No abstract

A method has been developed for the electrophoretic and quantitative analyses of human red cell delta-aminolevulinate dehydrase (ALADH). The enzyme is under the control of an autosomal gene, with two common codominant alleles. ALADH1 and ALADH2, with frequencies of 0.89 and 0.11, respectively, in the Italian population. Mean phenotypic enzyme activities are nearly identical: 52,. 49 and 55 mIU/g Hb for ALADH 1, 2-1 and 2 phenotypes respectively.

Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1

During detailed restriction enzyme mapping of the human X‐linked gene Gd, specifying the enzyme glucose 6‐phosphate dehydrogenase (G6PD), we have observed the presence, over a 14‐kb DNA region spanning across the 3′ end of the G6PD transcript, of a large number of methylatable sites. These include 60 HpaII sites, 13 SmaI sites, 22 AvaI sites and 46 HhaI sites. In male leukocyte DNA the majority of HpaII sites are resistant to digestion, indicating that they are in the Cm5CGG form. However, a few sites are found reproducibly unmethylated in 24 samples analyzed. By double and triple digestions we have mapped five unmethylated sites, four of which are within the gene transcript and one distal to the end of transcription. We have also identified a number of sites which are fully methylated, whereas for others the methylation status could not be positively assessed. Thus, in a housekeeping gene expressed in leukocytes, the 3′ end is extensively methylated, but some specific sites are unmethylated. In female leukocyte DNA, we found that all sites methylated in males were also methylated. However, of the five sites that are unmethylated in males two are partly methylated in females. This additional site‐specific methylation involves approximately 50% of the female leukocyte DNA, and we show evidence that it is associated with the inactive X‐chromosome.

The distribution of ADA activity has been studied for ADA 1 and ADA 2-1 individuals in intact and lysed erythrocytes and in lymphocyte lysates. ADA activity in intact RBC is 20-30% lower than in lysates, in lymphocytes it is 10 times higher. The difference between ADA 1 and ADA 2-1 mean enzyme activities which we previously described in RBC lysates has been found also in intact RBC and in lymphocyte lysates.

Glucose-6-phosphate dehydrogenase (G6PD) is a ubiquitous enzyme that supplies the cell with NADPH required for a variety of reductive reactions and biosynthetic processes. Therefore, the gene G6PD, located in mammals on the X chromosome, that specifies G6PD can be regarded as a typical housekeeping gene. We have investigated the expression of human G6PD in eight different fetal and adult tissues by determining the level of enzyme activity, the level of G6PD mRNA, and the methylation pattern of the 3' end of the gene, for which we have nucleic acid probes. By combining sequence information with results of Southern blot analysis of DNA samples digested with the methylation-sensitive restriction enzyme Hpa U, we have identified five specific sites that are unmethylated in all tissues examined, a number of sites that are uniformly methylated, and a number of sites that are sometimes methylated. A subset of Hpa H sites, designated on our restriction map as H37-H55, exhibit positive correlation between degree of methylation, level of mRNA, and level of G6PD activity. A comparison of these methylation patterns with those we previously have observed in the G6PD gene on the inactive X chromosome [Toniolo, D., D'Urso, M., Martini, G., Persico, M. G., Tufano, V., Battistu, G. & Luzzatto, L. (1984) EMBO J. 3, 1987EMBO J. 3, -1995 indicates that different sites are associated with X-inactivation and with the regulation of G6PD on the active X chromosome. We conclude that this housekeeping gene is subject to tissue-specific transcriptional regulation, which in turn correlates with methylation of specific sites located at and near the 3' end of the gene.

Nucleotide substitutions within a structural gene can cause two principal "drastic" phenotypic effects at the protein level: translatable -+ untranslatable and nonpolar hydrophobic + hydrophilic amino acid substitutions. The sequence of nucleotides in the structural human a-and f3-globin genes and their variants were examined to determine whether codon usage, patterns of nucleotide substitutions, or both, reduced the relative and absolute rates of these unfavorable mutations. Based on translation of abnormal hemoglobins, it is likely that all 61 nontermination codons are potentially translatable, though only 47 are normally used. Moreover, codons that can mutate to a termination codon are never used whenever the corresponding amino acid is specified also by triplets that cannot mutate to termination by a single-step mutation. Thus, the number of opportunities to mutate to an untranslatable codon is reduced to the minimum compatible with the amino acid composition of these chains. The relative rates of U + non-U substitutions were much lower than those of other substitutions. Because U residues must be involved in most termination mutations and in all nonpolar hydrophobic + hydrophilic amino acid substitutions, there is a considerable reduction of mutational events, causing drastic phenotypic effects. These findings are likely to be the end result of evolutionary selection by yet unknown mechanisms.It can be postulated that evolutionary forces have adjusted mutation rates to an optimal value, being a compromise between keeping mutational load at a minimum and preserving evolutionary plasticity. Thus, rates of various types of mutational events are expected to differ because the average phenotypic effects of different mutations vary. Specifically, the likely phenotypic consequences of single nucleotide substitutions in a structural gene might be observed in the following sequence: same-sense < missense (hydrophilic + hydrophilic and hydrophobic hydrophobic < hydrophilic + hydrophobic) < translatable untranslatable. It may be presumed that evolution has adjusted the rate of each type of mutation independently rather than fixed an overall mutation rate applicable to all substitutions. To some extent this hierarchy is already built in the genetic code because codons are clustered in a nonrandom fashion that favors substitutions involving identical or very similar amino acids. This is true for sense and nonsense synonymous codons and for codons of the most polar [Glu, Gln, Asp, Asn, Lys, Arg (1)] and nonpolar hydrophobic amino acids (Phe, Leu, Ile, Met, Val, Pro).Therefore, a search for additional evolutionary devices that avoid nucleotide substitutions which might entail drastic phenotypic consequences is justified. The present approach asks whether codon usage or patterns of nucleotide substitutions, or both, are biased to reduce the probability of mutations of nonpolar hydrophobic to hydrophilic residues (or the reverse) or of sense to nonsense codons.Only the a-and 83-globin genes in humans are suitable f...

No abstract