Search for methylation-sensitive amplification polymorphisms in mutant figs

Fig (Ficus carica) breeding programs that use conventional approaches to develop new cultivars are rare, owing to limited genetic variability and the difficulty in obtaining plants via gamete fusion. Cytosine methylation in plants leads to gene repression, thereby affecting transcription without changing the DNA sequence. Previous studies using random amplification of polymorphic DNA and amplified fragment length polymorphism markers revealed no polymorphisms among select fig mutants that originated from gamma-irradiated buds. Therefore, we conducted methylation-sensitive amplified polymorphism analysis to verify the existence of variability due to epigenetic DNA methylation among these mutant selections compared to the main cultivar 'Roxo-de-Valinhos'. Samples of genomic DNA were double-digested with either HpaII (methylation sensitive) or MspI (methylation insensitive) and with EcoRI. Fourteen primer combinations were tested, and on an average, non-methylated CCGG, symmetrically methylated CmCGG, and hemimethylated hmCCGG sites accounted for 87.9, 10.1, and 2.0%, respectively. MSAP analysis was effective in detecting differentially methylated sites in the genomic DNA of fig mutants, and methylation may be responsible for the phenotypic variation between treatments. Further analyses such as polymorphic DNA sequencing are necessary to validate these differences, standardize the regions of methylation, and analyze reads using bioinformatic tools. © FUNPEC-RP.

INHERITANCE of CUCUMBER TOLERANCE TO ZUCCHINI YELLOW MOSAIC VIRUS

Studies were carried out in Brazil to study the inheritance of tolerance to Zucchini yellow mosaic virus (ZYMV) in cucumber cv. Formosa. This cultivar was individually crossed with two cucumber lines from different varietal types (L(b) from a Brazilian type, and L(j) from a Japanese type), both susceptible to the virus. Two experiments, one for each line, were separately carried out, where 6 treatments (parents, generations F1, F2 and F1BC1 for both parents) were evaluated in a randomized block design with 5 repetitions. Cotyledons of 2-week-old cucumber seedlings were inoculated with ZYMV. Only the plants that did not show symptoms up to 63 days post inoculation were considered as tolerant. A chi-square (chi(2)) analysis for assessing segregation from F2 and both F1BC1, led to the conclusion that the tolerance found in cv. Formosa is determined by a recessive gene.

TRAIL-R3-related apoptosis: Epigenetic and expression analyses in women with ovarian neoplasia

Objective. To assess the expression of TRAIL-R3 and the methylation of a CpG island within the TRAIL-R3 promoter both in cystadenoma tumors and primary and metastatic epithelial ovarian carcinoma (EOC).Methods. RNA was obtained from women with normal ovarian (NO) tissues (n = 18), ovarian serous cystadenoma tumors (n = 11) and EOC (n = 16) using Trizol (R). Quantitative PCR (gRT-PCR) was performed to quantify the relative levels of TRAIL-R3. The methylation frequency of the CpG island in the TRAIL-R3 promoter was assessed using the methylation-specific PCR (MSP) assay after DNA bisulfite conversion. The differences between the groups were evaluated using the chi-square, Student's t, ANOVA, Mann-Whitney U, Wilcoxon or Kruskal-Wallis tests as indicated. The survival rates were calculated using the Kaplan-Meier method.Results. Cystadenoma and metastatic EOC tumors expressed significantly more TRAIL-R3 mRNA than primary EOC tumors. Methylation of the TRAIL-R3 promoter was absent in NO tissues, while hemimethylation of the TRAIL-R3 promoter was frequently found in the neoplasia samples with 45.4% of the cystadenoma tumors, 8.3% of the primary EOC samples and 11.1% of the metastatic EOC samples showing at least partial methylation (p = 0.018). Neither the expression of TRAIL-R3 nor alterations in the methylation profile were associated to cumulative progression-free survival or the overall survival in EOC patients.Conclusions. Primary EOC is associated to a lower TRAIL-R3 expression, which leads to a better understanding of the complex disease and highlighting potential therapeutic targets. Promoter DNA methylation was not related to this finding, suggesting the presence of other mechanisms to transcriptional control. (C) 2012 Elsevier B.V. All rights reserved.

Sulcus vocalis: evidence for autosomal dominant inheritance

We found evidence of autosomal dominant hereditary transmission of sulcus vocalis. Four dysphonic patients from three generations of the same family were submitted to videolaryngoscopic examination (three patients) and to direct laryngoscopy ( one patient) to diagnose the hoarseness. Sulcus vocalis was diagnosed in all four patients. The finding of four affected individuals in three generations, with vertical transmission affecting man and women, is more consistent with autosomal dominant inheritance pattern; it is an etiological model that we propose for the sulcus vocalis in this pedigree.

H19-DMR allele-specific methylation analysis reveals epigenetic heterogeneity of CTCF binding site 6 but not of site 5 in head-and-neck carcinomas: A pilot case-control analysis

Aberrant methylation of seven potential binding sites of the CTCF factor in the differentially methylated region upstream of the H19 gene (H19-DMR) has been suggested as critical for the regulation of IGF2 and H19 imprinted genes. In this study, we analyzed the allele-specific methylation pattern of CTCF binding sites 5 and 6 using methylationsensitive restriction enzyme PCR followed by RFLP analysis in matched tumoral and lymphocyte DNA from head-and-neck squamous cell carcinoma (HNSCC) patients, as well as in lymphocyte DNA from control individuals who were cancer-free. The monoallelic methylation pattern was maintained in CTCF binding site 5 in 22 heterozygous out of 91 samples analyzed. Nevertheless, a biallelic methylation pattern was detected in CTCF binding site 6 in a subgroup of HNSCC patients as a somatic acquired feature of tumor cells. An atypical biallelic methylation was also observed in both tumor and lymphocyte DNA from two patients, and at a high frequency in the control group (29 out of 64 informative controls). Additionally, we found that the C/T transition detected by HhaI RFLP suppressed one dinucleotide CpG in critical CTCF binding site 6, of a mutation showing polymorphic frequencies. Although a heterogeneous methylation pattern was observed after DNA sequencing modified by sodium bisulfite, the biallelic methylation pattern was confirmed in 9 out of 10 HNSCCs. These findings are likely to be relevant in the epigenetic regulation of the DMR, especially in pathological conditions in which the imprinting of IGF2 and H19 genes is disrupted.

Arabidopsis thaliana Uncoupling Proteins (AtUCPs): insights into gene expression during development and stress response and epigenetic regulation

Mitochondrial inner membrane uncoupling proteins (UCP) catalyze a proton conductance that dissipates the proton electrochemical gradient established by the respiratory chain, thus affecting the yield of ATP synthesis. UCPs are involved in mitochondrial energy flow regulation and have been implicated in oxidative stress tolerance. Based on the global gene expression profiling datasets available for Arabidopsis thaliana, in this review we discuss the regulation of UCP gene expression during development and in response to stress, and provide interesting insights on the possible existence of epigenetic regulation of UCP expression.

Evidence of epigenetic regulation of the tumor suppressor gene cluster flanking RASSF1 in breast cancer cell lines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

NORs inheritance analysis in crossings including individuals from two stocks of rainbow trout (Oncorhynchus mykiss)

Silver nitrate staining of rainbow trouts (Oncorhynchus mykiss) chromosomes, for the identification of the nucleolar organizing regions (NORs), revealed that in individuals from Nucleo Experimental de Salmonicultura de Campos do Jordao (Brazil) NORs were located in the long arms of a submetacentric pair while in specimens from Mount Shasta (USA) NORs were located in the short arms of a submetacentric pair. Cytogenetic analysis of the offspring, obtained through artificial crosses including individuals from both stocks, allowed the identification of NORs in two submetacentric chromosomes, one in the short arms and the other in the long arms, confirming the effectiveness of the hybridization process. Complementary results obtained using the FISH technique with 18S and 5S rDNA probes showed that NOR-bearing chromosomes exhibited a cluster of 5S genes located in tandem with the 18S gene cluster in both stocks. The results allow us to suggest that the difference in NOR-bearing chromosomes found between the two stocks is likely to be due to a pericentric inversion involving the chromosome segment where 18S and 5S rDNA genes are located. The presence of ribosomal genes in the long arms of a submetacentric chromosome is apparently a particular characteristic of the rainbow trout stock of Campos do Jordao and might be used as a chromosome marker in studies of controlled crosses in this species.

Supernumerary chromosome inheritance in the curimbata (Prochilodus lineatus) of the Mogi-Guacu River

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Epigenetic Silencing of CRABP2 and MX1 in Head and Neck Tumors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inheritance of resistance to powdery mildew in pea and pathogenesis-related aspects

A herança da resistência ao oídio na cultivar de ervilha MK-10 e alguns aspectos histológicos da infecção foram estudados. Para o estudo da herança, as gerações F1, F2, retrocuzamentos e geração F3 de MK-10 com duas populações suscetíveis foram avaliadas. Nas avaliações histológicas observou-se a porcentagem de conídios germinados, porcentagem de conídios que formaram apressório, porcentagem de conídios que estabeleceram colônia e número de haustórios por colônia. Para comparar as razões de segregação obtidas no estudo da herança da resistência, adotou-se o teste do Qui-quadrado (X²) e para os dados das análises histológicas, utilizou-se o teste Tukey a 5% de probabilidade. Concluiu-se que a resistência de MK-10 ao oídio é devida a um par de alelos recessivos e que a resistência é expressa na fase de pré-penetração, completada por uma morte celular localizada pós-penetração, característica da presença do par de alelos recessivos er1er1.

Variances of direct genetic effects, maternal genetic effects, and cytoplasmic inheritance effects for milk yield, fat yield, and fat percentage

Milk yield, fat yield, and fat percentage during the first three lactations were studied using New York Holsteins that were milked twice daily over a 305-d, mature equivalent lactation. Those data were used to estimate variances from direct and maternal genetic effects, cytoplasmic effects, sire by herd interaction, and cow permanent environmental effects. Cytoplasmic line was traced to the last female ancestor using DHI records from 1950 through 1991. Records were 138,869 lactations of 68,063 cows calving from 1980 through 1991. Ten random samples were based on herd code. Samples averaged 4926 dams and 2026 cytoplasmic lines. Model also included herd-year-seasons as fixed effects and genetic covariance for direct-maternal effects. Mean estimates of the effects of maternal genetic variances and direct-maternal covariances, as fractions of phenotypic variances, were 0.008 and 0.007 for milk yield, 0.010 and 0.010 for fat yield, and 0.006 and 0.025 for fat percentage, respectively. Average fractions of variance from cytoplasmic line were 0.011, 0.008, and 0.009 for milk yield, fat yield, and fat percentage. Removal of maternal genetic effects and covariance for maternal direct effects from the model increased the fraction of direct genetic variance by 0.014, 0.021, and 0.046 for milk yield, fat yield, and fat percentage; little change in the fraction was due to cytoplasmic line. Exclusion of cytoplasmic effects from the model increased the ratio of additive direct genetic variance to phenotypic variance by less than 2%. Similarly, when sire by herd interaction was excluded, the ratio of direct genetic variance to phenotypic variance increased 1% or less.