A new solid-phase synthesis of oligoribonucleotides by the phosphoro-p-anisidate method using tetrahydrofuranyl protection of 2''-hydroxyl groups.

Six nonaribonucleotides containing the 5'-splice site, one complementary nonamer and an octadecamer containing the 3'-splice site have been synthesized on a polymer support using the phosphoro-p-anisidate method. A 5'-linked 2'-O-tetrahydrofuranyl-N-protected nucleoside 3'-(o-chlorophenyl)phosphoro-p-anisidate was used as the starting nucleotide, and the chain elongated in the 3'-direction by removing the p-anisidate protecting group with isoamyl nitrite under neutral conditions. The octadecamer has been synthesized using dinucleotide blocks and a 3'-terminal trinucleotide.     

Cloning of a Vero toxin (VT2) gene from a VT2-converting phage isolated from Escherichia coli 0157: H7

Abstract A Vero toxin (VT2 or Shiga-like toxin II)-converting phage was isolated from Escherichia coli 0157: H7 strain J-2. Nontoxigenic E. coli C600 produced VT2 when lysogenized with the toxin-converting phage. Eco RI fragments of the phage DNA were ligated with Eco RI-digested pBR322 or pUC118 and were transformed into E. coli MC1061 or MV1184. Transformants exhibiting VT2 production commonly contained a 4.6 kb Eco RI fragment. It was found that a 2.3 kb Kpn I- Sph I fragment coded VT2 production and that this fragment hybridized weakly with the 2.1 kb fragment encoding VT1.     

Activation of mouse peritoneal macrophages by synthetic glyceroglycolipid liposomes

Summary Liposomes composed of chemically synthesized glyceroglycolipids, such as 1,2-dipalmityl-[-cellobiosyl-(1 3)]-glycerol (Cel-DAG), 1,2-dipalmityl-[-lactosyl-(1 3)]-glycerol, or 1,2-dipalmityl-[-maltosyl-(1 3)]-glycerol, were found to enhance protective immunity against transplantable tumor cells (sarcoma 180) in ICR mice. Peritoneal exudate cells prepared from mice treated in vivo with Cel-DAG showed cytostatic activity in vitro against the mouse leukemia cell line, EL-4. Adherent cells separated from this preparation showed similar activity. Peritoneal cells from polypeptone-injected mice acquired appreciable cytostatic activity when incubated in vitro in the presence of glyceroglycolipid liposomes. The adherent cell fraction alone showed rather weak cytostatic activity when pretreated with the glyceroglycolipids, and full activity was restored by supplementing with the nonadherent cell fraction. The ability of glycolipids to induce tumoricidal effects was affected by cholesterol content: with increasing cholesterol content, the activities decreased. Cholesterol-free glycolipid liposomes were taken more efficiently by macrophages than cholesterol-containing liposomes. Cholersterol modifies the surface property of glyceroglycolipid liposomes. Activation of macrophages is responsible for enhancement of protective immunity against tumor cells by injection of these glycolipids in vivo.This work was supported in part by Grants-in-Aid (Nos. 58010010, and 59870076) for Scientific Research from the Ministry of Education, Science and Culture of Japan     

Electron Transfer between Plastocyanin and P700 in Various Types of Photosystem I Complex

Three types of PS I Chl-protein complex, PS I 180, PS I 65,and PS I 30, have been prepared and the kinetic properties ofthe transfer of electrons from plastocyanin to P700 in the PSI complexes with different sized antennae were examined. ThePS I 180 complex, which consists of 180 Chi per P700, showedthe almost same rate constant and effects of cations for thetransfer of electrons from plastocyanin to P700 as those obtainedwith PS I-enriched membrane fragments. The rate constant increasedwith the addition of low concentrations of monovalent and divalentcations, but decreased with high concentrations of cations.However, the rate was severely reduced in the case of the PSI 65 and PS I 30 complexes, and quite different effects of cationswere observed. Given the presence of additional 25- to 28-kDapolypeptides in the PS I 180 complex as compared to the PS I65 and PS I 30 complexes, we discuss a possible function forthese polypeptides in the regulation of the reaction betweenplastocyanin and P700. ¹This work was supported in part by a Grant-in-Aid for ScientificResearch from the Ministry of Education, Science and Cultureof Japan. (Received May 27, 1988; Accepted November 7, 1988)     

Mutations in {beta}-Tubulin Block Transhyphal Migration of Nuclei in Dikaryosis in the Homobasidiomycete Coprinus cinereus

ß-Tubulins from fourteen benomyl-resistant strainsof the homobasidiomycete Coprinus cinereus, which carry thebenA, benB, benC or benD mutations, were analyzed by urea SDS-PAGEor isoelectric focusing and subsequent immunoblot analysis.Electrophoretic aberrations in a major ß-tubulin isotype,denoted ß1 were found in two strains, BEN154 and BEN215,both of which carry benomyl resistance mutations in benA ⁺ Theaberrations of ß1 in BEN154 and BEN215 cosegregatedwith benomyl resistance among the progeny of outcrosses of BEN154 and BEN215 to wild type, indicating that the ß1aberrations were caused by the benA mutations. Both the mutantand wild-type ß1 tubulins were present in the heterozygousdikaryons, BEN 154/wild-type and BEN215/wild-type, ruling outpost-translational modification as a possible cause for theaberrations in ß1. Thus, we conclude that benA isa structural gene for ß1. Transhyphal migration ofnuclei in dikaryosis was blocked in the mycelia of BEN 154 andin its progeny that carried benA (ß1 mutation), demonstratingthat microtubules are involved in the migration process. Nuclearmigration in dikaryosis seems to differ in terms of mechanism,at least in part, from the migration of tetrad nuclei from basidiainto prespores during formation of basidiospores and from themigration of nuclei from basidiospores into hyphae during germination,because a benA mutation blocked the former without affectingthe latter two processes. (Received May 19, 1989; Accepted August 30, 1989)     

The structure ofHLA-B35 suggests that it is derived fromHLA-Bw58 by two genetic mechanisms

The primary structure ofHLA-B51 andHLA-Bw52 suggested thatHLA-B51 was derived fromHLA-Bw52 by the combination of a genetic exchange withHLA-B8 and a point mutation. To investigate the evolution of theHLA-B5 cross reactive group, theHLA-B35 gene was cloned and the primary structure was determined.HLA-B35 is identical toHLA-Bw58 except in the α₁ domain. The α₁ domain ofHLA-B35 except Bw4/Bw6-associated amino acids is identical to that ofHLA-B51 ^*, which was suspected to be an intermediate gene betweenHLA-B51 andHLA-Bw52. These data suggest thatHLA-B35 has evolved fromHLA-Bw58 in two steps; an in vivo replacement of the α₁ domain withHLA-B51 and genetic exchange with one of theHLA-Bw6 genes. These three genes andHLA-Bw58 are postulated to share a common ancestor.     

The low C5 convertase activity of the C4A6 allotype of human complement component C4.

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.     

Conformational study of poly(L-lysine) interacting with acidic phospholipid vesicles

Circular dichroism measurements were carried out on poly(L-lysine) in the presence of vesicles of the negatively charged phospholipids, phosphatidylserine (PS; from bovine brain), phosphatidic acid (PA; prepared from egg yolk lecithin) and dimyristoylphosphatidylglycerol (DMPG). PS vesicles induced a conformational change in poly(L-lysine) from random coil to alpha-helix structure in 5 mM Tes (pH 7.0), whereas PA vesicles gave rise to beta-structure in the same buffer. The fraction of alpha-helix, F alpha (or beta-structure, F beta), increased with increasing PS (or PA) concentration, reaching a saturation value of about 0.7 (or about 1). Mixed vesicles comprising PS and dilauroylphosphatidylcholine (DLPC) also induced alpha-helix conformation, however, the saturation value of F alpha diminished with decreasing PS content in mixed vesicles. On the other hand, the spectral patterns for poly(L-lysine) in DMPG vesicle suspensions exhibited the coexistence of alpha-helix and beta-structure. Both F alpha and F beta increased with DMPG concentration and reached saturation values of about 0.5. Mixed vesicles composed of DMPG and dimyristoylphosphatidylcholine (DMPC) led to a reduction in F beta, while F alpha remained almost constant. The diversity in ordered structure induced by different phospholipid vesicles suggests the participation of lipid head groups in determining the secondary structure of poly(L-lysine) adsorbed on the vesicular surface.