BackA full-length eukaryotic gene is inserted into a bacterial chromosome. The gene contains a complete promoter sequence and a functional polyadenylation sequence, and it has wild-type nucleotides throughout the transcribed region. However, the gene fails to produce a functional protein. What changes would you recommend to permit expression of this eukaryotic gene in a bacterial cell?
A full-length eukaryotic gene is inserted into a bacterial chromosome. The gene contains a complete promoter sequence and a functional polyadenylation sequence, and it has wild-type nucleotides throughout the transcribed region. However, the gene fails to produce a functional protein. List at least three possible reasons why this eukaryotic gene is not expressed in bacteria.
Methylation of H3K9 by itself silences genes, but if H3K4 and H4K20 are also methylated, the combination of modifications stimulates transcription. What conclusions can you draw about this?
A particular type of anemia in humans, called β-thalassemia, results from a severe reduction or absence of the normal β-globin chain of hemoglobin. However, the γ-globin chain, normally only expressed during fetal development, can functionally substitute for β-globin. A variety of studies have explored the use of the nucleoside 5-azacytidine for the expression of γ-globin in adult patients with β-thalassemia.
How might 5-azacytidine lead to expression of γ-globin in adult patients?
A particular type of anemia in humans, called β-thalassemia, results from a severe reduction or absence of the normal β-globin chain of hemoglobin. However, the γ-globin chain, normally only expressed during fetal development, can functionally substitute for β-globin. A variety of studies have explored the use of the nucleoside 5-azacytidine for the expression of γ-globin in adult patients with β-thalassemia.
Explain why this drug may also have some adverse side effects.
DNA methylation is commonly associated with a reduction of transcription. The following data come from a study of the impact of the location and extent of DNA methylation on gene activity in eukaryotic cells. A bacterial gene, luciferase, was inserted into plasmids next to eukaryotic promoter fragments. CpG sequences, either within the promoter and coding sequence (transcription unit) or outside of the transcription unit, were methylated to various degrees, in vitro. The chimeric plasmids were then introduced into cultured cells, and luciferase activity was assayed. These data compare the degree of expression of luciferase with differences in the location of DNA methylation [Irvine et al. (2002). Mol. and Cell. Biol. 22:6689–6696]. What general conclusions can be drawn from these data?
Describe the organization of the interphase nucleus. Include in your presentation a description of chromosome territories and interchromatin compartments.
Present an overview of the manner in which chromatin can be remodeled. Describe the manner in which these remodeling processes influence transcription.
List three types of alternative splicing patterns and how they lead to the production of different protein isoforms.
Consider the CT/CGRP example of alternative splicing shown in Figure 16.9. Which different types of alternative splicing patterns are represented?
Nonsense-mediated decay is an mRNA surveillance pathway that eliminates mRNAs with premature stop codons. How does the cell distinguish between normal mRNAs and those with a premature stop?
Present an overview of RNA interference (RNAi). How does the silencing process begin, and what major components participate?
RNAi may be directed by small interfering RNAs (siRNAs) or microRNAs (miRNAs); how are these similar, and how are they different?
miRNAs target endogenous mRNAs in a sequence-specific manner. Explain, conceptually, how one might identify potential mRNA targets for a given miRNA if you only know the sequence of the miRNA and the sequence of all mRNAs in a cell or tissue of interest.
Competing endogenous RNAs act as molecular “sponges.” What does this mean, and what do they compete with?