Synchronization of drosophila cells in culture

Summary We synchronized Drosophila cell lines (Schneider's line 2 and K_c) by allowing the cells to enter the stationary phase of growth and then diluting them into fresh culture medium. The cells of both cell lines entered S phase, after an 8- to 14-hr delay, in a state of partial synchrony; 60 to 80% of the cell population accumulated in S phase. Measurements of the cell cycle phases of Schneider's line 2 cells (S=14 to 16 hr; G₂=6 to 8 hr; M=0.4 hr) were similar to those of K_c cells. This work was performed under the auspices of the U.S. Energy Research and Development Administration. A.R. was supported by an NIH post-doctoral fellowship, No. CA01060.     

Lipid binding by fragments of apolipoprotein C-III-1 obtained by thrombin cleavage.

We have used thrombin to cleave apolipoprotein C-III-1 into two fragments constituting residues 1-40 (apoLP-C-III-A) and 41-79 (apoLP-C-III-B). The lipid binding properties of these fragments with dimyristoyl- and 1-palmitoyl-2-oleoylphosphatidylcholines have been determined using circular dichroic and intrinsic tryptophan fluorescence spectroscopy. The peptide-phospholipid mixtures were fractionated by density gradients of cesium chloride. ApoLP-C-III-A showed disordered structure in the absence and presence of DMPC and no significant amount of peptide-phospholipid complex was isolated. ApoLP-C-III-B showed conformational changes in the circular dichroic spectrum and a shift in the intrinsic tryptophan fluorescence spectrum. Ultracentrifugation in cesium chloride gradients yielded peptide-phospholipid complexes isolated between density 1.10 and 1.18. The molar ratio of lipid to protein was 12:1. The results of these studies and the examination of space filling models of apoLP-C-III provide evidence that an amphipathic alpha helix which contains a nonpolar face and a polar face is the basic structural unit for binding of phospholipid by the plasma apolipoproteins. These results also provide direct evidence that the hydrophobicity of the nonpolar face is important in lipid binding since the nonpolar face of residues 1-40 is considerably less hydrophobic than the nonpolar face of residues 41-79.     

Charge clusters and the orientation of membrane proteins

Summary Although hydrophobic forces probably dominate in determining whether or not a protein will insert into a membrane, recent studies in our laboratory suggest that electrostatic forces may influence the final orientation of the inserted protein. A negatively charged hepatic receptor protein was found to respond totrans-positive membrane potentials as though electrophoresing into the bilayer. In the presence of ligand, the protein appeared to cross the membrane and expose binding sites on the opposite side. Similarly, a positively charged portion of the peptide melittin crosses a lipid membrane reversibly in response to atrans-negative potential. These findings, and others by Date and co-workers, have led us to postulate that transmembrane proteins would have hydrophobic transmembrane segments bracketed by positively charged residues on the cytoplasmic side and negatively charged residues on the extra-cytoplasmic side. In the thermodynamic sense, these asymmetrically placed charge clusters would create a compelling preference for correct orientation of the protein, given the inside-negative potential of most or all cells. This prediction is borne out by examination of the few transmembrane proteins (glycophorin, M13 coat protein, H-2K^b, HLA-A2, HLA-B7, and mouse Ig heavy chain) for which we have sufficient information on both sequence and orientation.In addition to the usual diffusion and pump potentials measurable with electrodes, the microscopic membrane potential reflects surface charge effects. Asymmetries in surface charge arising from either ionic or lipid asymmetries would be expected to enhance the bias for correct protein orientation, at least with respect to plasma membranes. We introduce a generalized form of Stern equation to assess surface charge and binding effects quantitatively. In the kinetic sense, dipole potentials within the membrane would tend to prevent positively charged residues from crossing the membrane to leave the cytoplasm. These considerations are consistent with the observed protein orientations. Finally, the electrostatic and hydrophobic factors noted here are combined in two hypothetical models of translocation, the first involving initial interaction of the presumptive transmembrane segment with the membrane; the second assuming initial interaction of a leader sequence.     

Thin-layer chromatography with agarose gels: A quick,simple method for evaluating liposome size

Thin-layer gels can be made with agarose and used to assess within a few minutes the efficiency with which multilamellar vesicles are converted to small unilamellar ones by sonication. A fluorescent lipid marker or vesicle-encapsulated solute permits continuous monitoring of the chromatography. Advantages over agarose gel column chromatography include speed of analysis, small sample size, the possibility of running multiple samples simultaneously, and direct accessibility to fluorescence microscopy. This approach should also be useful in the study of liposome-lipoprotein interactions and in affinity chromatography of liposomes.     

Host factor for coliphage Q beta RNA replication: presence in procaryotes and association with the 30S ribosomal subunit in Escherichia coli.

Summary The Host Factor required for in vitro coliphage Q RNA replication, a heat-stable RNA binding protein present in uninfectedEscherichia coli, has been detected by both immunological and functional tests inAcinetobacter calcoaceticus, Klebsiella pneumoniae, Pseudomonas aeruginosa andPseudomonas putida. It was not detectable by these criteria inBacillus stearothermophilus, Bacillus subtilis, Caulobacter crescentus, Micrococcus lysodeikticus, Rhodopseudomonas capsulata orSaccharomyces cerevisiae. InEscherichia coli the Host Factor protein has been shown to be associated with ribosomes. It is demostrated here that this association is specific for the 30S ribosomal subunit.     

Synchronization of Drosophila cells in culture.

We synchronized Drosophila cell lines (Schneider's line 2 and Kc) by allowing the cells to enter the stationary phase of growth and then diluting them into fresh culture medium. The cells of both cell lines entered S phase, after an 8- to 14-hr delay, in a state of partial synchrony; 60 to 80% of the cell population accumulated in S phase. Measurements of the cell cycle phases of Schneider's line 2 cells (S = 14 to 16 hr; G2 = 6 to 8 hr; M = 0.4 hr) were similar to those of Kc cells.     

Voltage-dependent orientation of membrane proteins

In order to study the influence of electrostatic forces on the disposition of proteins in membranes, we have examined the interaction of a receptor protein and of a membrane-active peptide with black lipid membranes. In the first study we show that the hepatic asialoglycoprotein receptor can insert spontaneously into lipid bilayers from the aqueous medium. Under the influence of a trans-positive membrane potential, the receptor, a negatively charged protein, appears to change its disposition with respect to the membrane. In the second study we consider melittin, an amphipathic peptide containing a generally hydrophobic stretch of 19 amino acids followed by a cluster of four positively charged residues at the carboxy terminus. The hydrophobic region contains two positively charged residues. In response to trans-negative electrical potential, melittin appears to assume a transbilayer position. These findings indicate that electrostatic forces can influence the disposition, and perhaps the orientation, of membrane proteins. Given the inside-negative potential of most or all cells, we would expect transmembrane proteins to have clusters of positively charged residues adjacent to the cytoplasmic ends of their hydrophobic transmembrane segments, and clusters of negatively charged residues just to the extracytoplasmic side. This expectation has been borne out by examination of the few transmembrane proteins for which there is sufficient information on both sequence and orientation. Surface and dipole potentials may similarly affect the orientation of membrane proteins.     

Fusion of Mycoplasma fermentans strain incognitus with T-lymphocytes.

The ability of Mycoplasma fermentans (strain incognitus) to fuse with cultured lymphocytes was investigated and the fusion process was characterized. Fusion was measured using an assay to determine lipid mixing based on the dequenching of the fluorescent probe, octadecylrhodamine (R18), that was incorporated into the mycoplasma cells. Fusion of M. fermentans was detected with both CD4+ (Molt 3) and CD4- (12-E1) cells. The amount of fusion induced was relatively low and ranged from 5-10% with either cell culture. When primary peripheral blood lymphocytes were used the fusion yield was somewhat higher, reaching 12% of the cell population. Similar findings were obtained with fluorescent microscopy analysis suggesting that a predetermined, but unidentified subpopulation of cultured lymphocytes, were being fused. The rate of fusion was temperature dependent. Following a short lag period fusion at 37 degrees C was virtually completed in 60 min. The lymphocytes remained intact throughout the fusion process, as determined by the Trypan blue staining procedure. Fusion was almost completely inhibited by anti-M. fermentans antisera and by pretreatment of M. fermentans cells with proteolytic enzymes, suggesting that a surface-exposed proteinaceous component is involved in the fusion process.