Pheromone 3 mRNA of the ciliate Euplotes octocarihatus contains three in-frame UGA codons that are translated as cysteines. This was revealed from cDNA sequencing and from plasma desorption mass spectrometry of cleaved pheromone 3 in connection with pyridylethylation of the fragments. N-terminal sequence analysis of carboxymethylated protein confirmed this conclusion for the first of the three UGA codons. Besides UGA the common cysteine codons UGU and UGC are also used to encode cysteine. UAA functions as a termination codon. Preparation of RNA. Total RNA was prepared by disruption of 1-3 x 107 cells in 8 M urea/4 M LiCl in a PotterElvehjem homogenizer, followed by precipitation on ice overnight. RNA was collected by centrifugation, dissolved in 10 mM Mops, pH 7.5/0.5% SDS, and extracted three times with phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform/isoamyl alcohol (24:1). Total RNA was then precipitated by addition of 0.1 volume of 4 M LiCl and 2.5 volumes of absolute ethanol.Poly(A)+ RNA was prepared by affinity chromatography on oligo(dT)-cellulose (Bethesda Research Laboratories) as recommended by the supplier with the exception that Mops was used as the buffer instead of Tris. Poly(A)+ RNA was precipitated by the addition of LiCI and ethanol as described above and redissolved in water. Quantity and quality were determined spectrophotometrically by measuring absorption at 260 and 280 nm (14).cDNA Synthesis and Cloning. The cDNA library was constructed (15) in the vector AgtlO. The cDNA was treated with S1 nuclease and ligated with EcoRI linkers prior to its insertion into the EcoRI site of the vector. The pheromone 3 gene was identified by plaque hybridization with the synthetic oligodeoxynucleotide 5'-GTRTANGGYTCYTCCCA-3', corresponding to the N terminus of the secreted pheromone, and was isolated by standard techniques (14).DNA Sequencing. Eight positively hybridizing plaques were obtained from 105 transformants. Five of them were further subcloned for sequencing by the dideoxy chain-termination method. Their nucleotide sequences were determined from double-stranded and single-stranded templates (pUC12, pT7T3, M13mpl8, and M13mp19 as sequencing vectors) according to the sequencing strategy outlined in Fig. 1 1To whom reprint requests should be addressed.

Adsorptive endocytosis of five different lysosomal enzymes from various human and non-human sources was susceptible to inhibition by mannose and l-fucose, methyl alpha-d-mannoside, alpha-anomeric p-nitrophenyl glycosides of mannose and l-fucose, mannose 6-phosphate and fructose 1-phosphate. A few exceptions from this general scheme were observed for particular enzymes, particularly for beta-glucuronidase from human urine. The inhibition of alpha-N-acetylglucosaminidase endocytosis by mannose, p-nitrophenyl alpha-d-mannoside and mannose 6-phosphate was shown to be competitive. The loss of endocytosis after alkaline phosphatase treatment of lysosomal enzymes supports the hypothesis that the phosphorylated sugars compete with a phosphorylated carbohydrate on the enzymes for binding to the cell-surface receptors [Kaplan, Achord & Sly (1977) Proc. Natl. Acad. Sci. U.S.A.74, 2026-2030]. Endocytosis of ;low-uptake' forms of alpha-N-acetylglucosaminidase and alpha-mannosidase was likewise susceptible to inhibition by sugar phosphates and by alkaline phosphatase treatment, suggesting that ;low-uptake' forms are either contaminated with ;high-uptake' forms or are internalized via the same route as ;high-uptake' forms. The existence of an alternative route for adsorptive endocytosis of lysosomal enzymes is indicated by the unaffected adsorptive endocytosis of rat liver beta-glucuronidase in the presence of phosphorylated sugars and after treatment with alkaline phosphatase.

A 16-kb fragment of human DNA containing the cathepsin D (CATD) gene was isolated. Nucleotide sequencing, primer extension, protection from mung bean nuclease, and promoter activity assays were used to characterize the gene. The transcribed portion of the gene is about 11,000 bp and is organized into 9 exons analogous with the human pepsinogen A gene. Human pepsinogen A and CATD proteins have 42% sequence identity, while the two cDNAs are 55.7% identical. The positions of the splice junctions are fully conserved in these two genes. The noncoding sequences of the two genes are dissimilar. We report the nucleotide sequence of an Eco RI-Bam HI fragment that contains the transcription initiation site. The promoter region contains no TATA and CCAAT boxes, but five potential Sp1 binding sites (one of them in the first intron) and four AP-2 binding sites (two of them in the first intron). In COS-1 cells, the region containing the three proximal Sp1 sites possesses the bulk of the promoter activity of the 5'-flanking sequence. The transcription start site of the CATD gene is localized within a CpG cluster. In the interval -390 through +450, the content of CpG is 5.8 times above the average throughout the human genome.

Complementary DNA clones for the human cation-dependent mannose 6-phosphate-specific receptor have been isolated from a human placenta library in Xgtll. The nucleotide sequence of the 2463-base-pair cDNA insert includes a 145-base-pair 5' untranslated region, an open reading frame of 831 base pairs corresponding to 277 amino acids (Mr = 30,993), and a 1487-base-pair 3' untranslated region. The deduced amino acid sequence is colinear with that determined by amino acid sequencing of the N-terminus peptide (41 residues) and nine tryptic peptides (93 additional residues). The receptor is synthesized as a precursor with a signal peptide of 20 amino acids. The hydrophobicity profile of the receptor indicates a single membrane-spanning domain, which separates an N-terminal region containing five potential Nglycosylation sites from a C-terminal region lacking Nglycosylation sites. Thus the N-terminal (Mr = 18,299) and C-terminal (Mr ' 7648) segments of the mature receptor are assumed to be exposed to the extracytosolic and cytosolic sides of the membrane, respectively. Analysis of a panel of somatic cell (mouse-human) hybrids shows that the gene for the receptor is located on human chromosome 12.

A 2112‐bp cDNA clone (lambda CT29) encoding the entire sequence of the human lysosomal acid phosphatase (EC 3.1.3.2) was isolated from a lambda gt11 human placenta cDNA library. The cDNA hybridized with a 2.3‐kb mRNA from human liver and HL‐60 promyelocytes. The gene for lysosomal acid phosphatase was localized to human chromosome 11. The cDNA includes a 12‐bp 5′ non‐coding region, an open reading frame of 1269 bp and an 831‐bp 3′ non‐coding region with a putative polyadenylation signal 25 bp upstream of a 3′ poly(A) tract. The deduced amino acid sequence reveals a putative signal sequence of 30 amino acids followed by a sequence of 393 amino acids that contains eight potential glycosylation sites and a hydrophobic region, which could function as a transmembrane domain. A 60% homology between the known 23 N‐terminal amino acid residues of human prostatic acid phosphatase and the N‐terminal sequence of lysosomal acid phosphatase suggests an evolutionary link between these two phosphatases. Insertion of the cDNA into the expression vector pSVL yielded a construct that encoded enzymatically active acid phosphatase in transfected monkey COS cells.