Transcription is a process by which a DNA segment is copied into an RNA complementary sequence which is used to be translated into proteins. Transcription involves promoters that RNA polymerase bind at, isomerization, elongation and termination. These processes are regulated by binding proteins. Many factors influence the productivity of transcription including the supercoiling of DNA. There are two types of supercoiling, positive and negative. Positive supercoiling is when the double helix, right-handed DNA is twisted tighter and begins to knot or warp. Negative supercoiling is twisted into a left-handed conformation and is usually the supercoiling present in most organism’s DNA. In the literature, supercoiling was investigated at different promoters on a plasmid by using a range of topoisomers in order to find out the amount of supercoiling that affects transcription and how it is affected.
In the experiment, a plasmid called pSA850 was used to investigate the transcription affects. This plasmid is comprised of multiple promoters that are followed by a transcription terminator that yields sequences in different lengths. Some promoters that were present on the plasmid included lacP, galP1 and galP2, pP, bP, and rP. In order to prepare the topoisomers of the plasmid pSA850, it was incubated in a mixture of calf thymus topoisomerase 1 and ethidium bromide. The reactions finished and proteins as well as the ethidium bromide were taken out by a phenol-chloroform extraction and then isoamyl alcohol was used twice afterwards to also extract. TAE-buffered agarose gel containing chloroquine was used to measure the mean linking number difference of the topoisomers that were produced. The linking number was determined to be 332 using the 10.5 base pair per turn and the superhelical densities were also concluded.
In vitro transcription was started in a blend comprising of DNA template, RNAP, Tris-acetate, pH 7.5, magnesium acetate, potassium glutamate, and rRNasin. The beginning blend was incubated for about five minutes and then added to a mixture of NTP containing ATP, GTP, CTP, UTP and [α-32P] UTP in order for the reactions to begin and then incubated. The reactions were then terminated using BRL which is a STOP solution. The mixtures were warmed for two minutes and in order to observe the resulting transcripts and the abandoned molecules that were prepared from the promoters, 3 microliters of each sample was put aboard polyacrylamide-urea sequencing gels. Gene regulatory proteins were present in varying degrees and therefore could determine whether different levels of topoisomers showed different effects.
The miniprep consisted of isolating the DNA plasmid from the bacterial cells. This was used to identify the success of EGFP ligation into pET41a(+) vector upon restriction digest and gel electrophoresis. Additionally, Polymerase Chain Reaction (PCR) was run on the isolated DNA plasmids with one of the primers specifically annealing to a part of pET41a(+) sequence and the other annealing to the EGFP gene.
Amplification reaction was done in a 25.0 µL reaction mixture containing 0.4 µL DNA (from DNA extraction), 5.0 µL of 10X PCR reaction buffer, 14.2 µL of sterelized dH2O, 2.0 µL of magnesium chloride (MgCl2, 25 mM), 1.0 µL nucleotide/dNTP mix (10 Mm), and 0.4 µL of 5 u/µL Taq DNA polymerase for each primer namely respectively. The components and the volume used for the amplification reactions are listed in Table 3.2. For the reaction, PCR reaction was performed in a programmable gradient-enabled thermocycler (Bio-Rad MyCycler™ Thermal Cycler).
The given DNA ladder sample and each individual ligation samples were run on 40ml of 0.8% agarose in 1x TAE buffer for approximately sixty minutes at 110V. The appropriate volume of 6x GelRed track dye was used after it was diluted to a final concentration of 1x and incubated for thirty minutes. Finally, the gel was illuminated under UV light and analyzed.
The expression of lac operon in each tube equals the amount of beta-galactosidase produced. Therefore, by looking at the amount of beta-galactosidase under different conditions collectively is a good way to understand the function of inducers and repressors in supervising the expression of lac operon and the control of gene expression generally.
Miller, Kenneth R. and Joseph S. Levine. “Chapter 12: DNA and RNA.” Biology. Upper Saddle River: Pearson Education, Inc., 2002. Print.
In order to do this a polymer of DNA “unzips” into its two strands, a coding strand (left strand) and a template strand (right strand). Nucleotides of a molecule known as mRNA (messenger RNA) then temporarily bonds to the template strand and join together in the same way as nucleotides of DNA. Messenger RNA has a similar structure to that of DNA only it is single stranded. Like DNA, mRNA is made up of nucleotides again consisting of a phosphate, a sugar, and an organic nitrogenous base. However, unlike in DNA, the sugar in a nucleotide of mRNA is different (Ribose) and the nitrogenous base Thymine is replaced by a new base found in RNA known as Uracil (U)3b and like Thymine can only bond to its complimentary base Adenine. As a result of how it bonds to the DNA’s template strand, the mRNA strand formed is almost identical to the coding strand of DNA apart from these
Hall, Linley Erin. “Understanding Genetics DNA and RNA.” New York: The Rosen Publishing Group, Inc., 2011. Print. 01 Apr. 2014.
In April of 1953, James Watson and Francis Crick published a game changing paper. It would blow the mind of the scientific community and reshape the entire landscape of science. DNA, fully knows as Deoxyribonucleic Acid is the molecule that all genes are made of. Though it is a relatively new term with regard to the age of science, the story of DNA and the path to its discovery covers a much broader timeframe and had many more contributors than James Watson and Francis Crick. After reading the paper the audience should have a better understanding of what DNA is, the most important experiments that contributed to its ultimate discovery and the names and contributions of the lesser-known scientists that helped Watson and Crick turn their idea
In bacteria, RNA polymerase attaches right to the DNA of the promoter. You can see how this process works, and how it can be regulated by transcription factors, in the lac operon and trp operon videos.
... starts relaxing the supercoils and altering of DNA and interacts with DNA helicase SGS1 and plays a role in DNA recombination, also cellular aging and maintenance of genome stability. Alternate splicing results in multiple transcript variants. Additional spliced variants of the gene have been described, but their complete length is unknown.
Modern techniques , rather than the gene map , maps the map of the DNA within the gene itself : the positions of short sequences " marker " are used as markers signaling over the cromosssomas . Once a gene is discovered, it is necessary to unravel its base sequence prior to its function being studied . The sequencing has become easier with the development of methods for cloning the DNA - producing large amounts of identical fragments. In the method most widely used DNA sequencing , the chain is denatured into single strands . These are then used as templates for DNA synthesis , but such that replication to as the double helix reaches a certain growth in the mold base . In addition to provide DNA polymerase and the four bases, A - G -C- T, also using small amounts of these dideoxynucleotide bases. This is incorporated , as the normal bases, the double helix growth but prevent the continuation of the chain. The fragments are then separated by gel electrophoresis and the base seq...
DNA testing has been the center of attention in many criminal justice cases. The United States corrections centers have utilized the DNA testing process. Seventeen death row inmates have been exonerated by the use of these tests. Earl Washington was convicted of rape and murder in 1984. Although he confessed to the rape, he was also diagnosed as being mentally retarded. In October of 2000 Mr., Washington was given a DNA test and was excluded as the rapist and murderer. The Virginia Governor pardoned Mr. Washington after he had served 16 years in prison with 14 of them being on death row (ACLU, 2011). DNA testing has become the rule rather than the exception; but what happens with the DNA after a person has been acquitted, dismissed, or exonerated. Where does DNA go to die or does it? Is the DNA destroyed, or is it retained in miscellaneous databanks for further retrieval and use? In 2010, the United States Congress began a campaign designed to encourage the states to require DNA to be taken from suspects whether they had been charged with a crime or not. In the case of S. and Marper v the United Kingdom found that the retention of the applicants' fingerprints, cellular samples and DNA profiles was in violation of Article 8 of the European Convention on Human Rights. Is creating a policy in the United States that demand DNA from suspects helps in finding subsequent criminals or is it just leading to a track and trace policy?
Citation: Philips, T. (2008) Regulation of Transcription and gene expression in Eukaryotes. Nature Education 1(1)
The Use of Recombinant DNA Technology Recombinant DNA technology is the technology of preparing recombinant DNA in vitro by cutting up DNA molecules and splicing together fragments from more than one organism.(1) This is the process of using recombinant DNA technology to enable the rapid production of human protein from a single gene of insulin. Firstly the single gene required must be isolated. This can be done three ways: Either by working backwards from the protein- Finding the amino acid sequence for the protein needed, the order of bases can be established using known genetic code. New DNA can be made from this sequence of bases resulting in artificial gene made from complementary DNA.
DNA (deoxyribonucleic acid) is a self-replicating molecule or material present in nearly all living organisms as the main constituent in chromosomes. It encodes the genetic instructions used in the development and functioning of all known living organisms and many viruses. Simply put, DNA contains the instructions needed for an organism to develop, survive and reproduce. The discovery and use of DNA has seen many changes and made great progress over many years. James Watson was a pioneer molecular biologist who is credited, along with Francis Crick and Maurice Wilkins, with discovering the double helix structure of the DNA molecule. The three won the Nobel Prize in Medicine in 1962 for their work (Bagley, 2013). Scientist use the term “double helix” to describe DNA’s winding, two-stranded chemical structure. This shape looks much like a twisted ladder and gives the DNA the power to pass along biological instructions with great precision.