Occurrence of pppApp-synthesizing activity in actinomycetes and isolation of purine nucleotide pyrophosphotransferase

The occurrence of adenosine 5-triphosphate-3-diphosphate-synthesizing activity was detected in five strains of actinomycetes; Streptomyces morookaensis, Streptomyces aspergilloides, Streptomyces hachijoensis, Actinomyces violascens and Streptoverticillium septatum, out of 825 strains of actinomycetes, bacteria, fungi and imperfecti. Purine nucleotide pyrophosphotransferase were extracellularly excreted associating with the cell growth, and were purified partially or to apparent homogeniety from the culture filtrate. The enzymes are a monomeric protein with a molecular weight of 18000–26000 and synthesize adenosine, guanosine and inosine 5-phosphate (mono, di or tri)-3-diphosphate such as pApp, ppApp, pppApp, pGpp, ppGpp, pppGpp and pppIpp by transferring a pyrophosphoryl group from the 5-position of ATP, dATP and pppApp to the 3-position of purine nucleotides in the presence of a divalent cation and in alkaline state.Abbreviations pppApp adenosine 5-triphosphate 3-diphosphate - ppApp adenosine 5-diphosphate 3-diphosphate - pApp adenosine 5-monophosphate 3-diphosphate - pppGpp guanosine 5-triphosphate 3-diphosphate     

Transfer of Ogu cytoplasmic male sterility to Brassica juncea and improvement of the male sterile line through somatic cell fusion

Male sterility conferred by ogu cytoplasm of Raphanus sativus has been transferred to Brassica juncea cv RLM 198 from male-sterile B. napus through repeated backcrossing and selection. The male-sterile B. juncea is, however, highly chlorotic and late. It has low female (seed) fertility and small contorted pods. To rectify these defects, protoplasts of the male sterile were fused with normal RLM 198 (green, self fertile). Four dark green, completely male-sterile plants were obtained and identified as putative cybrids. All the plants were backcrossed three times with RLM 198. Mitochondrial and chloroplast DNA analysis of backcross progeny confirmed hybridity of the cytoplasm. The restriction pattern of the chloroplast DNA of progeny plants of three cybrids (Og 1, Og 2, Og 3) was similar to that of the green self-fertile RLM 198 and indicated that the correction of chlorosis resulted from chloroplast substitution. The chloroplast DNA of the lone progeny plant of the fourth cybrid (Og 10) could not be analyzed because the plant was stunted and had only a few leaves. When total cellular DNA was probed with mitochondrial probes coxI and atpA it was found that the cybrids had recombinant mitochondria. The chlorosis-corrected plants were early flowering and had vastly improved seed fertility.     

Expression and genomic organization of a dinoflagellate gene family

The luciferin-binding protein (LBP) of the dinoflagellate Gonyaulax polyedra is encoded by large gene family with at least two different members. To more fully understand the expression and genomic organization of this gene family, 40 full-length LBP cDNAs were isolated and mapped with the restriction enzymes Xho I, Eco RI, Pvu II and Hind III. All cDNAs isolated could be placed into one of two groups called LBP and LBP. Two LBP group cDNAs were completely sequenced and were found to share 99% identity at both the nucleotide and protein levels. One LBP cDNA was sequenced and was found to share only 86% sequence identity with the LBP group at both the nucleotide and protein levels. Both groups of message appear to be expressed at nearly equal levels since (1) two-dimensional gels of purified LBP show two protein isoforms present in roughly equal amounts and (2) northern blots using group-specific probes suggest that cellular levels of LBP and LBP mRNAs are identical. Genomic Southern blots using group-specific probes suggests that the copy number of both gene groups is very similar and that LBP gene loci are organized as tandem repeats of either LBP or LBP sequences.     

Sources and inheritance of resistance to leaf curl virus in Lycopersicon

Summary One hundred and twenty-two varieties, lines and wild accessions of Lycopersicon were screened under three different regimes during the autumn/winter season of 1982–83 and 1983–84 for resistance to tomato leaf curl virus (TLCV). L. hirsutum f. glabratum (B6013) and L. hirsutum f. typicum (A1904) proved to be highly resistant to TLCV in all three environments. Various accessions of L. peruvianum were also highly resistant. L. pimpinellifolium (A1921) exhibited no TLCV symptoms within 90 days. Of the cultivated varieties, Acc 99 exhibited the minimim score for susceptibility; AC 142, Collection No. 2, Kalyanpur Angurlata and HS 101 had a low rating for virus incidence. The inheritance of resistance was studied in the interspecific crosses between a TLCV resistant line of L. pimpinellifolium (A1921) and five (HS 101, HS 102, HS 110, Pusa Ruby and Punjab Chhuhara) susceptible cultivars of L. esculentum. Parents, F₁, F₂ and backcross progenies were artificially inoculated with local strains of TLCV using vector the viruliferious whitefly, Bemisia tabaci (Genn.). Data indicated that the resistance of L. pimpinellifolium (A 1921) is monogenic and incompletely dominant over susceptibility.     

Evidence for maternal effect in the inheritance of grain protein in crosses between cultivated and wild tetraploid wheats

Summary Reciprocal crosses were made between cultivated wheat (Triticum turgidum var. durum) and a high-protein line of wild tetraploid wheat (T. turgidum var. dicoccoides). F₁ grains (on maternal spikes) were very similar to the selfed grains on the maternal parent in protein percentage, weight and protein content. These traits were also analyzed in F₃ grains developed on F₂ spikes of segregating populations derived from reciprocal crosses between the same cultivated parent and another high-protein line of var. dicoccoides. No significant differences in the mean values of these traits were found between the reciprocal crosses, indicating no cytoplasmic effect. It has been concluded that these grain characteristics are largely determined by the maternal plant.Recipient of Sir Charles Clore Postdoctoral FellowshipThe Marshall and Edith Korshak Professor of Plant Cytogenetics     

The transport of class I major histocompatibility complex antigens is determined by sequences in the α1 and α2 protein domains

The transport of human-mouse hybrid class I histocompatibility antigens has been studied in a mutant human cell line, 174 × CEM.T2 (T2). T2, a somatic cell hybrid of human B- and T-lymphoblastoid cell lines (B-LCL and T-LCL, respectively), synthesizes HLA-A2 and HLA-B5 glycoproteins, but expresses only low levels of A2 and undetectable levels of B5 at the cell surface. We have previously shown that the products of human class I genes introduced into T2 by transfection behave like the endogenous HLA-B5 glycoproteins, while the products of mouse class I alleles similarly introduced are transported normally to the cell surface. We have now determined that the surface expression of class I glycoproteins in T2 depends on the origin of the ₁ and ₂ domains. Human (HLA-B7) and mouse (H-2D ^p ) hybrid class I genes, encoding the leader, ₁, and ₂ sequences of one species fused to the ₃, transmembrane, and cytoplasmic domains of the other, were transfected into T2. Normal surface expression of the hybrid class I molecule was observed in T2 only when the leader, ₁, and ₂-encoding exons were derived from the mouse gene. The reciprocal construct, encoding human leader, ₁, and ₂ domains fused to the mouse ₃, transmembrane, and cytoplasmic regions, resulted in biosynthesis of a hybrid glycoprotein which was not transported to the cell surface. The products of both constructs were expressed normally in control cells. The effects of glycosylation on class I antigen transport were also studied using mutant class I constructs with altered glycosylation sites. Two mutant B7 genes encoding either an extra glycosylation site at position 176 or no glycosylation sites were transfected into T2. These mutant products were expressed at the cell surface in control cells, but were synthesized and not surface-expressed in T2. These data demonstrate that the HLA/H-2 transport dichotomy in T2 is a function of the origin of the ₁ and/or ₂ domains of the class I glycoprotein, and is not a reflection of glycosylation differences between the human and mouse molecules. Offprint requests to: P. Cresswell.     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Preparations ofN,N′-ethylene-bridged dipeptides(eXX) constructed from (S)-methionine, -tryptophan, -tyrosine and-N(ɛ)-benzyloxycarbonyllysine through acid-catalyzed cyclization

Summary N, N-Ethylene-bridged bis-(S)-methionine[(2S, 7S)-2, 7-bis(2-methyl-thioethyl)-3,6-diazaoctanedioic acid] derived from (S)-methionine and 1,2-dibromoethane was cyclized and esterified simultaneously in boiling ethanol in the presence of an appropriate amount of strong acid such asp-toluenesulfonic acid, affording a cyclic compound,N, N-ethylene-bridged (S)-methionyl-(S)-methionine ethyl ester {ethyl(2S, 3S)-4-(methylthio)-2-[2-oxo-3-(2-methylthioethyl)-1-piperazinyl] butanoate}, exclusively in 80–90% yields. It was also found that, by applying this method, 70–80% yields of the otherN, N-ethylenebridged dipeptides containing (S)-tryptophan, -tyrosine and -N()-benzyloxycarbonyllysine were obtained.     

Sialyltransferase activity in FR3T3 cells transformed withras oncogene: decreased CMP-Neu5Ac:Galβ1-3GalNAc α-2,3-sialyltransferase

We have investigated the activity of CMP-Neu5Ac:Gal\1-3GalNAc -2,3-sialyltransferase (EC 2.4.99.4) in FR3T3 cells transformed by the Ha-ras oncogene in which we have previously demonstrated the higher expression of the -galactosidase -2,6-sialyltransferase (EC 2.4.99.1) [21]. We demonstrate that the presence of the activatedras gene decreases the activity of this specific -2,3-sialyltransferase fourfold. According to the kinetic parameters and to mixing experiments, we can assume that this decreased enzymatic activity reflects a decrease in the number of activeO-glycan -2,3-sialyltransferase polypeptides inras-transformed cells. However, no change in the binding of Peanut agglutinin was observed on the cell surface ofras-transformed FR3T3 suggesting that no change in the sialylation ofO-glycan core 1 appeared in these cells, although the activity of the -2,3-sialyltransferase was decreased.Abbreviations -2,3-ST(O) CMP-Neu5Ac:Gal1-3GalNAc-R -2,3-sialyltransferase - -2,3-ST(N/O) CMP-Neu5Ac:Gal1-3/4GlcNAc-R -2,3-sialyltransferase - -2,6-ST(N) CMP-Neu5Ac:Gal1-4GlcNAc-R -2,6-sialyltransferase - -2,6-ST(O)I CMP-Neu5Ac:R-GalNAc(1-O)Ser -2,6-sialyltransferase - -2,6-ST(O)II CMP-Neu5Ac:Neu5Ac2-3Gal1-3GalNAc-R -2,6-sialyltransferase - ASFet asialofetuin - FR3T3 Fisher rat fibroblast - FRras Ha-ras-transfected FR3T3 fibroblasts - NaCl/P_i sodium phosphate 10mm, NaCl 0.15m, pH 7.4, buffer - pNp p-nitrophenol     

Gene structure of the ‘large’ sialidase isoenzyme fromClostridium perfringens A99 and its relationship with other clostridialnanH proteins

Clostridium perfringens possesses two sialidase isoenzymes of different molecular weight. Almost 90% of the gene encoding the large form was found on a 3.1 kb chromosomal fragment (Sau3AI) of strain A99 by hybridization with probes developed from the N-terminal protein sequence and from commonly conserved sialidase motifs (Asp-boxes), whereas the remaining 3-terminal part was detected on a 2.1 kb fragment (Hind III) of chromosomal DNA. After combination of both fragments, the resultingE. coli clones expressed sialidase activity, the properties of the recombinant sialidase corresponding with those of the wild type enzyme. The entire chromosomal fragment of 3665 bp encompasses the complete sialidase gene of 2082 bp corresponding to 694 amino aids, from which a molecular weight of 72956 for the mature protein can be deduced. The first 41 amino acids are mostly hydrophobic and probably represent a signal peptide. The sialidase structural gene follows a non-coding region with an inverted repeat and a ribosome-binding site. Upstream from the regulatory region, another open reading frame (ORF) was detected. The 3-terminus of the sialidase structural gene is directly followed by a further ORF of unknown function, which possibly encodes a putative permease or the acylneuraminate pyruvate-lyase involved in sialic acid catabolism. The primary structure of the large isoenzyme is very similar to the sialidase ofClostridium septicum (55% identical amino acids), whereas the homology with the small form of the same species is comparatively low (26%).