Somatic cell mutation. Detection and quantification of x-ray-induced mutation in cultured, diploid human fibroblasts

We describe a system for detecting somatic cell mutation to 8-azaguanine (8AG) resistance in cultured, diploid human fibroblasts. Hypoxanthine-guanine phosphoribosyltransferase (HG-PRT)-deficient, AG-resistant fibroblasts from boys with the X-chromosomal, Lesch-Nyhan (L-N) mutation served as one type of prototype mutant cells. Both spontaneous and X-ray-induced mutation were studied. Recovery of L-N cells was a function both of density of normal cells and of the AG concentration used for selection. Optimum recovery was achieved at an initial inoculum of 2·10⁴ normal cells per 60 mm diameter culture dish and an AG concentration of 8·10^?6M. Efficiency of recovery was between 39 and 90% and controls to determine this efficiency were included in mutagenesis experiments.Attempts to free normal cell populations of pre-existing AG-resistant mutant cells by pregrowth in HAT medium failed because, unlike L-N mutants, most spontaneous AG-resistant mutants can grow in HAT medium. Although pre-existing mutants probably caused overestimation, the average spontaneous mutation rate derived from our experiments was 4.5·10^?6 per cell generation. Eliminating one large-yieldv experiment reduced this estimate to 1.9·10^?6. Clonal survival of cultured human fibroblasts as a function of X-ray dose was studied. X-Irradiation increased the mutation rate above spontaneous background. Minimum estimates of the increases were 1.13·10^?9 per R per cell at 75 R, 7.49·10^?8 per R per cell at 125 R, 6.87·10^?8 per R per cell at 150 R and 2.16·10^?7 per R per cell at 250 R. The total mutagenic effect and the induced mutation rate appeared to be dose-dependent. Normal parental cell strains and their derived AG-resistant mutants had similar X-ray sensitivities indicating that X-rays induced mutations rather than selected for pre-existing mutants.Because of the realism of the cultured diploid, human fibroblast model vis-a-vis in vivohuman cellular events, the mutation detection system described herein is proposed as being potentially useful for environmental monitoring.     

A novel frameshift mutation in BLM gene associated with high sister chromatid exchanges (SCE) in heterozygous family members

The Bloom syndrome (BS) is an autosomic recessive disorder comprising a wide range of abnormalities, including stunted growth, immunodeficiency, sun sensitivity and increased frequency of various types of cancer. Bloom syndrome cells display a high level of genetic instability, including a 10-fold increase in the sister chromatid exchanges (SCE) level. Bloom syndrome arises through mutations in both alleles of the BLM gene, which was identified as a member of the RecQ helicase family. In this study, we screened a Tunisian family with three BS patients. Cytogenetic analysis showed several chromosomal aberrations, and an approximately 14-fold elevated SCE frequency in BS cells. A significant increase in SCE frequency was observed in some family members but not reaching the BS patients values, leading to suggest that this could be due to the heterozygous profile. Microsatellite genotyping using four fluorescent dye-labeled microsatellite markers revealed evidence of linkage to BLM locus and the healthy members, sharing higher SCE frequency, showed heterozygous haplotypes as expected. Additionally, the direct BLM gene sequencing identified a novel homozygous frameshift mutation c.3617–3619delAA (p.K1207fsX9) in BS patients and a heterozygous BLM mutation in the family members with higher SCE frequency. Our findings suggest that this latter mutation likely leads to a reduced BLM activity explaining the homologous recombination repair defect and, therefore, the increase in SCE. Based on the present data, the screening of this mutation could contribute to the rapid diagnosis of BS. The genetic confirmation of the mutation in BLM gene provides crucial information for genetic counseling and prenatal diagnosis.     

Role of GDF15 in radiosensitivity of breast cancer cells

The Growth Differentiation Factor-15 gene (GDF15) is a member of TGF-b superfamily and this cytokine family is considered to be a promising target for cancer therapy. The purpose of this study was to investigate the effect of tumor derived GDF15 on proliferation and radiosensitivity of breast cancer cells in vitro and in vivo. A mouse breast cancer LM2 cell line with stable transfection of full-length mouse GDF15 cDNA was established. Cell growth and proliferation was observed using WST assay and impedance-based method. Radiation induced GDF15 and TGF-b1 expression was determined by qRT-PCR. Radiosensitivity was measured by a colony formation assay in vitro and by a tumor growth delay assay in vivo. Cells with more than a 10-fold increase in GDF15 expression had a higher growth rate than parental control cells in vitro and in vivo. The radiation induced elevation of the expression of TGFb1 was reduced in GDF15 overexpressing cells. GDF15 may play a role in the radiation response of breast cancer cells by effecting cell survival, inhibiting radiation-induced cell death, and inhibiting the TGF-b1 related cytotoxic action.     

Localization and phenotypic expression of a Tol marker in Citrobacter freundii

Among colicin-A-tolerant mutants of Citrobacter freundii we characterized some as Tol-2 mutants. The Tol-2 mutation results in insensitivity to bacteriocin S6 and an enhanced sensitivity to deoxycholate (DOC), ethylenediaminetetraacetic acid (EDTA) and to ampicillin. The Tol-2 mutation was mapped close near gal and the gene order pro-tol-gal was established in crosses between C. freundii Hfr tol ⁺ donor strains and C. freundii tol ^- acceptor strains. In these crosses a difference was observed in phenotypic expression of the pleiotropic properties of this tol ⁺ gene. Expression of resistance to DOC is substantially slower than the expression of EDTA-resistance. This phenomenon may play a disturbing role in those studies on cell envelope mutants, in which resistance to DOC is used as a selected marker. The differences in expression of DOC-and EDTA-resistance are discussed.     

Expression status of let-7a and miR-335 among breast tumors in patients with and without germ-line BRCA mutations

The genetic factors of cancer predisposition remain elusive in the majority of familial and/or early-onset cases of breast cancer (BC). This type of BC is promoted by germ-line mutations that inactivate BRCA1 or BRCA2. On the other hand, recent studies have indicated that alterations in the levels of miRNA expression are linked to this disease. Although BRCA1 and BRCA2 gene mutations have been reported to commonly lead to alterations in genes that encode cancer-related proteins, little is known regarding the putative impact of these mutations on noncoding miRNAs. In the present study, we aimed to determine whether miRNA dysregulation is involved in the pathogenesis of BRCA-mutated BC. An expression analysis of 14 human miRNAs previously shown to be related to BC diagnosis, prognosis, and drug resistance was conducted using tissues from 60 familial and/or early-onset patients whose peripheral blood samples had been screened for BRCA1 and BRCA2 mutations through sequence analysis. Let-7a and miR-335 expression levels were significantly downregulated in the tumors of patients with a BRCA mutation compared with those of patients without a BRCA mutation (P = 0.04 and P = 0.02, respectively). Our results defined the associations between the expression status of let-7a and miR-335 and BRCA mutations. The expression analysis of these miRNAs might be used as biomarkers of the BRCA mutation status of early-onset and/or familial BC.     

Microsporogenesis,pollen mitosis and pollen tube growth in Gagea villosa (Liliaceae)

In this study, Gagea villosa (Bieb.) Duby was investigated by using light microscopy methods in cytological and cytoembryological respects. Anthers were tetrasporangiate. Anther wall was formed with an epidermis, endothecium, middle layer and tapetum. Tapetum was glandular type and it began to degenerate when microspores released from tetrads. Tapetum cells generally have one or two nuclei. Mitosis seen in tapetum cells was generally normal but micronuclei were found in some of them. Fibrous thickenings were determined in endothecium. Microsporogenesis and pollen mitosis were generally regular. Cytokinesis was successive type. Meiosis in pollen mother cells was asynchronous in one anther locus. Mature pollen grains were 2-celled. Pollen sterility was found to be 24%. Some of the fertile pollen grains, smaller than the normal were seen at the end of the pollen mitosis. Microgametophyte development was examined in vivo and in vitro. Germination ratio of pollen grains in vitro was 4%. Generally swollen pollen tube tips and weak development of some curled pollen tubes were seen. Callose plug formation was seen only in vivo pollen tube growth.     

Characterization of a Corynebacterium glutamicum dnaB mutant that shows temperature-sensitive growth and mini-cell formation

Corynebacterium glutamicum is known to perform a unique form of cell division called post-fission snapping division. In order to investigate the mechanism of cell division of this bacterium, we isolated temperature-sensitive mutants from C. glutamicum wild-type strain ATCC 31831, and found that one of them, M45, produced high frequencies of mini-cells with no nucleoids. Cell pairs composed of an elongated cell, with one nucleoid, connected to a mini-cell, with no nucleoids, were occasionally observed. The temperature sensitivity and mini-cell formation of M45 was complemented by a 2-kb DraI-EcoRI fragment derived from the ATCC 31831 chromosomal DNA, which carried a dnaB homolog encoding a replicative DNA helicase. DNA sequence analysis revealed that M45 carried a missense mutation in the dnaB gene, which caused a substitution of Thr364 to Ile. Microscopic observation after 4?,6-diamidino-2-phenylindole staining revealed that the DNA content of single cells was decreased by culturing at the restrictive temperature, suggesting that the mutation affects chromosomal replication. These results suggest that the C. glutamicum dnaB mutant performs an asymmetric cell division even after DNA replication is inhibited, which results in the production of mini-cells.     

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.     

Monoclonal antibody reveals H-2-linked quantitative and qualitative variation in the expression of a Qa-2 region determinant

We have produced a monoclonal antibody, Y-7, that reacts with a Qa-2 region-controlled determinant. Cellular and strain distribution analyses, coupled with quantitative variation in the amount of Y-7 antigen expressed among strains, provide overwhelming evidence that Y-7 reacts with the Qa-2^a determinant. The determinant detected by Y-7 is differentially expressed in T and B lymphocytes in a strain specific manner. Y-7 reacts with the majority of T lymphocytes (> 95%) and approximately one-half of B lymphocytes in certain strains (++ strains), and with the majority of T lymphocytes (> 95%) and no B lymphocytes in other strains (+ strains). T lymphocytes in + strains express approximately three fold less of the Y-7 determinant than T lymphocytes from ++ strains. In addition, we show that the Y-7 determinant is expressed in approximately one-third to one-half of Lyb-3^?, 5^? B lymphocytes. Possible mechanisms determining quantitative and qualitative variation in the expression of the Y-7 determinant in T and B lymphocytes are discussed.     

Genetic polymorphisms of glutathione S-transferases and cytochrome P450 enzymes as susceptibility factors to systemic lupus erythematosus in southern Brazilian patients

Systemic lupus erythematosus (SLE) is an autoimmune chronic inflammatory disease that presents several clinical manifestations, affecting multiple organs and systems. Immunological, environmental, hormonal and genetic factors may contribute to disease. Genes and proteins involved in metabolism and detoxification of xenobiotics are often used as susceptibility markers to diseases with environmental risk factors. Cytochrome P450 (CYP) enzymes activate the xenobiotic making it more reactive, while the Glutathione S-transferases (GST) enzymes conjugate the reduced glutathione with electrophilic compounds, facilitating the toxic products excretion. CYP and GST polymorphisms can alter the expression and catalytic activity of enzymes. This study aimed to investigate the role of genetic variants of CYP and GST in susceptibility and clinical expression of SLE, through the analysis of GSTM1 null, GSTT1 null, GSTP1*Ile105Val, CYP1A1*2C and CYP2E1*5B polymorphisms. 371 SLE patients from Hospital de Clínicas de Porto Alegre and 522 healthy blood donors from southern Brazil were evaluated. GSTP1 and CYP variants were genotyped using PCR–RFLP and GSTT1 and GSTM1 variants were analyzed by multiplex PCR. Among European-derived individuals, a lower frequency of GSTP1*Val heterozygous genotypes was found in SLE patients when compared to controls (p = 0.005). In African-derived SLE patients, the CYP2E1*5B allelic frequency was higher in relation to controls (p = 0.054). We did not observe any clinical implication of the CYP and GST polymorphisms in patients with SLE. Our data suggest a protective role of the GSTP1*Ile/Val heterozygous genotype against the SLE in European-derived and a possible influence of the CYP2E1*5B allele in SLE susceptibility among African-derived individuals.     

Study of effect of substitution of the penultimate amino acid residue on expression,structure, and functional properties of Yersinia pseudotuberculosis OmpY porin

The purpose of the study was to compare the expression of two Yersinia pseudotuberculosis proteins, wild-type porin OmpY and the mutant porin OmpY designated as OmpY-Q having the uncharged amino acid residue Gln instead of positively charged Arg at the penultimate position in the same heterologous host. According to the literature, a similar substitution (Lys to Gln) of the penultimate amino acid residue in Neisseria meningitidis porin PorA drastically improved the assembly of the protein in the E. coli outer membrane in vivo. Site-directed mutagenesis was used to replace Arg by Gln (R338Q) in OmpY, and the conditions for optimal expression and maturation of OmpY-Q were selected. It was found that the growth rates of E. coli strains producing OmpY and OmpY-Q and the expression levels of the porins were approximately equal. Comparative analysis of recombinant OmpY and OmpY-Q did not show significant differences in structure, antigenic, and functional properties of the porins, or any noticeable effect of the R338Q substitution in OmpY on its assembly in the E. coli outer membrane in vivo. The probable causes of discrepancies between our results and the previous data on porin PorA are discussed considering the known mechanisms of biogenesis of porins at the periplasmic stage.     

A micro-culture technique for the mitogen stimulation of lymphocytes fromMacaca mulatta (rhesus) andMacaca fascicularis (cynomolgus)

A micro-culture technique was developed to determine the optimum culture conditions for the mitogen stimulation of lymphocytes fromMacaca mulatta (rhesus) andMacaca fascicularis (cynomolgus). The optimum concentrations of PHA and Con A ranged from 10–50 µg/culture, those of lymphocytes from 1?2×10⁵ cells/culture, and those of serum from 10–20%. Tritiated thymidine was used at a concentration of .04µCi/culture.     

Microbial strain improvement for enhanced polygalacturonase production by Aspergillus sojae

Strain improvement is a powerful tool in commercial development of microbial fermentation processes. Strains of Aspergillus sojae which were previously identified as polygalacturonase producers were subjected to the cost-effective mutagenesis and selection method, the so-called random screening. Physical (ultraviolet irradiation at 254 nm) and chemical mutagens (N-methyl-N′-nitro-N-nitrosoguanidine) were used in the development and implementation of a classical mutation and selection strategy for the improved production of pectic acid-degrading enzymes. Three mutation cycles of both mutagenic treatments and also the combination of them were performed to generate mutants descending from A. sojae ATCC 20235 and mutants of A. sojae CBS 100928. Pectinolytic enzyme production of the mutants was compared to their wild types in submerged and solid-state fermentation. Comparing both strains, higher pectinase activity was obtained by A. sojae ATCC 20235 and mutants thereof. The highest polygalacturonase activity (1,087.2?±?151.9 U/g) in solid-state culture was obtained by mutant M3, which was 1.7 times increased in comparison to the wild strain, A. sojae ATCC 20235. Additional, further mutation of mutant M3 for two more cycles of treatment by UV irradiation generated mutant DH56 with the highest polygalacturonase activity (98.8?±?8.7 U/mL) in submerged culture. This corresponded to 2.4-fold enhanced polygalacturonase production in comparison to the wild strain. The results of this study indicated the development of a classical mutation and selection strategy as a promising tool to improve pectinolytic enzyme production by both fungal strains.     

Significant expressivity of Wolfram syndrome: phenotypic assessment of two known and one novel mutation in the WFS1 gene in three Iranian families

Wolfram syndrome also known as DIDMOAD (Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy, and Deafness) is a rare neurodegenerative autosomal recessive disorder. There is evidence of variable expressivity both in patients and heterozygous carriers. In this study, we describe three Persian Wolfram syndrome families with differences in the age of onset, signs and symptoms of the disease. We clinically evaluated affected families for verifying WS clinical diagnosis. After linkage analysis via 5 STR markers, molecular analysis for WFS1 was performed by direct sequencing for patients and available family members. Three homozygous mutations were identified including c.1885 C>T, c.2205C>A both in exon 8 and c.460+1G>A in intron 4. The mutation c.2205C>A was found to be novel. We report interesting phenotype-genotype correlations: homozygous c.1885C>T and c.2205C>A variants were correlated with quite different disease severity and onset in the siblings. We report a rare case of WS with homozygous c.1885C>T who is married and has a healthy child. c.460+1G>A showed a possible partial dominant inheritance put forth by a heterozygous parent showing partial WS symptoms while her daughter displayed typical WS symptoms. Due to variable expressivity, detailed clinical examination and molecular diagnostics should be used to confirm WS and a more exact recurrence risk data.     

Oogenesis and chromosomal heterozygosity in the thelytokous midge,Lundstroemia parthenogenetica (Diptera,Chironomidae)

The thelytokous triploid midge Lundstroemia parthenogenetica (Diptera: Chironomidae:Chironominae), 3n = 9, is always found to be heterozygous for two inversions and a deletion. Parthenogenesis is maintained by an apomiotic restitutional oogenesis as found in the subfamily Orthocladiinae. This mechanism is compared with other groups exhibiting a restitutional or disordered first division. Possible modes of evolution of the apomictic restitutional system, origin of triploidy in apomictic forms, and chromosomal polymorphisms are discussed.     

Cytophysiological characteristics of Arabidopsis thaliana cultivated cells with disable perception of ethylene signal by the ETR1 receptor

Contradictory data about ethylene influence on cell growth and division prompted us to investigate cytophysiological characteristics of suspension cultures of Arabidopsis thaliana of wild type Col-0 and ert1-1 mutant carrying a point mutation in the site of ethylene binding by the ETR1 receptor. Some cytophysiological characteristics of the etr1-1 cultivated cells differed from those of Col-0: the growth rate of mutant cells was less and cell sizes were smaller, the culture was committed to the formation of tracheary elements (TE), had a pronounced modal class of nuclei (54%) with the amount of DNA 8C and a tendency to expand the ploidy toward 32C. Despite the absence of ethylene perception by the ETR1 receptor, the cell culture of mutant responded to treatment with ethylene by growth acceleration, an increase in cell viability and in the number of cells in the S-phase of the cell cycle. The inhibitor of ethylene binding to receptors, 1-methylcyclopropene, suppressed growth and viability of the cells of both genotypes. In the etr1-1 cell culture, the inhibitor reduced the number of S-phase nuclei and activated TE formation. All data obtained indicate that ethylene perception and transduction of ethylene signal are required for the maintenance of cell viability and active in vitro growth. It is supposed that the functional activity of the ETR1 receptor is necessary for optimal cell expansion, whereas other receptors are responsible for cell proliferation.     

A novel MVK missense mutation in one Chinese family with disseminated superficial actinic porokeratosis

Disseminated superficial actinic porokeratosis (DSAP) is a severe chronic autosomal dominant cutaneous disorder with high genetic heterogeneity. mevalonate kinase, (MVK) a gene know to play an important role in regulation of calcium-induced keratinocyte differentiation and proliferation, has recently been suggested as the disease-causing gene for DSAP. Here we report a direct sequencing analysis of this gene in 3 DSAP families, 6 sporadic cases, and 100 unrelated healthy controls. We detected a heterozygous T to A transition at nucleotide 205 in exon 3 of MVK gene in one familial case. This mutation will result in an amino acid change at codon 69 (P.Ser69Thr), which is from a serine codon (TCA) to a threonine codon (ACA). No such mutation was detected in the unaffected family members or the 100 unrelated healthy controls. Our results demonstrated a novel missense mutation in MVK gene. This will be valuable for the diagnosis of DSAP as well as for genetic counseling and prenatal diagnosis of affected families.