Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis

Background  

Recent studies point to a great diversity of non-ribosomal peptide synthesis systems with major roles in amino acid and co-factor biosynthesis, secondary metabolism, and post-translational modifications of proteins by peptide tags. The least studied of these systems are those utilizing tRNAs or aminoacyl-tRNA synthetases (AAtRS) in non-ribosomal peptide ligation.     

Reconstitution of RecBC DNase activity from purified Escherichia coli RecB and RecC proteins

The Escherichia coli RecB protein, normally synthesized in low amounts, has been amplified by linkage of the recB gene to the phage lambda leftward promoter in an expression plasmid. From strains harboring this plasmid, RecB protein has been purified to homogeneity by a simple procedure which includes affinity chromatography on a column of RecC protein bound to agarose. The purified RecB protein has DNA-dependent ATPase activity but no exonuclease activity. RecC protein alone has neither ATPase nor exonuclease activity. However, when combined together, the RecB and RecC proteins show the ATP-dependent double-stranded exonuclease properties characteristic of the RecBC DNase.     

Choline transport in Fusarium graminearum A 3 5

Fusarium graminearum A 3/5 possesses a high affinity system (Km = 32 +/- 8 microM; mean +/- SE) for uptake of choline, which was shown to be energy-dependent and constitutive. The maximum rate of choline uptake by this system was repressed by ammonia and glucose, showing a three-fold increase in maximum activity after nitrogen (2 h) or carbon (4 h) starvation. The system was highly specific for choline with only dimethylethanolamine (Ki = 198 +/- 29 microM), betaine aldehyde (Ki = 95 +/- 14 microM) and chlorocholine (Ki = 352 +/- 40 microM) acting as competitive inhibitors. Hemicholinium-3 acted as a mixed (non-competitive) inhibitor (KIES = 1.9 +/- 0.6 microM; KIE = 3.6 +/- 1.9 microM).     

Genetic tagging of tumor cells with retrovirus vectors: clonal analysis of tumor growth and metastasis in vivo.

Retrovirus vector infection was used to introduce large numbers of unique genetic markers into tumor cell populations for the purpose of analyzing comparative changes in the clonal composition of metastatic versus that of nonmetastatic tumors during their progressive growth in vivo. The cell lines used were SP1, a nonmetastatic, aneuploid mouse mammary adenocarcinoma, and SP1HU9L, a metastatic variant of SP1. Cells were infected with delta e delta pMoTN, a replication-defective retrovirus vector which possesses the dominant selectable neo gene and crippled long terminal repeats. G418r colonies were obtained at a frequency of 4 x 10(-3). Southern blot analysis of a number of clones provided evidence of random and heritable integration of one or two copies of the proviral DNA. Clonal evolution of primary tumor growth and the nature of lineage relationships among spontaneous metastases and primary tumors were analyzed by subcutaneously injecting 10(5) cells from a pooled mixture of 3.6 x 10(2) G418r SP1HU9L or 10(4) G418r SP1 colonies into syngeneic CBA/J mice. The most striking finding was the relative clonal homogeneity of advanced primary tumors; they invariably consisted of a small number (less than 10) of distinct clones despite the fact that hundreds or thousands of uniquely marked clones had been injected. In the case of the metastatic SP1HU9L cells, the nature of these "dominant" clones varied from one tumor to another. Analysis of a number of lung metastases revealed that a proportion of them were derived from dominant primary tumor clones and were composed of one, and sometimes two, distinct progenitors. In some animals, all the lung metastases were derived from a common progenitor clone, whereas in others, each metastatic nodule had a different progenitor. The results show the following. (i) Retrovirus vector infection can be used to introduce large numbers of unique and stable clonal markers into tumor cell populations. (ii) The progeny of a very limited number of clones dominate in advanced primary tumors. (iii) Mammary carcinoma metastases are of mono- or biclonal origin. The significance of the results is discussed.     

Antigenic determinants expressed by human C4 allotypes; a study of 325 families provides evidence for the structural antigenic model

The antigenic determinants of human C4 have been defined by human IgG antisera, Rodgers (Rg) and Chido (Ch), in hemagglutination-inhibition assays (HAI). Eight (2 Rg and 6 Ch) are of high frequency, > 90% , and 1, WH, is of low frequency, 15 %. The phenotypic combinations are complex; generally, C4A expresses Rg, and C4B has Ch, but reverse antigenicities have been established both by HAI and by sequence data of selected C4 allotypes. A study of 325 families provides data on the antigenic expression of each C4 allotype and demonstrates strong associations. A structural model for the antigenic determinants of C4 proteins has been proposed and is completely supported by the family material. Of the 16 possible antigenic combinations for C4 proteins, only 3 are undetected. A new Ch combination has been recorded in two French families. The reported sequence variation within the C4d region can account for the antigenic determinants but leaves the location of electrophoretic variation in C4 still unclear.     

Bleomycin and X-ray-hypersensitive Chinese hamster ovary cell mutants: genetic analysis and cross-resistance to neocarzinostatin

We have previously reported the isolation of 3 mutants of Chinese hamster ovary cells which exhibit hypersensitivity to bleomycin. 2 mutants were isolated on the basis of bleomycin-sensitivity [designated BLM-1 and BLM-2, Robson et al., Cancer Res., 45 (1985) 5304-5309] and 1 as adriamycin-sensitive [ADR-1, Robson et al., Cancer Res., 47 (1987) 1560-1565]. Because bleomycin generates DNA-strand breaks via a free-radical mechanism, we have studied the survival response of these mutants to a range of drugs which also generate free radicals and consequently DNA-strand breaks. The mutants are all hypersensitive to phleomycin, which differs from bleomycin in being unable to intercalate due to a modified bithiazole moiety. However, BLM-2 cells alone are hypersensitive to pepleomycin, a semi-synthetic bleomycin analogue. In contrast, BLM-1 cells are more sensitive than BLM-2 to streptonigrin (which operates via a hydroquinone intermediate). ADR-1 cells show wild-type resistance to streptonigrin. The results obtained with neocarzinostatin, an antibiotic requiring thiol activation, are unusual in that both BLM-1 and BLM-2 are approximately 3-fold more resistant than parental cells. However, the steady-state intracellular level of the major non-protein thiol, glutathione, is not altered in BLM-1 or BLM-2 cells. ADR-1 cells show essentially wild-type resistance to neocarzinostatin. Analysis of cell hybrids shows that BLM-1 and BLM-2 cells are phenotypically recessive in combination with parental CHO-K1 cells and represent different genetic complementation groups not only from one another, but also from the bleomycin-sensitive mutant xrs-6, isolated on the basis of X-ray sensitivity by Jeggo and Kemp [Mutation Res., 112 (1983) 313-319]. These results indicate that at least 3 gene products are involved in cellular protection against bleomycin toxicity in mammalian cells.