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Genetics: Molecular Cloning, Transgenics, and Gene Regulation

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  • What are regulatory transcription factors (RTFs)?

    RTFs regulate the rate of transcription in target genes by binding to regulatory elements, distinct from basal transcription factors needed for basal transcription.

  • How does chromatin status affect transcription?

    Open chromatin allows RTFs and polymerase access for transcription; closed chromatin restricts access, inhibiting transcription.

  • Name factors that influence chromatin accessibility.

    Nucleosome positioning, histone variants, histone modifications, and DNA methylation affect chromatin accessibility and transcription.

  • What is the role of histone variants in gene expression?

    Histone variants replace standard histones in some nucleosomes and affect centromeres, telomeres, DNA repair, and X-chromosome inactivation.

  • Define the open complex in transcription.

    The open complex forms when DNA strands separate at the promoter, allowing RNA polymerase II to initiate transcription.

  • What is epigenetics?

    Epigenetics studies heritable, reversible gene expression changes without altering DNA sequence, involving DNA methylation, chromatin remodeling, and histone modifications.

  • How do noncoding RNAs participate in gene regulation?

    Noncoding RNAs can bind DNA, RNA, proteins, or small molecules to regulate gene expression.

  • Describe gene silencing by interfering RNAs.

    miRNA or siRNA are processed by Dicer into ~25 nt fragments, loaded onto RISC, which binds mRNA to degrade it or block translation.

  • What is recombinant DNA technology?

    It involves combining DNA fragments from different sources into vectors to create novel genotypes and phenotypes.

  • What features make plasmids useful as cloning vectors?

    Plasmids have an origin of replication, unique cloning sites, and selectable markers like antibiotic resistance genes.

  • How do restriction enzymes facilitate cloning?

    Restriction enzymes cleave DNA at specific sequences, generating sticky or blunt ends for ligation of DNA fragments.

  • What is blue-white selection in cloning?

    Insertion of DNA disrupts the lacZ gene in plasmids, preventing metabolism of X-gal and resulting in white colonies, indicating successful cloning.

  • Why use cDNA instead of genomic DNA for cloning eukaryotic genes?

    cDNA lacks introns, representing only exons, making it suitable for protein expression studies.

  • What is Gibson Assembly?

    An exonuclease-based method that ligates multiple DNA fragments with overlapping ends in one reaction without restriction enzymes.

  • Outline the general steps to create a transgenic corn plant expressing Bt toxin.

    Amplify Bt gene and corn promoter, clone into vector, ligate fragments, transform bacteria, verify recombinant DNA, then transform corn.

  • What is reproductive cloning?

    Cloning an entire organism to produce genetically identical individuals, e.g., Dolly the sheep.

  • What was a notable issue with Dolly the sheep's cloning?

    Dolly had shortened telomeres, possibly due to the donor somatic cell's age, affecting longevity.

  • How are transgenic animals made in mice?

    By germline transformation using embryonic stem cells injected into blastocysts, then implanted into foster mothers.

  • What is gene targeting in embryonic stem cells?

    Introducing specific changes into a gene via homologous recombination to insert or replace DNA sequences.

  • How does the P element system work for gene insertion in Drosophila?

    A two-plasmid system where transposase excises and inserts the gene of interest into the genome at specific sites.