was reduced in the knock-out mutant cells. The N-terminal region of Protein X is 100% conserved between mouse and humans at both the amino acid and the nucleotide level. The predicted mouse mRNA sequence is shown below where the AUG corresponds to the translational start site (AUG). 5’-AUGUUUACAGAGGGGAAU... -3’ Q3a) Using a total RNA sample obtained from human cells, what is the first primer you would use in an RT reaction to generate a single stranded cDNA that corresponds to this mRNA? (Write a 10 nt sequence of the primer from 5'-3') Q3b) Design a 12 nt upstream primer that would allow you to amplify a full length double stranded cDNA that corresponds to Human Protein X by PCR after having performed the initial RT reaction (Write the 12 nt sequence of the primer from 5'-3') Q3c) In what cellular compartment is the mouse Protein X required in order to fulfill its essential function?
Genetic Recombination
Recombination is crucial to this process because it allows genes to be reassorted into diverse combinations. Genetic recombination is the process of combining genetic components from two different origins into a single unit. In prokaryotes, genetic recombination takes place by the unilateral transfer of deoxyribonucleic acid. It includes transduction, transformation, and conjugation. The genetic exchange occurring between homologous deoxyribonucleic acid sequences (DNA) from two different sources is termed general recombination. For this to happen, an identical sequence of the two recombining molecules is required. The process of genetic exchange which occurs in eukaryotes during sexual reproduction such as meiosis is an example of this type of genetic recombination.
Microbial Genetics
Genes are the functional units of heredity. They transfer characteristic information from parents to the offspring.
Knock out mice that are mutant for a gene product X die. Variants of the X gene that were reintroduced through homologous recombination indicate that variant proteins that lack the C-terminal region of the protein cannot rescue the lethality (did not allow them to live), and always localized to the cytoplasm. Those that retained the C-terminal region rescued the lethality (conferred viability!) and were consistently localized within the nucleus. RNA-seq analysis of the mutant cells vs the wild-type cells indicated that the expression of many genes that are essential for neural function was reduced in the knock-out mutant cells.
The N-terminal region of Protein X is 100% conserved between mouse and humans at both the amino acid and the
5’-AUGUUUACAGAGGGGAAU... -3’
Q3a) Using a total RNA sample obtained from human cells, what is the first primer you would use in an RT reaction to generate a single stranded cDNA that corresponds to this mRNA? (Write a 10 nt sequence of the primer from 5'-3')
Q3b) Design a 12 nt upstream primer that would allow you to amplify a full length double stranded cDNA that corresponds to Human Protein X by PCR after having performed the initial RT reaction (Write the 12 nt sequence of the primer from 5'-3')
Q3c) In what cellular compartment is the mouse Protein X required in order to fulfill its essential function?
Q3d) What motif could be present to direct this protein to its correct destination?
Q3e) From the cellular localization data and the RNA-seq analysis, what is the likely function of Protein X in regulating the expression of these neuronal genes?
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