techniques have been employed to characterize the covalent inhibitory ternary complex of thymidylate synthetase consisting of enzyme, 5-fluorodeoxyuridylate, and 5,10-methylenetetrahydrofolate. 19F NMR studies of this covalent ternary complex reveal a single, broad resonance centered at 12.7 ppm to higher shielding of free nucleotide, while the 5-fluorodexyuridylate-enzyme binary complex exhibits two resonances to higher shielding of free nucleotide, one at 1.4 ppm representing noncovalently bound ligand and the other at 34.5 ppm indicative of covalently bound 5,6-dihydro-5-fluorodeoxyuridylate. In order to follow the transformation of the latter binary complex to a ternary complex, we have employed 19F NMR to profile changes in the environment of the nucleotide which result from the interaction of folates with the coenzyme binding site. At low molar excesses of folates (5-fold), the effects observed in the 19F NMR spectrum fall into three major classes. (1) 5-Methyltetrahydrofolate exhibited a weak interaction with the binary complex. (2) Methotrexate and aminopterin, antifolate drugs, were observed to increase the exchange rate among the species detected in the 19F NMR spectrum of the binary complex. (3) Folate, dihydrofolate, and a series of tetrahydrofolate derivatives were found to shift the equilibrium of the binary complex toward the covalent 5,6-dihydro-5-fluorodeoxyuridylate-enzyme complex. With the latter folates the chemical shifts for the covalent species of these ternary complexes were found in the range of 35-40 ppm to higher shielding and are interpreted to reflect subtle differences in the strength and steric nature of the interaction of the folate ligand with the binary complex. These data illustrate that the latter folates promote the conversion of the enzyme-bound nucleotide to a species which would be poised to form the second covalent bond of the ternary complex, namely the linkage of the methylene group of the coenzyme with carbon 5 of the nucleotide.