Eukaryotes have much larger genomes than prokaryotes, and therefore, must condense their DNA into chromatin. Chromatin is composed of histone proteins that help to condense and organize the DNA forming chromosomes. The basic unit of this chromatin is a nucleosome, which contains about 150 base pairs of DNA that are wrapped 1.7 times around the core histone proteins. However, this tightly wrapped chromatin becomes a problem when regulatory proteins need access to specific sections of the DNA. The genes are “turned off” to transcription by this physical structure until the needed section of chromatin is unwrapped, thus removing the obstacle. Once this unraveling occurs, the gene is turned on and transcription can begin [14].
Transcription, the first step in the process of creating a protein, copies the nucleotide sequence of DNA into a strand of RNA. RNA polymerase (Pol II) attaches to the chromosome and unwinds the two strands of DNA as it moves across the gene. This protein reads one strand of DNA, using it as a template, and attaches the complementary ribonucleotides. After Pol II completes transcription in one section, it rewinds the two strands back together. This process continues until the gene is completely transcribed, and a strand of RNA is created [14]. Our experiment studied the proteins involved in the FACT complex. The FACT complex is composed of Spt16 and Pob3 proteins that work with RNA polymerase II during transcription elongation. FACT travels across the genes with Pol II, helping to disassemble the nucleosomes before this enzyme reaches the next section of the chromatin [15]. This allows Pol II to access the template DNA and complete its process of transcription. Then, the FACT complex reassembles the nucleosomes after Pol II travels across a specific section. Therefore, the FACT complex is vital to the process of transcription [1]. The organism that we will focus on in our experiment is Saccharomyces cerevisiae (S. cerevisiae), commonly known as baker’s yeast. This organism is a good model for our experimentation for many reasons. Firstly, the cells are very easy to work with because they are inexpensive and easy to grow (one yeast cell divides every 90 minutes in optimal lab conditions). In addition, genes are easily moved in and out of yeast cells. This fact is very important to our experiment, which requires specific mutations to be added to the genome. Lastly, yeast is a good model system because it can be generalized to human biology and processes [13]. Within S. cerevisiae, a mutation has been found in H3-L61. In H3, the gene that makes histone when there is none in the environment, there is a mutation on the 61st nucleotide. At this location, the amino acid leucine should be made but is replaced with another amino acid. If the mutation is H3-L61W, leucine is replaced with tryptophan. This mutation causes Spt16 to be heavily distributed toward the 3’ end of a strand. Researchers have discovered that due to this mutation, less Spt16 is found at the 5’ end and much more is found at the 3’ end. Currently, there is a model to explain these observations in which the mutants impair the ability of Spt16 to dissociate, causing less availability of Spt16 in the nucleoplasm [15]. We hypothesize that there may be proteins, or other factors, that are involved with the FACT complexes. Through our experimentation, we searched for additional proteins that are necessary for proper Spt16-gene interactions or that aid Spt16 in dissociation. Our genetic approach comprised of screening for gene deletions in the CSGI (Controls Spt16-Gene Interaction) mutant that is sick or lethal, using Synthetic Gene Array …show more content…
coli cells were purified. The plasmid mini-preparation was done using the Qiagen kit to create a solution with purified plasmid DNA. Restriction enzyme digestion reactions used 8 μl of purified plasmid DNA, the EcoRV enzyme, and a cocktail [2]. Then agarose gel electrophoresis, using 1% agarose gel, was done on the digested plasmids [3]. Agarose gel electrophoresis displays the distances that the products had traveled, thus revealing the size of the products. This technique determined if the plasmid digestion worked properly, and the expected plasmids were obtained. Two bands, one at 603 bp and one at 2046 bp, should have
Transcription, the first step in the process of creating a protein, copies the nucleotide sequence of DNA into a strand of RNA. RNA polymerase (Pol II) attaches to the chromosome and unwinds the two strands of DNA as it moves across the gene. This protein reads one strand of DNA, using it as a template, and attaches the complementary ribonucleotides. After Pol II completes transcription in one section, it rewinds the two strands back together. This process continues until the gene is completely transcribed, and a strand of RNA is created [14]. Our experiment studied the proteins involved in the FACT complex. The FACT complex is composed of Spt16 and Pob3 proteins that work with RNA polymerase II during transcription elongation. FACT travels across the genes with Pol II, helping to disassemble the nucleosomes before this enzyme reaches the next section of the chromatin [15]. This allows Pol II to access the template DNA and complete its process of transcription. Then, the FACT complex reassembles the nucleosomes after Pol II travels across a specific section. Therefore, the FACT complex is vital to the process of transcription [1]. The organism that we will focus on in our experiment is Saccharomyces cerevisiae (S. cerevisiae), commonly known as baker’s yeast. This organism is a good model for our experimentation for many reasons. Firstly, the cells are very easy to work with because they are inexpensive and easy to grow (one yeast cell divides every 90 minutes in optimal lab conditions). In addition, genes are easily moved in and out of yeast cells. This fact is very important to our experiment, which requires specific mutations to be added to the genome. Lastly, yeast is a good model system because it can be generalized to human biology and processes [13]. Within S. cerevisiae, a mutation has been found in H3-L61. In H3, the gene that makes histone when there is none in the environment, there is a mutation on the 61st nucleotide. At this location, the amino acid leucine should be made but is replaced with another amino acid. If the mutation is H3-L61W, leucine is replaced with tryptophan. This mutation causes Spt16 to be heavily distributed toward the 3’ end of a strand. Researchers have discovered that due to this mutation, less Spt16 is found at the 5’ end and much more is found at the 3’ end. Currently, there is a model to explain these observations in which the mutants impair the ability of Spt16 to dissociate, causing less availability of Spt16 in the nucleoplasm [15]. We hypothesize that there may be proteins, or other factors, that are involved with the FACT complexes. Through our experimentation, we searched for additional proteins that are necessary for proper Spt16-gene interactions or that aid Spt16 in dissociation. Our genetic approach comprised of screening for gene deletions in the CSGI (Controls Spt16-Gene Interaction) mutant that is sick or lethal, using Synthetic Gene Array …show more content…
coli cells were purified. The plasmid mini-preparation was done using the Qiagen kit to create a solution with purified plasmid DNA. Restriction enzyme digestion reactions used 8 μl of purified plasmid DNA, the EcoRV enzyme, and a cocktail [2]. Then agarose gel electrophoresis, using 1% agarose gel, was done on the digested plasmids [3]. Agarose gel electrophoresis displays the distances that the products had traveled, thus revealing the size of the products. This technique determined if the plasmid digestion worked properly, and the expected plasmids were obtained. Two bands, one at 603 bp and one at 2046 bp, should have