Ferritin is composed of two subunits, H and L. cDNA's coding for these proteins from human liver (1,2,3), lymphocytes (4) and from the monocyte-like cell line U937 (5) have been cloned and sequenced. Southern blot analysis on total human DNA reveals that there are many DNA segments hybridizing to the apoferritin H and L cDNA probes (1,2,4,6). In view of the tissue heterogeneity of ferritin molecules (7,8), it appeared possible that apoferritin molecules could be coded by a family of genes differentially expressed in various tissues (1,2). In this paper we describe the cloning and sequencing of the gene coding for human apoferritin H. This gene has three introns; the exon sequence is identical to that of cDNA's isolated from human liver, lymphocytes, HeLa cells and endothelial cells. In addition we show that at least 15 intronless pseudogenes exist, with features suggesting that they were originated by reverse transcription and insertion. On the basis of these results we conclude that only one gene is responsible for the synthesis of the majority of apoferritin H mRNA in various tissues examined, and that probably all the other DNA segments hybridizing with apoferritin cDNA are pseudogenes.

The structure of the gene for protein C, an anticoagulant serine protease, was analyzed in 29 unrelated patients with hereditary thrombophilia and protein C deficiency. Gene deletion(s) or gross rearrangement(s) was not demonstrable by Southern blot hybridization to cDNA probes. However, two unrelated patients showed a variant restriction pattern after Pvu U or BamHI digestion, due to mutations in the last exon: analysis of their pedigrees, including three or seven heterozygotes, respectively, with -50% reduction of both enzymatic and antigen level, showed the abnormal restriction pattern in all heterozygous individuals, but not in normal relatives. Cloning of protein C gene and sequencing of the last exon allowed us to identify a nonsense and a missense mutation, respectively. In the first case, codon 306 (CGA, arginine) is mutated to an inframe stop codon, thus generating a new Pvu II recognition site. In the second case, a missense mutation in the BamHI palindrome (GGATCC --GCATCC) leads to substitution of a key amino acid (a tryptophan to cysteine substitution at position 402), invariantly conserved in eukaryotic serine proteases. These point mutations may explain the protein C-deficiency phenotype of heterozygotes in the two pedigrees.

A polypeptide chain-terminating mutation in the yeast mitochondrial oxi 1 gene has been shown to be an ochre (TAA) mutation by DNA sequence analysis. Mitochondrially inherited revertants of this mutation include two types: In the first, the ochre codon has been changed to a sense codon by further mutation in the oxi 1 gene while, in the second, the ochre codon is still present, indicating the occurrence of an extrageneic ochre suppressor mutation. This mitochondrial ochre suppressor, termed MSU1, has been "cloned" in rho-strains ofyeast and tested against other oxi 1 mutations. Several additional mutations are also suppressible, and those examined so far are also ochre mutations. MSU1 does not suppress known frameshift or missense mutations at oxi 1. Isoelectric focusing of the gene product (cytochrome oxidase subunit II) from a suppressed-mutant strain indicates that suppression does not involve insertion of charged amino acids. Physical mapping of the mtDNA retained in the MSUl-carrying rhoF clones localizes the suppressor mutation to the gene coding the 15S rRNA or a site not more than 300 base pairs from it. No known tRNA genes occur this close to the 15S rRNA gene, and mtDNA from a suppressor-carrying rho-does not hybridize detectably to mitochondrial tRNAs. These results suggest that MSU1 may be an alteration in the 15S rRNA.Nonsense mutations and their suppressors have been of great value for the study of both protein-coding structural genes and the genetic systems that express them. Although the mitochondrial genetic system of yeast (Saccharomyces cerevisiae) has been intensively studied over the past few years (1, 2), no defined set ofnonsense mutations and suppressors for them has been described. Suppressors of yeast mitochondrial mutations have indeed been reported. They arise as a result of either mutations in nuclear genes (3-6) or second-site mutations in mtDNA (4, 5, 7). However, in the previously described cases, the nature of the suppressible mutations was not determined precisely.We have been engaged in a study ofmutants at the oxi 1 gene in mtDNA (8-10), a simple uninterrupted gene that codes for the polypeptide cytochrome c oxidase subunit II (9,11,12). To examine suppression in this gene, we have now analyzed the DNA sequence alteration in the mutant oxi l-V25, which has been reported to be a chain-terminating mutation (13) that can be suppressed by several recessive nuclear mutations (A. Putrament and T. Zoladek, personal communication). We report here that oxi l-V25 is an ochre (TAA) mutation.Among the revertants isolated from this mutant, we have identified a strain that carries an extrageneic ochre suppressor mutation in mtDNA, termed MSUI. MSUI Table 3 were obtained from B. Weiss-Brummer (16), with the exception of M13-249 (9). Media and Genetic Techniques. YPEG medium contained 1% yeast extract, 2% Bactopeptone, 3% ethanol, and 3% glycerol. Glucose-containing medium (YPD) and genetic techniques were as described (17).Isolation of mtDNA and Sequence Analysis. The procedures ...