Question

molecular biology

Section C (40 marks) Answer ALL questions from this Section 5. You have isolated total RNA from muscle cells and constructeda muscle cDNA library. You wish to study the regulatory region of a muscle-specific cDNA gene (gene M) that you have previously identified. 6 (a) For your study, you need to isolate a genomic clone of gene M. Why isa cDNA clone of gene M not appropriate for your study? (2 marks) (b) Outline the steps you would take to isolate the genomic gene M. (4 marks) (c) You have isolated a genomic clone for gene M which contains the entire coding region as well as 1.2 kb of the upstream regulatory (promoter) region. A map of genomic gene M is shown below. You have also prepared a reporter lacZ fusion gene construct by cloning a 1.3 kb BamHl upstream fragment of gene M into a plasmid vector containing the lacz gene. This fusion gene, when injected into cultured muscle cells, is capable of expressing β-galactosidase activity. What experiment would you carry out next to determine if the fusion construct is developmentally regulated'? (3 marks) (d) Using an antibody that is specific for the B-galactosidase protein, you analyzed the tissues of a "gene M-lacZ" transgenic mouse and found that "protein M" is also expressed in bone cells of the mouse. How would you determine whether the same transcript is expressed in muscle and bone cells? If different size mRNA transcripts were obtained, suggest some possible reasons for the observation? (3 marks) (e) You decided to analyze the promoter region of gene M to identify regulatory DNA elements that control gene M expression in muscle and bone cells. The results obtained with the lacZ fusion constructs containing different fragments of the 5' promoter region of gene M are shown below. Which DNA region(s) are important for specific expression in muscle? and in bone? Explain. (8 marks) BamH BamHI 100 Expression Expression in bone DNA in subclone in muscle 2 3 4. 6 7

Answer 1

The isolation of high-quality RNA lacking polysaccharides, proteins, phenolic compounds, genomic DNA or secondary metabolites is crucial for the multiple techniques used to investigate gene expression patterns and functions during plant growth and development. Such techniques are reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR (RT-qPCR), complementary DNA (cDNA) library construction, Northern blotting and RNA sequencing. Phenolic compounds are oxidized to form quinones, which bind irreversibly to nucleic acids and proteins.[6] Polysaccharides can co-precipitate and degrade RNA, which renders RNA unsuitable for downstream applications. In addition, N. cadamba contains abundant alkaloids that bind RNA, increasing the difficulty of isolation.Currently commercially available kits, such as those manufactured by Takara, Promega, Omega and Qiagen, did not enable extraction of RNA from all N. cadamba tissues (data not shown), may be because the efficiency of the spin columns used in these kits decreases significantly in the presence of alkaloid compounds.Ouyang et al. isolated RNA from four young tissues to clone genes, using an RNeasy Plant Mini Kit (Qiagen), but the concentration and/or quantity of the extracted RNA were not sufficient for cDNA library construction, Northern blotting or RNA-sequencing. The only exception was the RNA extracted from the cambium region of the tree. Тo our knowledge, there is no report on total RNA extraction from the other tissues of N. cadamba.

RNA has been successfully extracted from various plant species, rich in phenolic compounds and polysaccharides, using the cetyltrimethyl ammonium bromide (CTAB) protocol, but this method is time consuming. A combination of the CTAB-based RNA extraction method and a commercial plant RNA extraction kit has been successfully utilized. However, RNA extracted from some N. cadambatissues, according to these protocols, smeared severely (data not shown). Here, we combined a modified CTAB lysis buffer and purification on RNA mini columns (Omega), which not only reduced the required time but also yielded large quantities of high-quality total RNA.

cDNA synthesis and real-time RT-PCR

Total RNA (0.5 μg) was reverse-transcribed to first-strand cDNA, according to the manufacturer's instructions in the PrimeScript™ RT Master Mix kit (Takara, Japan). According to Xiao et al. for further confirmation of the quality of total RNA extracted by this protocol, serial dilutions (1:1, 1:5, 1:25, 1:125 and 1:625) of the single-stranded cDNA were subjected to quantitative real-time RT-PCR. Real-time PCR was performed following the standard SYBR Premix Ex Taq™ kit (Takara, Japan) protocol, using a final volume of 20 μL which includes 2 μL of reverse-transcribed cDNA and 2 μL of 5 μmol/L forward and reverse primers. Thermocycling conditions were as follows: an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 58 °C annealing for 30 s and 72 °C extension for 15 s and an infinite hold at 10 °C. The specificity of the PCR amplification procedures was checked using a heat dissociation protocol (from 65 to 95 °C) after the final PCR cycle and was examined by electrophoresis in 2% agarose gel. The sequences of forward and reverse primers for the cyclophilin gene (JX902587) were 5′-GACAGGAGGAGAATCTATCTATGG-3′ and 5′-AACCTGCCCAAACACCACAT-3′, respectively.