This activity will help students understand the role of DNA, mRNA, tRNA, and amino acids in protein synthesis. This activity will also introduce the concept of mutations.
The brain stem auditory system of the chick is an advantageous model for examining changes that occur as a result of deafness. Elimination of acoustic input through cochlear ablation results in the eventual death of approximately 30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM). One early change following deafness is an alteration in NM ribosomes, evidenced both by a decrease in protein synthesis and reduction in antigenicity for Y10B, a monoclonal antibody that recognizes a ribosomal epitope. Previous studies have shown that mGluR activation is necessary to maintain Y10B antigenicity and NM viability. What is still unclear, however, is whether or not mGluR activation is sufficient to prevent deafness-induced changes in these neurons, or if other activity-dependent factors are also necessary. The current study investigated the ability of mGluR activation to regulate cochlear nucleus ribosomes in the absence of auditory nerve input. In vitro methods were employed to periodically pressure eject glutamate or mGluR agonists over neurons on one side of a slice preparation leaving the opposite side of the same slice untreated. Immunohistochemistry was then performed using Y10B in order to assess ribosomal changes. Application of glutamate and both group I and II selective mGluR agonists effectively rescued ribosomal antigenicity on the treated side of the slice in comparison to ribosomes on the untreated side. These findings suggest that administration of mGluR agonists is sufficient to reduce the early interruption of normal ribosomal integrity that is typically seen following loss of auditory nerve activity.
Studies directed at identifying the trans-synaptic signals necessary for preventing the early changes that occur in NM neurons following cochlea removal have made use of an in vitro slice preparation of the chick auditory brain stem. In this condition, NM activity is eliminated bilaterally since both cochleae are destroyed. In order to mimic the case of unilateral deafening, auditory nerve fibers are electrically stimulated on one side of the slice while fibers on the opposite side of the same slice remain unstimulated. Within 1 hr, NM neurons on the stimulated side of the slice show greater protein synthesis and Y10B labeling , , than the neurons on the unstimulated side. Through the use of the in vitro slice preparation, it has been shown that glutamate's action on ionotropic glutamate receptors (iGluRs) is responsible for the electrical activity in postsynaptic NM neurons , , and glutamate's action on metabotropic glutamate receptors (mGluRs) is responsible for providing trophic support to NM neurons , , . Previous in vivo studies have also shown that mGluR activation is necessary for maintaining ribosomal antigenicity of Y10B and for the ultimate survival of NM neurons . For example, application of selective group I or group II mGluR antagonists results in NM neuronal cell death even with an intact cochlea.
Following sectioning, endogenous peroxidase activity was quenched by placing sections in 0.03% H2O2 in methanol for 20 min. After quenching, sections were rinsed 3×10 min in PBS, and were then preincubated in a 4% normal horse serum solution for 10 min. Sections were then incubated on a rotator overnight at room temperature in 1500 Y10B, a mouse monoclonal antibody that recognizes a ribosomal epitope. The Y10B antiserum was raised from a clone originally developed by J. Steitz and subsequently supplied to our laboratory by E. Rubel. This antibody is an established marker of changes in ribosomes , and has been used extensively as an indicator of the early changes resulting from deafferentation (e.g., , , , , ). Changes in antigenicity appear to match changes in overall protein synthesis and a deafferentation-induced reduction in Y10B labeling has been confirmed at the EM level . Control sections processed without primary antibody show no labeling. The following day, sections were washed 3×10 min in PBS then incubated for 1 hr in 1200 biotinylated horse anti-mouse secondary antibody. Sections were washed 3×10 min in PBS and then incubated in avidin-biotin-peroxidase complex (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, USA) for 1 hr. Following 2×10 min washes in PBS and a 10 min wash in 0.1 M phosphate buffer (PB), sections were reacted with diaminobenzidine tetrahydrochloride and 0.03% H2O2 for 15 min. Sections were mounted onto slides following 2 10-min washes in 0.1 M PB, and allowed to dry overnight. The next day, slides were cleared though graded ethanols and xylenes and then coverslipped using DPX mounting medium (Sigma-Aldrich, St. Louis, MO, USA).
The factors that determine which cells die and which cells survive still remain unclear; however, the chain of events that occur within NM as a consequence of deafening are well documented. Within 1 to 3 hrs following cochlear ablation, NM neurons show a threefold increase in intracellular calcium ([Ca2+]i) , and a decrease in RNA and protein synthesis , , . At 6–12 hrs, deafened NM neurons appear to segregate into two populations: one population suffers a complete cessation of protein synthesis and eventually goes on to die, while the other population continues to synthesize proteins, albeit at a reduced level, and goes on to survive . The cessation of protein synthesis appears to be due to the dissociation of polyribosomes in NM neurons following cochlear ablation . One way to visualize the rapid activity-dependent changes that occur in ribosomes following deafening is by using Y10B, a monoclonal antibody that recognizes a ribosomal epitope , , , . Changes in Y10B immunoreactivity match the changes observed in overall protein synthesis , and these changes correspond with changes observed in ribosomes at the EM level , . Consequently, Y10B immunoreactivity provides an efficient and meaningful assay for examining changes in NM ribosomes, one of the earliest cellular changes to occur following deafferentation.
Ribosomes, also known as ribonucleoprotein performs the activity of translating genetic code. These are made up of two subunits that associate when the synthesis is initiated. A typical ribosome of eubacteria has been found to constitute 57 various molecules (54 and 2 rRNAs) and then dissociate into large (50S) and small (30S) subunit. The small subunit carries out the initiation complex formation, genetic information decoding and also restrains the codon anticodon interactions. The large subunit carries out the catalyzing of bond formation and also creates a path for budding polypeptide chain.
While the sequence of RNA and proteins are encoded for by the nucleotide sequence in DNA (the genes and genomes), polysaccharides which play important roles in physiology are not encoded for by genetic information, but rather by the spatial and temporal activity of enzymes that synthesize these polysaccharides.
Ribosomes play a major role of accumulating amino acid to prepare specific type of . These are very essential for carrying out cell activities. The DNA or deoxyribonucleic acid first creates RNA (messenger RNA or mRNA) via DNA transcription process. This is followed by the translation of genetic message from mRNA into in the process of DNA translation. More elaborately, the whole process can be expressed as the synthesis carried out by the ribosomes in which the assembling sequence of amino acids for synthesis is carried out and are specified by mRNA. This synthesized mRNA is then forwarded to cytoplasm for further synthesis continuation. The two subunits of ribosomes bind or coil around mRNA polymers in the cytoplasm and then synthesize by taking the help of RNA transfer according to the code of genetic. This entire synthesis process is also known as central dogma.