Specificity of mutation by UV light and delayed photoreversal in umuC-defective Escherichia coli K-12: a targeting intermediate at pyrimidine dimers.

Prototrophic mutants produced by UV light in Escherichia coli K-12 strains with argE3(Oc) and hisG4(Oc) defects are distinguished as backmutations and specific nonsense suppressor mutations. In strains carrying a umuC defect, mutants are not produced unless irradiated cells are incubated and then exposed to photoreversing light (delayed photoreversal mutagenesis). The mutants thus produced are found to be specifically suppressor mutations and not backmutations. The suppressor mutations are primarily glutamine tRNA ochre suppressor mutations, which have been attributed previously to mutation targeted at T = C pyrimidine dimers. In a lexA51 recA441 strain, where the SOS mutagenesis functions are constitutive, targeting at dimers is confirmed by demonstrating that the induction of glutamine tRNA suppressor mutations is susceptible to photoreversal. In the same strain induction of backmutations is not susceptible to photoreversal. Thus delayed photoreversal mutagenesis produces suppressor mutations that can be targeted at pyrimidine dimers and does not produce backmutations that are not targeted at pyrimidine dimers. This correlation supports the idea that delayed photoreversal mutagenesis in umuC defective cells reflects a mutation process arrested at a targeting pyrimidine dimer photoproduct, which is the immediate cause of both the alteration in DNA sequence and the obstruction (unless repaired) to mutation fixation and ultimate expression.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Ultraviolet Mutagenesis and Its Repair in an ESCHERICHIA COLI Strain Containing a Nonsense Codon

Ultraviolet mutagenesis and its repair were studied mainly in WU36-10-89, a uvr(-) strain of Escherichia coli containing a UAG mutation in a gene for leucine biosynthesis. Following ultraviolet (UV) irradiation revertants appearing with or without direct photoreactivation (PR) were classified according to the presence and type of suppressor they contained. We find UV mutation production to be quite specific. An analysis of revertants produced by UV indicates they are formed mainly from GC --> AT and that the miscoding is due to a cytosine residue at the site of mutation in a cytosine-thymine (CT) dimer. We propose that the dimer serves as template during some aspects of repair replication and at the time of replication the C in the dimer directs the insertion of A in the complementary strand. We also note that C --> A and T -->G changes caused by a CT dimer occur much less frequently.     

RexAB proteins of bacteriophage lambda enhance the effect of photolyase-dimer complexes on lacZ gene expression in Escherichia coli.

Summary Expression of the lacZ gene in Escherichia coli is inactivated by exposure to ultraviolet light (UV). Inactivation is exceptionally effective when cells contain amplified levels of DNA photolyase (which forms complexes with pyrimidine dimers in the absence of light for actual photoreversal) and a prophage. Without amplified photolyase, the prophage or both, inactivation rates are similar and much lower. UV-inactivation of lacZ gene expression in the presence of both amplified photolyase and is even more effective if cI857 is used in place of the wildtype prophage but is wholly unexceptional if the prophage carries defects in the genes rexA or rexB. When Rex AB proteins are provided by expression from a plasmid and the cell also contains amplified photolyase, exceptional inactivation rates again obtain; in fact inactivation is most effective under these conditions. The data are considered to reveal a role for Rex AB proteins, which mediate superinfection exclusion, in the exceptional inactivation of gene expression by photolyase bound to pyrimidine dimers in DNA. Photolyase-dimer complexes may mimic the structure of certain complexes that arise during phage development and thus influence Rex A and/or B proteins, thereby shutting down cell metabolism.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

DNA damage-processing in E. coli: on-going protein synthesis is required for fixation of UV-induced lethality and mutation

UV irradiation of E. coli produces photoproducts in the DNA genome. In consequence, some bacteria lose viability (colony-forming ability) or remain viable as mutant cells. However, the end-points of viability inactivation (lethality) or mutation are determined by cellular processes that act on the UV-damaged DNA. We have investigated the in vivo time course for processes that deal with cyclobutane pyrimidine dimers (CPD) which can be specifically removed by photoreactivation (PR). At different times during post-UV incubation, samples were challenged with PR and assayed for viability or mutation. We used excision-defective E. coli B/r cells and worked under yellow light to avoid background PR. During post-UV incubation (0-100min) in fully supplemented defined medium, inactivation and mutation were initially significantly reversed by PR but the extent of this reversal decreased during continued incubation defining "fixation" of lethality or mutation, respectively. In contrast, if protein synthesis was restricted during the post-UV incubation, no fixation developed. When chloramphenicol was added to inhibit protein synthesis after 30min of supplemented post-UV incubation, at a time sufficient for expression of UV-induced protein(s), fixation of lethality or mutation was still annulled (no change in the effectiveness of PR developed). Lethality fixation did progress when protein synthesis was restricted and the cells were incubated in the presence of puromycin or were either clpP or clpX defective. We discuss these and related results to suggest (1) on-going protein synthesis is required in the fixation process for lethality and mutation to sustain an effective level of a hypothetical protein sensitive to ClpXP proteolysis and (2) this protein plays a critical role in the process leading to exchange between Pol III activity and alternative polymerase activities required as each cell deals with damage in template DNA.     

Physiological Streptomycin Resistance in a Multiauxotroph of Escherichia coli Strain 15 T

Escherichia coli strain WWU was found to be moderately resistant to streptomycin when grown in a minimal medium, although the strain was sensitive if grown in nutrient broth. Transfer experiments showed that cells grown in minimal medium retain the resistant state for a period of time after dilution into nutrient broth; and conversely, sensitive cells grown in nutrient broth were sensitive after dilution into minimal medium for a period of time. The kinetics of transition from resistant to sensitive and from sensitive to resistant were observed, and kinetics of ³H-dihydrostreptomycin accumulation by resistant and sensitive cells were compared. The data suggested that cells grown in minimal medium were physiologically resistant because they accumulated streptomycin poorly. Inactivation per incorporated antibiotic molecule was the same in resistant and sensitive cells.     

Nonsense suppression in a multiauxotrophic derivative of Escherichia coli 15T-: identification and consequences of an amber triplet in the deoxyribomutase gene

Previously, arginine revertants of Escherichia coli WWU, a derivative of E. coli 15T(-), have been subdivided by two independent methods: (i) the streak morphology on nutrient agar, and (ii) the pattern of phage growth using amber and ochre mutants of bacteriophage T4. In the first assay, revertants were subdivided into two classes according to the appearance of streaks after incubation on nutrient agar, a thick, even line of growth defining normal revertants and a thin, irregular line defining aberrant revertants. In the second assay, revertants were classified by the suppressors they contained. The present work demonstrates that revertants containing an amber suppressor show the aberrant morphology and are also able to catabolize thymidine for energy and carbon. This is in contrast to the parent WWU containing no suppressor, which shows a normal morphology and cannot utilize thymidine as an energy source. Revertants containing no suppressor, isolated specifically for their ability to catabolize thymidine, show an aberrant morphology. Together, these results indicate that the aberrant morphology results from suppression of an amber triplet in a gene of the thymidine catabolic pathway. Enzyme assays show the amber triplet to be in the gene specifying deoxyribomutase. It is suggested that the aberrant arginine revertants are analogous to high thymine-requiring mutants and that, in general, high and low thymine-requiring mutants differ from one another in their ability to catabolize deoxyribose-1-phosphate.