Analysis of the human ferrochelatase promoter in transgenic mice.

Ferrochelatase catalyzes the chelation of ferrous iron and protoporphyrin to form heme. It is expressed as a housekeeping gene in all cells, but is upregulated during erythropoiesis. Ferrochelatase activity is deficient in the inherited disease protoporphyria as a result of heterogeneous mutations. Although human ferrochelatase is transcribed from a single promoter in both nonerythroid and erythroid cells, previous studies using transient transfection assays failed to demonstrate erythroid-specific increased expression from 4.0 kb of the human ferrochelatase promoter containing the erythroid cis-elements, GATA and NF-E2. The present study analyzes the in vivo regulation of the ferrochelatase gene to provide insights into the mechanism of its erythroid-specific enhancement. Transgenic (TG) mouse lines were generated in which the luciferase reporter gene was driven by either a 150-bp ferrochelatase minimal promoter (-0.15 TG) or by a 4.0 kb extended 5' upstream region (-4.0 TG). Expression of the -4.0 TG transgene was generally consistent with the endogenous gene during embryonic development and in nonerythroid and erythroid tissues as demonstrated by Northern blotting and mRNA in situ hybridization. The -4.0 TG was expressed at a higher level than the -0.15 TG in nonerythroid and erythroid tissues, including during extramedullary erythropoiesis induced by n-acetylphenylhydrazine injection. The enhanced erythroid expression of the -4.0 TG correlates with the appearance of a DNase I hypersensitive site in the 5' flanking region of the transgene. Therefore, in the context of chromosomal integration, the 5' flanking region of the ferrochelatase gene is necessary and sufficient to confer high levels of transgene expression in erythroid tissue.

Site-specific incorporation of nonnatural residues during in vitro protein biosynthesis with semisynthetic aminoacyl-tRNAs.

A method is presented for the incorporation of nonnatural amino acids into proteins during in vitro cell-free translation. A combination of chemical synthesis and run-off transcription was employed to prepare a semisynthetic, nonhypermodified tRNA(Gly) nonsense suppressor acylated with L-3-[125I]iodotyrosine. The presence of this synthetic tRNA during in vitro translation of mRNA containing a nonsense suppression site (e.g., a UAG termination codon) results in the incorporation of the nonnatural amino acid L-3-iodotyrosine into the polypeptide exclusively at the position corresponding to that site. Incorporation of the nonnatural amino acid L-3-[125I]iodotyrosine into the model polypeptide was assessed by quantitative and unambiguous determination of suppression efficiency, read-through, and site specificity of incorporation. Minor modifications of the method employed in this initial experiment also allow the rapid analysis of unlabeled acylated tRNA analogues. Under optimum conditions, the unlabeled amino acid L-3-iodotyrosine was found to be incorporated with a suppression efficiency of 65%. Other nonnatural residues, including N-methylphenylalanine, D-phenylalanine, and phenyllactic acid, were tested in the assay under these same conditions. Suppression efficiencies for this series ranged from 0 to 72% depending on the structure of the residue incorporated. Several other aspects of this methodology, such as tRNA structure and context effects, are briefly discussed.

A clinically homogeneous group of families with facioscapulohumeral (Landouzy-Déjérine) muscular dystrophy: linkage analysis of six autosomes.

Facioscapulohumeral muscular dystrophy (FSHMD) is a neuromuscular disorder characterized by autosomal dominant inheritance and clinical onset in the muscles of the face and shoulder girdle. Using a set of RFLP markers spaced at approximately 20 centimorgans, we have begun a systematic search for markers linked to the disease. A total of 81 RFLP loci on six autosomes (1, 2, 5, 7, 10, and 16) have been examined for linkage to FSHMD in 13 families. With the computer program CRI-MAP, two-point and multipoint analyses have not resulted in any LOD score indicative of linkage to FSHMD. However, these analyses have allowed us to exclude 909 centimorgans (sex average) of our genetic maps in intervals where the LOD score is less than -2.0. We estimate our data have excluded 23% of the human genome.

Synergistic activity of purine metabolism inhibitors in cultured human tumor cells.

Cytotoxic effects of 3-deazaguanosine (3-DGUO) result from the inhibition of DNA synthesis and incorporation of the drug into DNA. Synergistic antiproliferative effects of a combination of 3-deazaguanosine and 2-beta-D-ribofuranosylthiazole-4-carboxamide, a potent inhibitor of inosine monophosphate dehydrogenase, was observed in human tumor cells. Inosine reversed the antiproliferative effects of the 3-deazaguanosine but not 2-beta-D-ribofuranosylthiazole-4-carboxamide. 3-Deazaguanosine monophosphate was shown to inhibit the activity of the de novo purine synthesis enzyme, 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide transformylase. The data suggested a cytotoxic effect of 3-DGUO associated with the inhibition of de novo purine synthesis by drug nucleotides, an effect which may account for the synergistic action noted.

Extent of DNA methylation in human tumor cells.

The total genomic DNA methylation, i.e., the percentage of methylated cytosines, was measured in 20 cell lines derived from different types of human tumors. The measurements were obtained by cation-exchange liquid chromatography of bases released by formic acid hydrolysis. These experiments were done to determine if altered methylation is a prevalent and large defect in oncogenic transformation. A majority of the tumor cells measured had decreased levels of methylated DNA in comparison to our laboratory's and other laboratories' published measurements of normal cells and tissues. In fact, tumor cell DNA ranged as low as 1.2% of cytosines methylated compared to a value of 3% or more for normal cells and tissues. HpaII and MspI DNA restriction enzyme analysis confirmed for all tumor cell lines tested that their DNA was hypomethylated in comparison to the DNA from normal diploid fibroblasts tested. The results obtained by liquid chromatography and restriction enzyme analysis were strikingly similar. The reduced methylation of the tumor and DNA correlated with the recent observation of other laboratories that individual genes are undermethylated in human cancer cells and that a number of different carcinogens can lower DNA methylation directly.

Epstein-Barr HR-1 virion DNA is very highly methylated.

The virion DNA of Epstein-Barr virus strain HR-1 was found to be methylated to a very large extent, approaching 15%. This extent of methylation is in great contrast to other classes of tumor viruses, and it is the highest that we know of in any mammalian system.

Mitochondrial control of cell surface characteristics in Saccharomyces cerevisiae.

Defects in the inner mitochondrial membrane of petite mutants of yeast resulted not only in respiratory deficiency, but also in changes in cell surface characteristics. These were (1) concanavalin A agglutinability, (2) cell movement in a biphasic polymer system, (3) cell adhesion. Genetic analysis indicated that the control exerted by the mitochondria was on nuclear genes or on the products of these genes which were presumably specifying cell surface components. These findings ascribe a new role to mitochondria but also have implications for neoplastic transformation.

Primary antimitochondrial activity of the cancer drug methylglyoxal bis(guanylhydrazone) in yeast cells.

Primary action of methylglyoxal bis(guanylhydrazone) (MGBG) on the yeast mitochondrial system was demonstrated by (1) selective inhibition of cell growth in non-fermentable medium, (2) blockage of mitochondrial synthesis of cytochromes aa3 and b and (3) ultrastructural aberration. The drug caused extensive deletions in mitochondrial DNA detected by an increase in the frequency of the mitochondrial mutant petite but had little or no effect on cell viability. Growth inhibition by MGBG had any effect on growth inhibition by ethidium bromide. Strains showed no cross-correlation in their tolerance to MGBG and ethidium bromide.