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We have developed a way to fit yeast artificial chromosomes (YACs) with markers that permit the selection of stably transformed mammalian cells, and have determined the fate and expression of such YACs containing the genes for human ribosomal RNA (rDNA) or glucose-6-phosphate dehydrogenase (G6PD). The YACs in the yeast cell are "retrofitted" with selectable markers by homologous recombination with the URA3 gene of one vector arm. The DNA fragment introduced contains a LYS2 marker selective in yeast and a thymidine kinase (TK) marker selective in TK-deficient cells, bracketed by portions of the URA3 sequence that disrupt the endogenous gene during the recombination event. Analyses of transformed L-M TK- mouse cells showed that YACs containing rDNA or G6PD were incorporated in essentially intact form into the mammalian cell DNA. For G6PD, a single copy of the transfected YAC was found in each of two transformants analyzed and was fully expressed, producing the expected human isozyme as well as the heterodimer composed of the human gene product and the endogenous mouse gene product.

From the collection described by Abidi et al., 102 yeast artificial chromosomes (YACs) with human DNA inserts more than 300 kb in length were assigned to chromosomal band positions on early metaphase chromosomes by in situ hybridization using the biotin-avidin method. All the YACs hybridized within the Xq24-Xqter region, supporting the origin of the vast majority of the YACs from single human X-chromosomal sites. With assignments precise to +/- 0.5 bands, YACs were distributed among cytogenetic bands to roughly equal extents. Thus, there is no gross bias in the cloning of DNA from different bands into large YACs. To test band assignments further, hybridizations were carried out blind, and band positions were then compared with (1) probe localizations in cases in which a reported location was present in one of the YACs; (2) cross-hybridization of a labeled YAC with others in the collection; and (3) hybridization to a panel of DNAs from a series of hybrid cells containing Xq DNA truncated at various regions. Of 31 cases in which YACs contained a probe with a previously reported location, 28 in situ assignments were in agreement, and 14 other assignments, including one of the three discordant with probe localization, were confirmed by YAC cross-hybridization studies. Results with a group of nine YACs were further confirmed with a panel of somatic cell hybrid DNAs from that region. Five YACs hybridized both to Xq25 and to a second site (four in Xq27 and one in Xq28), suggestive of some duplication of DNA of the hybrid cell and perhaps in normal X chromosomes. The in situ assignments are thus sufficient to place YACs easily and systematically within bins of about 7-10 Mb and to detect some possible anomalies. Furthermore, on the basis of expectations for random cloning of DNA in YACs, the assigned YACs probably cover more than 50% of the total Xq24-Xq28 region. This provides one way to initiate the assembly of YAC contigs over extended chromosomal regions.

Yeast artificial chromosomes (YACs) permit the cloning of large tracts of human DNA. A YAC containing the human glucose-6-phosphate dehydrogenase gene is shown to encode active enzyme, supporting the inference that the YAC conserves the structure of the genomic DNA.

Sequences corresponding to the Xq28 loci DXS15, DXS52, DXS134, and DXS130 were shown to be present in a 140-kb yeast artificial chromosome (YAC XY58, isolated by Little et al.). This YAC clone appears to contain a faithful copy of this genomic region, as shown by comparison with human DNA and with a cosmid clone that contains probes St14c (part of the DXS52 sequences) and cpX67 (DXS134). cpX67 and St14c are contained in 11 kb and detect the same MspI RFLP polymorphism. A comparison of the YAC restriction map and pulsed-field gel electrophoresis data leads us to propose the following order of loci: DXS52(VNTR)-DXS33-DXF22S3-DXS130-DXS134 -DXS52-DXS15-DXS52, this whole cluster being comprised within 575 kb. The physical proximity of the DXS15, DXS52, and DXS134 loci led us to reinvestigate recombination events that had been reported between these loci in families from the Centre d'Etude du Polymorphisme Humain. Our results do not support the assumption that this region shows increased recombination.

The purpose of this study was to establish whether exposure to intense lighting favors the development or aggravates experimental oxygen-induced retinopathy in the newborn rat. Five groups of Wistar rats were studied. The control group was maintained for the first 14 days of life under conditions of cyclical (12L:12D) lighting at 12 Lx in room air. Two other groups were subjected, for the same amount of time, to semi-darkness (2 Lx; 12L: 12D), one with room air and the other with supplemental 80% oxygen. The final two groups were exposed to the same room air and hyperoxic treatments under intense lighting conditions (600 Lx; 12L:12D). After the treatment period, four rats were randomly chosen from each group, sacrificed and their retinas examined under electron microscope. Marked structural changes were seen only in the photoreceptor outer segments of those rats exposed to intense light. In eighty-five of the remaining rats retinal vascular morphology was examined in retinal flat mounts after intracardiac injection of India ink. Retinopathy was observed in rats treated with hyperoxia but no significant differences could be attributed to the light conditions under which the retinopathic rats had been maintained. In the rest of the rats, axonal transport along the optical pathways was evaluated after intravitreal injection of (3H) taurine. In the two groups exposed to hyperoxia, axonal transport was altered, but less markedly in those exposed to intense lighting than in those exposed to semi-darkness. Intense illumination under conditions of normoxia favors axonal transport. Exposure to intense lighting does not seem to aggravate oxygen induced retinopathy in the rat though it does produce structural lesions of the photoreceptors.

Some authors have reported that vitamin D treatment worsens renal function. We studied 10 patients with stable renal function, who were given calcitriol (0.5 micrograms/day) for a 4-month period. Creatinine and inulin clearance were performed at the beginning and at the end of the treatment. Although serum creatinine increased and creatinine clearance decreased, inulin clearance did not show significant variations. Furthermore, serum creatinine fell to the baseline value within 60 days after discontinuation of vitamin D therapy. The increased serum creatinine may be explained by an augmented release from muscular tissue, probably due to the improvement of uremic myopathy induced by calcitriol.

By biochemical characterization of glucose-6-phosphate dehydrogenase (G6PD) from the red cells of seventeen subjects of the population of Matera (Southern Italy) we have identified six genetically determined common variants. Among these, G6PD Metaponto and G6PD A(-) Matera had been already fully characterized. We have now found that A(-) Matera is genetically heterogeneous since one of two subjects examined had the two mutations at codons 68 and 126 characteristic of a typical A(-) variant, while the other subject had only the codon 126 mutation. G6PD Pisticci and G6PD Tursi are two new variants whose molecular lesion is not yet known. G6PD Cagliari-like has biochemical characteristics reminiscent of G6PD Cagliari, isolated in Sardinia, and was found to have the same nucleotide substitution as G6PD Mediterranean. G6PD Montalbano is a new variant, with nearly normal properties, due to a G----A transition which causes an Arg----His amino acid replacement at position 285.

One hundred twenty-seven yeast strains with artificial chromosomes containing Xq24-Xqter human DNA were obtained starting from a human/hamster somatic cell hybrid. The clones were characterized with respect to their insert size, stability, and representation of a set of Xq24-Xqter DNA probes. The inserts of the clones add up to 19.3 megabase (Mb) content, or about 0.4 genomic equivalents of that portion of the X chromosome, with a range of 40-650 kb in individual YACs. Eleven clones contained more than one YAC, the additional ones usually having hamster DNA inserts; the individual YACs could be separated by extracting the total DNA from such strains and using it to retransform yeast cells. One of the YACs, containing the probe for the DXS49 locus, was grossly unstable, throwing off smaller versions of an initial 300-kb YAC during subculture; the other YACs appeared to breed true on subculture. Of 52 probes tested, 12 found cognate YACs; the YACs included one with the glucose-6-phosphate dehydrogense gene and another containing four anonymous probe sequences (DX13, St14, cpx67, and cpx6). Xq location of YACs is being verified by in situ hybridization to metaphase chromosomes, and fingerprinting and hybridization methods are being used to detect YACs that overlap.

Lactoperoxidase-catalyzed cell surface radioiodination was employed to radiolabel membrane polypeptides of a murine macrophage cell line P388D1. Optimal conditions for radioiodination of P388D1 cells were determined and were found to differ from conditions used to label lymphoid cells. SDS-polyacrylamide gel electrophoresis of detergent soluble membrane polypeptides revealed that 9 to 10 molecular species from 1 X 10(5) to 0.15 X 10(5) daltons were labeled. Radioiodinated, Triton X-100 extracted P388D1 membrane polypeptides were subjected to affinity chromatography on aggregated IgG:Sepharose columns. Elution of the bound polypeptides and analysis by SDS-polyacrylamide gel electrophoresis revealed polypeptides with an apparent molecular size of 8 X 10(4), which possess binding affinity for the Fc portion of aggregated IgG. The 8 X 10(4) dalton membrane polypeptides do not readily aggregate, are resistant to degradation, are not composed of disulfide-linked subunits, and do not appear to contain much carbohydrate. Cellular binding characteristics paralleled the binding of soluble receptor for sieved fractions of aggregated IgG suggesting that these polypeptides may be responsible for the in situ binding of aggregated IgG to P388D1 cells.

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 II, 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 II 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., Battistuzzi, G. & Luzzatto, L. (1984) EMBO J. 3, 1987-1995] 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.

A patient with primary thrombocythemia, who was heterozygous for glucose-6-phosphate dehydrogenase deficiency (GdB/GdMed), was investigated to test for the clonal origin of this myeloproliferative disorder. In order to assess somatic cell mosaicism in various tissues, we have made use of the different rate of utilization of 2-deoxyglucose-6-phosphate, an analog of glucose-6-phosphate, by normal glucose-6-phosphate dehydrogenase and by the Mediterranean variant: the results demonstrate that essential thrombocythemia is a clonal disease involving the erythrocytic, granulocytic, and megakaryocytic series, without affecting monocytes, T lymphocytes, and non-T lymphocytes.

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 genetic polymorphism of an approximately 100-kb DNA region comprising and flanking the glucose-6-phosphate dehydrogenase (G6PD) gene on human chromosome Xq28 has been analyzed in detail. By using 14 unique sequence probes and 18 restriction enzymes, we have characterized 257 restriction fragments or 370 restriction sites. On testing 12-57 individual X chromosomes, all sites but one were nonpolymorphic. However, a PstI site that maps to exon 10 of the G6PD gene, which is still monomorphic in all British and Italian subjects tested, is polymorphic in west-African people. Specifically, it is absent from 22% of Nigerian X chromosomes. By sequence analysis we have shown that the absence of this PstI site results from a G----A replacement at position 1116, corresponding to the third base of a glutamine codon; no amino acid change is produced in the protein. Thus, a polymorphic silent mutation is demonstrated in a human gene.

Human--hamster somatic cell hybrids have been obtained by fusion of a CHO line (NA31) doubly deficient in hypoxanthine guanine phosphoribosyltransferase and glucose 6-phosphate dehydrogenase (G6PD) with normal G6PD(+) human fibroblasts. Analysis of NA31 extracts has revealed that, although G6PD activity is nearly absent, significant activity can be detected with 2-deoxyglucose 6-phosphate as substrate, so that the mutant and normal forms of the enzyme can both be easily detected. The cell hybrids obtained express human G6PD. The human G6PD subunits are distributed in homodimeric molecules as well as in human--hamster heterodimeric molecules. However, whereas the amount of hamster G6PD subunits present in the hybrid is similar to that in the hamster parental cells, the amount of human G6PD subunits is decreased by 3- to 10-fold when compared to the human parental cell. These results indicate that either the expression of the G6PD gene or the stability of the gene product is altered in the hybrid. By mutagenesis and selection in diamide (a substance that oxidizes intracellular glutathione), we have isolated a clone with a 3- to 5-fold increase in human G6PD activity. This derivative may have an increased rate of expression of the human G6PD structural gene.

We report the isolation and analysis of human genomic DNA clones spanning about 100 kb of the X chromosome and comprising the entire gene coding for the enzyme glucose 6-phosphate dehydrogenase (G6PD). The G6PD gene is 18 kb long and consists of 13 exons: the protein-coding region is divided into 12 segments ranging in size from 12 to 236 bp; an intron is present in the 5' untranslated region. Mature G6PD mRNA has a single polyadenylation site in HeLa cells. The major 5' end of mature G6PD mRNA in several cell lines is located 177 bp upstream of the translation initiating codon; longer mRNA molecules extending further in the 5' direction could be identified by S1 mapping and by comparing genomic and cDNA sequences. The DNA sequence around the major mRNA start is very GC rich; as to putative transcription regulatory sequences, a non-canonical TATA box and 9 CCGCCC elements are present, but no CAAT element could be identified. The genomic DNA we have isolated includes another ubiquitously transcribed region, provisionally named the GdX gene. Although the function of GdX is as yet unknown, we have established that this gene is located about 40 kb downstream of G6PD and is transcribed in the same direction. A comparative analysis of the promoter region of G6PD and 10 other housekeeping enzyme genes has confirmed the presence of a number of common features. In particular, in the eight cases in which a 'TATA' box is present, a conserved sequence of 25 bp is seen immediately downstream.

The present study compares the effects of bicarbonate hemodialysis (Bic. HD) and biofiltration (BF), a new hemodiafiltration technique, on plasma volume (PV) changes and extravascular fluid mobilization (Vfm). Ten uremic patients underwent one experimental session of Bic. HD and, one week later, one of BF, both on the second dialysis of the week. Net ultrafiltration rate was limited to 700 ml/min. At the start of each session, whole blood volume (WBV), PV and red cell volume (RCV) were determined using 5 mu Ci of radioiodinated serum albumin (RISA). PV and Vfm were calculated at hourly intervals using a serial hematocrit method. On Bic. HD, PV increased at 60 min. then decreased at 120 and 180 min., with efficient Vfm only during the first hour. On BF, PV increased throughout treatment, with greater Vfm. It would appear that PV is better preserved in BF, on account of more efficient Vfm.

Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is a common genetic abnormality affecting an estimated 400 million people worldwide. Clinical and biochemical analyses have identified many variants exhibiting a range of phenotypes, which have been well characterized from the hematological point of view. However, until now, their precise molecular basis has remained unknown. We have cloned and sequenced seven mutant G6PD alleles. In the nondeficient polymorphic African variant G6PD A we have found a single point mutation. The other six mutants investigated were all associated with enzyme deficiency. In one of the commonest, G6PD Mediterranean, which is associated with favism among other clinical manifestations, a single amino acid replacement was found (serine----phenylalanine): it must be responsible for the decreased stability and the reduced catalytic efficiency of this enzyme. Single point mutations were also found in G6PD Metaponto (Southern Italy) and in G6PD Ilesha (Nigeria), which are asymptomatic, and in G6PD Chatham, which was observed in an Indian boy with neonatal jaundice. In G6PD "Matera," which is now known to be the same as G6PD A-, two separate point mutations were found, one of which is the same as in G6PD A. In G6PD Santiago, a de novo mutation (glycine----arginine) is associated with severe chronic hemolytic anemia. The mutations observed show a striking predominance of C----T transitions, with CG doublets involved in four of seven cases. Thus, diverse point mutations may account largely for the phenotypic heterogeneity of G6PD deficiency.

Sequences located several kilobases both 5' and 3' of the stably transcribed portion of several genes hybridize to radio-labeled pure fragments of the alternating sequence poly (dG-dT) (dC-dA) ["poly(GT)"]. The genes include the ribosomal DNA of mouse, rat, and human, and also human glucose-6-phosphate dehydrogenase (G6PD) and mouse hypoxanthine-guanine phosphoribosyl transferase (HPRT). HPRT has additional hybridizing sequences in introns. Fragments that include the hybridizing sequences and up to 300 bp of adjoining DNA show perfect runs of poly(GT) (greater than 30bp) in all but the human 5' region of rDNA, which shows a somewhat different alternating purine:pyrimidine sequence, poly(GTAT) (36bp). Within 150 bp of these sequences in various instances are found a number of other sequences reported to affect DNA conformation in model systems. Most marked is an enhancement of sequences matching at least 67% to the consensus binding sequence for topoisomerase II. Two to ten-fold less of such sequences were found in other sequenced portions of the nontranscribed spacer or in the transcribed portion of rDNA. The conservation of the locations of tracts of alternating purine:pyrimidine between evolutionarily diverse species is consistent with a possible functional role for these sequences.

Pyruvate kinase (PK) has been purified from the red blood cells of two sisters who had suffered severe chronic non-spherocytic haemolytic anaemia since infancy, and of one patient who had haemolytic anaemia during pregnancy. The two sisters showed remarkable clinical improvement following splenectomy. The enzyme from their red cells was found to exhibit low activity (about 25% of normal) in crude haemolysates, low affinity for the substrate, phosphoenol pyruvate (PEP), and high sensitivity to fructose-1,6-diphosphate (FDP) activation. This PK differs from previously reported variants and it is provisionally designated PK 'Torre Annunziata'. The enzyme from the other patient had near-normal activity in crude haemolysates, slight changes in kinetics with respect to the substrate, PEP, and with respect to the effects of FDP, ATP and pH, and a markedly reduced thermostability. This PK also differs from previously reported variants and it is provisionally designated PK 'Torre del Greco'. During the course of this study an improved method for purification of PK using Cibacron blue sepharose has been developed.

Glucose-6-phosphate dehydrogenase (G6PD) is an ubiquitous enzyme which by determining the NADPH level has a crucial role in NADPH-mediated reductive processes in all cells (1). The structural gene for G6PD, Gd, is X-linked in mammals and on the basis of its expression in many tissues, it can be regarded as a typical "housekeeping" gene (2). Over 300 variants of the protein are known, many of which have deficient enzyme activity. Nearly 100 of these variants are polymorphic in various populations (3). The mammalian enzyme is a homodimer or a homotetramer with a subunit molecular weight of approximately 56000 daltons (4). Here we report the isolation of cDNA clones from HeLa cells, SV40-transformed human fibroblasts, human placenta and human teratocarcinoma cell lines. These clones have enabled us to sequence the entire coding region of Gd. Thus, the entire amino acid sequence of human G6PD is provided for the first time. This work is the first step for structural analysis of G6PD variants and for an understanding of the biological features of this enzyme at the molecular level.

It has been previously reported that following neuraminidase treatment alpha-galactosidase A is converted into the B form, as revealed by electrophoresis. By a variety of techniques such as isoelectrofocusing, DEAE-chromatography and by enzyme kinetic parameters, no conversion of alpha-galactosidase A into B, or the reverse, could be detected after neuraminidase treatment. Only an apparent transformation of alpha-galactosidase A into B was revealed by Cellogel electrophoresis. In addition, a discrepancy was noticed between the pattern of electrophoretic migration on starch gel and Cellogel and the net electrical charges of the two alpha-galactosidases as deduced by isoelectrofocusing and DEAE-cellulose. Neuraminidase treatment did not affect the activity of alpha-galactosidase A towards the natural substrate, ceramidetrihexoside, but the activity of alpha-galactosidase B decreased by about 30% under the same conditions. The two forms of alpha-galactosidases A and B used in this study were extensively purified by classical procedures.

During investigation of folate polyglutamate biosynthesis in rat liver slices utilizing [2-(14)C]folic acid, a folate compound that behaved like a polyglutamate form in the Sephadex G-15 gel filtration system was found to accumulate. Subsequent chromatographic, spectral, chemical, and enzymic studies have indicated that the compound formed in liver slices incubated with [(14)C]folic acid with and without methotrexate was 10-formyl folate. This folate is of interest in that it is the most potent natural inhibitor of dihydrofolate reductase known and may be capable of serving a regulatory function within the cell.

Sequences hybridizing to several human gene probes have been recovered as cloned inserts in yeast artificial chromosomes (YACs). Among 2300 YACs made from human leukocyte DNA (totaling about 0.1 genomic equivalent of human DNA) we have found two, 200 and 780 kilobases (kb), containing sequences of V kappa I immunoglobulin (V = variable); one, 240 kb, with class I HLA; and 11, 200-800 kb, with 5S rRNA-encoding DNA (rDNA). Fifty human YACs from a hamster-human cell hybrid with only the Xq24-Xq28 portion of the X chromosome include one that contains two anonymous probe sequences, DX13 and St14, previously inferred by indirect means to lie within about 70 kb of one another in Xq28. The YACs specific for human DNA arise at a frequency equivalent to the fraction of cellular DNA that is human-specific. Furthermore, the human YACs, formed in a 280-fold excess of hamster DNA, do not hybridize to a hamster DNA probe, indicating that individual YACs do not contain a combination of human and hamster DNA. To confirm that sequences are not scrambled, the YACs containing V kappa I or DX13 and St14 sequences were shown to produce restriction fragments identical in mobility to fragments detected by the same probes in total human DNA digested with the same enzymes. YACs may therefore provide large clones to bridge gene mapping at the chromosome level to molecular analyses of small fragments of genomic DNA.

The alpha-galactosidase A activity from fibroblasts of five Fabry patients and five controls has been separated from alpha-galactosidase B through small DEAE-cellulose columns and in some experiments by treatment of the fibroblast extracts with Sepharose coupled to anti-alpha-galactosidase B antibodies. By these independent methods, it has been shown that there is a residual alpha-galactosidase A in Fabry's disease, which is immunologically similar to the alpha-galactosidase A from the controls. The alpha-galactosidase A from all of the patients and controls has the same apparent Km value for the synthetic substrate 4-methylumbelliferyl-alpha-galactosidase A, while the fifth has a thermolabile enzyme like that from the controls. The amount of immunologically active alpha-galactosidase A seems to be decreased in the patients tested.