DNA repair gene polymorphisms, pre-natal factors and the frequency of somatic mutations in the glycophorin-A gene among healthy newborns

This study investigates variation in somatic mutation frequency, as measured by the glycophorin-A (GPA) somatic mutation assay, in relation to polymorphic variation among 435 newborn babies in DNA repair genes XRCC1, XRCC3 and XRCC4 and gender, parental age, social class and smoking habits. The three polymorphisms under investigation were an Arg --> Gln substitution at codon 399 in exon 10 of XRCC1, a Thr --> Met substitution at codon 241 in exon 7 of XRCC3 and an Ile --> Thr substitution at codon 401 in exon 4 of XRCC4. The study population is an extension of that previously analysed for GPA mutations and XRCC1 polymorphisms. A significant difference was seen in the earlier work in the genotype distribution for the XRCC1 Arg399Gln polymorphism between the main population and the small number with extreme values for NN variant frequency and this was maintained in the larger study group (OR 3.20 [95% CI: 1.16, 8.81]) P = 0.043). No such association was seen for XRCC3 or XRCC4 polymorphisms. When adjustments were made for multiple testing, neither N0 nor NN variant frequencies in the main study population were found to be influenced by the polymorphisms in XRCC1, XRCC3, or XRCC4. In addition, neither maternal or paternal smoking, age or social class nor the gender of the offspring were found to affect variant frequencies nor were variant frequencies influenced by any interaction between any of these factors and genotype. It is concluded that the genotypic variation in DNA repair genes examined in this study has no discernable effect on the genesis of the somatic mutations observed at birth.     

Evidence for an elevated frequency of in vivo somatic cell mutations in ataxia telangiectasia.

Somatic cell mutation frequency in vivo was measured in individuals with high cancer risk who were from ataxia telangiectasia (A-T) families. The assay for somatic mutation measures the frequency of variant erythrocytes which are progeny of erythroid precursor cells with mutations that result in a loss of gene expression at the polymorphic glycophorin A (GPA) locus. Samples from 14 of 15 A-T homozygotes showed high frequencies of GPA gene expression-loss variant cells with normal expression of only one of the two alleles at the GPA locus (i.e., GPA hemizygous variant cells). The mean elevation of the frequency of hemizygous variant cells over those in normal controls and unaffected family members was 7-14-fold. A-T homozygotes also showed an increase in the frequency of cells in which one allele at the GPA locus had lost expression and in which the remaining allele was expressed at a homozygous level (i.e., GPA homozygous variant cells). Family members who are obligate A-T heterozygotes did not appear to have a significantly elevated frequency of GPA hemizygous or homozygous variant cells. These indications of elevated in vivo frequencies of variant erythrocytes in A-T homozygotes support a causal link between susceptibility to somatic mutation and susceptibility to cancer.     

The yeast G-protein homolog is involved in the mating pheromone signal transduction system.

I have isolated a new type of sterile mutant of Saccharomyces cerevisiae, carrying a single mutant allele, designated dac1, which was mapped near the centromere on chromosome VIII. The dac1 mutation caused specific defects in the pheromone responsiveness of both a and alpha cells and did not seem to be associated with any pleiotropic phenotypes. Thus, in contrast to the ste4, ste5, ste7, ste11, and ste12 mutations, the dac1 mutation had no significant effect on such constitutive functions of haploid cells as pheromone production and alpha-factor destruction. The characteristics of this phenotype suggest that the DAC1 gene encodes a component of the pheromone response pathway common to both a and alpha cells. Introduction of the GPA1 gene encoding an S. cerevisiae homolog of the alpha subunit of mammalian guanine nucleotide-binding regulatory proteins (G proteins) into sterile dac1 mutants resulted in restoration of pheromone responsiveness and mating competence to both a and alpha cells. These results suggest that the dac1 mutation is an allele of the GPA1 gene and thus provide genetic evidence that the yeast G protein homolog is directly involved in the mating pheromone signal transduction pathway.     

Suppressors of a Gpa1 Mutation Cause Sterility in Saccharomyces Cerevisiae

The Saccharomyces cerevisiae GPA1 gene encodes a protein highly homologous to the α subunit of mammalian G proteins and is essential for haploid cell growth. We have selected 77 mutants able to suppress the lethality resulting from disruption of GPA1 (gpa1::HIS3). Two strains bearing either of two recessive mutations, sgp1 and sgp2, in combination with the disruption mutation, showed a cell type nonspecific sterile phenotype, yet expressed the major α-factor gene (MFα1) as judged by the ability to express a MFα1-lacZ fusion gene. The sgp1 mutation was closely linked to gpa1::HIS3 and probably occurred at the GPA1 locus. The sgp2 mutation was not linked to GPA1 and was different from the previously identified cell type nonspecific sterile mutations (ste4, ste5, ste7, ste11 and ste12). sgp2 GPA1 cells showed a fertile phenotype, indicating that the mating defect caused by sgp2 is associated with the loss of GPA1 function. While expression of a FUS1-lacZ fusion gene was induced in wild-type cells by the addition of α-factor, mutants bearing sgp1 or sgp2 as well as gpa1::HIS3 constitutively expressed FUS1-lacZ. These observations suggest that GPA1 (SGP1) and SGP2 are involved in mating factor-mediated signal transduction, which causes both cell cycle arrest in the late G(1) phase and induction of genes necessary for mating such as FUS1.     

Interactions among the subunits of the G protein involved in Saccharomyces cerevisiae mating.

The SCG1 (GPA1), STE4, and STE18 genes of Saccharomyces cerevisiae encode mating-pathway components whose amino acid sequences are similar to those of the alpha, beta, and gamma subunits, respectively, of mammalian G proteins. Genetic evidence suggests that the STE4 and STE18 gene products interact. The mating defects of a set of ste4 mutants were partially suppressed by the overexpression of STE18, and, moreover, a combination of partially defective ste4 and ste18 alleles created a totally sterile phenotype, whereas such synthetic sterility was not observed when the ste18 allele was combined with a weakly sterile ste11 allele. Others have provided genetic evidence consistent with an interaction between the SCG1 (GPA1) and STE4 gene products. We have examined the physical interactions of these subunits by using an in vivo protein association assay. The STE4 and STE18 gene products associated with each other, and this association was disrupted by a mutation in the STE4 gene product whose phenotype was partially suppressed by overexpression of STE18. The STE4 and SCG1 (GPA1) gene products also interacted in the assay, whereas we detected no association of the SCG1 (GPA1) and STE18 gene products.     

Effect of target gene CpG content on spontaneous mutation in transgenic mice

Transgenic mutation assays utilizing bacterial target genes display a high frequency of spontaneous mutation at CpG sequences. This is believed to result from the fact that: (1) the prokaryotic genes currently being used as transgenic mutation targets have a high CpG content and (2) these sequences are methylated by mammalian cells to produce 5-methylcytosine (5MC), a known promutagenic base. To study the effect of CpG content on the frequency and type of spontaneous mutation, we have synthesized an analogue of the bacterial lacI target gene (mrkII) that contains a reduced number of CpG sequences. This gene was inserted into a lambda vector and used to construct trangenic mice that undergo vector rescue from genomic DNA upon in vitro packaging. Results on spontaneous mutation frequency and spectrum have been collected and compared to those observed at the lacI gene in Big Blue™ transgenic mice. Spontaneous mutations at the mrkII gene occurred at a frequency in the mid-10⁻⁵ range and were predominantly base pair substitutions, similar to results seen in Big Blue™. However, mrkII mutations were distributed toward the carboxyl end of the gene instead of the bias toward the amino terminus seen in lacI. Unexpectedly, 23% of the spontaneous mrkII mutations were GC → AT transitions at CpG sequences (compared to 32% in lacI), despite the reduction in CpG number from 95 in lacI to only 13 in mrkII. Nine of the CpG bases undergoing transition mutations in mrkII have not been recorded previously as spontaneous sites in Big Blue™. Therefore, substantial reduction of the number of CpG sequences in the lacI transgene did not significantly reduce the rate of spontaneous mutation or alter the contribution of CpG-related events. This suggests that other factors are also operating to establish frequency and composition of spontaneous mutations in transgenic targets.     

Population genetics of polymorphisms in Cardiff newborn. Relationship between blood group and allozyme heterozygosity and birth weight

A newborn population of Cardiff, Wales, was screened for variation at three blood group loci (ABO, Rhesus and MN) and four enzyme loci (ACP-1, PGM-1, ADA and EST-D). Birth weights were measured. There were no significant differences between mean birth weights or birth weight variances for individuals homozygous or heterozygous at the MN and the four enzyme loci. (ABO and Rhesus loci cannot be used in these tests.) There was no significant heterogeneity in contingency tables relating phenotypes at the seven loci to birth weight. There were no significant differences in mean heterozygosity per locus between babies placed in different birth weight categories, ranging from 2.5 to 4.2 kg. The genetic variation screened appears therefore to be neutral with respect to this character.     

Multiple biomarkers study in painters in a shipyard in Korea

Shipbuilding workers are exposed to a variety of genotoxic compounds including polycyclic aromatic hydrocarbons (PAHs). A limited number of studies have been conducted to evaluate biomarkers related to PAH exposure in painters in the shipyard industry. We examined this in 208 workers recruited from a shipyard located in South Korea. Employees were grouped into three exposure groups: (1) 111 painters using coal tar paints, (2) 70 painters using general paints, and (3) 27 on-site controls using no paints. Levels of urinary 1-hydroxypyrene glucuronide (1-OHPG), as internal dose of PAH exposure, were measured by synchronous fluorescence spectroscopy. Glutathione S-transferase (GST) M1 and T1 genotypes were assessed by a multiplex polymerase chain reaction (PCR)-based method, aromatic-DNA adducts in peripheral white blood cells were measured by 32P-postlabeling, and glycophorin A (GPA) variant frequencies in red blood cells were assessed by flow cytometry. Information on demographic characteristics, smoking habits, diet, job title and use of personal protective equipment (e.g. respiratory and dermal) were collected by self-administered questionnaire. Average urinary 1-OHPG levels in coal tar paint (2.24 micromol/mol creatinine) and general paint (1.38 micromol/mol creatinine) users were significantly higher than in on-site controls (0.62 micromol/mol creatinine) (P<0.001). Paint use, irrespective of the type of paints, and smoking (yes/no) were positively associated with urinary 1-OHPG levels, whereas green tea consumption (yes/no) was negatively associated with the 1-OHPG levels. No significant effect in the 1-OHPG levels were observed for the GSTM1 and GSTT1 genotypes. Aromatic-DNA adduct levels tended to be higher in coal tar paint users (P = 0.06) and painters (P = 0.07) compared to on-site controls. No differences in adduct levels were observed, between the two groups of painters, and the combined group showed greater adduct levels than on-site controls (P = 0.05). GPA mutation frequencies measured in 55 individuals with MN heterozygote genotypes were not significantly different among the three exposure groups, and no correlation was observed between urinary 1-OHPG levels and aromatic-DNA adducts or GPA mutation frequency. These results suggest that painters in the shipyard were exposed to significant amounts of PAHs and possibly to other genotoxic aromatic compounds, and that urinary 1-OHPG may be a potential biomarker of PAH exposure in this population.     

Chromosome aberrations in premature infant lymphocytes]

A cytogenetic investigation of a group of prematurely born babies was carried out during the first months of their life (at ages of 0 days, 5-7 days, 2-4 weeks), as well as of a group of infants born in proper time and having a normal weight. As it was shown by the analysis of chromosome aberrations, frequencies of aberrant cells in babies at ages of 0 days, 5-7 days, 2-4 weeks and in those that have endured some bacterial or viral infection were 1,96% (0.22 aberrations per cell), 3,38% (0,037 aberrations per cell), 4,92% (0,053 aberrations per cell) and 6,73% respectively. The role of infection of drugs in the increase of the frequency of aberrant cells is also indicated by the investigation of babies born in proper time and having normal weight, that have endured an acute respiratory disease. In this group of children the frequency of aberrant cells was 5,3%. However, it is impossible to assess the role of each of these factors separately, since their effect on the organism of prematurely born babies is simultaneous from the very moment of birth.     

GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p Galpha subunit and functions in a Ras-independent pathway.

The yeast RAS1 and RAS2 genes appear to be involved in control of cell growth in response to nutrients. Here we show that this growth control also involves a signal mediated by the heterotrimeric G protein alpha subunit homolog encoded by GPA2. A GPA2 null allele conferred a severe growth defect on cells containing a null allele of RAS2, although either mutation alone had little effect on growth rate. A constitutive allele of GPA2 could stimulate growth of a strain lacking both RAS genes. Constitutive GPA2 conferred heat shock sensitivity on both wild-type cells and cells lacking RAS function, but had no effect in a strain containing a null allele of SCH9, which encodes a kinase related to protein kinase A. The GPR1 gene was isolated and was found to encode a protein with the characteristics of a G protein-coupled receptor. Double Deltagpr1 Deltaras2 mutants displayed a severe growth defect that was suppressed by expression of the constitutive allele of GPA2, confirming that GPR1 acts upstream of GPA2. Gpr1p is expressed on the cell surface and requires sequences in the membrane-proximal region of its third cytoplasmic loop for function, as expected for a G protein-coupled receptor. GPR1 RNA was induced when cells were starved for nitrogen and amino acids. These results are consistent with a model in which the GPR1/GPA2 pathway activates the Sch9p kinase to generate a response that acts in parallel with that generated by the Ras/cAMP pathway, resulting in the integration of nutrient signals.     

Quality of antenatal care and its dose-response relationship with birth weight in a maternal and child health training institute in Bangladesh

Four hundred and sixty-five pregnant women and their newborn babies were studied at a maternal and child health training institute in Dhaka, Bangladesh, between July 2002 and June 2003 with the objective of (1) examining the relationship between birth weight and maternal factors, and, if there was a dose-response relationship between quality of antenatal care and birth weight, (2) predicting the number of antenatal visits required for women with different significant characteristics to reduce the incidence of low-birth-weight babies. The study revealed that 23.2% of the babies were of low birth weight according to the WHO cut-off point of <2500 g. Mean birth weight was 2674.19+/-425.31 g. A low birth weight was more common in younger (<20 years) and older (> or =30 years) mothers, the low-income group and those with little or no education. The mean birth weight of the babies increased with an increase in quality of antenatal care. The babies of the mothers who had 6+ antenatal visits were found to be 727.26 g heavier than those who had 1-3 visits and 325.88 g heavier than those who had 4-5 visits. No significant relationship was found between number of conception, birth-to-conception interval, BMI at first visit, sex of the newborn and birth weight. Further, from multiple regression analysis (stepwise), it was revealed that number of antenatal visits, educational level of the mother and per capita yearly income had independent effects on birth weight after controlling the effect of each variable. Using multiple regression analysis, the estimated number of antenatal visits required to reduce the incidence of low-birth-weight babies for women with no education and below-average per capita income status was 6; the number required for women with no education and above-average per capita income status was 5; and that for women with education and with any category of income status was 4 visits. So there is a need to stratify women according to their income and educational status so that, along with other measures, the required number of antenatal visits can be estimated beforehand to reduce the incidence of low-birth-weight babies.     

GPA1Val-50 mutation in the mating-factor signaling pathway in Saccharomyces cerevisiae.

The GPA1 gene of Saccharomyces cerevisiae encodes a protein that is highly homologous to the alpha subunit of mammalian hetrotrimeric G proteins and is essential for haploid cell growth. A mutation of the GPA1 protein, GPA1Val-50, in which Gly-50 was replaced by valine, could complement the growth defect of a GPA1 disruption, gpal::HIS3. However, cells with gpa1::HIS3 expressing the GPA1Val-50 protein were supersensitive to alpha-factor in a short-term incubation but resumed growth after long-term incubation even after exposure to high concentrations of alpha-factor. The former phenotype associated with GPA1Val-50 is recessive, and the latter phenotype is dominant to GPA1+. The supersensitivity of GPA1Val-50 to alpha-factor was dependent on STE2 and STE4, which demonstrates that this GPA1Val-50-produced phenotype requires the mating-factor receptor and the beta subunit of the G protein. The double mutant of sst2-1 GPA1Val-50 recovered from division arrest, which suggested that SST2 is not required for recovery of the GPA1Val-50 mutant.     

16S rRNA/DNA-PCR对初生新生儿肠道乳酸杆菌和大肠埃希菌的动态检测

目的观察新生儿生后肠道菌群的动态变化。方法采用16S rRNA/DNA荧光定量PCR技术,分别对40例足月儿和40例早产儿生后第1（〉12h）、4、7天粪便标本中的乳酸杆菌和大肠埃希菌进行定量分析。结果不同日龄足月儿粪便标本中乳酸杆菌数量的对数值分别为5.50±0.81、6.87±0.81、9.20±0.87,早产儿分别为4.89±0.46、6.05±0.46、8.06±0.18;不同日龄足月儿粪便标本中大肠埃希菌数量的对数值分别为6.49±0.40、7.59±0.58、7.15±0.55,早产儿分别为6.32±0.51、7.39±0.78、7.05±0.63;2种细菌对数值分别在组间行单因素方差分析,差异有显著性（P〈0.05）;组内行配对t检验,差异也有显著性（P〈0.05）。结论新生儿肠道菌群的建立是动态变化的,个体间差异受多因素影响,早产儿落后于足月儿。     

Rapid evolution of enormous, multichromosomal genomes in flowering plant mitochondria with exceptionally high mutation rates

Genome size and complexity vary tremendously among eukaryotic species and their organelles. Comparisons across deeply divergent eukaryotic lineages have suggested that variation in mutation rates may explain this diversity, with increased mutational burdens favoring reduced genome size and complexity. The discovery that mitochondrial mutation rates can differ by orders of magnitude among closely related angiosperm species presents a unique opportunity to test this hypothesis. We sequenced the mitochondrial genomes from two species in the angiosperm genus Silene with recent and dramatic accelerations in their mitochondrial mutation rates. Contrary to theoretical predictions, these genomes have experienced a massive proliferation of noncoding content. At 6.7 and 11.3 Mb, they are by far the largest known mitochondrial genomes, larger than most bacterial genomes and even some nuclear genomes. In contrast, two slowly evolving Silene mitochondrial genomes are smaller than average for angiosperms. Consequently, this genus captures approximately 98% of known variation in organelle genome size. The expanded genomes reveal several architectural changes, including the evolution of complex multichromosomal structures (with 59 and 128 circular-mapping chromosomes, ranging in size from 44 to 192 kb). They also exhibit a substantial reduction in recombination and gene conversion activity as measured by the relative frequency of alternative genome conformations and the level of sequence divergence between repeat copies. The evolution of mutation rate, genome size, and chromosome structure can therefore be extremely rapid and interrelated in ways not predicted by current evolutionary theories. Our results raise the hypothesis that changes in recombinational processes, including gene conversion, may be a central force driving the evolution of both mutation rate and genome structure.     

SGV1 encodes a CDC28/cdc2-related kinase required for a G alpha subunit-mediated adaptive response to pheromone in S. cerevisiae.

The GPA1 gene of S. cerevisiae encodes a G alpha subunit that plays a positive role in the transduction of signals stimulating recovery from pheromone-induced cell cycle arrest. The GPA1Val50 mutation, in which Gly-50 is replaced by valine, causes hyperadaptation to pheromone. However, GPA1Val50 cells do not recover from division arrest in the absence of both CLN1 and CLN3, which encode G1 cyclins, indicating that the recovery-promoting activity of GPA1Val50 requires the function of G1 cyclins. An sgv1 mutation suppresses the hyperadaptive response caused by GPA1Val50 and also confers cold- and temperature-sensitive growth. The SGV1 gene encodes an apparent protein kinase homologous to CDC28/cdc2 kinase: SGV1 is 42% identical to CDC28. The activated mutation, CLN3-2, partially suppresses the growth defect of sgv1, suggesting that the SGV1 and CLN3 proteins may act in the same growth control pathway.     

Interindividual Variation in Mitotic Recombination

Mitotic recombination (MR) between homologous chromosomes is a mutational event that results in loss of heterozygosity in half of the segregants at mitosis. Loss of heterozygosity may have important biological consequences. The purpose of this study was to describe human variation in the spontaneous frequency of MR. Using an immunoselection technique for isolating the somatic mutations that result in loss of expression of one of the codominant alleles at the HLA-A locus, we have measured the frequency and molecular basis of somatic mutations in lymphocytes from a population of young adults. Mutations were classified as being the result of intragenic changes, major deletions, or MR. Here we show that the MR mutation frequency in females was significantly greater than that in males but that intragenic mutation frequency showed no association with sex. Individual variation in MR frequency ranged over more than two orders of magnitude and was not normally distributed. Furthermore, the observed number of individuals from whom no mutants resulting from MR were obtained was significantly greater than was expected. The endogenous level of MR may be under genetic control. Given the association of loss of heterozygosity with cancer initiation and progression, low endogenous MR may confer a reduced lifetime risk of cancer, and the converse may apply.     

Evidence for long-distance dispersal in a sedentary passerine, Gymnorhina tibicen (Artamidae)

Australian magpies (Gymnorhina tibicen) are group-living birds found across much of mainland Australia. Adults commonly remain in a breeding territory until death. Young of the year either remain on the natal (birth) site or are forced by their parents to disperse. Observational studies in south-eastern Australia suggest that most dispersing juveniles settle within 7 km of their natal territory. Therefore, despite potential for considerable gene flow (via flight), social organization predisposes magpies towards local population structuring. In this study, we measured genetic variation at both nuclear (allozyme) and mitochondrial loci and found evidence of substantial gene flow over very large distances (up to 1599 km). Thus, some juvenile magpies may disperse much greater distances than was previously thought. For mtDNA, geographic and genetic distance were strongly correlated, consistent with a pattern of isolation by distance. Therefore, although female gene flow is substantial it is apparently geographically restricted over large distances, in approximately a stepping-stone fashion. We conclude that a strong relationship between gene flow and geographic distance can develop even over large distances if populations have experienced no major historical disturbances to gene flow.     

The Nature and Extent of Mutational Pleiotropy in Gene Expression of Male Drosophila serrata

The nature and extent of mutational pleiotropy remain largely unknown, despite the central role that pleiotropy plays in many areas of biology, including human disease, agricultural production, and evolution. Here, we investigate the variation in 11,604 gene expression traits among 41 mutation accumulation (MA) lines of Drosophila serrata. We first confirmed that these expression phenotypes were heritable, detecting genetic variation in 96% of them in an outbred, natural population of D. serrata. Among the MA lines, 3385 (29%) of expression traits were variable, with a mean mutational heritability of 0.0005. In most traits, variation was generated by mutations of relatively small phenotypic effect; putative mutations with effects of greater than one phenotypic standard deviation were observed for only 8% of traits. With most (71%) traits unaffected by any mutation, our data provide no support for universal pleiotropy. We further characterized mutational pleiotropy in the 3385 variable traits, using sets of 5, randomly assigned, traits. Covariance among traits chosen at random with respect to their biological function is expected only if pleiotropy is extensive. Taking an analytical approach in which the variance unique to each trait in the random 5-trait sets was partitioned from variance shared among traits, we detected significant (at 5% false discovery rate) mutational covariance in 21% of sets. This frequency of statistically supported covariance implied that at least some mutations must pleiotropically affect a substantial number of traits (>70; 0.6% of all measured traits).