To determine whether a gene is located in a compact region of the chromatin fibe

Question

To determine whether a gene is located in a compact region of the chromatin fiber, scientists use a hybridization assay using genomic DNA digested with DNAseI as a template a radiolabeled single stranded cDNA probe that matches a portion of the studied gene.

1) The hybridization curve showing the fraction of unbound radiolabeled probe (C/C0) as a function of the product C0 x t has a specific sigmoid shape that can be divided into 3 portions (1, 2, and 3). C is the concentration of radiolabeled probe at time t and C0 is the initial concentration of the radiolabeled probe (at time t=0)

Explain how the kinetics of hybridizations leads to these 3 portions of the curve.

2) The following figure shows the re-association curves for genomic DNA extracted from liver and muscle. The Y-axis represents the percentage of free (single-stranded) myoglobin cDNA.

The myoglobin gene is expressed in muscle and repressed in liver. Identify the curve (A or B) was obtained by mixing DNAse I treated-genomic DNA from muscle and myoglobin cDNA? Explain your reasoning.

Explanation / Answer

1).
This given graph shows the individual hybridization curve showing the fraction of unbound radiolabeled probe. The hybridization curve of the target always increases monotonically; since the target is at a lower concentration than other species, it does not control the initial phase of hybridization.

In the presence of the target, the competitor will always be displaced, while in the absence of the target, the competitor will be the highest affinity species and monotonically increase. The low affinity background is at higher concentration, so it initially grows rapidly, but, because of its low affinity, it is quickly displaced.

However, the high affinity background grows more slowly and is displaced more slowly. Even in a complex environment, the equilibrium distribution can be predicted via thermodynamic models, but it is not practical to measure experimentally.

In the kinetic regime, the distribution of bound species is time-dependent, as should be clear here. Also shown in the figure is the composite background signal as determined by fitting with the three-component model.

2).

The myoglobin gene is regulated stringently in muscle fibers, such that high myoglobin expression is observed in mitochondria-rich, oxidative myofibers compared with glycolytic fibers.

In addition, we demonstrated a previously unsuspected role for an intragenic E-box motif as a negative regulatory element contributing to the tightly regulated variation in myoglobin gene expression among particular myofiber subtypes.

Human myoglobin gene promoter fragments linking either 2 or 0.4 kb of 5? flanking sequences to a luciferase reporter gene were expressed more abundantly in the soleus (SOL) than the EDL muscles, reflecting the pattern of the endogenous myoglobin gene.

Related Questions

Navigate

• Browse (All)
• Browse T
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.