expressed sequence tag

Examples of expressed sequence tag in the following topics:

• Physical Maps and Integration with Genetic Maps

  • Physical maps display the physical distance between genes and can be constructed using cytogenetic, radiation hybrid, or sequence mapping.
  • There are three methods used to create a physical map: cytogenetic mapping, radiation hybrid mapping, and sequence mapping.
  • Sequence mapping resulted from DNA sequencing technology that allowed for the creation of detailed physical maps with distances measured in terms of the number of base pairs.
  • A genetic site used to generate a physical map with sequencing technology (a sequence-tagged site, or STS) is a unique sequence in the genome with a known exact chromosomal location.
  • An expressed sequence tag (EST) and a single sequence length polymorphism (SSLP) are common STSs.

• Plasmids as Cloning Vectors

  • Vectors called expression vectors (expression constructs) express the transgene in the target cell, and they generally have a promoter sequence that drives expression of the transgene.
  • Epitope: A vector containing a sequence for a specific epitope that is incorporated into the expressed protein.
  • Targeting sequence: Expression vectors may include encoding for a targeting sequence in the finished protein that directs the expressed protein to a specific organelle in the cell or specific location such as the periplasmic space of bacteria.
  • Protein purification tags: Some expression vectors include proteins or peptide sequences that allows for easier purification of the expressed protein.
  • The target protein is fused to the protein tag, but a protease cleavage site positioned in the polypeptide linker region between the protein and the tag allows the tag to be removed later.

• Purifying Proteins by Affinity Tag

  • Protein tags are peptide sequences genetically grafted onto a recombinant protein.
  • Protein tags are peptide sequences genetically grafted onto a recombinant protein.
  • Solubilization tags are used, especially for recombinant proteins expressed in chaperone-deficient species such as E. coli, so as to assist in the proper folding in proteins and keep them from precipitating.
  • Epitope tags are short peptide sequences which are chosen because high-affinity antibodies can be reliably produced in many different species.
  • Epitope tags include V5-tag, c-myc-tag, and HA-tag.

• Shuttle Vectors and Expression Vectors

  • An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell .
  • The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.
  • Therefore, to make the purification process easy, the cloned gene should have a tag.
  • This tag could be histidine (His) tag or any other marker peptide.
  • Expression vectors must have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).

• The FISH Technique

  • It is used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
  • FISH can be used to detect RNA or DNA sequences of interest.
  • In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.
  • Tagging can be done in various ways, such as nick translation, or PCR using tagged nucleotides.
  • Probes can vary in length from 20 to 30 nucleotides to much longer sequences.

• Tracking Cells with Light

  • It was then cloned and its sequence identified in 1992 by Douglas Prasher.
  • This gene's regulatory sequence now controls the production of GFP or luciferase, in addition to the protein of interest.
  • In cells where the gene is expressed, and the tagged proteins are produced, GFP or luciferase are produced at the same time.
  • Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy , or bioluminesce (emit light) when luciferin, the substrate for luciferase is added.
  • Reporter gene used as an indication of the regulatory sequence expression in the cell.

• DNA Sequencing Techniques

  • As each differently-sized fragment exits the capillary column, a laser excites the flourescent tag on its terminal nucleotide.
  • Each sequencing reaction is a modified replication reaction involving flourescently-tagged nucleotides, but no chain-terminating dideoxy nucleotides are needed.
  • Sanger sequence can only produce several hundred nucleotides of sequence per reaction.
  • Most next-generation sequencing techniques generate even smaller blocks of sequence.
  • The smallest fragments were terminated earliest, and they come out of the column first, so the order in which different fluorescent tags exit the column is also the sequence of the strand.

• Epigenetic Control: Regulating Access to Genes within the Chromosome

  • These tags are not permanent, but may be added or removed as needed.
  • The tags do not alter the DNA base sequence, but they do alter how tightly wound the DNA is around the histone proteins .
  • These changes to DNA are inherited from parent to offspring, such that while the DNA sequence is not altered, the pattern of gene expression is passed to the next generation.
  • Transcription factors can bind, allowing gene expression to occur.
  • Modifications affect nucleosome spacing and gene expression.

• DNA Sequencing Based on Sanger Dideoxynucleotides

  • Sanger sequencing, also known as chain-termination sequencing, refers to a method of DNA sequencing developed by Frederick Sanger in 1977.
  • Technical variations of chain-termination sequencing include tagging with nucleotides containing radioactive phosphorus for radiolabelling, or using a primer labeled at the 5' end with a fluorescent dye.
  • Automated DNA-sequencing instruments (DNA sequencers) can sequence up to 384 DNA samples in a single batch (run) in up to 24 runs a day.
  • Automation has lead to the sequencing of entire genomes.
  • Different types of Sanger sequencing, all of which depend on the sequence being stopped by a terminating dideoxynucleotide (black bars).

• Proteolytic Degradation

  • This ubiquitin sequence is a modification to proteins that are targeted for degradation.
  • It is necessary for homeostasis functioning in controlling cell cycle and gene expression, for example.
  • The protein is tagged with several ubiquitin signals that target the proteasome.