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 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