Greetings, dear colleagues!
Our team conducts research on newly discovered SIRC elements in plant genomes (Article Short Interrupted Repeat Cassette (SIRC)-Novel Type of Repet...
, which are thought to be MITE transposons losing inverted repeats products, which could influence genome regulation) using bioinformatics, and we plan to conduct experimental molecular biology studies to elucidate the functions of SIRC. The problem is - our team is specialized in molecular bology experiments aiming to reveal the functions of genes, not non-coding DNA elements. That's why I want to ask your expert opinion - what experimental techniques would help to reveal the functions of abundant DNA elements of repetitive nature?What comes to mind is the creation of mutant lines without several of these elements, but such experiments are too large-scale and can last for years, which is too complicated at the moment.
Another technique that comes to mind is the amplification of certain sequences and examination using circular dichroism spectroscopy to reveal whether given elements have unusual secondary structure like G-quadruplex of triplex DNA etc that could influence processes of genome transcription or replication.
And one more - we thought it could be possible to capture and identify plant proteins that specifically recognize SIRC via some modification of EMSA (electroforetic mobility shift assay) method. Unfortunatelly, up to date we didn't find any mentions of EMSA variant that uses not single purified protein, but whole DNA-free nuclear lysate, with subsequent identification of binding proteins via MALDI-TOF.
What other in vitro experiments could be useful?
Would it not be easier to just use a plasmid based approach where you insert your elements around/in a promoter of known strength that expresses a marker gene like LacZ?
The main difference between IRES (Internal Ribosome Entry Site) and IRES2 lies in their ability to regulate gene expression in a bicistronic mRNA construct.
1. **IRES (Internal Ribosome Entry Site):** IRES is a sequence element found in some viral and cellular mRNAs that allows for cap-independent translation initiation. When an IRES sequence is present in an mRNA, it enables ribosomes to bind and initiate translation internally, bypassing the need for a 5' cap structure. This allows for the translation of multiple open reading frames (ORFs) from a single mRNA molecule. However, the efficiency of translation initiation mediated by IRES elements can vary depending on the specific sequence and context.
2. **IRES2:** IRES2 is a modified version of the IRES sequence that has been engineered or optimized for improved translation initiation efficiency compared to the original IRES sequence. Through rational design or directed evolution approaches, researchers may modify the sequence or structure of the IRES element to enhance its activity. The goal is to increase the expression level of downstream genes in a bicistronic mRNA construct.
In summary, while both IRES and IRES2 are used to achieve cap-independent translation initiation in bicistronic mRNA constructs, IRES2 typically refers to an optimized or engineered version of the IRES sequence with improved translation efficiency.
The plasmid based approach undoubtedly deserves attention, but encounters such complexity that the element in question is of eukaryotic origin. As a result, there is some doubt that we will receive the correct answer to our question.
@Robert Adolf Brinzer The question is, what is the possible structural and functional role of newly discovered SIRC (Short Interrupted Repeat Cassettes) elements in plant genome. The point is that in bacterial genomes there are CRISPR cassettes which are looking similar to SIRC but have nothing common.
Studying DNA elements involves several strategies, including:DNA Sequencing: This technique allows scientists to determine the precise sequence of nucleotides in a DNA molecule, providing insight into the genetic code and identifying specific DNA elements.Functional Assays: These experiments involve manipulating DNA sequences to study their function. For example, researchers can insert DNA elements into a host organism and observe the resulting phenotype to understand the element's role.Bioinformatics: Computational methods are used to analyze DNA sequences and predict the presence and function of elements such as promoters, enhancers, and regulatory sequences.Chromatin Immunoprecipitation (ChIP): This method allows researchers to identify DNA elements bound by specific proteins by immunoprecipitating the protein-DNA complexes and analyzing the associated DNA.CRISPR/Cas9 Genome Editing: This technology enables precise manipulation of DNA sequences, allowing researchers to delete, insert, or modify specific DNA elements to study their function.By combining these strategies, researchers can gain a comprehensive understanding of DNA elements and their roles in gene regulation, genome organization, and other cellular processes.