Retinoblastoma-related Gene Research Articles

The Retinoblastoma-related gene RBL901 can trigger drought response actions in potato

Key MessageThis study provided new insights into response of potato to drought stress.

Higher CO2 Assimilation in Selected Rice Recombinant Inbred Lines Is Driven by Higher CO2 Diffusion and Light Use Efficiency Related to Leaf Anatomy and Mesophyll Cell Density.

Leaf anatomy determining the light distribution within the leaf and exerting influence on CO2 diffusion is considered to have dramatic potential for photosynthesis performance increase. In this study, we observed that two rice recombinant inbred lines, H138 and H217 (RILF11 plants from Sasanishiki × IRAT10), have higher net CO2 assimilation (An) than their parent Sasanishiki due mainly to the improvement of leaf anatomy. Our results showed that An positively correlated with anatomy traits’ mesophyll cell number per cross-sectional area (NO.mescell/Acros) and mesophyll area (Ames). NO.mescell/Acros exert direct and indirect effects on An. Compared to Sasanishiki flag leaves, IRAT10, H138, and H217 have higher mesophyll cell numbers. Simultaneously, higher chlorophyll content and expression of genes encoding the light-harvesting protein of PSII and PSI (Lhcb1, 2, 3 and Lhca1, 2, 3) were recorded in IRAT10, H138, and H217, which facilitates light use efficiency. Higher electron transport rate and RuBP concentration were recorded in IRAT10, H138, and H217 flag leaves. Retinoblastoma-related gene (OsRBR1), exerting effects on mesophyll cell density, can be used to modify leaf anatomy for improving leaf photosynthesis. Additionally, higher stomatal conductance and mesophyll conductance were also recorded in H138 and H217 than in Sasanishiki. Furthermore, we modeled mesophyll conductance through anatomical traits, and the results revealed that chloroplast thickness was the dominant factor restricting CO2 diffusion within mesophyll cells rather than cell wall thickness. Higher RuBP content accompanied by higher CO2 concentration within the carboxylation set in H138 and H217 flag leaves contributed to higher CO2 assimilation.

Retinoblastoma Related Gene RB2/P130 Are Rarely Mutated in Burkitt's Lymphoma from Brazil.

Human RB2/p130 is a member of the retinoblastoma (Rb) family which also includes the retinoblastoma tumor suppressor gene RB/p105 (RB1) and the retinoblastoma related gene, p107. RB1 and other members of Rb family, p107 and p130 perform overlapping, but not fully redundant functions in cell cycle regulation and cell growth inhibition. Moreover, all three Rb family members display suppressive activities, which result in cell cycle G1 phase blocking. It has been suggested that alterations in genes controlling cell cycle probably contribute to Burkitt's lymphoma pathogenesis, in association to the 8:14 translocation that leads to c-myc protein up regulation. Mutations in tumor suppressor gene RB2/p130 were detected in cell lines and primary tumors of patients with endemic Burkitt's lymphoma (eBL). In addition, RB2/p130 mutational inactivation has been reported as a common feature in lung and nasopharyngeal carcinomas. RB2/p130 gene mutation screening was performed in 26 BL tumor pediatric samples and in mononuclear cells from 2 healthy blood donors by single strand conformation polymorphism (PCR-SSCP) analysis. Exons 19, 20, 21 and 22 were amplified separately and denatured PCR products were heated at 95°C for 5 min, chilled on ice, and immediately electrophoresed in a native 10% polyacrilamide gel, with or without the addition of glycerol at 80 volts for 20–26 hours, and silver stained. In this series, 26 children were diagnosed as BL according to histopathologic and clinical criteria. The median age was 4.5 years (range: 2 to 9 years). There were 7 females and 19 males. According to the St Jude system 19% of patients had stage I/II and 81% stage III/IV disease. Burkitt's lymphoma HIV-associated was diagnosed only in 2 patients. All four exons (19–22) were analyzed by PCR-SSCP. No changes were found upon SSCP analysis. Our results suggest that mutations in exons 19–22 of the RB2/p130 are unlikely to be involved directly in the pathogenesis of Brazilian BL. In conclusion, the characterization of tumor cells continues to emphasize the important role of Rb gene in tumor suppression but the evidence linking p107 or p130 inactivation to tumor development remains limited and controversial in Burkitt's lymphomas and other malignant tumors.

Low Temperature Affects Stem Cell Maintenance in Brassica oleracea Seedlings.

Most of the above ground tissues in higher plants originate from stem cells located in the shoot apical meristem (SAM). Several plant species can suffer from spontaneous stem cell arrest resulting in lack of further shoot development. In Brassica oleracea this SAM arrest is known as blindness and occurs in an unpredictable manner leading to considerable economic losses for plant raisers and farmers. Detailed analyses of seedlings showed that stem cell arrest is triggered by low temperatures during germination. To induce this arrest reproducibly and to study the effect of the environment, an assay was developed. The role of genetic variation on the susceptibility to develop blind seedlings was analyzed by a quantitative genetic mapping approach, using seeds from a double haploid population from a cross between broccoli and Chinese kale, produced at three locations. The analysis revealed, besides an effect of the seed production location, a region on linkage group C3 associated with blindness sensitivity. A subsequent dynamic genome-wide transcriptome analysis resulted in the identification of around 3000 differentially expressed genes early after blindness induction. A large number of cell cycle genes were en masse induced early during the development of blindness, whereas shortly after, all were down-regulated. This miss-regulation of core cell cycle genes is accompanied with a strong reduction of cells reaching the DNA replication phase. From the differentially expressed genes, 90 were located in the QTL region C3. Among them are two genes belonging to the MINICHROMOSOMAL MAINTENANCE gene family, known to be involved in DNA replication, a RETINOBLASTOMA-RELATED gene, a key regulator for cell cycle initiation, and several MutS homologs genes, involved in DNA repair. These genes are potential candidates for being involved in the development of blindness in Brassica oleracea sensitive genotypes.

Characterization and Expression Analysis of a Retinoblastoma-Related Gene from Chinese Wild Vitis pseudoreticulata

Retinoblastoma-related (RBR) genes, a conserved gene family in higher eukaryotes, play important roles in cell differentiation, development, and mammalian cell death; however, little is known of their function in plants. In this study, a RBR gene was isolated from the Chinese wild grape, Vitis pseudoreticulata W. T. Wang clone “Baihe-35-1”, and designated as VpRBR. The cDNA sequence of VpRBR was 3,030 bp and contained an open reading frame of 3,024 bp. Conceptual translation of this gene indicated a composition of 1,007 amino acids with a predicted molecular mass of 117.3 kDa. The predicted protein showed a retinoblastoma-associated protein domain A from amino acid residues 416 to 579, and domain B from residues 726 to 855. The result of expression analysis indicated that VpRBR was expressed in tissues, leaves, stem, tendrils, flower, and grape skin at different expression levels. Further quantitative reverse transcription-PCR (qRT-PCR) data indicated that VpRBR levels were higher in Erysiphe necator-treated “Baihe-35-1” and “Baihe-13-1”, two resistant clones of Chinese wild V. pseudoreticulata, than in E. necator-treated “Hunan-1”, a susceptible clone of V. pseudoreticulata. Furthermore, the expression of VpRBR in response to salicylic acid (SA), methyl jasmonate (MeJA), and ethylene (Eth) in grape leaves was also investigated. Taken together, these data indicate that VpRBR may contribute to some aspect of powdery mildew resistance in grape.

Influence of retinoblastoma-related gene silencing on the initiation of DNA replication by African cassava mosaic virus Rep in cells of mature leaves in Nicotiana benthamiana plants

BackgroundGeminiviruses mainly infect terminally differentiated tissues and cells in plants. They need to reprogramme host cellular machinery for DNA replication. This process is thought to be mediated by inactivation of cell-cycle repressor proteins and by induction of host DNA synthesis protein expression through actions of the geminviral replication initiator protein (Rep).FindingsExploiting a Nicotiana benthamiana pOri2 line, which is transformed with a transgene consisting of a direct repeat of the African cassava mosaic virus (ACMV)-replication origin (Ori) flanking a non-viral DNA region, and virus-induced RNA silencing (VIGS), the impact of host gene expression on replication of the ACMV-derived replicon was investigated. The ACMV Rep trans-replicated the viral episomal replicon in leaves of young but not older pOri2 plants. Upon VIGS-mediated down-regulation of N. benthamiana NbRBR1, the retinoblastoma-related protein gene coding for a negative cell-cycle suppressor, recovered the ability of ACMV Rep for trans DNA replication, whereas the silencing of NbPCNA coding for the sliding clamp of DNA polymerase had no effect.ConclusionsThese results suggest that the cellular machinery for DNA replication in differentiated tissues of older leaves cannot be reprogrammed by Rep alone but may need other uncharacterised viral and plant factors.

Expression of the Retinoblastoma-related Gene Rb2/p130 in the Pathogenesis of Serous Carcinoma of the Ovary

The purpose of this study was to evaluate the immunohistochemical Rb2/p130 expression in a series of benign, borderline, and malignant ovarian tumors. Seventy-six operative specimens were analyzed. Frequencies and percents were calculated for variables by each of the 5 disease stage groups in increasing severity and again for the disease stage groups collapsed into benign (serous cystadenoma or endosalpingosis), low-grade [serous borderline tumors (SBT) or low-grade serous carcinoma (LGSC)] and high-grade serous carcinoma. There was no loss of expression in benign serous cystadenomas (0/18). Ten percent of SBT's (2/20), 47% of LGSC's (9/19), and 16% of HGSC's (3/16) had loss of expression. There was a significant difference in the expression during the progression from cystadenoma to SBT to LGSC. Loss of Rb2/p130 expression is a rare event in benign cystadenoma. The progression to LGSC is associated with a decrease in gene expression.

C4 protein of Beet severe curly top virus is a pathomorphogenetic factor in Arabidopsis

The Curtovirus C4 protein is required for symptom development during infection of Arabidopsis. Transgenic Arabidopsis plants expressing C4 from either Beet curly top virus or Beet severe curly top virus produced phenotypes that were similar to symptoms seen during infection with wild-type viruses. The pseudosymptoms caused by C4 protein alone were novel to transgenic Arabidopsis and included bumpy trichomes, severe enations, disorientation of vascular bundles and stomata, swelling, callus-like structure formation, and twisted siliques. C4 induced abnormal cell division and altered cell fate in a variety of tissues depending on the C4 expression level. C4 protein expression increased the expression levels of cell-cycle-related genes CYCs, CDKs and PCNA, and suppressed ICK1 and the retinoblastoma-related gene RBR1, resulting in activation of host cell division. These results suggest that the Curtovirus C4 proteins are involved actively in host cell-cycle regulation to recruit host factors for virus replication and symptom development.

Positive regulation of minichromosome maintenance gene expression, DNA replication, and cell transformation by a plant retinoblastoma gene

Retinoblastoma-related (RBR) genes inhibit the cell cycle primarily by repressing adenovirus E2 promoter binding factor (E2F) transcription factors, which drive the expression of numerous genes required for DNA synthesis and cell cycle progression. The RBR-E2F pathway is conserved in plants, but cereals such as maize are characterized by having a complex RBR gene family with at least 2 functionally distinct members, RBR1 and RBR3. Although RBR1 has a clear cell cycle inhibitory function, it is not known whether RBR3 has a positive or negative role. By uncoupling RBR3 from the negative regulation of RBR1 in cultured maize embryos through a combination of approaches, we demonstrate that RBR3 has a positive and critical role in the expression of E2F targets required for the initiation of DNA synthesis, DNA replication, and the efficiency with which transformed plants can be obtained. Titration of endogenous RBR3 activity through expression of a dominant-negative allele with a compromised pocket domain suggests that these RBR3 functions require an activity distinct from its pocket domain. Our results indicate a cell cycle pathway in maize, in which 2 RBR genes have specific and opposing functions. Thus, the paradigm that RBR genes are negative cell cycle regulators cannot be considered universal.

Cloning and molecular characterisation of the maize retinoblastoma gene ( ZmRBR2)

Retinoblastoma-related (RBR) proteins have been described in plant cells, and have been shown to be important in controlling plant growth, gametophyte and leaf development, and maintenance of stem cell identity. Whereas all dicotyledonous plants studied so far contain a single RBR gene, monocotyledonous species contain at least two genes and maize is the only known plant species with three RBR genes in its genome suggesting a recent gene duplication event. We report the genomic structure of the ZmRBR2 gene and its expression pattern. The ZmRBR2 transcript contained a 283-bp 5′-UTR and a putative canonical TATA box. The gene had a low GC content and showed conserved and unique features with other plant RBR genes. ZmRBR2 gene promoter-GUS fusions, introduced in Arabidopsis thaliana plants, drove high GUS activity in cotyledons, the shoot apical meristem and in flowering tissue, especially in anthers and pollen. ZmRBR2 transcript levels were consistently 50% lower in several tissues than those of ZmRBR1, suggesting a differential regulation of these two genes. Our results are consistent with the ubiquitous tissue distribution and function of plant RBR proteins and emphasize differences between the monocot and dicot RBR pathways.

Mutation detection using ENDO1: application to disease diagnostics in humans and TILLING and Eco-TILLING in plants.

BackgroundMost enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA by a mismatch-specific endonuclease at mismatch sites and the analysis of the digestion product on a DNA sequencer. Important limitations of these methods are the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in pool of DNA, the cost of the analysis and the ease by which the technique could be implemented in a standard molecular biology laboratory.ResultsThe co-agroinfiltration of ENDO1 and p19 constructs into N. benthamiana leaves allowed high level of transient expression of a mismatch-specific and sensitive endonuclease, ENDO1 from Arabidopsis thaliana. We demonstrate the broad range of uses of the produced enzyme in detection of mutations. In human, we report the diagnosis of the G1691A mutation in Leiden factor-V gene associated with venous thrombosis and the fingerprinting of HIV-1 quasispecies in patients subjected to antiretroviral treatments. In plants, we report the use of ENDO1 system for detection of mutant alleles of Retinoblastoma-related gene by TILLING in Pisum sativum and discovery of natural sequence variations by Eco-TILLING in Arabidopsis thaliana.ConclusionWe introduce a cost-effective tool based on a simplified purification protocol of a mismatch-specific and sensitive endonuclease, ENDO1. Especially, we report the successful applications of ENDO1 in mutation diagnostics in humans, fingerprinting of complex population of viruses, and in TILLING and Eco-TILLING in plants.

Geminivirus-induced gene silencing of the tobacco retinoblastoma-related gene results in cell death and altered development

The retinoblastoma-related protein (RBR) is required for cell cycle control and differentiation and is expressed throughout the life of plants and animals. In this study, the tomato golden mosaic virus (TGMV) geminivirus vector was used to silence NbRBR1 in Nicotiana benthamiana by microprojectile bombardment into fully developed leaves. Similar to previous results using agroinoculation of a tobacco rattle virus silencing vector [Park et al. (Plant J 42:153, 2005)], developmental defects caused by disruptions in cell size and number were seen in new growth. Leaf midvein cross-sections showed tissue-specific differences in size, cell number, and cell morphology. While cortical cell numbers decreased, size increased to maintain overall shape. In contrast, xylem parenchyma cells increased approximately three fold but remained small. Normally straight flowers often curved up to 360 degrees without a significant change in size. However, the most striking phenotype was cell death in mature cells after a delay of 3-4 weeks. Trypan blue staining confirmed cell death and demonstrated that cell death was absent in similarly treated leaves of wild type TGMV-inoculated plants. Quantitative RT-PCR confirmed that the mature TGMV:RBR-inoculated leaves still maintained reduced accumulation of RBR transcript at 4 weeks compared to controls. The results suggest that either inappropriate activation of the cell cycle is lethal in plants or that RBR has other functions, unrelated to the cell cycle. The results also demonstrate that continual transcription of RBR is necessary for cell survival.

Retinoblastoma‐related geneRb2/p130 are rarely mutated in Burkitt's lymphoma from Brazil

It has been suggested that alterations of cell cycle genes probably contribute to the pathogenesis of endemic Burkitt's lymphoma (BL) in addition to c-MYC translocation. Mutations disrupting the normal nuclear localization signal of the retinoblastoma-related gene Rb2/p130 have been documented in BL cell lines and primary tumors from endemic areas. The aim of this study was to investigate the involvement of Rb2/p130 gene in the pathogenesis of sporadic BL in Brazil. DNA samples from 26 pediatric BL tumors and two healthy blood donors were screened by PCR amplification followed by single strand conformation polymorphism analysis of exons 19 and 20 (B domain) and exons 21 and 22 (C-terminus), where most of the point mutations in the Rb2/p130 gene were identified. No abnormal band shifts were present in the samples analyzed. We concluded that mutations in exons 19-22 of the Rb2/p130 are unlikely to be involved directly in the pathogenesis of sporadic Brazilian BL.

Cell type-specific role of the retinoblastoma/E2F pathway during Arabidopsis leaf development.

Organogenesis in plants is almost entirely a postembryonic process. This unique feature implies a strict coupling of cell proliferation and differentiation, including cell division, arrest, cell cycle reactivation, endoreplication, and differentiation. The plant retinoblastoma-related (RBR) protein modulates the activity of E2F transcription factors to restrict cell proliferation. Arabidopsis contains a single RBR gene, and its loss of function precludes gamete formation and early development. To determine the relevance of the RBR/E2F pathway during organogenesis, outside its involvement in cell division, we have used an inducible system to inactivate RBR function and release E2F activity. Here, we have focused on leaves where cell proliferation and differentiation are temporally and developmentally regulated. Our results reveal that RBR restricts cell division early during leaf development when cell proliferation predominates, while it regulates endocycle occurrence at later stages. Moreover, shortly after leaving the cell cycle, most of leaf epidermal pavement cells retain the ability to reenter the cell cycle and proliferate, but maintain epidermal cell fate. On the contrary, mesophyll cells in the inner layers do not respond in this way to RBR loss of activity. We conclude that there exists a distinct response of different cells to RBR inactivation in terms of maintaining the balance between cell division and endoreplication during Arabidopsis (Arabidopsis thaliana) leaf development.

The RETINOBLASTOMA-RELATED Gene Regulates Stem Cell Maintenance in Arabidopsis Roots

The maintenance of stem cells in defined locations is crucial for all multicellular organisms. Although intrinsic factors and signals for stem cell fate have been identified in several species, it has remained unclear how these connect to the ability to reenter the cell cycle that is one of the defining properties of stem cells. We show that local reduction of expression of the RETINOBLASTOMA-RELATED (RBR) gene in Arabidopsis roots increases the amount of stem cells without affecting cell cycle duration in mitotically active cells. Conversely, induced RBR overexpression dissipates stem cells prior to arresting other mitotic cells. Overexpression of D cyclins, KIP-related proteins, and E2F factors also affects root stem cell pool size, and genetic interactions suggest that these factors function in a canonical RBR pathway to regulate somatic stem cells. Expression analysis and genetic interactions position RBR-mediated regulation of the stem cell state downstream of the patterning gene SCARECROW.

Mutations in the Retinoblastoma-related Gene RB2 / p130 in Adult T-cell Leukaemia/Lymphoma

The retinoblastoma (Rb) family consists of the tumor suppressor Rb/p105 and related proteins p107 and Rb2/p130. Although the involvement of the RB / p105 gene in Adult T-cell leukaemia/lymphoma (ATL) has been studied, no mutational data is reported regarding the RB2 / p130 gene in ATL. We screened for mutations of the RB2 / p130 gene. Mutation was detected in 1 of 41 primary ATL sample. This is the first report describing mutation of the RB2 / p130 gene in ATL, suggesting that RB2/p130 may be involved in the development of ATL, and may behave as a tumor suppressor gene in T lymphocytes.

P130 expression in thyroid neoplasms: its linkage with tumor size and dedifferentiation

p130 belongs to the retinoblastoma-related gene family, and its gene product works to negatively regulate cell cycle progression in the G1 phase. In this study, we investigated p130 expression in thyroid neoplasms. p130 overexpression was observed in 33.3% of follicular adenoma and 50% of follicular carcinoma and the incidences were not significantly different. In papillary carcinoma, it was overexpressed in 36.5% of cases, but in microcarcinoma, the incidence was significantly lower (14.3%). Furthermore, in anapalstic carcinoma, p130 overexpression was less frequently seen than in papillary carcinoma except for microcarcinoma and follicular carcinoma, and only 14.3% of cases overexpressed this protein. These findings suggest that: (1) reduced p130 expression may contribute to the aggressive character of anplastic carcinoma; and (2) p130 may specify the growth characteristics of microcarcinoma.

Alterations of the retinoblastoma-related gene RB2/p130 in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine in rats.

Alteration of the retinoblastoma-related gene RB2/p130 was investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in male Wistar rats. At 6 wk of age, 21 animals were given 2000 ppm of BHP in their drinking water for 12 wk and then maintained without further treatment until they were killed at the end of week 25. A total of 21 lung adenocarcinomas were obtained, and total RNAs were extracted from each for mutation analysis of RB2/p130 by the reverse transcription-polymerase chain reaction-single-strand comformation polymorphism approach. No mutations were found in exons 19-22. However, examination of the expression of the RB2/p130 gene by Northern blot analysis showed mRNA levels to be significantly lower than those of normal lung tissues. Western blot analysis showed reduction of the pRb2/p130 protein in all of the adenocarcinomas examined. These results suggest that alteration of the RB2/p130 gene may play important roles in the development of lung adenocarcinomas induced by BHP in rats.

Retinoblastoma-related gene RB2/p130 exons 19-22 are rarely mutated in glioblastomas

The retinoblastoma gene family RB1, p107 and RB2/p130 cooperate to regulate cell cycle progression through the G1 phase of the cell cycle. Previous data demonstrated that RB2/p130 inhibits proliferation of the glioblastoma cell line T98G, which is resistant to the growth suppressive effects of both RB1 and p107, and that RB2/p130 gene overexpresion induces astrocyte differentiation. We screened by single-strand conformation polymorphism and sequence analysis the structure of exons 19 through 22 of the RB2/p130 gene, which encodes the B domain and C terminus, in a series of 42 glioblastomas (32 primary and 10 secondary). Sequence variations were identified in one tumor, suggesting that mutation inactivation of RB2/p130 is a rare event in glioblastoma.

Stimulation of the cell cycle and maize transformation by disruption of the plant retinoblastoma pathway.

The genome of the Mastreviruses encodes a replication-associated protein (RepA) that interacts with members of the plant retinoblastoma-related protein family, which are putative cell cycle regulators. Expression of ZmRb1, a maize retinoblastoma-related gene, and RepA inhibited and stimulated, respectively, cell division in tobacco cell cultures. The effect of RepA was mitigated by over-expression of ZmRb1. RepA increased transformation frequency and callus growth rate of high type II maize germplasm. RepA-containing transgenic maize calli remained embryogenic, were readily regenerable, and produced fertile plants that transmitted transgene expression in a Mendelian fashion. In high type II, transformation frequency increased with the strength of the promoter driving RepA expression. When a construct in which RepA was expressed behind its native LIR promoter was used, primary transformation frequencies did not improve for two elite Pioneer maize inbreds. However, when LIR:RepA-containing transgenic embryos were used in subsequent rounds of transformation, frequencies were higher in the RepA+ embryos. These data demonstrate that RepA can stimulate cell division and callus growth in culture, and improve maize transformation.