Tissue-specific expression of porphobilinogen deaminase. Two isoenzymes from a single gene

Porphobilinogen deaminase (hydroxymethylbilane synthase; EC 4.3.1.8), the third enzyme of the heme biosynthetic pathway, catalyzes the stepwise condensation of four porphobilinogen units to yield hydroxymethylbilane, which is in turn converted to uroporphyrinogen III by cosynthetase. We compared the apparent molecular mass of porphobilinogen deaminase from erythropoietic and from non-erythropoietic cells by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immune-blotting. The results indicate that two isoforms of porphobilinogen deaminase can be distinguished and differ by 2000 Da. Analysis of cell-free translation products directed by mRNAs from human erythropoietic spleen and from human liver demonstrates that the two isoforms of porphobilinogen deaminase are encoded by distinct messenger RNAs. We cloned and sequenced cDNAs complementary to the non-erythropoietic form of porphobilinogen deaminase encoding RNA. Comparison of these sequences to that of human erythropoietic mRNA [Raich et al. (1986) Nucleic Acids Res. 14, 5955-5968] revealed that the two mRNA species differ by their 5' extremity. From the mRNA sequences we could deduce that an additional peptide of 17 amino acid residues at the NH2 terminus of the non-erythropoietic isoform of porphobilinogen deaminase accounts for its higher molecular mass. RNase mapping experiments demonstrate that the two porphobilinogen deaminase mRNAs are distributed according to a strict tissue-specificity, the erythropoietic form being restricted to erythropoietic cells. We propose that a single porphobilinogen deaminase gene is transcribed from two different promoters, yielding the two forms of porphobilinogen deaminase mRNAs. Our present finding may have some relevance for further understanding the porphobilinogen deaminase deficiency in certain cases of acute intermittent porphyria with an enzymatic defect restricted in non-erythropoietic cells.     

Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.     

Binding and precipitating activities ofErythrina lectins with complex type carbohydrates and synthetic cluster glycosides. A comparative study of the lectins fromE. corallodendron,E. cristagalli,E. flabelliformis,andE. indica

Erythrina lectins possess similar structural and carbohydrate binding properties. Recently, tri- and tetra-antennary complex type carbohydrates with non-reducing terminal galactose residues have been shown to be precipitated as tri- and tetravalent ligands, respectively, with certainErythrina lectins [Bhattacharyya L, Haraldsson M, Brewer CF (1988) Biochemistry 271034-41]. The present work describes a comparative study of the binding and precipitating activities of fourErythrina lectins,viz. E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica, with multi-antennary complex type carbohydrates and synthetic cluster glycosides. The results show that though their binding affinities are very similar, theErythrina lectins show large differences in their precipitating activities with the carbohydrates. The results also indicate significant dependence of the precipitating activities of the lectins on the core structure of the carbohydrates. These findings provide a new dimension to the structure-activity relationship of the lectins and their interactions with asparagine-linked carbohydrates.Abbreviations EAL, ECorL, ECL, EFL, and EIL represent the lectins from the seeds ofErythrina arborescens, - E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica respectively - AFOS thetri-antennary complex type oligosaccharide from asialofetuin - AFGP the tri-antennary glycopeptide from asialofetuin - MeGal methyl -d-galactopyranoside Unless stated otherwise all sugars are in thed-configuration.     

Intrinsic potential for high fetal hemoglobin production in a Druze family with β-thalassemia is due to an unlinked genetic determinant

Summary The mechanism for elevated production of fetal hemoglobin (Hb F) in a Druze patient with °-thalassemia intermedia was investigated. Heterozygous family members exhibited normal Hb F levels, suggesting that the increase in -gene expression in the propositus may be partly due to anemic stress. Erythroid progenitors of these family members cultured in vitro [burst forming units (erythroid); (BFUe)] showed elevated synthesis of Hb F, indicating the existence of a genetically determined intrinsic capacity for high Hb F production in this family. The propositus was found to be homozygous for a IVS2-position 1 mutation, on the background of Mediterranean haplotype I, which is not known to be linked to high Hb F production. Moreover, extensive molecular studies of the -globin gene cluster, including sequence analysis of the promoter regions of the -globin genes, did not reveal any cisacting mechanism that could account for the high Hb F production in the propositus. A young niece of the propositus with °-thalassemia major was recently discovered, who was homozygous for the same -globin allele and haplotype as the propositus. However, unlike her uncle, she does not have a high Hb F level and presents with a severe clinical course. Her inability to produce high Hb F suggests that the genetic determinant for increased -gene expression in the propositus is unlinked to the -globin gene cluster.     

Characterization oftrans-acting regulatory elements affecting the expression of Mn-superoxide dismutase (sodA) inEscherichia coli

Escherichia coli strains harboringtrans-acting mutations affecting the expression of Mn-superoxide dismutase (SOD) gene (sodA) were used to studysodA regulation. Complementation studies revealed that eitherarc (aerobic respiratory control) orfur (ferric uptake regulation) loci independently complemented anaerobic expression of asodA::lacZ protein fusion in one mutant strain (UV16). This mutant exhibited phenotypes (i.e., elevated outer membrane proteins, enzyme activity, and dye sensitivity) typical offur andarc mutants. When these mutations were introduced into an otherwise wild-type background, anaerobicsodA expression occurred only when botharc andfur mutations were present simultaneously, suggesting cooperative roles of Fur and Arc insodA repression. The reconstructedfur arcA andfur arcB double mutants were still inducible by iron chelators, suggesting the possible involvement of another iron-containing repressor protein. A second independent mutant strain harboring atrans-acting regulatory mutation (UV14) was only partially complemented by multicopy plasmids carryingfur ⁺ orarc ⁺ genes, implicating other genetic elements insodA regulation.     

Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts.

The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.     

Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Summary Spinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector gt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting apparatus of the photosystem II reaction center, the ATP synthase subunits gamma, delta and CF_o-II, the Rieske Fe/S protein of the cytochrome b/f complex, subunits 2, 3, 5 and 6 of the photosystem I reaction center, plastocyanin, ferredoxin oxidoreductase, chlorophyll a/b-binding apoproteins of the lightharvesting complex associated with photosystem II, and the small subunit of the stromal enzyme ribulose bisphosphate corboxylase/oxygenase. The cDNA inserts lack complementarity to plastid DNA but hybridize to restricted nuclear DNA as well as to discrete poly A⁺-mRNA species. The precursor products obtained after translation of hybrid selected RNA fractions in a wheat germ assay are imported and processed by isolated unbroken spinach chloroplasts. The imported components comigrate with the respective authentic proteins.     

Isolation of a human stromal cell strain secreting hemopoietic growth factors

A diploid fibroblastoid cell strain, termed "ST-1," has been established from a long-term liquid culture of human fetal liver cells. ST-1 cells are nonphagocytic, nonspecific esterase negative and do not possess factor VIII-related antigen but stain with antibodies specific for fibronectin and type I collagen. The ST-1 cells produce nondialyzable hemopoietic growth factors capable of stimulating the development of erythroid bursts, mixed granulocyte-macrophage colonies, pure granulocyte colonies, and pure macrophage colonies. These factors are active on both human fetal liver and human adult bone marrow progenitors. When liquid cultures of human fetal liver hemopoietic progenitors are established with a preformed monolayer of ST-1 cells, the yields of nonadherent cells, erythroid progenitors, and myeloid progenitors are greatly increased. These studies demonstrate that the fibroblastoid ST-1 cells support hemopoiesis in vitro and may be a critical element in the stromal microenviroment in vivo.     

Activation of mouse peritoneal macrophages in vitro and in vivo by interferon-gamma

To determine the role of IFN-gamma in the activation of resident mouse peritoneal macrophages, crude macrophage-activating lymphokines were incubated with a monoclonal anti-murine IFN-gamma antibody. This treatment abolished the capacity of mitogen-induced lymphokines to enhance either H2O2 release or activity against the intracellular protozoa Toxoplasma gondii and Leishmania donovani. All macrophage-activating factor detected by these assays was also removed by passing the lymphokines over a Sepharose column to which the monoclonal anti-IFN-gamma antibody had been coupled. Therefore, pure murine rIFN-gamma was tested both in vitro and in vivo as a single activating agent. After 48 hr of pretreatment in vitro with 0.01 to 1 antiviral U/ml, macrophage H2O2-releasing capacity was enhanced an average of 6.4-fold; half-maximal stimulation was induced by 0.03 U/ml. Resident macrophages infected with T. gondii half-maximally inhibited parasite replication after 24 hr of preincubation with 0.14 U/ml of rIFN-gamma, and near complete inhibition was achieved by pretreatment with 100 U/ml. Half-maximal leishmanicidal activity was induced by 0.08 U/ml of rIFN-gamma, and 67 to 75% of intracellular L. donovani amastigotes were killed after macrophages were preincubated with 10 to 100 U/ml. Eighteen hours after parenteral injection of rIFN-gamma, peritoneal macrophages displayed a dose-dependent enhancement of H2O2-releasing capacity and antiprotozoal activity. Half-maximal enhancement required 85 to 250 U or rIFN-gamma given i.p. Peritoneal macrophages were also activated by rIFN-gamma injected i.v. and intramuscularly. These results suggest that, in the mouse model, IFN-gamma is likely to be a primary factor within mitogen-induced lymphokines responsible for activating macrophage oxidative metabolism and antiprotozoal activity, and indicate that rIFN-gamma is a potent activator of these effector functions both in vitro and in vivo. These findings provide a rationale for evaluating rIFN-gamma in the treatment of systemic intracellular infections, and indicate that murine models are appropriate for such studies.