Tpp1 Lab Report

Second, in order to further confirm the information about characteristics and function of the targeting protein that we have

The filter flask, vacuum, and SNAP i.d. 2.0 were securely connected with tubing. The proteins were transferred to a membrane and was incubated with primary antibody. The primary antibody was then washed three times. Afterwards a secondary antibody was added and the same procedure was repeated. 10mL of 5-bromo-4-chloro-3-indoyl phosphate/ nitro blue tetrazolium chloride chromogenic substrate solution was added to the blot which was then wrapped with aluminum foil and labeled.

Telomerase (or terminal transferase) is an enzyme made of proteins, specifically reverse transcriptase (Telomerase Reverse Transcriptase-TERT), and RNA subunits. Nonetheless, chicken TERT (chTERT) shares 45% amino acid identity with human TERT (hTERT). However, chTERT is larger than the hTERT because of an extensive N-terminal flexible linker region. Also, chTERT is found on chromosome 2q21 near an interstitial telomere site. Generally, the role of this enzyme is to elongate the end of chromosomes by adding the repeated base sequence of TTAGGG, as described above. This enzyme can be found in fetal tissue, germ cells, and tumor cells. Telomerase activity is critical high on development because of the continued growth and division of the cells.

Both proteins have signalling pathways that are very alike, and both bind activin type 2

The PBGD polypeptide chain consists of 313 amino acids and is approximately 57 x 43 x 32 Å in size. Crystal structure of PBGD revealed a highly flexible protein with three equally sized α/β domains. The N terminal domain (domain 1) and the central domain (domain 2) have similar structures, both being doubly wound parallel beta sheets. Domain 3 (C terminal) is an open faced, anti-parallel, three stranded beta sheet with one side covered in 3 alpha helices, this domain interacts with both 1 and 2. In domains 1 and 2, each sheet has 4 parallel and 1 anti parallel strand with alpha helical segments packed against each face and are orientated parallel to one another. Domains 1 & 2 are motifs and they are both related by a two-fold axis. Domain

Telomeres hold the important DNA code intact, preventing it from fraying as the molecules replicate over time.

Chromatin Assembly Factor 1 (CAF1) interacts with the proliferating cell nuclear antigen region and H3 & H4 proteins. CAF-1 aids in the first step of tetramerization for nucleosome formation and also aids with depositing new H3/H4 onto DNA behind replication forks. If CAF1 was deemed non-functional, the cell may not to be able to form the nucleosome structures, so the DNA wouldn’t be packaged correctly. Cheloufi and colleagues have found that CAF-1 suppression slightly help with reprogramming and develops decent iPS cells.

Chromatin rearrangement takes place during repair to increase accessibility of damage to repair proteins. Activity of histone acetyltransferases and chromatin remodeling complexes seems to be essential for nucleosome rearrangement during repair. Once repair is completed, reconstitution of nucleosomes is required to recover primary chromatin structure. Such chromatin assembly is coupled to repair DNA synthesis. DNA damages induce some modifications to chromatin structure (e.g. phosphorylation of a histone H2A variant in response to DNA double-strand breaks). Such chromatin modifications may serve as signals recognized by DNA repair

Protamines are 50 amino acid long, cysteine and arginine-rich proteins that bind tightly to the negative DNA backbone. As with TPs, PRMs are transcribed in round spermatids and only appear in late spermiogenesis to complete chromatin condensation. Their positive charge stabilizes the chromatin as it abolishes electrostatic repulsion between proximal chromatin strands, forming toroids loops (Cho et al., 2003). Further chromatin compaction occurs during epididymal transit during which PRMs form disulfide bridges between cysteine residues to form a semi-crystalline chromatin (Carrell et al., 2007). Two PRMs have been identified, PRM1 and PRM2, both of which are present in human and mouse germ cells, but only PRM1 exists in the rat. There are two critical differences dissociate the two protamines, both structurally and functionally. First, PRM2 binds longer stretches of DNA, which renders the chromatin less compacted. Second, PRM2 coordinates one zinc atom, which in the case of humans makes zinc deficiency an important issue (Balhorn, 2007). However, this difference does not make one PRM more essential than the other. In fact, the PRMs must be present in equal ratio, as a change in this ratio leads to diminished semen quality and low fertility potential in humans (Balhorn, 2007). Xenobiotics are chemicals that an organism is exposed to that are

Targeting these Telomere/Shelterin compounds may be useful to suppress the tumour cells, killing it off.Grueber CE et al. 2015

Telomerase (TERT) is an enzyme responsible for maintaining the ends of telomeres and reversing the process of telomere shortening in humans by adding a “TTAGGG” polynucleotide to the 3’ end of the telomere. The addition of the “TTAGGG” compensates for inefficiencies in replication of the ends of DNA in eukaryotes. Telomerase is limited to highly proliferative cells such as germ cells and lymphocytes where the telomerase is necessary to maintain telomere length, and the telomeres themselves are not sufficiently long to facilitate such rapid cell division without incurring damage to the DNA. Most somatic cells exhibit no telomerase activity and in cells where telomerase is present telomerase activity is strictly regulated. In cells that exhibit

The TATA box is located at a regulatory region of the DNA and is positioned 25 nucleotides upstream from the starting point of transcription. Once TBP is attached, it allows TFIID to bind. The recruitment of three additional TFs, TFIIA, TFIIB, and TFIIF is necessary to prepare the DNA for the binding of RNA polymerase II, thus forming the protein complex. RNA polymerase II must arrive with TFIIE and TFIIH to gather around the TATA box and form the pre-initiation complex. TFIIH utilizes the energy from ATP to unwind the double helical structure of DNA and widens the minor groove. RNA polymerase II becomes phosphorylated by TFIIH and causes a conformational change that initiates RNA synthesis and elongation begins (Berg JM,.et al, 2002). However, preRNAs need to be processed before translation takes place through splicing, capping, and polyadenylation, which ultimately form the mRNA

Part A: Describe the structural components of DNA and outline mammalian DNA replication and repair.

The class results displayed in Table 2 indicate which of the four ‘prey’ inserts produce proteins able to undergo protein-protein interactions with each strain of Bub1B. A high Leu, Trp, Ade colony count indicates that protein-protein interactions occurred, allowing for yeast growth. From Table 2, BUB3 interacts with strains Bub1B (186-613), Bub1B (324-667) and Bub1B (328-1052). CDC20 interacts with strain Bub1B (186-613). Ppp2rc interacts with Bub1B (328-1052), and Bub1B (588-1052). Zfp207 interacts with no strains of Bub1B, according to Table 2.

The 393 amino acid long protein has an amino terminal transactivation domain, a core domain around 200 amino acid which recognizes p53 DNA-binding sites (DBSs) and a carboxyl terminal tetramerization domain.