Biology

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Simple allele-discriminating PCR for cost-effective and rapid genotyping and mapping
(Springer Nature, 2009-01-08) Bui, Minh; Liu, Zhongchi
Single nucleotide polymorphisms (SNPs) are widely observed between individuals, ecotypes, and species, serving as an invaluable molecular marker for genetic, genomic, ecological and evolutionary studies. Although, a large number of SNP-discriminating methods are currently available, few are suited for low-throughput and low-cost applications. Here, we describe a genotyping method named S imple A llele-discriminating P CR (SAP), which is ideally suited for the small-scale genotyping and gene mapping routinely performed in small to medium research or teaching laboratories. We demonstrate the feasibility and application of SAP to discriminate wild type alleles from their respective mutant alleles in Arabidopsis thaliana. Although the design principle was previously described, it is unclear if the method is technically robust, reliable, and applicable. Three primers were designed for each individual SNP or allele with two allele-discriminating forward primers (one for wild type and one for the mutant allele) and a common reverse primer. The two allele-discriminating forward primers are designed so that each incorporates one additional mismatch at the adjacent (penultimate) site from the SNP, resulting in two mismatches between the primer and its non-target template and one mismatch between the primer and its target template. The presence or absence of the wild type or the mutant allele correlates with the presence or absence of respective PCR product. The presence of both wild type-specific and mutant-specific PCR products would indicate heterozygosity. SAP is shown here to discriminate three mutant alleles (lug-3, lug-16, and luh-1) from their respective wild type alleles. In addition, the SAP principle is shown to work in conjunction with fluorophore-labeled primers, demonstrating the feasibility of applying SAP to high throughput SNP analyses. SAP offers an excellent alternative to existing SNP-discrimination methods such as Cleaved Amplified Polymorphic Sequence (CAPS) or derived CAPS (dCAPS). It can also be adapted for high throughput SNP analyses by incorporating fluorophore-labeled primers. SAP is reliable, cost-effective, fast, and simple, and can be applied to all organisms not limited to Arabidopsis thaliana.
Internal modifications in the CENP-A nucleosome modulate centromeric dynamics
(Springer Nature, 2017-04-04) Bui, Minh; Pitman, Mary; Nuccio, Arthur; Roque, Serene; Donlin-Asp, Paul Gregory; Nita-Lazar, Aleksandra; Papoian, Garegin A.; Dalal, Yamini
Posttranslational modifications of core histones are correlated with changes in transcriptional status, chromatin fiber folding, and nucleosome dynamics. However, within the centromere-specific histone H3 variant CENP-A, few modifications have been reported, and their functions remain largely unexplored. In this multidisciplinary report, we utilize in silico computational and in vivo approaches to dissect lysine 124 of human CENP-A, which was previously reported to be acetylated in advance of replication. Computational modeling demonstrates that acetylation of K124 causes tightening of the histone core and hinders accessibility to its C-terminus, which in turn diminishes CENP-C binding. Additionally, CENP-A K124ac/H4 K79ac containing nucleosomes are prone to DNA sliding. In vivo experiments using a CENP-A acetyl or unacetylatable mimic (K124Q and K124A, respectively) reveal alterations in CENP-C levels and a modest increase in mitotic errors. Furthermore, mutation of K124 results in alterations in centromeric replication timing. Purification of native CENP-A proteins followed by mass spectrometry analysis reveals that while CENP-A K124 is acetylated at G1/S, it switches to monomethylation during early S and mid-S phases. Finally, we provide evidence implicating the histone acetyltransferase (HAT) p300 in this cycle. Taken together, our data suggest that cyclical modifications within the CENP-A nucleosome contribute to the binding of key kinetochore proteins, the integrity of mitosis, and centromeric replication. These data support the paradigm that modifications in histone variants can influence key biological processes.
LEUNIG, LEUNIG HOMOLOG, AND SEUSS ARE TRANSCRIPTIONAL CO-REPRESSORS THAT REGULATE FLOWER DEVELOPMENT, MUCILAGE SECRETION, AND PATHOGEN RESISTANCE
(2009) Bui, Minh; Higgins, William J; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Transcriptional repression is an important regulatory mechanism for development. My thesis focuses on dissecting the function of Groucho (Gro)/Transducin-Like Enhancer of split (TLE) family of transcriptional repressors in plant development. My work characterizes two Arabidopsis thaliana genes, LEUNIG (LUG), first discovered to repress transcription of the floral homeotic gene AGAMOUS (AG), and LEUNIG_HOMOLOG (LUH), a gene with the highest sequence similarity to LUG. To investigate the functional redundancy between LUG and LUH, I constructed and analyzed lug; luh double mutants, and concluded that both LUG and LUH repress AG expression in the flower, with LUG playing a more prominent role than LUH. The double mutant also revealed a previously unknown function of LUG and LUH in embryogenesis because lug-3; luh-1 double mutants are embryo lethal, while the single mutants develop normal embryos. During the course of this study, I developed a new genotyping method called Simple Allele-discriminating PCR (SAP), which is cost-effective, quick, and easy to perform. This method has greatly facilitated my research as well as others in the lab. A second part of my thesis addresses the role of LUG and LUH in other developmental processes besides flower development. My data indicate that these two genes, like their counter parts in fungi and animals, act as "global co-repressors" in various developmental and physiological processes. My thesis work revealed that both co-repressors, together with its interacting protein SEUSS (SEU), repress the Salicylic Acid (SA) pathogen defense pathway. Although lug-3, luh-1, and seu-1> mutants induced PR1 expression at higher levels than wild-type, only lug-3 and seu-1 mutants were pathogen resistant. Furthermore, LUH functions as a positive regulator in seed mucilage secretion, a process important for proper seed germination, hydration, and dispersal. I propose a possible connection between the defect in mucilage secretion and pathogen defense in luh-1 mutant plants and seeds, which places the foundation for further investigation and may uncover mucilage secretion as a major defense mechanism. My thesis has provided important insights into how transcriptional co-repressors regulate diverse developmental and physiological pathways in higher plants.

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