Determination of Direct Binding between FMRP and Cyclin E

The specificity of albumin binding experiment was to determine the binding interactions that occur between serum albumin and three synthetic dyes with the use of electrophoretic procedure. Whole blood, or plasma. Clots upon standing and if the clot is removed, the remaining straw colored fluid is called serum. The major protein in serum is albumin which functions as a carrier molecule for the transport of certain small molecular weight compounds in blood. Molecules that bind to serum albumin are fatty acids, hormones and some synthetic dyes. In this experiment the synthetic dyes used are Bromophenol Blue, Ponceau S and Orange G. we observed that free dyes not bound to albumin migrate faster that albumin or dyes bound to albumin. This

Molecular Cell Biology, 7th Edition, 2013, Lodish, Berk, Kaiser, Krieger, Bretscher. Ploegh, Amon, and Scott. W.H. Freeman and Company (ISBN-13: 978-1-4292-3413-9)

At the 12-cell stage of embryogenesis, new P1 decendants(MS and E) will become signalling cells. GLP-1/Notch still remains on the surfaces of the ABa descendants. According to genetic studies done by Shelton and Bowerman (2005), signals from MS and E are identified as products of embryonically-transcribed genes. Since MS will be in contact with 2 out of 4 ABa descendants GLP-1 will be activated. Thus, ref-1 will be expressed. On the other hand, for ABp descendants, they are refractive to second Notch interaction even though they contact MS or E but still continue the GLP-1/Notch

When studying macromolecular interactions in vivo, it is essential to use methods that rapidly ‘‘freeze’’ these interactions as well as prevent the re-assortment of protein and RNA components during cell lysis. Cross-linking agents have been exploited for this purpose. In particular, crosslinking agents that are reversible are the most useful because they simplify subsequent characterization of the interacting molecules. Cross-linking to stabilize RNA and protein complexes has proven to be a useful tool for site-specific interactions. Such linkages can generally be achieved by UV or chemical means (Pashev et al., 1991; Matsunaga et al., 2001). The interactions or mere proximity of macromolecules can be studied by the clever use of crosslinking

Gene expression is an essential factor in cell biology research. Reverse transcriptase-polymerase chain reaction, also known as RT-PCR, is used to express specific genes. RT-PCR is a type of PCR in which it used to analyze RNA expression by transcribing RNA into complementary DNA (cDNA) (6). The specific genes focused on throughout this experiment are pax6a and pax2a. These genes are known as transcription factors and aid in various functions such as neural cell maintenance, neural cell migration, and differentiation. Transcription factors are proteins that bind to DNA that have the ability of turning the expression of a gene on or off (1). The pax6a

Cheung uses many different approaches throughout her research. It is common to see the Cheung Lab prove their findings extensively using multiple approaches to the same problem to ensure the production of high quality work. Working with plants, the mapping of genes and generating mutants plays a critical role in how plants are studied. By inserting genes of interest such as FER with a GFP tag, fluorescence microscopy becomes a solution in observing the localization of FER during different stages in the plants life as well as different conditions of its environment. In addition to tagging proteins, the Cheung lab heavily relies on fluorescence microscopy to visualize many different structures and molecules in different types of cells throughout the plant. With the combination of fluorescence, confocal, and electron microscopy, the Cheung Lab can utilize different approaches in overcoming the scientific challenges they face. Dr. Cheung clearly demonstrated the interactions with FER to permit localization with LLG1 as well as membrane embedding with LRE. The next step she would like to take in her research is to identify which ligands interact with FER at the membrane and what effect do they have on FER localization, as well the signaling effects downstream. Also, she will explore how these processes change in different cell types throughout the

There is a place in the FMR1 gene where the DNA pattern CGG is repeated over and over again. In most people, the number of repeats is small (5 to 44 repeats), which is normal. If the number of repeats is too large (more than 200 repeats), the gene turns off. When the gene is turned off, no protein is made.

Accepted 9 April 2008 Journal of Cell Science 121, 1771 Published by The Company of Biologists 2008 doi:10.1242/jcs.033340

BRAF fusions, kinase activation is the resultant effect. Due to their size and location, pLGG biopsies are generally

Oocyte development in Drosophila proceeds through fourteen stages. The first seven stages are pre-vitellogenic, or without any yolk protein. The last seven stages are called vitellogenic because yolk is being deposited and the egg chamber is growing. In an ovary from Drosophila melanogaster, there are many egg chambers. Each egg chamber is made up of sixteen individual cells. Of these sixteen cells, fifteen cells are nurse cells. The other cell is the developing oocyte. The function of these nurse cells is to utilize non-specific proteins such as clathrin and adaptin as well as specific receptors to sequester yolk protein from the hemolymph. Yolk protein is trafficked from the nurse cells into the developing oocyte (Shilling, 2004).

of an H2A.Z/H2B dimer from nucleosomes when encountered by RNA Polymerase II (Weber et al., 2014). Therefore, accumulation of H2A.Z around TSSs promotes transcriptional initiation. In Arabidopsis, H2A.Z is required for activation of Flowering Locus C (FLC), in that way ensuring the proper timing of the transition from vegetative growth to flowering (Deal et al., 2007). It is shown that the TSS of FLC locus is enriched with H2A.Z and its expression is strongly up-regulated by FRIGIDA (FRI). FRI acts as a scaffold protein in a transcription activator complex that binds to the FLC locus and directly associates with the SWR1 complex, suggesting that FLC up-regulation is mediated by direct modulation of H2A.Z deposition by FRI (Choi et al., 2011).

During the development of multicellular organisms, the fate of a cell is often determined by the influence of neighboring cells or tissues. The molecular mechanisms by which such inductive signals cause changes in the genetic program of the responding cell remain largely unknown. In the early stages of the response, signals from the cell surface must lead to modifications in the activity of one or more pre-existing transcription factors, which then set in motion the appropriate cascade of gene activation. Post-translational activation of transcription factors has been demonstrated in a number of cases, including steroid hormone receptors (Glineur et al. 1990), the yeast heat shock response factor (Sorger and Pelham 1988), and the mammalian factor AP-1 (Angel et al. 1987; Lee et al. 1987). The activation of transcription factors in response to inductive signals during development has proved more difficult to demonstrate, largely because the critical transcription factors have not been identified. Cell identities in the developing eye of Drosophila are determined by induction, and mutations in several genes that encode putative transcription factors have been shown to disrupt normal eye development (Tomlinson 1988; Banerjee and Zipursky 1990). Here, it is shown that one of these genes, glass, encodes a site-specific DNA-binding protein and that glass function, in its broadest sense, is regulated at the protein level. The glass gene is required for the normal development of

The study of Drosophila oogenesis provides invaluable information about signaling pathway regulation and cell cycle programming. During Drosophila oogenesis, a string of egg chambers in each ovariole progressively develops toward maturity. Egg chamber development consists of 14 stages. From stage 1 to stage 6 (mitotic cycle), mainbody follicle cells undergo mitotic divisions. From stage 7 to stage 10a (endocycle), follicle cells cease mitosis but continue another three rounds of endoreduplication. From stage 10a to stage 13 (gene amplification), instead of whole genome duplication, follicle cells selectively amplify specific genomic regions, mostly for chorion production. So far, Drosophila oogenesis is one of the most well-studied model systems to understand cell cycle switches, which furthers our knowledge about cell cycle control machinery and sheds new light on potential cancer treatments. Here, we give a brief summary of cell cycle switches, their involved signaling pathways and factors, and detailed experimental procedures to

Using a combination of IHC and IF experiments, we confirmed that the protein level of E2F3A and E2F8 during cell cycle, which reached peak at G1-early S and late S-G2, respectively, while E2F4 translocates from cytoplasm to nuclear when cells exit cell cycle. The activator E2Fs are required for transcriptional activation of S phase genes, while pocket protein needs to be phosphorylated by cyclin/CDK complexes to release its inhibitory effect on the activator E2Fs [10, 12, 29, 30]. The atypical repressor E2Fs function as transcriptional repressors of genes involved in DNA replication, inactivation of atypical repressor E2F activity would extend the progression of the DNA synthesis machinery [4, 5, 9, 16]. The typical repressor

To explore regions of the Drosophila genome that weren’t categorized by Stark lab fragments, we used a molecular approach to design novel candidate enhancers from the ChIP sequence peaks. This approach also allowed us to create narrower fragments (approximately 1000 bp), focusing on enhancer activity and reducing the chance of background activity. Fragments containing in vivo Bcd and Otd binding signals were selected by searching between 500 bp upstream to 500 bp downstream of the primary binding peaks from ChIP sequence data (Datta, Unpublished) for Bcd Early (BcdE), Otd Late (OtdL), Otd Early (OtdE), or both BcdE and OtdL. We designed primers for the top candidate enhancers using Primer3, calculating for appropriate GC content and melting temperature (primers ordered through Integrated DNA Technologies, Iowa). Restriction enzyme sites (AscI at 5` and BglII at 3’) were incorporated into the primers. Standard PCR amplification (Chen et al.) was performed with wildtype fly genomic DNA and candidate primers. Qiagen™ kits were used for PCR purification and gel extraction.