Mutations induced by ionizing radiation in a plasmid replicated in human cells. II. Sequence analysis of alpha-particle-induced point mutations

The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells. Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E. coli. The mutations were characterized by sequencing the tRNA gene. The mutant frequency increased linearly with the alpha-particle dose and, at 259 Gy, it was 16 times (0.29%) that observed in unirradiated controls (0.018%). The distribution of alpha-particle-induced point mutations was highly nonrandom and similar to that observed in the unirradiated or X-irradiated plasmid DNAs. The majority of the mutations were G.C----A.T transitions and occurred selectively at most 5'-TC (3'-AG) and 5'-CC (3'-GG) sequences. For the unirradiated control DNA, these mutations at C's (G's) were preferentially located in the nontranscribed strand, similar to the observation previously made for mutations in X-irradiated DNA. Such a strand bias was not observed for mutations in the alpha-particle-irradiated DNA. The data suggest that, although similar types of point mutations are induced in unirradiated, X-irradiated, and alpha-particle-irradiated DNAs, the mechanisms of their induction and the exact nature of the lesions involved may be quite different.     

Mutations induced by ionizing radiation in a plasmid replicated in human cells. I. Similar, nonrandom distribution of mutations in unirradiated and X-irradiated DNA

The Escherichia coli supF gene encoding the suppressor tyrosine tRNA in a human shuttle plasmid, pZ189, was used as a target for molecular analysis of X-ray-induced mutations in human lymphoblastoid cells. Following replication of the in vitro-irradiated plasmid in human cells, the mutant supF-containing molecules were cloned by phenotypic screening in E. coli and the nature of the mutations was determined by direct sequencing of the tRNA gene. At 160 Gy the mutant frequency was 13 times (0.39%) that observed in unirradiated controls (0.031%). When control plasmid was replicated directly in E. coli, the mutant frequency was 16 times less than that of the plasmid passaged through the human cells. The distribution of mutations was highly nonrandom and remarkably similar in both irradiated and control DNAs. The majority of the mutations were transitions involving G.C pairs and occurred selectively at most 5'-TC (3'-AG) sequences. These mutations at C's were preferentially distributed in the nontranscribed strand. We propose that mutations in the control plasmid result from oxidative damages that occur during and/or after its incorporation into human cells and that these damages are similar to those induced by ionizing radiation. The hot spots for mutations suggest that the proximate nucleotide sequence and the overall conformation of the target DNA are important in the production and/or processing of these damages during repair and replication.     

Inherited X-chromosome inverted tandem duplication in a male traced to a grandparental mitotic error.

A male infant was referred for cytogenetic evaluation because of dysmorphic features and developmental delay. In both lymphocytes and skin fibroblasts, a modal number of 46 chromosomes was obtained with an obvious elongation of the long arm of the X chromosome (Xq+). Studies of seven members in 3 generations of this family showed that the proband's mother, sister, and maternal grandmother were phenotypically normal carriers of this abnormal X chromosome. High resolution GTG- and RBG-banding defined the extra chromatin material as an inverted duplication of Xq21----Xq24. This was supported by an approximate twofold increase in alpha-galactosidase A activity, localized to Xq21----q24, observed in the proband's lymphocytes and fibroblasts. BrdU-incorporation studies of the mother's lymphocytes showed the abnormal X to be late replicating in all 100 cells studied and normal alpha-galactosidase A levels. Cytogenetic analysis of the maternal grandmother revealed cytogenetic mosaicism with one cell line containing the abnormal X (37%), and the other, a normal female karyotype (63%). This family is instructive since: (1) it represents only the second case of a dysmorphic male demonstrating a confirmed interstitial partial Xq duplication, and (2) the origin of this familial structural rearrangement has been traced to a grandparental mitotic error.     

DNA environment of the aerobactin iron uptake system genes in prototypic ColV plasmids.

The aerobactin iron uptake system genes in the prototypic plasmid pColV-K30 are flanked by inverted copies of insertion sequence IS1 and by two distinct replication regions. To address the question of how these flanking regions may facilitate the maintenance and spread of the aerobactin system among the plasmids and chromosomes of enteric species, we investigated the DNA environment of 12 ColV plasmids. We found that the aerobactin system-specific genes are conserved in every plasmid phenotypically positive for the aerobactin system. The upstream IS1 and its overlapping replication region (REPI) are also conserved. This replication region was cloned from several ColV plasmids and found to be functional by transforming these cloned derivatives into a polA bacterial host. In contrast, the downstream flanking region is variable. This includes the downstream copy of IS1 and the downstream replication region (REPII). We infer from these results that sequences in addition to the two flanking copies of IS1, in particular the upstream region including REPI, have been instrumental in the preservation and possible spread of aerobactin genes among ColV plasmids and other members of the FI incompatibility group.     

Purification and Characterization of an Iminopeptidase from the Primary Leaf of Wheat (Triticum aestivum L.)

Iminopeptidase (EC 3.4.11.5) was substantially purified from the primary leaves of 7-day-old wheat seedlings (Triticum aestivum L.). The purification procedure consisted of five steps: acid precipitation, molecular exclusion chromatography on Sephacryl S-200, Ultrogel AcA 44, Sepharose 2B and ion-exchange chromatography on DEAE-cellulose. Iminopeptidase isolated in this manner was only active against the β-naphthylamides of proline and hydroxyproline. For each substrate, the pH optimum was 7.4 and activity was sensitive to sulfhydryl group inhibitors. The iminopeptidase hydrolyzed the dipeptides Pro-Leu, Pro-Gly, Hyp-Gly, and Pro-Tyr. Iminopeptidase activity against the dipeptide Pro-Gly was higher than against Hyp-Gly. The molecular weight was estimated to be about 400,000. Evidence was obtained for the existence of endogenous inhibitors of iminopeptidase activity.     

Asymptomatic Significant Bacteriuria in the Non-pregnant Woman: II. Response to Treatment and Follow-up

Short courses of nitrofurantoin and ampicillin produced an immediate cure in 80% of adult non-pregnant bacteriuric women. Of the subjects so treated, 55% remained cured at the end of one year. Over the same follow-up period 36% of untreated bacteriuric women developed a spontaneous remission of bacteriuria. Treatment failed to prevent the development of symptomatic infection, and the reinfections which followed successful treatment were more commonly associated with the development of symptoms than the persistent or relapsing infections in untreated or unsuccessfully treated subjects.It is concluded that a search for bacteriuria in non-pregnant women is unlikely to be of value as a preventive measure, since in many instances it fails to detect urinary tract infection at an early stage and since treatment by methods suitable for large-scale use is ineffective.     

Effect of Nitrofurans and Chlortetracycline on Microorganisms Associated with Shrimp

Nitrofuran AF-2 displayed greater inhibitory effect than did nitrofuran Z when a mixed bacterial culture, including several proteolytic bacteria, isolated from shrimp was subjected to these compounds in vitro. Nitrofuran Z exhibited greater bactericidal properties than did chlortetracycline in all cultures used. Only 10 mug of nitrofuran AF-2 per ml was sufficient to inhibit the growth of mixed bacteria in nutrient broth, whereas 50 mug of nitrofuran Z per ml was necessary to accomplish the same inhibition. A 50-mug amount of chlortetracycline per ml displayed about the same inhibitory effect as either 10 mug of AF-2 per ml or 20 mug of Z per ml. The isolated proteolytic bacteria showed greater suppression of growth when subjected to AF-2 than when subjected to Z; however, both nitrofurans were effective in preventing growth. The addition of either 1 mug of AF-2 per ml or 5 mug of Z per ml to nutrient broth inhibited the growth of Achromobacter aquarmarinus, whereas chlortetracycline was less effective, requiring about 20 mug to suppress growth to the same degree.     

Biased gene conversion is not occurring among rDNA repeats in the Brassica triangle.

Hybridization is a common phenomenon that results in complex genomes. How ancestral genomes interact in hybrids has long been of great interest. Recombination among ancestral genomes may increase or decrease genetic variation. This study examines rDNA from members of the Brassica triangle for evidence of gene conversion across ancestral genomes. Gene conversion is a powerful force in the evolution of multigene families. It has previously been shown that biased gene conversion can act to homogenize rDNA repeats within hybrid genomes. Here, we find no evidence for biased gene conversion or unequal crossing over across ancestral genomes in allotetraploid Brassica species. We suggest that, while basic genomic processes are shared by all organisms, the relative frequency of these processes and their evolutionary importance may differ among lineages. Key words : Brassica, rDNA, gene conversion, allotetraploids.