Expanding the Benefits of Tnt1 for the Identification of Dominant Mutations in Polyploid Crops: A Single Allelic Mutation in the MsNAC39 Gene Produces Multifoliated Alfalfa

Most major crops are polyploid species and the production of genetically engineered cultivars normally requires the introgression of transgenic or gene-edited traits into elite germplasm. Thus, a main goal of plant research is the search of systems to identify dominant mutations. In this article, we show that the Tnt1 element can be used to identify dominant mutations in allogamous tetraploid cultivated alfalfa. Specifically, we show that a single allelic mutation in the MsNAC39 gene produces multifoliate leaves (mfl) alfalfa plants, a pivot trait of breeding programs of this forage species. Finally, we discuss the potential application of a combination of preliminary screening of beneficial dominant mutants using Tnt1 mutant libraries and genome editing via the CRISPR/Cas9 system to identify target genes and to rapidly improve both autogamous and allogamous polyploid crops.

Download Full-text

3131 ONCOSTREAMS: NOVEL DYNAMICS PATHOLOGICAL MULTICELLULAR STRUCTURES INVOLVED IN GLIOBLATOMA GROWTH AND INVASION

Journal of Clinical and Translational Science ◽

10.1017/cts.2019.253 ◽

2019 ◽

Vol 3 (s1) ◽

pp. 111-111 ◽

Cited By ~ 2

Author(s):

Andrea Comba ◽

Patrick Dunn ◽

Anna E Argento ◽

Padma Kadiyala ◽

Sebastien Motsch ◽

...

Keyword(s):

Extracellular Matrix ◽

Target Genes ◽

Collective Motion ◽

Malignant Tumors ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Genetically Engineered ◽

Functional Enrichment ◽

Low Grade ◽

Normal Brain

OBJECTIVES/SPECIFIC AIMS: Oncostreams represent a novel growth pattern of GBM. In this study we uncovered the cellular and molecular mechanism that regulates the oncostreams function in GBM growth and invasion. METHODS/STUDY POPULATION: We studied oncostreams organization and function using genetically engineered mouse gliomas models (GEMM), mouse primary patient derived GBM model and human glioma biopsies. We evaluated the molecular landscape of oncostreams by laser capture microdissection (LCM) followed by RNA-Sequencing and bioinformatics analysis. RESULTS/ANTICIPATED RESULTS: Oncostreams are multicellular structures of 10-20 cells wide and 2-400 μm long. They are distributed throughout the tumors in mouse and human GBM. Oncostreams are heterogeneous structures positive for GFAP, Nestin, Olig2 and Iba1 cells and negative for Neurofilament. Using GEMM we found a negative correlation between oncostream density and animal survival. Moreover, examination of patient’s glioma biopsies evidenced that oncostreams are present in high grade but no in low grade gliomas. This suggests that oncostreams may play a role in tumor malignancy. Our data also indicated that oncostreams aid local invasion of normal brain. Transcriptome analysis of oncostreams revealed 43 differentially expressed (DE) genes. Functional enrichment analysis of DE genes showed that “collagen catabolic processes”, “positive regulation of cell migration”, and “extracellular matrix organization” were the most over-represented GO biological process. Network analysis indicated that Col1a1, ACTA2, MMP9 and MMP10 are primary target genes. These genes were also overexpressed in more malignant tumors (WT-IDH) compared to the less malignant (IDH1- R132H) tumors. Confocal time lapse imagining of 3D tumor slices demonstrated that oncostreams display a collective motion pattern within gliomas that has not been seen before. DISCUSSION/SIGNIFICANCE OF IMPACT: In summary, oncostreams are anatomically and molecularly distinctive, regulate glioma growth and invasion, display collective motion and are regulated by the extracellular matrix. We propose oncostreams as novel pathological markers valuable for diagnosis, prognosis and designing therapeutics for GBM patients.

Download Full-text

A set of microRNAs coordinately controls tumorigenesis, invasion, and metastasis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913307116 ◽

2019 ◽

Vol 116 (48) ◽

pp. 24184-24195 ◽

Cited By ~ 6

Author(s):

Iacovos P. Michael ◽

Sadegh Saghafinia ◽

Douglas Hanahan

Keyword(s):

Tumor Growth ◽

Target Genes ◽

Genetically Engineered ◽

Malignant Progression ◽

Metastasis Suppressor ◽

Invasion And Metastasis ◽

Biologically Relevant ◽

Genetically Engineered Mouse Model ◽

Functional Involvement

MicroRNA-mediated gene regulation has been implicated in various diseases, including cancer. This study examined the role of microRNAs (miRNAs) during tumorigenesis and malignant progression of pancreatic neuroendocrine tumors (PanNETs) in a genetically engineered mouse model. Previously, a set of miRNAs was observed to be specifically up-regulated in a highly invasive and metastatic subtype of mouse and human PanNET. Using functional assays, we now implicate different miRNAs in distinct phenotypes: miR-137 stimulates tumor growth and local invasion, whereas the miR-23b cluster enables metastasis. An algorithm, Bio-miRTa, has been developed to facilitate the identification of biologically relevant miRNA target genes and applied to these miRNAs. We show that a top-ranked miR-137 candidate gene, Sorl1, has a tumor suppressor function in primary PanNETs. Among the top targets for the miR-23b cluster, Acvr1c/ALK7 has recently been described to be a metastasis suppressor, and we establish herein that it is down-regulated by the miR-23b cluster, which is crucial for its prometastatic activity. Two other miR-23b targets, Robo2 and P2ry1, also have demonstrable antimetastatic effects. Finally, we have used the Bio-miRTa algorithm in reverse to identify candidate miRNAs that might regulate activin B, the principal ligand for ALK7, identifying thereby a third family of miRNAs—miRNA-130/301—that is congruently up-regulated concomitant with down-regulation of activin B during tumorigenesis, suggestive of functional involvement in evasion of the proapoptotic barrier. Thus, dynamic up-regulation of miRNAs during multistep tumorigenesis and malignant progression serves to down-regulate distinctive suppressor mechanisms of tumor growth, invasion, and metastasis.

Download Full-text

Legume biology: the basis for crop improvement

Functional Plant Biology ◽

10.1071/fpv40n12_fo ◽

2013 ◽

Vol 40 (12) ◽

pp. v ◽

Cited By ~ 5

Author(s):

Rajeev K. Varshney ◽

Himabindu Kudapa

Keyword(s):

Target Genes ◽

Crop Improvement ◽

Crop Productivity ◽

Food Sources ◽

Yield Potential ◽

Systematic Research ◽

Breeding Programs ◽

Biotic Stresses ◽

Legume Crop ◽

Precise Understanding

Legumes represent the most valued food sources in agriculture after cereals. Despite the advances made in breeding food legumes, there is a need to develop and further improve legume productivity to meet increasing food demand worldwide. Several biotic and abiotic stresses affect legume crop productivity throughout the world. The study of legume genetics, genomics and biology are all important in order to understand the limitations of yield of legume crops and to support our legume breeding programs. With the advent of huge genomic resources and modern technologies, legume research can be directed towards precise understanding of the target genes responsible for controlling important traits for yield potential, and for resistance to abiotic and biotic stresses. Programmed and systematic research will lead to developing high yielding, stress tolerant and early maturing varieties. This issue of Functional Plant Biology is dedicated to ‘Legume Biology’ research covering part of the work presented at VI International Conference on Legume Genetics and Genomics held at Hyderabad, India, in 2012. The 13 contributions cover recent advances in legume research in the context of plant architecture and trait mapping, functional genomics, biotic stress and abiotic stress.

Download Full-text

White-box Deep Neural Network Prediction of Genome-Wide Transcriptome Signatures

10.1101/2021.02.11.430730 ◽

2021 ◽

Author(s):

Rasmus Magnusson ◽

Jesper N. Tegnér ◽

Mika Gustafsson

Keyword(s):

Neural Network ◽

Target Genes ◽

Hierarchical Organization ◽

Rapid Decline ◽

Sequence Information ◽

Neural Network Prediction ◽

Gene Structures ◽

Or Gene ◽

Hidden Layer ◽

Tf Gene

AbstractPrediction algorithms for protein or gene structures, including transcription factor binding from sequence information, have been transformative in understanding gene regulation. Here we ask whether human transcriptomic profiles can be predicted solely from the expression of transcription factors (TFs). To this end, we explore whether a neural network (NN) could predict the transcriptome from TFs. Using at least one hidden layer, we find that the expression of 1,600 TFs can explain >95% of variance in 25,000 genes. Using the light-up technique to inspect the trained NN, we find an overrepresentation of known TF-gene regulations. Furthermore, the learned prediction network has a hierarchical organization. A smaller set of around 125 core TFs could explain close to 80% of the variance. Interestingly, reducing the number of TFs below 500 induces a rapid decline in prediction performance. Next, we evaluated the prediction model using transcriptional data from 22 human diseases. The TFs were sufficient to predict the target genes’ dysregulation (rho=0.61, P < 10−216). By inspecting the model, key causative TFs could be extracted for subsequent validation using disease-associated genetic variants. In conclusion, we demonstrate the construction of an interpretable neural network predictor. Analysis of the predictors revealed key TFs that were inducing transcriptional changes during disease.

Download Full-text

Creating Biotechnological Hybrids of Sugar Beet

International Journal of Engineering and Advanced Technology - Regular Issue ◽

10.35940/ijeat.f9103.088619 ◽

2019 ◽

Vol 8 (6) ◽

pp. 5167-5175 ◽

Cited By ~ 1

Keyword(s):

Gene Expression ◽

Sugar Beet ◽

Target Genes ◽

Mendelian Segregation ◽

Breeding Programs ◽

Its Gene ◽

Pair Mating ◽

The Stability ◽

High Level

Currently, implementation of the breeding programs, including the commonly recognized areas and classic breeding methods, cannot sufficiently ensure a quick and significant increase in the productivity of sugar beet hybrids, since its gene pool is almost exhausted. Based on the achievements in the field of genetics, new approaches to and opportunities in creating highly productive agrocoenoses of sugar beet have become popular. As a result of many years of work, results have been obtained about the nature of inheriting the resistance to glyphosate in individual heterozygous apo- and syncarpous forms in case of inbreeding and pair mating with the MC tester. The expression of target genes in the generations was monitored by the survival rate of sugar beet plants after the treatment with glyphosate. During the research, individuals with a high level of gene expression were selected. Upon self-pollination of initial heterozygous original forms, deviations from Mendelian segregation were observed in most cases. The criterion for assessing the stability of expression of glyphosate resistance genes in case of seed breeding was the compliance with the laws of Mendel among the analyzed descendants. In the initial stages of the research, the level of stability gene expression had been 10 – 15 % of the total number of analyzed plants. After four self-pollinations, the stability gene expression significantly increased, and genotypes with the resistance of 91 – 100 % were selected. The first apo- and syncarpous self-pollinating lines of sugar beet with high tolerance in the role of resistance donors have been created. The positive results of preliminary tests of the first glyphosate-tolerant hybrids need confirmation. Seeds and roots of resistant forms have been obtained for further research.

Download Full-text

Targeted Delivery of Gene Silencing in Fungi Using Genetically Engineered Bacteria

Journal of Fungi ◽

10.3390/jof7020125 ◽

2021 ◽

Vol 7 (2) ◽

pp. 125

Author(s):

Jonatan Niño-Sánchez ◽

Li-Hung Chen ◽

Jorge Teodoro De Souza ◽

Sandra Mosquera ◽

Ioannis Stergiopoulos

Keyword(s):

Targeted Delivery ◽

Pathogenic Bacteria ◽

Target Genes ◽

Genetically Engineered ◽

Biologically Active ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Whole Cell ◽

Thaw Cycle ◽

Living Bacteria

Exploiting RNA interference (RNAi) in disease control through non-transformative methods that overcome the hurdle of producing transgenic plants has attracted much attention over the last years. Here, we explored such a method and used non-pathogenic bacteria as a versatile system for delivering RNAi to fungi. Specifically, the RNaseIII-null mutant strain of Escherichia coli HT115(DE3) was transformed with two plasmid vectors that enabled the constitutive or IPTG-inducible production of double-stranded RNAs (dsRNAs) against genes involved in aflatoxins production in Aspergillus flavus (AflC) or virulence of Botrytis cinerea (BcSAS1). To facilitate the release of the dsRNAs, the bacterial cells were further genetically engineered to undergo a bacteriophage endolysin R-mediated autolysis, following a freeze-thaw cycle. Exposure under in vitro conditions of A. flavus or B. cinerea to living bacteria or their whole-cell autolysates induced silencing of AflC and BcSAS1 in a bacteria concentration-dependent manner, and instigated a reduction in aflatoxins production and mycelial growth, respectively. In planta applications of the living bacteria or their crude whole-cell autolysates produced similar results, thus creating a basis for translational research. These results demonstrate that bacteria can produce biologically active dsRNA against target genes in fungi and that bacteria-mediated RNAi can be used to control fungal pathogens.

Download Full-text

Genetic divergence among accessions of Manihot spp.

Acta Scientiarum Agronomy ◽

10.4025/actasciagron.v42i1.44076 ◽

2020 ◽

Vol 42 ◽

pp. e44076

Author(s):

Ana Paula Gomes da Silva ◽

Divan Soares da Silva ◽

Mailson Monteiro do Rego ◽

Albericio Pereira de Andrade ◽

Fleming Sena Campos ◽

...

Keyword(s):

Genetic Diversity ◽

Morphological Characters ◽

Semiarid Region ◽

Dissimilarity Matrix ◽

Breeding Programs ◽

Morphometric Characters ◽

Relative Contribution ◽

Brazilian Semiarid ◽

Forage Species ◽

Generalized Distance

The aim of this study was to perform a morphological and morphoagronomic analysis of wild Manihot species from a Brazilian semiarid region for inclusion in the collection at the Center for Agricultural Sciences, Federal University of Paraíba. To characterize the 55 accessions, 12 quantitative and 18 qualitative descriptors were used. A dissimilarity matrix was generated by Mahalanobis generalized distance (D²), and clusters were identified by the UPGMA method. It was possible to verify the formation of 8 dissimilar groups based on morphological characters and 5 groups based on morphometric characters, indicating the presence of genetic diversity among accessions. The evaluated morphometric variable with the greatest relative contribution was the length between the central lobe. Based on the dissimilarity matrix, the accessions 16 x 48 were the most genetically distant accessions, followed by 47 x 49. The accessions 4 Monteiro, 16 Soledad, 38 Boa Vista, 3 Pedra Lavrada, 7 Junco, 10 Barra de Santa Rosa, 21 Monteiro, and 39 Junco are the most promising and can be used as parents in breeding programs for this forage species.

Download Full-text

Dissection of the genetic programs of p53-mediated G1 growth arrest and apoptosis: blocking p53-induced apoptosis unmasks G1 arrest

Blood ◽

10.1182/blood.v85.10.2691.bloodjournal85102691 ◽

1995 ◽

Vol 85 (10) ◽

pp. 2691-2698 ◽

Cited By ~ 62

Author(s):

C Guillouf ◽

X Grana ◽

M Selvakumaran ◽

A De Luca ◽

A Giordano ◽

...

Keyword(s):

Cell Cycle ◽

Target Genes ◽

Ectopic Expression ◽

Growth Arrest ◽

Genetically Engineered ◽

Temperature Sensitive ◽

G1 Arrest ◽

Induced Apoptosis ◽

High Level ◽

And Function

Employing the myeloblastic leukemia M1 cell line, which does not express endogenous p53, and genetically engineered variants, it was recently shown that activation of p53, using a p53 temperature-sensitive mutant transgene (p53ts), resulted in rapid apoptosis that was delayed by high level ectopic expression of bcl-2. In this report, advantage has been taken of these M1 variants to investigate the relationship between p53-mediated G1 arrest and apoptosis. Flow cytometric cell cycle analysis has provided evidence that activation of wild-type (wt) p53 function in M1 cells resulted in the induction of G1 growth arrest; this was clearly seen in the M1p53/bcl-2 cells because of the delay in apoptosis that unmasked p53-induced G1 growth arrest. This finding was further corroborated at the molecular level by analysis of the expression and function of key cell cycle regulatory genes in M1p53 versus M1p53/bcl-2 cells after the activation of wt p53 function; events that take place at early times during the p53-induced G1 arrest occur in both the M1p53 and the M1p53/bcl-2 cells, whereas later events occur only in the M1p53/bcl-2 cells, which undergo delayed apoptosis, thereby allowing the cells to complete G1 arrest. Finally, it was observed that a spectrum of p53 target genes implicated in p53-induced growth suppression and apoptosis were similarly regulated, either induced (gadd45, waf1, mdm2, and bax) or suppressed (c-myc and bcl-2), after activation of wt p53 function in M1p53 and M1p53/bcl-2 cells. Taken together, these findings show that wt p53 can simultaneously induce the genetic programs of both G1 growth arrest and apoptosis within the same cell type, in which the genetic program of cell death can proceed in either G1-arrested (M1p53/bcl-2) or cycling (M1p53) cells. These findings increase our understanding of the functions of p53 as a tumor suppressor and how alterations in these functions could contribute to malignancy.

Download Full-text

EZH2-induced lysine K362 methylation enhances TMPRSS2-ERG oncogenic activity in prostate cancer

Nature Communications ◽

10.1038/s41467-021-24380-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Marita Zoma ◽

Laura Curti ◽

Dheeraj Shinde ◽

Domenico Albino ◽

Abhishek Mitra ◽

...

Keyword(s):

Prostate Cancer ◽

Disease Progression ◽

Target Genes ◽

Genetically Engineered ◽

Post Translational Modification ◽

Pten Loss ◽

Genetically Engineered Mouse Model ◽

Oncogenic Activity ◽

Domain Interactions ◽

Prostate Cancers

AbstractThe TMPRSS2-ERG gene fusion is the most frequent alteration observed in human prostate cancer. However, its role in disease progression is still unclear. In this study, we uncover an important mechanism promoting ERG oncogenic activity. We show that ERG is methylated by Enhancer of zest homolog 2 (EZH2) at a specific lysine residue (K362) located within the internal auto-inhibitory domain. Mechanistically, K362 methylation modifies intra-domain interactions, favors DNA binding and enhances ERG transcriptional activity. In a genetically engineered mouse model of ERG fusion-positive prostate cancer (Pb-Cre4 Ptenflox/floxRosa26-ERG, ERG/PTEN), ERG K362 methylation is associated with PTEN loss and progression to invasive adenocarcinomas. In both ERG positive VCaP cells and ERG/PTEN mice, PTEN loss results in AKT activation and EZH2 phosphorylation at serine 21 that favors ERG methylation. We find that ERG and EZH2 interact and co-occupy several sites in the genome forming trans-activating complexes. Consistently, ERG/EZH2 co-regulated target genes are deregulated preferentially in tumors with concomitant ERG gain and PTEN loss and in castration-resistant prostate cancers. Collectively, these findings identify ERG methylation as a post-translational modification sustaining disease progression in ERG-positive prostate cancers.

Download Full-text

CSIG-20. L3MBTL3 SUPPRESSES MEDULLOBLASTOMA TUMORIGENESIS THROUGH MODULATION OF THE NOTCH/RBPJ SIGNALING PATHWAY

Neuro-Oncology ◽

10.1093/neuonc/noaa215.132 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii32-ii32

Author(s):

Honglai Zhang ◽

Tao Xu ◽

Claire Peabody ◽

Ester Calvo Fernández ◽

Rashmi Budhathoki ◽

...

Keyword(s):

Survival Analysis ◽

Tumor Suppressor ◽

Signaling Pathway ◽

Molecular Mechanism ◽

Cell Fate ◽

Target Genes ◽

Genetically Engineered ◽

Rank Test ◽

Rna Seq

Abstract The NOTCH/RBPJ pathway governs cell proliferation in many biological contexts, including SHH and Group#3medulloblastoma (MB) tumorigenesis. Using our proteomic platform, we discovered an interaction between RBPJ, a key co-factor of NOTCH for the modulation of the NOTCH/RBPJ signaling pathway, and L3MBTL3, a methyllysine reader. L3MBTL3 is recruited by RBPJ on chromatin at the enhancers of NOTCH/RBPJ target genes to repress their expression. Deletions of the L3MBTL3 locus are observed in patients with WNT and Group#3 MB and expression of L3MBTL3 in the SHH MB-derived cell DAOY inhibits cell growth, suggesting a putative tumor suppressor role for L3MBTL3 in MB. To further investigate the putative role of L3MBTL3 as a suppressor of MB tumorigenesis, we used our novel L3mbtl3 KO mouse in combination with a genetically engineered ND2:SmoA1 mouse model of SHH MB in a survival analysis. Furthermore, to identify the biological processes regulated by L3mbtl3 in MB, we analyzed by RNA-seq the transcriptome of L3mbtl3 KO mouse cerebella. Our survival analysis validated in vivo our hypothesis that L3mbtl3 is a tumor suppressor in this disease context. Indeed, our data show that [ND2:SmoA1; L3mbtl3+/-] mice have a significantly lower survival rate than ND2:SmoA1 mice (P = 0.032; Log-rank test). Moreover, our RNA-seq studies showed that L3MBTL3 regulates cell fate in the cerebellum via modulation the NOTCH/RBPJ signaling pathway. Hence, the RBPJ-L3MBTL3 interaction is at the heart of a molecular mechanism governing the repression of NOTCH/RBPJ target genes and malfunction of this molecular mechanism contributes to L3MBTL3’s tumor suppressor role in MB through aberrant “de-repression” of NOTCH/RBPJ target genes. Our discovery provides insights into the tumor suppressor role of the L3MBTL3 in MB that could be harnessed in the future for the therapeutic benefit of patients with MB.

Download Full-text