The wild-type mouse hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) gene has been isolated from genomic libraries and its structure has been determined. This X chromosome-linked gene is >33 kilobases long and is split into nine exons. All the exon sequences have been determined, and a single-base substitution in the HPRT cDNA coding sequence from a mouse neuroblastoma cell line that overproduces a mutant HPRT protein has been identified. The 5' end of the gene has been defined, both by nuclease S1 protection and primer extension studies and by a functional assay in which an HPRT minigene, capable of expression in cultured cells, was created by ligating the 5' end of the gene onto wild-type human HPRT cDNA. Sequences normally associated with eukaryotic promoters are not present in the immediate 5'-flanking region of the HPRT gene, which is instead highly G+C rich. This observation is discussed in relation to the possible link between DNA methylation and X-chromosome inactivation.The enzyme hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, 2.4.2.8) catalyzes one of the first steps in the salvage pathway for the purine bases hypoxanthine and guanine in mammalian cells. HPRT deficiency in man results in the clinical disorders of Lesch-Nyhan syndrome and gouty arthritis. Although this X-linked locus has been much studied by somatic cell geneticists because of its functional haploidy and the ability to select for cells both expressing and lacking enzyme activity (for review, see ref. 1), the analysis of mutations was hampered by the lack of nucleic acid probes. We recently reported the isolation of cloned cDNA sequences to the HPRT gene from a mouse neuroblastoma revertant cell line (NBR4), which overproduces a mutant HPRT protein as a consequence of HPRT gene amplification (2-4), and from a similarly amplified Chinese hamster cell line (5) as well as from wild-type Chinese hamster and human cells (6). Cloned cDNA sequences of the human HPRT gene have also been isolated independently (7). We now describe the isolation and characterization of the entire wild-type mouse HPRT gene and the nature of the mutation in the NBR4 cell line.EXPERIMENTAL PROCEDURES Mammalian Cell Lines. The origin and characterization of wild-type mouse neuroblastoma (NB+), 6-thioguanine resistant (NB-), HPRT-overproducing derivative (NBR4), and HPRT-deficient Chinese hamster (RJK88) cell lines have been described (2)(3)(4)8).Isolation and Characterization of Genomic Clones. The genomic libraries used were kindly provided by John Seidman (Harvard University). One was constructed as a Hae III partial digest of mouse myeloma DNA, cloned into bacteriophage Charon 4A using synthetic EcoRI linkers. The second was a Mbo I partial digest of BALB/c mouse embryo DNA cloned directly into the BamHI site of Charon 28. Phage were plated on lawns of Dp5OsupF (9) and those with homology to HPRT cDNA were identified by using the plaque-hybridization procedure ...

We have determined the primary structure of bovine chromogranin A as a first step in the elucidation of the function of this widespread protein. After oligonucleotide screening of a cDNA library of bovine adrenal medulla, a clone (insert length 1.9 kb) containing the entire coding region for chromogranin A was isolated and sequenced. The authenticity of the sequence was verified by comparison with N‐terminal, several internal, and C‐terminal amino acid sequences as well as the amino acid composition of chromogranin A. The cDNA clone hybridized to an mRNA of 2.1 kb and, after in vitro transcription‐translation, yielded a polypeptide with a similar electrophoretic mobility in SDS gels to chromogranin A. The polypeptide chain of chromogranin A comprises 431 amino acid residues, corresponding to an unmodified protein of 48 kd, and is preceded by a cleaved signal peptide of 18 amino acid residues. Interesting features of the chromogranin A structure include repeated clusters of glutamic acid residues, the occurrence of eight potential dibasic cleavage sites, six of which are located in the C‐terminal domain, and the presence, in the N‐terminal domain, of ‐Arg‐Gly‐Asp‐ (RGD), a three amino acid sequence involved in the binding of several constitutively secreted proteins to cell membranes.

Our results strongly support the hypothesis that there is a molecular basis for phenotypic heterogeneity in phenylketonuria. The establishment of genotype will therefore aid in the prediction of biochemical and clinical phenotypes in patients with this disease.

Communicated by B.DobbersteinWe have determined and analyzed the primary structure of human secretogranin I (chromogranin B), a tyrosine-sulfated secretory protein found in a wide variety of peptidergic endocrine cells. A 2.5-kb cDNA clone, hybridizing to an mRNA of similar length, was isolated from a cDNA library of human pheochromocytoma. The identity of the clone was established by comparison of its deduced amino acid sequence with N-terminal and several internal secretogranin I sequences as well as by immunoprecipitation of the protein produced by in vitro transcription-translation of the cloned cDNA.Secretogranin I is a 657 amino acid long polypeptide of 76 kd and is preceded by a cleaved N-terminal signal peptide of 20 residues. Comparison of the predicted amino acid sequence of human secretogranin I with that of bovine chromogranin A reveals significant homologies near the N termini and at the C termini. The N-terminal homologous domains contain the only two cysteine residues of both proteins and form disulfide-stabilized loop structures. The sequences between the homologous terminal domains in both proteins differ but are characterized by a remarkable hydrophilicity, an abundance of acidic amino acids and potential dibasic cleavage sites for the generation of smaller, perhaps hormone-like, peptides.

Recombinant plasmids containing DNA inserts complementary to mRNA coding for hypoxanthine-guanine phosphoribosyltransferase (HPRT) from mouse and Chinese hamster cell lines have been isolated from cDNA libraries and characterized by DNA sequence analysis. A total of 1292 nucleotides of the mouse cDNA sequence and 1301 nucleotides of the Chinese hamster cDNA sequence has been determined. Each of these sequences includes an open reading frame of 654 nucleotides (218 amino acids) corresponding to the HPRT protein coding region. The deduced amino acid sequences for the mouse and Chinese hamster enzymes are presented and compared to that of human HPRT. At least 95% of the amino acids are conserved in the three species. In addition, we present evidence that two species of HPRT mRNA, which differ in the site of polyadenylation that is utilized during processing of the RNA transcripts, exist in Chinese hamster cells.