Somatic crossing over in glycine max (L). Merrill: Possible potentiation of the recombinogenic effect of caffeine by 5-fluorodeoxyuridine and cytosine-β-D-arabinofuranoside

The leaves of variety T219 of Glycine max (soybean) exhibit yellow, dark green and twin or double spots on the two simple leaves and the first compound leaf of heterozygous Y₁₁y₁₁ plants. These spots mimic the phenotypes controlled by Y₁₁y₁₁, Y₁₁Y₁₁ and Y₁₁Y₁₁−y₁₁y₁₁ genotypes. It has been argued that p recombination is responsible for the origin of twin spots. At least some of the single spots appear due to failure of one of the components of the double spots.Treatment of seeds with caffeine solutions increases the frequency of all types of spots, particularly those of doubles. 5-Fluorodeoxyuridine (FUdR) and cytosine-β-D-aranibofuranoside (CA), both inhibitors of DNA synthesis, cause potentiation of the effects of caffeine by increasing all three types of spots. Since the relative frequencies of the different types of spots do not differ much in caffeine plus FUdR (or CA) treated materials from those observed in case of caffeine treatment alone, it is suggested that (1) the increase in the frequency of spots in caffeine plus FUdR (or CA) treated material is not due simply to additive effect of the two chemical, and (2) there is true synergism between caffeine and FUdR (or CA) which somehow leads to enhancement of the phenomenon of compementary reunions.     

Studies on the expression of somatic crossing over in Glycine max L.

Summary Variety T219 of Glycine max L. has spontaneous yellow, dark green and double (yellow-dark green) spots on the leaves of plants of genotype Y ₁₁ y ₁₁ but no such spots are found on leaves of Y ₁₁ Y ₁₁ or y₁₁y₁₁ plants. It was suggested (Vig and Paddock, 1968) that the double spots result from somatic crossing over whereas the two types of single spots primarily originate from chromosomal disturbances.Cold shocks disproportionately increased the frequency of double spots, but ethyl methanesulfonate (EMS) did not do so. However, in most cases of each treatment, the frequency of single spots increased. It is suggested that EMS is not very potent in bringing about somatic recombination whereas cold shocks are. Plants from a sample of seeds of variety L65-1237 that had been harvested in 1968 at Urbana, Illinois, did not express the spotting phenomenon, but plants from seeds harvested in 1969 at Reno did have spots. Application of mitomycin C(MC) to the seeds of this variety as well as of T219 increased the incidence of double spots manyfold indicating that MC can reveal the potential for somatic crossing over in a variety which might not otherwise express it. Soaking dry seeds of L65-1237 in aqueous solutions of MC for intervals as short as 2 hours was found effective in increasing the frequency of double spots. The role of MC in relation to DNA synthesis and somatic crossing over is discussed. Application of the chromosome-breaking agent, claunomycin (DM) was ineffective in causing double spots.     

Somatic Crossing over in GLYCINE MAX (L.) Merrill: Mutagenicity of Sodium Azide and Lack of Synergistic Effect with Caffeine and Mitomycin C

Glycine max (soybean) is one angiosperm which lends itself to the study of somatic crossing over. This is made possible because some varieties have gene combinations Y(11)Y(11), Y(11)y(11) and y(11)y(11) in the segregating populations from Y(11)y(11) plants. The gene in question is responsible for chlorophyll synthesis. The Y(11)Y(11) plants have dark green leaves, Y(11)y(11) are light green and y(11)y(11) plants are golden yellow. The heterozygous plants have dark green, yellow and dark green-yellow (double) spots on the leaves of the untreated control material, whereas the two homozygotes are almost always devoid of somatic sectoring. Application of caffeine, or mitomycin C, to the seeds increased the frequency of double, dark green and yellow spots on the Y(11)y(11) background. Possibly, some dark green or yellow spots originate by failure of one of the two components of what might start as a double spot due to somatic crossing over. The application of NaN(3) increases the frequency of dark green or yellow spots, almost exclusively. The two spots increase in equal frequency. The y(11)y(11) plants so treated do not have any light green sectors, but dark green, Y(11)Y(11), plants do develop a few light green or very dark green spots. The data indicate that NaN(3) is capable of inducing nondisjunction, but does not cause mutations (at this locus), chromosome fragmentations (segmental losses) or somatic crossing over to an appreciable degree. It has previously been shown that caffeine-induced chromosome rejoining in Vicia faba can be inhibited by treating the roots with NaN(3). In the present experiments NaN(3) did not affect the processes of somatic crossing over as induced by caffeine or mitomycin C. The effect was additive. This system offers advantages for studying chemical mutagens in that somatic crossing over, point mutations, segmental losses through chromosome breakage and nondisjunction can all be studied in a single treatment to the seeds.     

The ineffectiveness of hycanthone methanesulfonate in inducing somatic crossing over and mutations in Glycine max

Seeds of varieties T219 and L65-1237 of Glycine max heterozygous for the Y₁₁y₁₁ gene combination (controlling chlorophyll development) were soaked for 0–24 h in aqueous solutions of hycanthone in concentration of 125 ppm to 1000 ppm and hycanthone plus FUdR in concentration of 5 × 10^?7 to 2 × 10^?6. Analysis of the simple leaves and the first compound leaf of the heterozygotes indicated that the drug did not increase the frequency of mutations or somatic crossing over in this system.     

The inability of aminoazotoluene to induce somatic crossing-over or mutation in Glycine max (L.) merrill

Seeds from varieties T219 and L65–1237 of Glycine max heterozygous for the gene combination Y₁₁y₁₁ controlling chlorophyll development were soaked in aqueous solutions of aminoazotoluene (AAT) for varying time periods. Analysis of the simple leaves and the first compound leaf for spots indicated that this drug did not increase the frequency of mutation or somatic crossing-over in this system.     

Relations between Factors Controlling Crossing over and Mutability in Males of DROSOPHILA ANANASSAE

Several stocks, selected because they carried previously identified factors governing either crossing over in males or mutability, were examined to determine whether the effects of these factors are restricted to one or the other process. Neither of two dominant enhancers of male crossing over had detectable effects on Minute mutation frequencies among progenies of assayed F₁ males. Genetically equivalent F₁ males monitored for crossing over showed no unequivocal effect of either of three mutators (two dominant and one extrachromosomal) or of a suppressor of mutability. However, one combination of a dominant crossover enhancer with a dominant mutator showed synergistic increases in both crossover and Minute frequencies, and the possibility exists that a single extrachromosomally transmitted element suppresses both male crossing over and mutability. This suppressor element (or elements) had been previously diagnosed in the pc stock which, in this study, was discovered to have also a dominant enhancer of male crossing over and a dominant mutator occupying separable loci in the third chromosome. The pc enhancer of male crossing over differs from the dominant enhancer in another stock with respect to the regional distribution of crossovers, and the pc mutator is distinguished from another 3-linked mutator by its preferential induction of mutations at the Delta locus.     

Oxidative Stress Is Not a Major Contributor to Somatic Mitochondrial DNA Mutations

The accumulation of somatic mitochondrial DNA (mtDNA) mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC) assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10⁻⁵), an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2′-deoxyguanosine), did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations.     

Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae

A plasmid gap repair assay was used to assess the role of three known nucleases, Exo1, Mre11 and Rad1, in the processing of DNA ends and resolution of recombination intermediates during double-strand gap repair. In this assay, alterations in end processing or branch migration are reflected by the frequency of co-conversion of a chromosomal marker 200 bp from the gap. Gap repair associated with crossing over results in integration at the homologous chromosomal locus, whereas the plasmid remains episomal for non-crossover repair events. In mre11 strains, the frequency of gap repair was reduced 3- to 10-fold and conversion tracts were shorter than in the wild-type strain, consistent with a role for this nuclease in processing double-strand breaks. However, conversion tracts were longer in a strain containing the nuclease deficient allele, mre11-H125N, suggesting increased end processing by redundant nucleases. The frequency of gap repair was reduced 2-fold in rad1 mutants and crossing over was reduced, consistent with a role for Rad1 in cleaving recombination intermediates. The frequency of gap repair was increased in exo1 mutants with a significant increase in crossing over. In exo1 mre11 double mutants gap repair was reduced to below the mre11 single mutant level.     

Somatic Mutation Analysis in Salix suchowensis Reveals Early-Segregated Cell Lineages

Long-lived plants face the challenge of ever-increasing mutational burden across their long lifespan. Early sequestration of meristematic stem cells is supposed to efficiently slow down this process, but direct measurement of somatic mutations that accompanies segregated cell lineages in plants is still rare. Here, we tracked somatic mutations in 33 leaves and 22 adventitious roots from 22 stem-cuttings across eight major branches of a shrub willow (Salix suchowensis). We found that most mutations propagated separately in leaves and roots, providing clear evidence for early segregation of underlying cell lineages. By combining lineage tracking with allele frequency analysis, our results revealed a set of mutations shared by distinct branches, but were exclusively present in leaves and not in roots. These mutations were likely propagated by rapidly dividing somatic cell lineages which survive several iterations of branching, distinct from the slowly dividing axillary stem cell lineages. Leaf is thus contributed by both slowly and rapidly dividing cell lineages, leading to varied fixation chances of propagated mutations. By contrast, each root likely arises from a single founder cell within the adventitious stem cell lineages. Our findings give straightforward evidence that early segregation of meristems slows down mutation accumulation in axillary meristems, implying a plant “germline” paralog to the germline of animals through convergent evolution.     

Unequal Crossing over at the Rrna Tandon as a Source of Quantitative Genetic Variation in Drosophila

Abdominal bristle selection lines (three high and three low) and controls were founded from a marked homozygous line to measure the contribution of sex-linked "mutations" to selection response. Two of the low lines exhibited a period of rapid response to selection in females, but not in males. There were corresponding changes in female variance, in heritabilities in females, in the sex ratio (a deficiency of females) and in fitness, as well as the appearance of a mutant phenotype in females of one line. All of these changes were due to bb alleles (partial deficiencies for the rRNA tandon) in the X chromosomes of these lines, while the Y chromosomes remained wild-type bb⁺. We argue that the bb alleles arose by unequal crossing over in the rRNA tandon.—A prediction of this hypothesis is that further changes can occur in the rRNA tandon as selection is continued. This has now been shown to occur.—Our minimum estimate of the rate of occurrence of changes at the rRNA tandon is 3 x 10^-4. As this is substantially higher than conventional mutation rates, the questions of the mechanisms and rates of origin of new quantitative genetic variation require careful re-examination.     

The Cytogenetic Study of Crossing over in Interspecific Hybrids between DROSOPHILA VIRILIS and D. TEXANA

Spontaneous crossing over was studied by means of combined cytological and genetic methods in F₁ Drosophila virilis x D. texana females (series I) and in D. virilis females carrying a D. texana fifth chromosome in heterozygous condition (series II). The main criterion utilized to distinguish the oogonial crossovers from the meiotic ones is the identity of cytological positions of genetic exchange in crossovers constituting a cluster. Five clusters of crossovers with identical positions of exchange were found in the first series of experiments. In the second series of experiments not a single cluster of crossovers resulting from oogonial crossing over was found.     

Massive synthesis of ribonucleic Acid and cellulase in the pea epicotyl in response to indoleacetic Acid, with and without concurrent cell division

Measurements were made over a 4-day period of the effect of added indoleacetic acid (IAA), puromycin, actinomycin D and 5-fluorodeoxyuridine (FUdR) on growth and the levels of total DNA, RNA, protein and cellulase in segments of tissue at the apex of decapitated etiolated epicotyls of Pisum sativum, L. var. Alaska.

The hormone induced swelling of parenchyma cells and cell division. By 3 days after IAA application, the amounts of DNA and protein were approximately double, RNA triple and cellulase 12 to 16 times the levels in controls. All of these changes were prevented by both puromycin and actinomycin D. FUdR prevented DNA synthesis and cell division but not swelling or synthesis of RNA, protein and cellulase.

It is concluded that IAA-induced RNA synthesis is required for cellulase synthesis and lateral cell expansion, whether or not cell division takes place.

     

Somatic recombination in the common-AB diploid of Schizophyllum commune

Summary A recessive suppressor, su-1, of arg-2 was used to detect somatic recombination in common-AB diploids of S. commune. Recombinants were recovered from dense, fastgrowing sectors on arginine-deficient medium. The majority of spontaneous recombinants (129/154) recovered in this study were apparently haploid. Strains which scored as aneuploid and diploid were also recovered and analyzed. Genetic analysis of spontaneous recombinants indicated that crossing over is rare and that haploidization very likely proceeds via stages of aneuploidy. No increase in the frequency of crossing over was detected in recombinants derived following treatment with UV light. The preliminary results favor a parasexual mechanism of recombination in common-AB diploids.Journal Article No. 5126, Michigan Agricultural Experiment Station. This resarch was supported in part by Contract AT-(11-1) 1301 from the United States Atomic Energy Commission and Grant No. GB-13654 from the National Science Foundation.     

Integrative Analysis of Somatic Mutations Altering MicroRNA Targeting in Cancer Genomes

Determining the functional impact of somatic mutations is crucial to understanding tumorigenesis and metastasis. Recent sequences of several cancers have provided comprehensive lists of somatic mutations across entire genomes, enabling investigation of the functional impact of somatic mutations in non-coding regions. Here, we study somatic mutations in 3′UTRs of genes that have been identified in four cancers and computationally predict how they may alter miRNA targeting, potentially resulting in dysregulation of the expression of the genes harboring these mutations. We find that somatic mutations create or disrupt putative miRNA target sites in the 3′UTRs of many genes, including several genes, such as MITF, EPHA3, TAL1, SCG3, and GSDMA, which have been previously associated with cancer. We also integrate the somatic mutations with germline mutations and results of association studies. Specifically, we identify putative miRNA target sites in the 3′UTRs of BMPR1B, KLK3, and SPRY4 that are disrupted by both somatic and germline mutations and, also, are in linkage disequilibrium blocks with high scoring markers from cancer association studies. The somatic mutation in BMPR1B is located in a target site of miR-125b; germline mutations in this target site have previously been both shown to disrupt regulation of BMPR1B by miR-125b and linked with cancer.     

Immunolocalization of Non-Symbiotic Hemoglobins During Somatic Embryogenesis in Chicory

Hemoglobins are ancient O₂-binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum × C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.Key Words: chicory, immunolocalization, nonsymbiotic hemoglobin, somatic embryogenesis     

The Occurrence of Double Strand DNA Breaks is not the Sole Condition for Meiotic Crossing over in Drosophila Melanogaster

Analysis of the interchromosomal effects of In(2L+2R)Cy, In(3L+3R)LVMand their joint effect on the frequencies of single and double crossovers in the cv-v-fregion of the X chromosome as well as interference showed that both inversions, occurring separately, increased the frequency of single as well as double crossovers and the coefficient of coincidence. However, when the inversions occurred together the frequencies of single crossovers no longer increased, but the frequency of double crossovers, as well as the coefficient of coincidence did increase. These results indicate firstly that the interchromosomal effects influence some precondition of exchange, but that this precondition is not an occurrence of double strand DNA breaks. Thus, the occurrence of double strand DNA breaks is not the sole condition for crossing over in Drosophila melanogaster.     

Increase induced by colchicine in the incidence of somatic crossing over in Glycine max

Summary The frequency of somatic crossing over in Glycine max has been significantly increased by soaking the dry seeds in aqueous solutions of 0.0025, 0.005 and 0.01% colchicine. This increase was quite consistent for several treatments involving time × concentration interaction as well as in cases where post-treatment with mitomycin C was given. Results indicate that colchicine is inefficient in disturbing the arrangements of segments of homologous chromosomes involved in the process of somatic exchanges before the onset of the process of somatic crossing over.     

A Pathway-Centric Survey of Somatic Mutations in Chinese Patients with Colorectal Carcinomas

Previous genetic studies on colorectal carcinomas (CRC) have identified multiple somatic mutations in four candidate pathways (TGF-β, Wnt, P53 and RTK-RAS pathways) on populations of European ancestry. However, it is under-studied whether other populations harbor different sets of hot-spot somatic mutations in these pathways and other oncogenes. In this study, to evaluate the mutational spectrum of novel somatic mutations, we assessed 41 pairs of tumor-stroma tissues from Chinese patients with CRC, including 29 colon carcinomas and 12 rectal carcinomas. We designed Illumina Custom Amplicon panel to target 43 genes, including genes in the four candidate pathways, as well as several known oncogenes for other cancers. Candidate mutations were validated by Sanger sequencing, and we further used SIFT and PolyPhen-2 to assess potentially functional mutations. We discovered 3 new somatic mutations in gene APC, TCF7L2, and PIK3CA that had never been reported in the COSMIC or NCI-60 databases. Additionally, we confirmed 6 known somatic mutations in gene SMAD4, APC, FBXW7, BRAF and PTEN in Chinese CRC patients. While most were previously reported in CRC, one mutation in PTEN was reported only in malignant endometrium cancer. Our study confirmed the existence of known somatic mutations in the four candidate pathways for CRC in Chinese patients. We also discovered a number of novel somatic mutations in these pathways, which may have implications for the pathogenesis of CRC.     

Potato virus X induces DNA damage in leaf nuclei of the host plant Nicotiana tabacum L. var. xanthi

We employed the comet assay (single cell gel electrophoresis) to evaluate induced DNA damage in nuclei isolated from tobacco leaves (Nicotiana tabacum var. xanthi) inoculated with Potato virus X (PVX). The highest DNA damage, expressed by the tail moment value, was observed in the inoculated leaves and decreased in the 1^st to 4^th systemic leaves. DNA damage increased with the time after the inoculation (from day 3 to day 21) and was higher in nuclei isolated from a part of the leaf at the petiole compared to nuclei isolated from the leaf tip. A Pearson moment correlation (r = 0.94) between the induced DNA damage and the PVX titres expressed by ELISA absorbance values was observed. The PVX infection did not induce a significant increase in the rate of somatic mutations evaluated by appearance of dark green, yellow, and double green/yellow sectors on the heterozygous pale green leaves of N. tabacum var. xanthi.     

Clinical Significance in Oral Cavity Squamous Cell Carcinoma of Pathogenic Somatic Mitochondrial Mutations

Somatic mutations affecting the mitochondrial DNA (mtDNA) have been frequently observed in human cancers and proposed as important oncological biomarkers. However, the clinical significance of mtDNA mutations in cancer remains unclear. This study was therefore performed to explore the possible clinical use in assessing oral squamous cell carcinoma (OSCC) of pathogenic mtDNA mutations. The entire mitochondrial genome of 300 OSCC with their matched control DNAs was screened by direct sequencing and criteria were set to define a pathogenic somatic mutation. The patients'' TP53 R72P genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. The relationships between pathogenic somatic mutations, clinicopathogical features, TP53 R72P genotype and clinical prognosis were analyzed. Overall, 645 somatic mtDNA mutations were identified and 91 of these mutations were defined as pathogenic. About one quarter (74/300) of the OSCC tumor samples contained pathogenic mutations. Individuals with the TP53 R allele had a higher frequency of pathogenic somatic mutation than those with the PP genotype. Kaplan-Meier analysis indicated that TP53 R allele patients with pathogenic somatic mutations demonstrated a significant association with a poorer disease-free survival than other individuals (HR = 1.71; 95% CI, 1.15–2.57; p = 0.009) and this phenomenon still existed after adjusting for mtDNA haplogroup, tumor stage with treatment regimens, differentiation and age at diagnosis (HR = 1.59; 95% CI, 1.06–2.40; p = 0.03). Subgroup analyses showed that this phenomenon was limited to patients who received adjuvant radiotherapy/chemo-radiotherapy after surgery. The results strongly indicated that pathogenic mtDNA mutations are a potential prognostic marker for OSCCs. Furthermore, functional mitochondria may play an active role in cancer development and the patient''s response to radiotherapy/chemo-radiotherapy.