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The A TPase eomplex has been isolated from mitoehondria of N eurospora crassa by immunologieal teehniques. The protein ean be obtained rapidly and qua ntitatively in high purity by miero- or large-seale immunopreeipitation. Immunopreeipitation has been applied to labeled and doubly labeled mitoehondrial proteins in order to investigate the number and moleeular weights of subunit polypeptides , the site of synthesis of subunit polypeptides, and the dieycIohexyIcarbodiimide-binding protein . The A TPase complex obtained by large-seale immunopreeipitation has been used as starting ma terial for the isolation of hydrophobie polypeptides.
Three F0 subunits and the F\(_1\) subunit P of the ATP synthase from Neurospora crassa were labeled with the lipophilic photoactivatable reagent 3-(trifluoromethyl)-3-(m-[\(^{125}\)I]iodophenyl)diazirine ([\(^{125}\)I]TID). In the proteolipid subunit which was the most heavily labeled polypeptide labeling was confmed to five residues at the NH2-terminus and five residues at the C-terminus ofthe protein. Labeling occurred at similar positions compared with the homologaus protein (subunit c) in the ATP synthase from Escherichia coli, indicating a similar structure of the proteolipid subunits in their respective organisms. The inhibitors oligomycin and dicyclohexylcarbodiimide did not change the pattern of accessible surface residues in the proteolipid, suggesting that neither inhibitor induces gross conformational changes. However, in the presence of oligomycin, the extent oflabeling in some residues was reduced. Apparently, these residues provide part of the binding site for the inhibitor. After reaction with dicyclohexylcarbodiimide an additional labeled amino acid was found at position 65 corresponding to the invariant carbodümide-binding glutamic acid. These results and previous observations indicate that the carboxyl side chain of Glu-65 is located at the protein-lipid interphase. The idea is discussed that proton translocation occurs at the interphase between different types if F\(_0\) subunits. Dicyclohexylcarbodiimide or oligomycin might disturb this essential interaction between the F\(_0\) subunits.
Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo
(1985)
A method has been developed that allows the isolation of genomic clones from a cosmid library by homologaus recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene. The genomic cosmid library was packaged in vivo into A. phage particles. A recombination-proficient host strain carrying IL2 cDNA sequences in a non-homologaus plasmid vector was infected by the packaged cosmid library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both vectors were selected. From a recombinant cosmid clone the chromosomal IL2 genewas restored. After DNA mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively.
The effects of the interlenkins IL-7 and IL-9 on cell cycle progression were investigated by conventional [3H]thymidine incorporation and by the bivariate BrdU/Hoechst technique. 8oth IL· 7 and IL-9 drive phytohemagglutinin-activated T cells through more than one cell cycle, but IL-7 wasmorepotent on cell cycle progression than IL-9. Neither synergistic nor inhibitory effects were seen between various combinations of the lymphokines IL-7, IL-9 and IL-4 compared to each lymphokine alone. When T cells are activated with phytohemagglutinin for 3 days, all or most IL-4 responsive cells respond to IL-7 as weil, whereas only a part of IL-7 responders are IL-4 responders. In contrast, when T cells are activated with phytohemagglutinin for 7 days, the quantitative data of the cell cycle distribution soggest that the population of IL-7 responders is at least an overlapping, if not a real subset of the population of the IL-4 responders.