Prediction of beta-turns.

An automated computer prediction of the chain reversal regions of globular proteins is described herein using bend frequencies and beta-turn conformational parameters (Pt) determined from 408 beta-turns in 29 proteins calculated from x-ray atomic coordinates. The probability of bend occurrence at residue i is pt = fi X fi+1 X fi+2 X fi+3 with the average bend probability less than Pt greater than = 0.55 X 10(-4). Tetrapeptides with pt greater than 0.75 X 10(-4) ( approximately to 1.5 X less than pt greater than) as well as less than Pt greater than 1.00 and less than Pa greater than less than less than Pt greater than greater than less than P beta greater than are selected by the computer as probable bends. Adjacent probable bends (i.e., 11-14, 12-15, 13-16) are compared pairwise by the computer, and the tetrapeptide with the higher pt value is predicted as a beta-turn. The percentage of bend and nonbend residues predicted correctly for 29 proteins by this computer algorithm is %t+nt = 70%, whereas 78% of the beta-turns were localized correctly within +/- 2 residues. The average beta-turn content in the 29 proteins is 32%, with helical proteins having fewer bends (17%) than beta-sheet proteins (41%). Three proteins having iron-sulfur clusters were found with the highest percentages of beta-turns: Chromatium high potential iron protein (65%), ferredoxin (57%), and rubredoxin (65%). Finally, the bend frequencies at all 12 positions from 457 beta-turns in 29 proteins (Chou and Fasman, 1977) were used to test the effectiveness of predicting bends using 2, 4, 8, and 12 residues as well as different cut-off pt values. The computer analysis showed that 1.25 less than pt greater than to be the best cut-off yielding 70% accuracy in %t+nt for 4 residues and %t+nt = 73% for 12 residues in predicting the bend and nonbend regions of proteins.     

Conservation of chain reversal regions in proteins.

Using the bend frequencies based on 29 proteins in the previous paper (Chou and Fasman, 1979), beta-turn probability profiles were calculated for the C-peptides of 10 mammalian proinsulins, for 7 proteinase inhibitors, and for 12 species of pancreatic ribonucleases. Beta-turn correlation coefficient matrix tables were also computed to obtain the statistical mean between 45 pairs of proinsulin C-peptides, less than Ct greater than = 0.57 +/- 0.31; 21 pairs of proteinase inhibitors, less than Ct greater than = 0.73 +/- 0.13; and 66 pairs of ribonucleases, less than Ct greater than = 0.83 +/- 0.08. Despite relatively low sequence conservation in these three sets of proteins, beta-turns were predicted to be highly conserved: 33% sequence vs. 78% bend for the proinsulins, 20% sequence vs. 85% bend for the proteinase inhibitors, and 65% sequence vs. 92% bend for the ribonucleases. These results suggest that chain reversal regions play an essential role in keeping the active structural domains in hormones and enzymes intact for their specific biological function.     

Absence of Detectable Mitochondrial Recombination in Paramecium

An extensive search for recombination between mitochondrial markers was carried out in Paramecium tetraurelia. Thirty-two combinations, altogether involving 24 different markers, were studied. The markers belonged to the three main categories of mitochondrial mutations presently available in this organism, (a) Spontaneous or UV-induced antibiotic resistance mutations, most probably affecting mitochondrial ribosomes, (b) nitrosoguanidine-induced antibiotic resistance markers displaying thermosensitivity or slow growth, enabling easy selection of possible wild-type recombinants, and (c) mitochondrial partial suppressors of a nuclear gene, probably corresponding to molecular alterations distinct from the preceding two categories. In addition, different genetic configurations were analyzed (i.e., mutant X mutant, double-mutant X wild-type, etc.).--None of the combinations yielded any evidence for the occurrence of recombined genomes despite the fact that: (1) all of them were studied on a large scale involving the screening of at least several thousand mitochondrial genomes (often several millions), (2) in many of them the detection level was sufficiently high to enable the isolation of spontaneous mutants in control cells, and (3) in several of them, reconstitution experiments carried out in parallel show that the conditions were fully adequate to detect recombinant genotypes. The results are in marked contrast with those obtained on the few other organisms in which mitochondrial recombination has been studied, particularly Saccharomyces cerevisiae, in which mitochondrial recombination is intense.--The most likely basis for the various manifestations of mitochondrial genetic autonomy in Paramecium, described in this as well as in previous publications, is that the chondriome of this organism is made up of thousands of structurally discrete, noninteracting units.     

Studies on normal human bone marrow cultures in controlled environment thin-film culture systems

Summary Two thin film culture systems, the controlled environment steady state system (SS) and the rocker tube configuration of that system (RT), were used to identify some of the conditions that appear to maintain morphologic and functional characteristics of cells of human bone marrow explants in vitro. The systems configuration assured continual gassing, control and easy monitoring of the cultures. Cytocentrifuge preparations of media of specimens cultured in RT disclosed, though in decreasing numbers, various hematopoietic cells for periods exceeding one month. Hematopoietic cells shed from specimens cultured in the SS system were retained in the culture tubes; cells of the myelocytic series predominated for the first 2 weeks while an increasing number of monocytes and macrophages appeared in the media of older cultures. Histologic examination of cultured explants disclosed preservation of the marrow architecture and the persistence of hematopoietic cells. Specimens cultured in RT tubes tended to be less cellular than similar cultures placed in dialysis bags or as cultured in the SS system. Immunoglobulins (Ig) were released into the culture media at a constant rate throughout the period of culture. Specimens that were cultured at a controlled pH of 7.4 released 2 to more than 4 times as much Ig as similar specimens maintained at a pH level of 7.1. There were no definitive differences in Ig levels in the cultures maintained at comparable pH levels and overlaid with various CO₂ concentrations, i.e. 2%, 5%, 10%; similarly, no differences in Ig levels were found in specimens cultured in media containing fetal bovine sera as opposed to horse sera. Supported by U.S.P.H.S. Grant CA-5834 from the National Cancer Institute. Department of Medicine A. Department of Cell Physiology Department of Immunology and Immunochemistry.     

Synthesis of alpha subunit of human chorionic gonadotrophin by presumptive HeLa cells

Summary Several cell lines, originally thought to be derived from a human placenta at term but possibly HeLa-contaminated, have been studied. These cells secrete a protein indistinguishable immunochemically from the alpha subunit of chorionic gonadotropin but not the beta subunit of chorionic gonadotropin or placental lactogen. Complete chorionic gonadotropin was detected but amounted to less than 1% of the level of the alpha subunit. The cells also produce an alkaline phosphatase similar to placental alkaline phosphatase in immunochemical, gel-electrophoretic, and heat-denaturation properties. They induce tumor growth when inoculated into nude mice. These cells are aneuploid and have a model chromosome number of 66. The common HeLa karyologic markers, designated 1, 2, and 3, and A-type glucose-6-phosphate dehydrogenase are present in these cells. HeLa cells have not previously been shown to secrete theα subunit of hCG.     

The conformation of glucagon: predictions and consequences.

It is proposed that glucagon, a polypeptide hormone, is delicately balanced between two major conformational states. Utilizing a new predictive model [Chou, P.Y., and Fasman, G.D. (1974), Biochemistry 13, 222] which considers all the conformational states in proteins (helix, beta sheet, random coil, and beta turns), the secondary structural regions of glucagon are computed herein. The conformational sensitivity of glucagon may be due to residues 19-27 which have both alpha-helical potential (mean value of Palpha = 1.19) as well as beta-sheet potential (mean value of Pbeta = 1.25). Two conformational states are predicted for glucagon. In predicted form (a), residues 5-10 form a beta-sheet region while residues 19-27 form an alpha-helical region (31% alpha, 21% beta) agreeing well with the circular dichroism (CD) spectra of glucagon. The similarity in the CD spectra of glucagon and insulin further suggests the presence of beta structure in glucagon, since X-ray analysis of insulin showed 24% beta sheet. In predicted form (b), both regions, residues 5-10 and residues 19-27, are beta sheets sheets (0% alpha, 52% beta) in agreement with the infrared spectral evidence that glucagon gels and fibrils have a predominant beta-sheet conformation. Since three reverse beta turns are predicted at residues 2-5, 10-13, and 15-18, glucagon may possess tertiary structure in agreement with viscosity and tritium-hydrogen exchange experiments. A proposal is offered concerning an induced alpha yields beta transition at residues 22-27 in glucagon during receptor site binding. Amino acid substitutions are proposed which should disrupt the beta sheets of glucagon with concomitant loss of biological activity. The experimental findings that glucagon aggregates to form dimers, trimers, and hexamers can be explained in terms of beta-sheet interactions as outlined in the present predictive model. Thus the conflicting conclusions of previous workers, concerning the conformation of glucagon in different environments, can be rationalized by the suggested conformational transition occurring within the molecule.     

Products of complementation between temperature-sensitive mutants of simian virus 40.

Temperature-sensitive mutants of the D complementation group of simian virus 40 exhibit delayed complementation. Analysis of the thermal stability, kinetic profiles in temperature shift experiments, and progeny of complementation have led to the hypothesis that delayed complementation is not true complementation, but the result of a very low level of leakiness, followed by phenotypic mixing of the progeny D mutants. This hypothesis is consistent with the proposal that D mutants are defective in uncoating. In the course of these experiments, it was observed that fresh medium suppresses the growth of D mutants at the restrictive temperature.     

Plasmodium berghei leucine-rich repeat protein 1 downregulates protein phosphatase 1 activity and is required for efficient oocyst development

Protein phosphatase 1 (PP1) is a key enzyme for Plasmodium development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the functional characterization of the Plasmodium berghei leucine-rich repeat protein 1 (PbLRR1), an orthologue of SDS22, one of the most ancient and conserved PP1 interactors. Our study shows that PbLRR1 is expressed during intra-erythrocytic development of the parasite, and up to the zygote stage in mosquitoes. PbLRR1 can be found in complex with PbPP1 in both asexual and sexual stages and inhibits its phosphatase activity. Genetic analysis demonstrates that PbLRR1 depletion adversely affects the development of oocysts. PbLRR1 interactome analysis associated with phospho-proteomics studies identifies several novel putative PbLRR1/PbPP1 partners. Some of these partners have previously been characterized as essential for the parasite sexual development. Interestingly, and for the first time, Inhibitor 3 (I3), a well-known and direct interactant of Plasmodium PP1, was found to be drastically hypophosphorylated in PbLRR1-depleted parasites. These data, along with the detection of I3 with PP1 in the LRR1 interactome, strongly suggest that the phosphorylation status of PbI3 is under the control of the PP1–LRR1 complex and could contribute (in)directly to oocyst development. This study provides new insights into previously unrecognized PbPP1 fine regulation of Plasmodium oocyst development through its interaction with PbLRR1.