Gene Regulation and Transcription Mechanisms

Reagents/Assays:

First, clone β-globin & cDNA. Then, identify the TSS by comparing the cDNA and gene sequence of the DNA. The promoter is then mutated one base at a time using PCR site-directed mutagenesis, creating approximately 250 mutated DNAs. Transfect eukaryotic cells with the promoter and measure the transcriptional activity of a reporter gene via mRNA or protein activity.

Valleys observed in the results indicate mutations in the promoter that decrease transcriptional activity.

Conclusion:

The promoter is mapped by observing its upstream movement from the TTS (Transcription Start Site).

Reagents/Assays:

Incubate G1cN6P with and without Mg²⁺ at varying times. Also, incubate other sugars with similar structures to G1cN6P but different substituents, treating them with Mg²⁺. Run on an autoradiography with a denaturing gel to visualize mRNA cleavage and uncleavage.

Results show that over time, G1cN6P with Mg²⁺ induces cleavage, while other sugars do not.

Conclusion:

A metabolite (like Mg²⁺) may induce mRNA cleavage, which codes for an enzyme that produces the basis of negative feedback.

Background Information:

• TFIID: Recruits transcription machinery to the promoter. Binds to the TATA Box minor groove; a rate-limiting step.
• TFIIB: Orients the complex on the promoter and identifies the template strand.
• TriC: Identifies the non-template strand (CTD is not yet phosphorylated).
• TFIIE: Binds and recruits TFIIH.
• TFIIH: Kinases the CTD (C-terminus domain), allowing transcription to begin.

Hypothesis:

There is an ordered binding and assembly in the pre-initiation complex.

Reagents/Assays:

Use ³²P-DNA (with TATA Box) and combine it with TFIID, TFIIB, TFIIE, TFIIH, and TriC in a test tube. Employ autoradiography and EMSA on a non-denaturing gel to visualize binding.

Conclusion:

Binding occurs in the pre-initiation complex to a promoter in a specific order. Ordered assembly is necessary for binding to occur.

Reagents/Assays:

Treat normal HT, and closed chromatin immature RBC nuclei with a DNase sensitivity assay. Deactivate the DNase and isolate the stripped DNA. Use PCR with β-globulin oligo primer pairs to check for remaining DNA. Alternatively, use PCR with closed chromatin regions to quantify DNA presence. Analyze results using a non-denaturing gel.

Results indicate that higher amounts of closed chromatin correlate with less transcriptional activity.

Conclusion:

The lack of LCR in HT patients increases closed chromatin levels in the gene.

1) Epigenetic Marks in Chromatin:

• CH₃ DNA represses retrotransposon jumping.
• Writer, reader, and eraser proteins deregulate and upregulate gene expression.
• Nucleosomes help compact chromosomes.

2) Undifferentiated Cell Potential:

Replicated cells have similar cell types. Examples include:

• Germ stem cells (totipotent): Create placenta, embryo, and ~210 adult cell types.
• Embryonic stem cells (pluripotent): Create embryo and ~210 adult cell types.
• Adult stem cells (multipotent): Create all ~210 adult cell types.

3) Monoallelic Imprinting Gene Expression:

One allele is expressed due to hypermethylation in GC_n or C_n-DNA imprinting regions. With over ~100 imprinted genes, selection is not random.

X inactivation is random. X inactivation is mediated by XIST (X inactivation specific transcript), allowing for equal X expression in females and males.

1) Rett Syndrome:

Occurs on an X chromosome; MECP2 is mutated and binds to Arg 168 stop codon.

2) Fragile X Syndrome:

Characterized by (>200) CGG repeats due to hypermethylation of the 5′ UTR and the promoter.