In a previous paper, initiator and terminator trinucleotides were shown to stimulate sequentially the binding of f-Met-tRNA to ribosomes and the release of free f-methionine from the ribosomal intermediate.1 The formation of f-methionine was dependent upon a terminator codon, such as UGA, UAA, or UAG, and the release factor R discovered by Capecchi.2 The separation of R into two components is described in this report. R1 corresponds to the codons UAA and UAG; R2, to UAA and UGA. Methods.-R assay: The termination assay is described elsewhere.1 Each reaction contained the following components in a final volume of 50 Al: 0.05 M Tris-acetate, pH 7.2; 0.03 M magnesium acetate; 0.05 M potassium acetate; 4-6 AMmoles of the (f ['H]-Met-tRNAf ..AUG.-.Ribosome) complex (4.0-6.7 A/Amoles of TCA-precipitable f ['H]-Met-tRNAf, 0.02 A2W0 unit was present/reaction); 0.96 A260 unit-of E. coli B ribosomes (washed with 0.5 M ammonium chloride); 0.17 mumole of AUG; and, where indicated, 7-7.7 mInmoles of terminator trinucleotide, and an R preparation. Reactions were incubated for 16 min at 30°unless otherwise stated; hence, the rate of f [3H]-methionine formation was determined in all experiments. The preparation of tRNAfMet (E. coli B), fractionated by benzoylated-DEAE-cellulose column chromatography,' was the gift of Dr. Michael Wilcox. The tRNAfmet was acylated with ['H]-methyl-methionine (3.1 c/mmole, Schwarz BioResearch Corp.) and then converted to f['H]-Met-tRNAfMet.l Ribosomes were obtained from E. coli B as described by Lucas-Lenard and Lipmann,4 except that seven, rather than five, 0.5 M ammonium chloride washes were employed. R fractionation: Escherichia coli B supernatant and ribosome fractions, treated with DNase but not "preincubated," were prepared as described previously,5 except that cells were lysed with a French pressure cell at 18,000 psi, 0.005 M DTT replaced ,B-mercaptoethanol, and extracts were centrifuged for 5 hr at 137,000 X g. Release factors (prep. A, Table 1) were obtained as follows: R was precipitated by the addition of 116 gm of ammonium sulfate to 359 ml of the S-137 fraction; the pH was maintained at 7.8 by the addition of 0.5 N ammonium hydroxide. All steps were performed at 4°. The precipitate was collected by centrifugation at 30,000 X g for 15 min, dissolved in 139 ml of buffer A (0.05 M Tris-chloride, pH 8.0; 0.15 M potassium chloride; 0.001 M EDTA, adjusted to pH 7 with NaOH; and 0.003 M DTT) and dialyzed against 4 liters of buffer A for 4 hr (0-55% (NH4)2S04 fraction of Table 1). DEAE-Sephadex column: The 0-55% (NH4)2S04 fraction (138 ml, 2200 mg protein)

Genetic mapping has indicated that meiotic recombination occurs about 4 time more frequently in the dystrophin gene than expected on the basis of its length. To detect where recombinations occur within the gene, we have studied the CEPH families panel using highly polymorphic microsatellite markers located at the ends of the gene or flanking the major deletion hot spot in intron 44. We found a major hot spot of recombination between markers STR44 and STR50(1), i.e., between exons 44 and 51. Within this hot spot, a peak of recombination was located in the large intron 44. A second minor recombination prone region was found between DXS 206, (XJ, in the large intron 7) and the 5' end of the DMD gene. The distribution of the recombination events in the gene of healthy individuals was very similar to that of deletion breakpoints in DMD/BMD patients, suggesting that the two phenomenon may share a common mechanism. These results should also improve efficiency and accuracy of linkage analysis applied to carrier detection and prenatal diagnosis. In particular, if markers located at the very 3' end of the gene are not informative, the highly polymorphic ones located between exons 50 and 60 can be used instead of presently available extragenic markers, with a very low risk of diagnostic error due to recombination.

No abstract

We studied 5 boys, 2 to 10 years old, with marked or complete deficiency of hypoxanthine-guanine phosphoribosyltransferase and Lesch-Nyhan syndrome with varying degrees of mental retardation, dysarthria, chorea, dystonia, spasticity, and ataxia. Four patients had marked reduction of homovanillic acid in the cerebrospinal fluid (CSF) and all showed low CSF 3-methoxy-4-hydroxy phenylethylene glycol, indicating reduced dopamine and norepinephrine turnover. Three patients showed high CSF 5-hydroxyindoleacetic acid, suggesting increased serotonin turnover. Some patients improved with carbidopa-levodopa, but others benefited from tetrabenazine, a monoamine-depleting agent. This study provides support for the theory of abnormal central monoamine metabolism in Lesch-Nyhan syndrome.