ACHT操作引起黑麦(Secale cereale)染色体的畸变和重建

用ACHT处理黑麦萌动种子，对修复前后材料的观察和分析结果表明：1. ACHT操作引起染色体数目变化和染色体断裂损失。在一定 条件和范围内，不同处理引起的这种变化具有显著差异，条件越剧烈，染色体数目变化的范围和频率愈大，断片发生的数量和频率 也愈高，同时修复前后染色体数目的变化范围和频率与断片发生的数量和频率以及它们的修复频率均表现明显的相关性。2. ACHT 操作引起染色体畸变的多样性。经ACHT处理后，胚根细胞染色体有4种断裂方式，包括着丝粒断裂、次溢痕断裂、长臂断裂和短臂 断裂等，其中着丝断裂频率最高；产生6种断片类型，包括有着丝粒和端粒的、有着丝粒而无端粒的、有部分着丝粒和端粒的、有 部分着丝粒而无端粒的、只有端粒的、既无着丝粒也无端粒的断片等。3. ACHT操作引起遗传结构重建的多样性。经ACHT处理后， 对修复24-72小时材料进行核型比较(按Stebbins 和 Levan 标准)和随体分析，处理细胞在染色体数目、大小、形态、位置等方面 均发生显著变化，说明ACHT处理使这些细胞的染色体结构和染色体组型发生了深刻变化。进一步通过Giemsa C— 带分析，观察到 多种重建染色体类型，包括易位型染色体、附加型染色体、无着丝粒染色体、化染色体、增加的m染色体以及某些带型特异的染色 体等。4. RAPD 分析从分子水平上验证了ACHT能有效地引起遗传结构的改变。所用10种引物对处理和对照材料基因组DNA的扩增产 物在条带数目、条带位置及带型特征等方面均有明显差异，其中4种引物出现条带减少，6种引物出现条带增加，后者还包括一个带 位移动。这说明两种材料的基因组DNA具有明显的RAPD反应多态性差异。This paper descripes some results draw on the basis of the observation and analysis on the rye before and after repaired through treating its budding seeds by ACHT in contrast to without ACHT: 1. ACHT manipulation caused the number variation and breakage damage of rye chromosome. Within certain conditions and timits, this phenomenon caused by different treats had signifcant difference: the more the treatment condition is drastie, the more the chageable range and frequence of rye chromosomae number, and so is the produced fragments. Meanwhile, there existed striking relationship among the changeable range and frequence of rye chromosome, the produced number and frequence of fragments and repairing frequence. 2. ACHT manipulafion engendered the diversify of rye chromosomal aberration. Four breakage patterns and six sorts of fragment were observed by watching the chromosome of the rye radicle treated by ACHT, including centric breakage (occuring in the highest frequence), secondary constriction breakage, long arm breakage and short arm breakage to the former, Comprising that with both centromere and telomere, that with centromere and without telomere, that with partial centromere and with telomere, that with partfial ceetromere and without telomere, that only with telomere and that neither with centromere nor with telomere, etc. 3. ACHT manipulation engendered the diversify or rye genetic structs reconstruction. Karureotype analysis(according to Stebbins and Levan) and satellite anaeysis were carried out to rye radicle through 24-72-hour-long recoverage after ACHT manipulation, which showed remarkable change happened on the rye chromosomal number、shape、arm ration and pattern, etc. and also on the satellite number、size、shape and location etc. Those indicated that ACHT manipulation engendered violent changes to rye chromatin structure and chromosome type. Further Giemsa C-banding analysis showed many types of reconstructed chromosome, such as translocation、addition、without centromere、st and other chromosome. 4. RAPD analysis checked the validity of ACHT on changing genetic structure of rye on the level of molecular biology. The treated and recovered rye has different amplifying band pattern by using IO valid arbitary primers selected from 40 comparing with the control.

Chromosomal aberrations induced by C-12(6+) ions and Co-60 gamma-rays in mouse immature oocytes

The ovaries of Kun-Ming strain mice (3 weeks) were irradiated with different doses of C-12(6+) ion or Co-60 gamma-ray. Chromosomal aberrations were analyzed in metaphase II oocytes at 7 weeks after irradiation. The relative biological effectiveness (RBE) of C C-12(6+) ion was calculated with respect to Co-60 gamma-ray for the induction of chromosornal aberrations. The C-12(6+) ion and Co-60 gamma-ray dose-response relationships for chromosomal aberrations were plotted by linear quadratic models. The data showed that there was a dose-related increase in frequency of chromosomal aberrations in all the treated groups compared to controls. The RBE values for C-12(6+) ions relative to (CO)-C-60 gamma-rays were 2.49, 2.29, 1.57, 1.42 or 1.32 for the doses of 0.5, 1.0, 2.07 4.0 or 6.0 Gy, respectively. Moreover, a different distribution of the various types of aberrations has been found for C-12(6+) ion and Co-60 gamma-ray irradiations. The dose-response relationships for C-12(6+) ion and (CO)-C-60 gamma-ray exhibited positive correlations. The results from the present study may be helpful for assessing genetic damage following exposure of immature oocytes to ionizing radiation.

Comparison of clonogenic assay with premature chromosome condensation assay in prediction of human cell radiosensitivity

Aim: To determine whether the number of non-rejoining G2-chromatid breaks can predict the radiosensitivity of human cell lines. Methods: Cell lines of human ovary carcinoma cells (HO8910), human hepatoma cells (HepG2) and liver cells (L02) were irradiated with a range of doses and assessed both of cell survival and non-rejoining G2-chromatid breaks at 24 h after irradiation. Cell survival was documented by a colony assay. Non-rejoining G2-chromatid breaks were measured by counting the number of non-rejoining G2 chromatid breaks at 24 h after irradiation, detected by the prematurely chromosome condensed (PCC) technique. Results: A linear-quadratic survival curve was observed in three cell lines, and HepG2 was the most sensitive to gamma-radiation. A dose-dependent linear increase was observed in radiation-induced non-rejoining G2-PCC breaks measured at 24 h after irradiation in all cell lines, and HepG2 was the most susceptible to induction of non-rejoining G2-PCC breaks. A close correlation was found between the clonogenic radiosensitivity and the radiation-induced non-rejoining G2-PCC breaks (r=0.923). Furthermore, survival-aberration correlations for two or more than two doses lever were also significant. Conclusion: The number of non-rejoining G2 PCC breaks holds considerable promise for predicting the radiosensitivity of normal and tumor cells when two or more than two doses lever is tested.

波动方程偏移速度分析

On the subject of oil and gas exploration, migration is an efficacious technique for imagining structures underground. Wave-equation migration (WEM) dominates over other migration methods in accuracy, despite of higher computational cost. However, the advantages of WEM will emerge as the progress of computer technology. WEM is sensitive to velocity model more than others. Small velocity perturbations result in grate divergence in the image pad. Currently, Kirrchhoff method is still very popular in the exploration industry for the reason of difficult to provide precise velocity model. It is very urgent to figure out a way to migration velocity modeling. This dissertation is mainly devoted to migration velocity analysis method for WEM: 1. In this dissertation, we cataloged wave equation prestack depth migration. The concept of migration is introduced. Then, the analysis is applied to different kinds of extrapolate operator to demonstrate their accuracy and applicability. We derived the DSR and SSR migration method and apply both to 2D model. 2. The output of prestack WEM is in form of common image gathers (CIGs). Angle domain common image gathers (ADCIGs) gained by wave equation are proved to be free of artifacts. They are also the most potential candidates for migration velocity analysis. We discussed how to get ADCIGs by DSR and SSR, and obtained ADCIGs before and after imaging separately. The quality of post stack image is affected by CIGs, only the focused or flattened CIGs generate the correct image. Based on wave equation migration, image could be enhanced by special measures. In this dissertation we use both prestack depth residual migration and time shift imaging condition to improve the image quality. 3. Inaccurate velocities lead to errors of imaging depth and curvature of coherent events in CIGs. The ultimate goal of migration velocity analysis (MVA) is to focus scattered event to correct depth and flatten curving event by updating velocities. The kinematic figures are implicitly presented by focus depth aberration and kinetic figure by amplitude. The initial model of Wave-equation migration velocity analysis (WEMVA) is the output of RMO velocity analysis. For integrity of MVA, we review RMO method in this dissertation. The dissertation discusses the general ideal of RMO velocity analysis for flat and dipping events and the corresponding velocity update formula. Migration velocity analysis is a very time consuming work. Respect to computational convenience, we discus how RMO works for synthetic source record migration. In some extremely situation, RMO method fails. Especially in the areas of poorly illuminated or steep structure, it is very difficult to obtain enough angle information for RMO. WEMVA based on wave extrapolate theory, which successfully overcome the drawback of ray based methods. WEMVA inverses residual velocities with residual images. Based on migration regression, we studied the linearized scattering operator and linearized residual image. The key to WEMVA is the linearized residual image. Residual image obtained by Prestack residual migration, which based on DSR is very inefficient. In this dissertation, we proposed obtaining residual migration by time shift image condition, so that, WEMVA could be implemented by SSR. It evidently reduce the computational cost for this method.

地震波场延拓+菲涅尔体叠加方法及成像效果

In the increasingly enlarged exploration target, deep target layer(especially for the reservoir of lava) is a potential exploration area. As well known, the reflective energy becomes weak because the seismic signals of reflection in deep layer are absorbed and attenuate by upper layer. Caustics and multi-values traveltime in wavefield are aroused by the complexity of stratum. The ratio of signal to noise is not high and the fold numbers are finite(no more than 30). All the factors above affect the validity of conventional processing methods. So the high S/N section of stack can't always be got with the conventional stack methods even if the prestack depth migration is used. So it is inevitable to develop another kind of stack method instead. In the last a few years, the differential solution of wave equation was hold up by the condition of computation. Kirchhoff integral method rose in the initial stages of the ninetieth decade of last century. But there exist severe problems in it, which is are too difficult to resolve, so new method of stack is required for the oil and gas exploration. It is natural to think about upgrading the traditionally physic base of seismic exploration methods and improving those widely used techniques of stack. On the other hand, great progress is depended on the improvement in the wave differential equation prestack depth migration. The algorithm of wavefield continuation in it is utilized. In combination with the wavefield extrapolation and the Fresnel zone stack, new stack method is carried out It is well known that the seismic wavefield observed on surface comes from Fresnel zone physically, and doesn't comes from the same reflection points only. As to the more complex reflection in deep layer, it is difficult to describe the relationship between the reflective interface and the travel time. Extrapolation is used to eliminate caustic and simplify the expression of travel time. So the image quality is enhanced by Fresnel zone stack in target. Based on wave equation, high-frequency ray solution and its character are given to clarify theoretical foundation of the method. The hyperbolic and parabolic travel time of the reflection in layer media are presented in expression of matrix with paraxial ray theory. Because the reflective wave field mainly comes from the Fresnel Zone, thereby the conception of Fresnel Zone is explained. The matrix expression of Fresnel zone and projected Fresnel zone are given in sequence. With geometrical optics, the relationship between object point in model and image point in image space is built for the complex subsurface. The travel time formula of reflective point in the nonuniform media is deduced. Also the formula of reflective segment of zero-offset and nonzero offset section is provided. For convenient application, the interface model of subsurface and curve surface derived from conventional stacks DMO stack and prestack depth migration are analyzed, and the problem of these methods was pointed out in aspects of using data. Arc was put forward to describe the subsurface, thereby the amount of data to stack enlarged in Fresnel Zone. Based on the formula of hyperbolic travel time, the steps of implementation and the flow of Fresnel Zone stack were provided. The computation of three model data shows that the method of Fresnel Zone stack can enhance the signal energy and the ratio of signal to noise effectively. Practical data in Xui Jia Wei Zhi, a area in Daqing oilfield, was processed with this method. The processing results showed that the ability in increasing S/N ratio and enhancing the continuity of weak events as well as confirming the deep configuration of volcanic reservoir is better than others. In deeper target layer, there exists caustic caused by the complex media overburden and the great variation of velocity. Travel time of reflection can't be exactly described by the formula of travel time. Extrapolation is bring forward to resolve the questions above. With the combination of the phase operator and differential operator, extrapolating operator adaptable to the variation of lateral velocity is provided. With this method, seismic records were extrapolated from surface to any different deptlis below. Wave aberration and caustic caused by the inhomogenous layer overburden were eliminated and multi-value curve was transformed into the curve.of single value. The computation of Marmousi shows that it is feasible. Wave field continuation extends the Fresnel Zone stack's application.

各向异性与非线性介质中弹性波场传播特征研究

The topic of this study is about the propagation features of elastic waves in the anisotropic and nonlinear media by numerical methods with high accuracy and stability. The main achievements of this paper are as followings: Firstly, basing on the third order elastic energy formula, principle of energy conservation and circumvolved matrix method, we firstly reported the equations of non-linear elastic waves with two dimensions and three components in VTI media. Secondly, several conclusions about some numerical methods have been obtained in this paper. Namely, the minimum suitable sample stepth in space is about 1/8-1/12 of the main wavelength in order to distinctly reduce the numerical dispersion resulted from the numerical mehtod, at the same time, the higher order conventional finite difference (CFD) schemes will give little contribution to avoid the numerical solutions error accumulating with time. To get the similar accuracy with the fourth order center finite difference method, the half truncation length of SFFT should be no less than 7. The FDFCT method can present with the numerical solutions without obvious dispersion when the paprameters of FCT is suitable (we think they should be in the scope from 0.0001 to 0.07). Fortunately, the NADM method not only can reported us with the higher order accuracy solutions (higher than that of the fourth order finite difference method and lower than that of the sixth order finite difference method), but also can distinctly reduce the numerical dispersion. Thirdly, basing on the numerial and theoretical analysis, we reported such nonlinear response accumulating with time as waveform aberration, harmonic generation and resonant peak shift shown by the propagation of one- and two-dimensional non-linear elasticwaves in this paper. And then, we drew the conclusion that these nonlinear responses are controlled by the product between nonlinear strength (SN) and the amplitude of the source. At last, the modified FDFCT numerical method presented by this paper is used to model the two-dimensional non-linear elastic waves propagating in VTI media. Subsequently, the wavelet analysis and polarization are adopted to investigate and understand the numerical results. And then, we found the following principles (attention: the nonlinear strength presented by this paper is weak, the thickness of the -nonlinear media is thin (200m), the initial energy of the source is weak and the anisotropy of the media is weak too): The non-linear response shown by the elastic waves in VTI media is anisotropic too; The instantaneous main frequency sections of seismic records resulted from the media with a non-linear layer have about 1/4 to 1/2 changes of the initial main frequency of source with that resulted from the media without non-linear layer; The responses shown by the elasic waves about the anisotropy and nonlinearity have obvious mutual reformation, namely, the non-linear response will be stronger in some directions because of the anisotropy and the anisotropic strength shown by the elastic waves will be stronger when the media is nonlinear.

Peace and Peacemaking in Paul Against the Backdrop of Greco-Roman Conceptions of Peace

Peace in the ancient world has been studied primarily from the perspective of pacifism and questions related to war and peace. This study employs a socio-historical method to determine how peace was understood in itself, not just with respect to war. It demonstrates that the Greco-Roman world viewed peace as brief periods of tranquility in an existence where conflict was the norm, while Paul regarded peace as the norm and conflict as an intrusive aberration. Through a historical and literary survey of Greco-Roman thought and culture, this study shows that myth, legend, religion, education, philosophy, and science created and perpetuated the idea that conflict was necessary for existence. Wars were fought to attain peace, which meant periods of calm, quiet, and security with respect to the gods, one's inner self, nature, others who are insiders, and others who are outsiders. Despite the desirability of peace, genuine peace was seldom experienced, and even then, only briefly, as underlying enmity persisted without resolution. While Paul supports the prevailing conception of peace as tranquility and felicity in relation to God, self, nature, and others, he differs as to the origin, attainment, and maintenance of peace. In Paul, peace originates in God and is graciously given to those who are justified and reconciled to God through Jesus Christ. God removes the enmity caused by sin and provides the indwelling Spirit to empower believers to think and behave in ways that promote and maintain peace. This study also examines how three social dynamics (honor-shame, patron-client, friendship-enmity) affect Paul's approach to conflict resolution with Philemon and Onesimus, Euodia and Syntyche, believers who are prosecuting one another in civil courts, and Peter. Rather than giving specific procedures for resolving conflict, Paul reinforces the believer's new identity in Christ and the implications of God's grace, love, and peace upon their thoughts, words, and behavior toward one another. Paul uses these three social dynamics to encourage believers in the right direction, but their ultimate accountability is to God. The study concludes with four strategic principles for educating the church and developing an atmosphere and attitude within the church for peacemaking.

Enhancing imaging systems using transformation optics.

We apply the transformation optical technique to modify or improve conventional refractive and gradient index optical imaging devices. In particular, when it is known that a detector will terminate the paths of rays over some surface, more freedom is available in the transformation approach, since the wave behavior over a large portion of the domain becomes unimportant. For the analyzed configurations, quasi-conformal and conformal coordinate transformations can be used, leading to simplified constitutive parameter distributions that, in some cases, can be realized with isotropic index; index-only media can be low-loss and have broad bandwidth. We apply a coordinate transformation to flatten a Maxwell fish-eye lens, forming a near-perfect relay lens; and also flatten the focal surface associated with a conventional refractive lens, such that the system exhibits an ultra-wide field-of-view with reduced aberration.

Utility of a molecular prescreening program in advanced colorectal cancer for enrollment on biomarker-selected clinical trials.

BACKGROUND: Incorporation of multiple enrichment biomarkers into prospective clinical trials is an active area of investigation, but the factors that determine clinical trial enrollment following a molecular prescreening program have not been assessed. PATIENTS AND METHODS: Patients with 5-fluorouracil-refractory metastatic colorectal cancer at the MD Anderson Cancer Center were offered screening in the Assessment of Targeted Therapies Against Colorectal Cancer (ATTACC) program to identify eligibility for companion phase I or II clinical trials with a therapy targeted to an aberration detected in the patient, based on testing by immunohistochemistry, targeted gene sequencing panels, and CpG island methylation phenotype assays. RESULTS: Between August 2010 and December 2013, 484 patients were enrolled, 458 (95%) had a biomarker result, and 157 (32%) were enrolled on a clinical trial (92 on biomarker-selected and 65 on nonbiomarker selected). Of the 458 patients with a biomarker result, enrollment on biomarker-selected clinical trials was ninefold higher for predefined ATTACC-companion clinical trials as opposed to nonpredefined biomarker-selected clinical trials, 17.9% versus 2%, P < 0.001. Factors that correlated positively with trial enrollment in multivariate analysis were higher performance status, older age, lack of standard of care therapy, established patient at MD Anderson, and the presence of an eligible biomarker for an ATTACC-companion study. Early molecular screening did result in a higher rate of patients with remaining standard of care therapy enrolling on ATTACC-companion clinical trials, 45.1%, in contrast to nonpredefined clinical trials, 22.7%; odds ratio 3.1, P = 0.002. CONCLUSIONS: Though early molecular prescreening for predefined clinical trials resulted in an increase rate of trial enrollment of nonrefractory patients, the majority of patients enrolled on clinical trials were refractory to standard of care therapy. Within molecular prescreening programs, tailoring screening for preidentified and open clinical trials, temporally linking screening to treatment and optimizing both patient and physician engagement are efforts likely to improve enrollment on biomarker-selected clinical trials. CLINICAL TRIALS NUMBER: The study NCT number is NCT01196130.

DNA and chromosomal damage in response to intermittent extremely low-frequency magnetic fields.

Considerable controversy still exists as to whether electric and magnetic fields (MF) at extremely low frequencies are genotoxic to humans. The aim of this study was to test the ability of alternating magnetic fields to induce DNA and chromosomal damage in primary human fibroblasts. Single- and double-strand breaks were quantified using the alkaline comet assay and the gammaH2AX-foci assay, respectively. Chromosomal damage was assayed for unstable aberrations, sister chromatid exchange and micronuclei. Cells were exposed to switching fields - 5min on, 10min off - for 15h over the range 50-1000microT. Exposure to ionizing radiation was used as a positive-effect calibration. In this study two separate MF exposure systems were used. One was based on a custom-built solenoid coil system and the other on a commercial system almost identical to that used in previous studies by the EU REFLEX programme. With neither system could DNA damage or chromosomal damage be detected as a result of exposure of fibroblasts to switching MF. The sensitive gammaH2AX assay could also not detect significant DNA damage in the MF-exposed fibroblasts, although the minimum threshold for this assay was equivalent to an X-ray dose of 0.025Gy. Therefore, with comparable MF parameters employed, this study could not confirm previous studies reporting significant effects for both the alkaline and neutral comet assays and chromosomal aberration induction.

Non-nematode-derived double-stranded RNAs induce profound phenotypic changes in Meloidogyne incognita and Globodera pallida infective juveniles

Nine non-nematode-derived double-stranded RNAs (dsRNAs), designed for use as controls in RNA interference (RNAi) screens of neuropeptide targets, were found to induce aberrant phenotypes and an unexpected inhibitory effect on motility of root knot nematode Meloidogyne incognita J2s following 24 h soaks in 0.1 mg/ml dsRNA; a simple soaking procedure which we have found to elicit profound knockdown of neuronal targets in Globodera pallida J2s. We have established that this inhibitory phenomenon is both time- and concentration-dependent, as shorter 4 h soaks in 0.1 mg/ml dsRNA had no negative impact on M. incognita J2 stage worms, yet a 10-fold increase in concentration to 1 mg/ml for the same 4 h time period had an even greater qualitative and quantitative impact on worm phenotype and motility. Further, a 10-fold increase of J2s soaked in 0.1 mg/ml dsRNA did not significantly alter the observed phenotypic aberration, which suggests that dsRNA uptake of the soaked J2s is not saturated under these conditions. This phenomenon was not initially observed in potato cyst nematode G. pallida J2s, which displayed no aberrant phenotype, or diminution of migratory activity in response to the same 0.1 mg/ml dsRNA 24 h soaks. However, a 10-fold increase in dsRNA to 1 mg/ml was found to elicit comparable irregularity of phenotype and inhibition of motility in G. pallida, to that initially observed in M. incognita following a 24 h soak in 0.1 mg/ml dsRNA. Again, a 10-fold increase in the number of G. pallida J2s soaked in the same volume of 1 mg/ml dsRNA preparation did not significantly affect the observed phenotypic deviation. We do not observe any global impact on transcript abundance in either M. incognita or G. pallida J2s following 0.1 mg/ml dsRNA soaks, as revealed by reverse transcriptase-PCR and quantitative PCR data. This study aims to raise awareness of a phenomenon which we observe consistently and which we believe signifies a more expansive deficiency in our knowledge and understanding of the variables inherent to RNAi-based investigation.

Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality

Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control On silencing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (sORNA-soaked M incognita eggs Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH2)-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhibition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually leading to embryonic lethality To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cytosolic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abundance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberration of the J2s. which recovered within 24 h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48 h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-en-I transcript which encodes an RNAi-inhibiting siRNA exonuclease We observe a marked up-regulation of MI-en-I transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RN/NI-based reverse genetic screens in plant parasitic nematodes. (C) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Regulation of cyclin D1 by the BRCA1-BARD1 complex

Background BRCA1 and cyclin D₁ are both essential for normal breast development and mutation or aberration of their expression is associated with breast cancer [1,2]. Cyclin D₁ is best known as a G₁ cyclin where it regulates the G₁ to S phase transition by acting as a rate-limiting subunit of CDK4/6 kinase activity. More recently, however, Stacey has demonstrated that cyclin D₁ levels in G₂/M determine whether a cell continues to proliferate or exits the cell cycle [3]. The majority of BRCA1 in the cell is bound to BARD1 through their N-terminal RING domains. Heterodimerization is essential for the stability and correct localization of the complex and confers ubiquitin ligase activity to BRCA1. The importance of the ligase activity of BRCA1 to breast cancer development is inferred from the fact that N-terminal diseaseassociated mutations are proposed to reduce ligase activity [4]. Methods Protein–protein interactions were demonstrated using yeast-two-hybrid and coimmunoprecipitation. Protein levels were altered through overexpression, siRNA and antisense technology. The effect of proteasome inhibitors and cycloheximide treatment was also examined. Results We initially identified cyclin D₁ as a binding partner of BARD1 in a yeast-two-hybrid screen and defined the minimal binding region as the N-terminus of BARD1. This interaction was confirmed in vivo by coimmunoprecipitation. The N-terminus of BARD1 also binds BRCA1 and imparts ubiquitin ligase activity to the complex. Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Traditionally polyubiquitin chains linked through lysine 48 target proteins for degradation by the 26 S proteasome. We have demonstrated that cyclin D₁ protein levels are inversely related to BRCA1 and BARD1 levels in several model systems. Furthermore, regulation of cyclin D1 levels occurs through a post-transcriptional mechanism and requires the ligase activity of BRCA1. Interestingly, this phenomenon is cell-cycle regulated, occurring in G₂/M. Conclusion We propose that cyclin D₁ is a potential substrate for BRCA1 ubiquitination and that this targets cyclin D₁ for proteasomal-mediated degradation. Future work will focus on ascertaining the functional consequence of cyclin D₁ regulation by the BRCA1–BARD1 complex; in particular, the impact of BRCA1, mediated through regulation of cyclin D₁, on the proliferation versus differentiation decision.