The relationship of valine resistance .to the expression of the ilvGEDA operon of Escherichia cob K-12 has been determined. DNA sequence and in vivo protein analyses indicate that in wild-type E. coli K-12 there is a frameshift site within the gene (ilvG) for valine resistance. The ilvG+2096 (formerly designated ilv02096) mutation displaces this frameshift site, resulting in the~expression of ilvG and the relief of transcriptional-polarity on the distal genes of this operon. Thus, the "ilvO" mutation, which concomitantly confers valine resistance and increased expression of the ilvEDA genes, is, in fact, the "reversion" of a polar site within the first structural gene-of the.ilvGEDA operon.

The complete nucleotide sequence has been determined for a Kgtll cDNA done (XAG18) containing the full-length coding region for the mature lysosomal form of human a-galactosidase A (a-Gal A; EC 3.2.1.22). The XAG18 insert contained a 1226-base-pair sequence with an open reading frame encoding 398 amino acids of the mature polypeptide (predicted Mr = 45,356) and the last 5 amino acids of the propeptide sequence. The poly(A) signals AATACA and ATTAAA occurred 28 and 11 nucleotides prior to the TAA stop codon, respectively. There was no 3' untranslated region as the poly(A) sequence immediately followed the TAA termination codon; a second independently cloned cDNA confirmed this finding. The predicted amino acid sequence was colinear with 86 nonoverlapping residues (22% of the mature subunit) determined by microsequencing amino-terminal, tryptic, and cyanogen bromide peptides of the purified mature enzyme. Four potential N-glycosylation sites were identified, all of which occurred at predicted (3 turns in hydrophilic regions of secondary structure. RNA transfer hybridization analysis of HeLa poly(A)+ RNA demonstrated a single 1.45-kilobase band whose signal was decreased by prior immunoabsorption of polysomes with monospecific a-Gal A antibodies. Searches of nucleic acid and protein data bases did not reveal significant homology even with the limited sequences available for mammalian lysosomal enzymes.Human a-galactosidase A (a-D-galactoside galactohydrolase; EC 3.2

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Forty-four sulfa drugs were screened against crude preparations of recombinant Pneumocystis carinii dihydropteroate synthetase. The apparent Michaelis-Menten constants (K m ) for p-aminobenzoic acid and 7,8-dihydro-6-hydroxymethylpterin pyrophosphate were 0.34 ؎ 0.02 and 2.50 ؎ 0.71 M, respectively. Several sulfa drugs, including sulfathiazole, sulfachlorpyridazine, sulfamethoxypyridazine, and sulfathiourea, inhibited dihydropteroate synthetase approximately as well as sulfamethoxazole, as determined by the concentrations which cause 50% inhibition and/or by K i . For all sulfones and sulfonamides tested, unsubstituted p-amino groups were necessary for activity, and sulfonamides containing an N 1 -heterocyclic substituent were found to be the most effective inhibitors. Folate biosynthesis in isolated intact P. carinii was approximately equally sensitive to inhibition by sulfamethoxazole, sulfachlorpyridazine, sulfamethoxypyridazine, sulfisoxazole, and sulfathiazole. Two of these drugs, sulfamethoxypyridazine and sulfisoxazole, are known to be less toxic than sulfamethoxazole and should be further evaluated for the treatment of P. carinii pneumonia.

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