Direct estimate of the spontaneous germ line mutation rate in African green monkeys

Here, I provide the first direct estimate of the spontaneous mutation rate in an Old World monkey, using a seven individual, three‐generation pedigree of African green monkeys. Eight de novo mutations were identified within ～1.5 Gbp of accessible genome, corresponding to an estimated point mutation rate of 0.94 × 10^?8 per site per generation, suggesting an effective population size of ～12000 for the species. This estimation represents a significant improvement in our knowledge of the population genetics of the African green monkey, one of the most important nonhuman primate models in biomedical research. Furthermore, by comparing mutation rates in Old World monkeys with the only other direct estimates in primates to date–humans and chimpanzees–it is possible to uniquely address how mutation rates have evolved over longer time scales. While the estimated spontaneous mutation rate for African green monkeys is slightly lower than the rate of 1.2 × 10^?8 per base pair per generation reported in chimpanzees, it is similar to the lower range of rates of 0.96 × 10^?8–1.28 × 10^?8 per base pair per generation recently estimated from whole genome pedigrees in humans. This result suggests a long‐term constraint on mutation rate that is quite different from similar evidence pertaining to recombination rate evolution in primates.     

DNA sequence analysis of 1-nitropyrene-4,5-oxide and 1-nitropyrene-9,10-oxide induced mutations in the hprt gene of Chinese hamster ovary cells

Nitropyrene, the predominant nitropolycyclic hydrocarbon found in diesel exhaust, is a mutagenic and tumorigenic environmental pollutant that requires metabolic activation via nitroreduction and ring oxidation. In order to determine the role of ring oxidation in the mutagenicity of 1-nitropyrene, its oxidative metabolites, 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide, were synthesized and their mutation spectra were determined in the coding region of hprt gene of CHO cells by a PCR amplification of reverse-transcribed hprt mRNA, followed by a DNA sequence analysis. A comparison of the two metabolites for mutation frequencies showed that 1-nitropyrene 9,10-oxide was 2-times higher than 1-nitropyrene 4,5-oxide. The mutation spectrum for 1-nitropyrene 4,5-oxide was base substitutions (33/49), one base deletions (11/49) and exon deletions (5/49). In the case of 1-nitropyrene 9,10-oxide, base substitutions (27/50), one base deletions (15/50), and exon deletions (8/50) were observed. Base substitutions were distributed randomly throughout the hprt gene. The majority of the base substitutions in mutant from 1-nitropyrene 4,5-oxide treated cells were A-->G transition (15/33) and G-->A transition (8/33). The predominant base substitution, A-->G transition (11/27) and G-->A transition (8/27), were also observed in mutant from 1-nitropyrene 9,10-oxide treated cells. The mutation at the site of adenine and guanine was consistent with the previous results, where the sites of DNA adduct formed by these compounds were predominant at the sites of purines. A comparison of the mutational patterns between 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide showed that there were no significant differences in the overall mutational spectrum. These results indicate that each oxidative metabolite exhibits an equal contribution to the mutagenicity of 1-nitropyrene, and ring oxidation of 1-nitropyrene is an important metabolic pathway to the formation of significant lethal DNA lesions.     

Deamination of 5-methylcytosines within cyclobutane pyrimidine dimers is an important component of UVB mutagenesis

UVB mutagenesis is characterized by an abundance of C --> T and 5-methylcytosine --> T transitions at dipyrimidine sequences. It is not known how these mutations might arise. One hypothesis is that UV-induced mutations occur only after deamination of the cytosine or 5-methylcytosine within the pyrimidine dimer. It is not clear how methylation of cytosines at the 5-position influences deamination and how this affects mutagenesis. We have now conducted experiments with a CpG-methylated supF shuttle vector that was irradiated with UVB and then incubated at 37 degrees C to allow time for deamination before passage through a human cell line to establish mutations. This led to a significantly increased frequency of CC --> TT mutations and of transition mutations at 5'-PymCG-3' sequences. A spectrum of deaminated cis-syn cyclobutane pyrimidine dimers in the supF gene was determined using the mismatch glycosylase activities of MBD4 protein in combination with ligation-mediated PCR. Methylation at the C-5 position promoted the deamination of cytosines within cis-syn cyclobutane pyrimidine dimers, and these two events combined led to a significantly increased frequency of UVB-induced transition mutations at 5'-PymCG-3' sequences. Under these conditions, the majority of all supF mutations were transition mutations at 5'-PymCG-3', and they clustered at several mutational hot spots. Exactly these types of mutations are frequently observed in the p53 gene of nonmelanoma skin tumors. This particular mutagenic pathway may become prevalent under conditions of inefficient DNA repair and slow proliferation of cells in the human epidermis.     

Oxidative DNA damage induced by copper and hydrogen peroxide promotes CG-->TT tandem mutations at methylated CpG dinucleotides in nucleotide excision repair-deficient cells

Oxidative DNA damage may play an important role in human disease including cancer. Previously, mutational spectra have been determined using systems that include transition metal ions and hydrogen peroxide (H₂O₂). G→T transversions and C→T transitions were the most common mutations observed including some CC→TT tandem mutations. C→T transition mutations at methylated CpG dinucleotides are the most common mutations in human genetic diseases. It has been hypothesized that oxidative stress may increase the frequency of mutations at methylated CpG sequences. Here we have used a CpG-methylated shuttle vector to derive mutational spectra of copper/H₂O₂-induced DNA damage upon passage of the shuttle vector through human fibroblasts. We find that copper/H₂O₂ treatment produces higher numbers of CpG transition mutations when the CpGs are methylated but does not create clear C→T hotspots at these sites. More strikingly, we observed that this treatment produces a substantial frequency of mutations that were mCG→TT tandem mutations. Six of seven tandem mutations were of this type. mCG→TT mutations (6/63 = 10% of all mutations) were observed only in nucleotide excision repair-deficient (XP-A) cells but were not found in repair-proficient cells. The data suggest that this novel type of mutation may be produced by vicinal or cross-linked base damage involving 5-methylcytosine and a neighboring guanine, which is repaired by nucleotide excision repair. We suggest that the underlying oxidative lesions could be responsible for the progressive neurodegeneration seen in XP-A individuals.     

Methylation of the RASSF1A gene in human cancers

Loss of genetic material from chromosome 3p21.3 is one of the most common and earliest events in the pathogenesis of lung cancer and many other solid tumors. The chromosomal area 3p21.3 is thought to harbor at least one important tumor suppressor gene, which, despite many years of investigation, has remained elusive. In our previous studies, we have identified and cloned a gene from the common homozygous deletion area at 3p21.3. The gene, named RASSF1A (Ras ASSociation domain Family 1A), has homology to a mammalian Ras effector. The RASSF1A gene is epigenetically inactivated in a large percentage of human lung cancers, in particular small cell carcinomas. A high frequency of methylation of RASSF1A is found also in breast cancers, renal cell carcinomas, ovarian, gastric and bladder cancers, and in neuroblastomas. The RASSF1A gene is a candidate for a tumor suppressor gene in 3p21.3.     

Small-fragment genomic libraries for the display of putative epitopes from clinically significant pathogens

Taking advantage of whole genome sequences of bacterial pathogens in many thriving diseases with global impact, we developed a comprehensive screening procedure for the identification of putative vaccine candidate antigens. Importantly, this procedure relies on highly representative small-fragment genomic libraries that are expressed to display frame-selected epitope-size peptides on a bacterial cell surface and to interact directly with carefully selected disease-relevant high-titer sera. Here we describe the generation of small-fragment genomic libraries of Gram-positive and Gram-negative clinically significant pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae, Enterococcus faecalis, Helicobacter pylori, Chlamydia pneumoniae, the enterotoxigenic Escherichia coli, and Campylobacter jejuni. Large-scale sequencing revealed that the libraries, which provide an average of 20-fold coverage, were random and, as demonstrated with two S. aureus libraries, highly representative. Consistent with the comprehensive nature of this approach is the identification of epitopes that reside in both annotated and putatively novel open reading frames. The use of these libraries therefore allows for the rapid and direct identification of immunogenic epitopes with no apparent bias or difficulty that often associate with conventional expression methods.     

Interaction of sperm with purified native chicken ZP1 and ZPC proteins

The avian perivitelline membrane (PVM) is the site of initial contact between sperm and egg. It consists of only two major components, which are both homologues of the mammalian zona pellucida (ZP) proteins, and belong to the ZP1 and ZPC families, respectively. We have established a method to isolate large quantities of both native avian ZP proteins and have used these preparations to investigate their sperm-binding capacities. Chicken ZPC forms multimeric structures of defined size and binds to an approximately 180-kDa protein complex present in rooster sperm extracts. Based on experiments using both PVM and isolated proteins, we show that chicken ZP1 is proteolytically degraded by a sperm-associated protease but that chicken ZPC remains intact. An antiserum directed against chicken ZP1 is capable of inhibiting sperm binding to the PVM. Taken together, these data suggest that ZP1, in addition to ZPC, plays a major role in the initial interactions between sperm and egg.     

Similar mutagenicity of photoactivated porphyrins and ultraviolet A radiation in mouse embryonic fibroblasts: involvement of oxidative DNA lesions in mutagenesis

Ultraviolet A (UVA) radiation is implicated in the etiology of human skin cancer. However, the underlying mechanism of carcinogenicity for UVA is not fully delineated. A mutagenic role for UVA has been suggested, which involves activation of endogenous photosensitizers generating oxidative DNA damage. We investigated the mutagenicity of UVA alone and in combination with delta-aminolevulinic acid (delta-ALA), a precursor of the intracellular photosensitizers porphyrins, in transgenic Big Blue mouse embryonic fibroblasts. A significant generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a typical promutagenic oxidative DNA lesion, was observed in cells treated with a combination of delta-ALA (1 mM) and UVA (0.06 J/cm(2)) as quantified by high-pressure liquid chromatography-tandem mass spectrometry (p < 0.001; relative to the control). The steady-state level of 8-oxo-dG, however, remained unchanged in cells irradiated with UVA or treated with delta-ALA alone. Other photolesions including cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts were not detectable in cells treated with delta-ALA and/or irradiated with UVA as determined by terminal transferase-dependent polymerase chain reaction assay. Mutation analyses of the cII transgene in cells treated with a combination of delta-ALA and UVA showed an approximately 3-fold increase in mutant frequency relative to the control (p < 0.008), as well as a unique induced mutation spectrum as established by DNA sequence analysis (p < 0.005; 95% CI, 0.002-0.009). No mutagenic effects were observed in cells irradiated with UVA or treated with delta-ALA alone. The spectrum of mutations produced by delta-ALA plus UVA was characterized by a significantly increased frequency of G --> T transversions (p < 0.0003; relative to the control), which are the hallmark mutations induced by 8-oxo-dG. Notably, the 8-oxo-dG-mediated mutagenicity of UVA plus delta-ALA is similar to that established previously for UVA alone at a mutagenic dose of 18 J/cm(2). We conclude that, in the presence of exogenous photosensitizers, UVA at a nonmutagenic dose induces mutations through the same mechanism as does a mutagenic dose of UVA per se.     

Peroxyl radical mediated oxidative DNA base damage: implications for lipid peroxidation induced mutagenesis

Endogenous DNA damage induced by lipid peroxidation is believed to play a critical role in carcinogenesis. Lipid peroxidation generates free radical intermediates (primarily peroxyl radicals, ROO(*)) and electrophilic aldehydes as the principal genotoxicants. Although detailed information is available on the role of aldehyde base adducts in mutagenesis and carcinogenesis, the contribution of peroxyl radical mediated DNA base damage is less well understood. In the present study we have mapped oxidative base damage induced by peroxyl radicals in the supF tRNA gene and correlated this information with peroxidation-induced mutations in several human fibroblast cell lines. Nearly identical patterns of oxidative base damage were obtained from reaction of DNA with either peroxidizing arachidonic acid (20:4omega6) or peroxyl radicals generated by thermolysis of ABIP in the presence of oxygen. Oxidative base damage primarily occurred at G and C. Transversions at GC base pairs in the supF gene were the major base substitution detected in all cell lines. Peroxyl radical induced tandem mutations were also observed. Many mutation hot spots coincided with sites of mapped oxidative lesions, although in some cases hot spots occurred adjacent to the damaged base. Evidence is presented for the involvement of 8-oxodG in the oxidation of DNA by ROO(*). These results are used to interpret some key features of previously published mutation spectra induced by lipid peroxidation in human cells.     

Probing membrane protein orientation and structure using fast magic-angle-spinning solid-state NMR

One and two-dimensional solid-state NMR experiments are discussed that permit probing local structure and overall molecular conformation of membrane-embedded polypeptides under Magic Angle Spinning. The functional dependence of a series of anisotropic recoupling schemes is analyzed using theoretical and numerical methods. These studies lead to the construction of a set of polarization dephasing or transfer units that probe local backbone conformation and overall molecular orientation within the same NMR experiment. Experimental results are shown for a randomly oriented peptide and for two model membrane–peptides reconstituted into lipid bilayers and oriented on polymer films according to a method proposed by Bechinger etal. [J. Am. Chem. Soc., 124, (2002), 1146–1147].