Intermolecular disulfide bonds are not required for the expression of the dimeric state and functional activity of the transferrin receptor.

The human transferrin receptor is expressed as a disulfide-linked dimer at the cell surface. The sites of intermolecular disulfide bonds are Cys-89 and Cys-98. We have examined the functional significance of the covalent dimeric structure of the transferrin receptor by substitution of Cys-89 and Cys-98 with serine residues. Wild-type and mutated transferrin receptors were expressed in Chinese hamster ovary cells (clone TF-) that lack detectable endogenous transferrin receptors. The rates of receptor endocytosis and recycling were measured and the accumulation of iron by cells incubated with [59Fe]diferric transferrin was investigated. No significant differences between these rates were observed when cells expressing wild-type and mutated receptors were compared. The structure of the mutant receptor lacking intermolecular disulfide bonds was investigated. The presence of a population of mutant receptors with a non-covalent dimeric structure was indicated by cross-linking studies using diferric [125I]transferrin and the bifunctional reagent disuccinimidyl suberimidate. However, sucrose density gradient sedimentation analysis of Triton X-100 solubilized transferrin receptors demonstrated that the mutant receptor existed as a monomer in the absence of diferric transferrin and as an apparent dimer in the presence of this receptor ligand. We conclude that the covalent dimeric structure of the transferrin receptor is not required for the expression of the dimeric state and functional activity of the receptor.     

Sequence motifs characteristic of DNA[cytosine-N4]methyltransferases: similarity to adenine and cytosine-C5 DNA-methylases

The sequences coding for DNA[cytosine-N4]methyltransferases MvaI (from Micrococcus varians RFL19) and Cfr9I (from Citrobacter freundii RFL9) have been determined. The predicted methylases are proteins of 454 and 300 amino acids, respectively. Primary structure comparison of M.Cfr9I and another m4C-forming methylase, M.Pvu II, revealed extended regions of homology. The sequence comparison of the three DNA[cytosine-N4]-methylases using originally developed software revealed two conserved patterns, DPF-GSGT and TSPPY, which were found similar also to those of adenine and DNA[cytosine-C5]-methylases. These data provided a basis for global alignment and classification of DNA-methylase sequences. Structural considerations led us to suggest that the first region could be the binding site of AdoMet, while the second is thought to be directly involved in the modification of the exocyclic amino group.     

Extrachromosomal circular DNAs in Drosophila melanogaster: Comparison between embryos and Kc0% cells

We established the size distribution of extrachromosomal covalently closed circular DNA molecules from embryos of various Drosophila melanogaster strains and from Kc0% tissue culture cells. In embryos, more than 80% of the circular DNA molecules are smaller than 2.5 kb and all the distributions show a peak of molecules of between 200 and 400 bp. The Kc0% cell distribution differs mainly from that of embryos in that 48% of the molecules have a size between 4 and 8 kb. Correlating with this, circular molecules homologous to copia, 412 and 297 were detected only in Kc0% cells. The three tandemly repeated families containing the 5S genes, the histone genes and the 240 bp repeat of the ribosomal DNA intergenic spacer, which had previously been identified in circular DNAs from embryos, were also found in cultured cells. A fourth tandemly repeated family corresponding to the 1.688 g/cm³ satellite DNA was detected, both in embryos and Kc0% cells. It consists of circular multimeric molecules containing multiple copies of the 359 bp repeated unit. No circular DNA molecules homologous to the actin genes, the type I ribosomal DNA insertion, or the F and I transposable elements were found in embryos or Kc0% cells. Thus it appears that the extrachromosomal circular DNA molecules from embryos and from tissue culture cells differ mainly in the presence of circular copies of the copia-like transposable elements.     

Copy number and distribution of P and I mobile elements in Drosophila melanogaster populations

The distribution of the number of copies of P and I transposable elements per genome was investigated by in situ hybridization for a large set of Drosophila melanogaster strains. These included the P, Q and M types of the P-M system of hybrid dysgenesis. P element copy number varied widely (range 5–59). P and Q strains had around 40 copies whereas M strains generally had lower numbers (between 5 and 35) with one extreme value (52). The copy number of I elements appeared to be precisely regulated, as no strains were found outside the 15±5 range. The number of copies of the two families were independent. An excess of P copies on the X chromosome compared with the autosomes was found for the P and Q strains, but not for M strains. Among X-inserted P sites, a very high frequency of occupation was found at the tip of the X chromosome (cytological site 1A), especially for P and Q strains. The possible regulatory role in the P-M system of X-inserted P sites is discussed.     

The role of subunit 4, a nuclear-encoded protein of the F0 sector of yeast mitochondrial ATP synthase, in the assembly of the whole complex

The yeast nuclear gene ATP4, encoding the ATP synthase subunit 4, was disrupted by insertion into the middle of it the selective marker URA3. Transformation of the Saccharomyces cerevisiae strain D273-10B/A/U produced a mutant unable to grow on glycerol medium. The ATP4 gene is unique since subunit 4 was not present in mutant mitochondria; the hypothetical truncated subunit 4 was never detected. ATPase was rendered oligomycin-insensitive and the F1 sector of this mutant appeared loosely bound to the membrane. Analysis of mitochondrially translated hydrophobic subunits of F0 revealed that subunits 8 and 9 were present, unlike subunit 6. This indicated a structural relationship between subunits 4 and 6 during biogenesis of F0. It therefore appears that subunit 4 (also called subunit b in beef heart and Escherichia coli ATP synthases) plays at least a structural role in the assembly of the whole complex. Disruption of the ATP4 gene also had a dramatic effect on the assembly of other mitochondrial complexes. Thus, the cytochrome oxidase activity of the mutant strain was about five times lower than that of the wild type. In addition, a high percentage of spontaneous rho- mutants was detected.     

Effect of Growth Conditions and Trehalose Content on Cryotolerance of Bakers' Yeast in Frozen Doughs

The cryotolerance in frozen doughs and in water suspensions of bakers' yeast (Saccharomyces cerevisiae) previously grown under various industrial conditions was evaluated on a laboratory scale. Fed-batch cultures were very superior to batch cultures, and strong aeration enhanced cryoresistance in both cases for freezing rates of 1 to 56°C min⁻¹. Loss of cell viability in frozen dough or water was related to the duration of the dissolved-oxygen deficit during fed-batch growth. Strongly aerobic fed-batch cultures grown at a reduced average specific rate (μ = 0.088 h⁻¹ compared with 0.117 h⁻¹) also showed greater trehalose synthesis and improved frozen-dough stability. Insufficient aeration (dissolved-oxygen deficit) and lower growth temperature (20°C instead of 30°C) decreased both fed-batch-grown yeast cryoresistance and trehalose content. Although trehalose had a cryoprotective effect in S. cerevisiae, its effect was neutralized by even a momentary lack of excess dissolved oxygen in the fed-batch growth medium.     

Radiometric assay for carboxypeptidase H (EC 3.4.17.10) and other carboxypeptidase B-like enzymes

Carboxypeptidase H, EC 3.4.17.10, also known as enkephalin convertase, carboxypeptidase E, and crino carboxypeptidase B, is an important enzyme involved in the biosynthesis of bioactive peptides. To assay the enzyme, tissues are homogenized in at least 20 vol (ml/g) of 0.025 M Tris-HCl buffer, pH 8, with 5 mg/ml of bovine serum albumin. After centrifugation, the supernatant is brought to pH 5.6 and centrifuged again. Following a 20-min preincubation in 2 mM CoCl2, the supernatant is incubated with 0.1 mM (final concentration) of the radioactive substrate [3H]benzoyl-Phe-Ala-Arg. The 100-microliters assay is stopped by the addition of 680 microliters of acetonitrile/0.25 M HCl (0.7/1). The 1.5-ml tube is transferred into a scintillation vial and is flushed with 4 ml of Econofluor, a water-immiscible scintillation fluid. The product, [3H]benzoyl-Phe-Ala, recovered in the organic phase, is counted directly with no interference from the substrate remaining in the aqueous phase. The blank is below 1%. Expressed in nanomoles per minute per milligram of tissue, the activity of the soluble enzyme in rat is 0.34 for striatum, 21.0 for pancreatic islet, 16.6 for anterior pituitary, 46.0 for intermediate pituitary, and 10.9 for neural pituitary. In every case 25 microM guanidinoethylmercaptosuccinic acid, an active site-directed inhibitor of carboxypeptidase H, completely inhibits the activity.     

Direct segregation analysis of reciprocal translocations: a study of 283 sperm karyotypes from four carriers.

Using the technique of in vitro human-hamster fertilization, sperm of four men heterozygous for 4 reciprocal translocations--t(4;17),t(5;13),t(6;7), and t(9;18)--was studied. Frequencies of numerical abnormalities unrelated to the translocations range from 8.3% to 13.3%, and the incidence of imbalances ranges from 23.0% to 66.0%. Results are pooled with data from the nine other reciprocal translocations reported elsewhere, and the combined data demonstrate that male meiotic segregation is not random: whatever the type of translocation may be, the distribution of imbalances in sperm is constant, with approximately 72.0% adjacent 1, 18.5% adjacent 2, and 9.5% 3:1 segregations. The same prevalence of adjacent 1 segregations as that reported at term for translocations of paternal origin is observed. There is a strong postzygotic elimination process; for a given translocation it affects selectively the maximum-imbalance zygotes so that imbalanced segregations observed at term are always predetermined.     

Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in Ca2+-ATPase/lipid interaction

The solubilization and delipidation of sarcoplasmic reticulum Ca2+-ATPase by different nonionic detergents were measured from changes in turbidity and recovery of intrinsic fluorescence of reconstituted ATPase in which tryptophan residues had been quenched by replacement of endogenous phospholipids with brominated phospholipids. It was found that incorporation of C12E8 or dodecyl maltoside (DM) at low concentrations in the membrane, resulting in membrane "perturbation" without solubilization, displaced a few of the phospholipids in contact with the protein; perturbation was evidenced by a parallel drop in ATPase activity. As a result of further detergent addition leading to solubilization, the tendency toward delipidation of the immediate environment of the protein was stopped, and recovery of enzyme activity was observed, suggesting reorganization of phospholipid and detergent molecules in the solubilized ternary complex, as compared to the perturbed membrane. After further additions of C12E8 or DM to the already solubilized membrane, the protein again experienced progressive delipidation which was only completed at a detergent concentration about 100-fold higher than that necessary for solubilization. Delipidation was correlated with a decrease in enzyme activity toward a level similar to that observed during perturbation. On the other hand, Tween 80, Tween 20, and Lubrol WX failed to solubilize SR membranes and to induce further ATPase delipidation when added after preliminary SR solubilization by C12E8 or dodecyl maltoside. For Tween 80, this can be related to an inability to solubilize pure lipid membrane; in contrast, Tween 20 and Lubrol WX were able to solubilize liposomes but not efficiently to solubilize SR membranes. In all three cases, insertion of the detergent in SR membranes is, however, demonstrated by perturbation of enzyme activity. Correlation between detergent structure and ability to solubilize and delipidate the ATPase suggests that one parameter impeding ATPase solubilization might be the presence of a bulky detergent polar headgroup, which could not fit close to the protein surface. We also conclude that in the active protein/detergent/lipid ternary complexes, solubilized by C12E8 or dodecyl maltoside, most phospholipids remain closely associated with the ATPase hydrophobic surface as in the membranous form. Binding of only a few detergent molecules on this hydrophobic surface may be sufficient for inhibition of ATPase activity observed at high ATP concentration, both during perturbation and in the completely delipidated, solubilized protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

GPIIb and GPIIIa amino acid sequences deduced from human megakaryocyte cDNAs

Platelet GPIIbIIIa is only synthesized in megakaryocyte or in cell lines with megakaryocytic features. The sequence for GPIIb and GPIIIa have recently been derived from cDNAs obtained from HEL cells. The sequence of these proteins produced by the megakaryocyte, has however, not been determined yet. This study describes full length cDNAs for GPIIb and GPIIIa isolated from megakaryocyte cDNA libraries. The cDNA sequences indicate the presence of nucleotide differences, between the sequence of the GPIIIa cDNAs from HEL cells, endothelial cells and megakaryocytes. One difference was also observed between HEL and megakaryocyte GPIIb at position 633 where a cystein in the megakaryocyte GPIIb, is replaced by a serine in the HEL sequence. The mRNA species for GPIIb (3.4kb) and GPIIIa (6.1 kb) were of the same size in HEL cells and human megakaryocytes.