E2

How can gene exp be regl in the following processes (transcription, post transcription, translation, post translational)?

Act/ rep, DNA binding prot; RNA stability; rate of translation; prot mod

How do DNA binding prot affect gene exp?

Regl prot that bind to DNA and act as switch for transcription

Define operons and illustrate its structure Fig.16.3

group of bact genes that are transcribed and function together; 2 sections control region (promoter, operator) structural genes

negative inducible control; transcription usually on or off? Regl protein? What binds to switch?

Off, active repressor, inducer

negative repressible control; transcription usually on or off? Regl protein? What binds to switch?

On, inactive repressor, corepressor

positive inducible control; transcription usually on or off? Regl protein? What binds to switch?

Off, activator, inducer

positive repressible control; transcription usually on or off? Regl protein? What binds to switch?

On, activator, corepressor

Explain the control of lac operon in E. coli

Negative inducible control: lac absent repressor binds the operator, notranscription; lac present, some converted to allolactose which acts as an inducer and the repressor is inactive
Positive control: cAMP-CAP complex: act that binds to promoter to transcribe structural genes, occurs during low glucose

Organization of lac operon: regl gene, promoter, operator, structural genes (encode enzymes for metabolism of lactose)

lacI; lacP; lacO; lacZ, lacY and lacA

What does positive control mechanism (cAMP-CAP) do when glucose is present?

override lactose control to decrease transcription of lactose meta genes

Describe the control of trp operon Fig. 16.14

Negative repressible: low W, transcription on; high W repressor binds to operator, transcription off

Chromosome structure organization eukaryotes

Made up of folded nucleosomes

Nucleosome

core particle that consists of chromatin wrapped twice around histone octamer

Chromatin types

Euchromatin: transcriptionally active; heterochromatin: dense and inactive

How is gene expression regl at DNA level?

Chromatin structure remodeling: histone acetylation to open chromatin, activate; deacetylation to return to condensed state, inhibition

How is gene expression regl at Transcription level?

transcriptional activator; GAL4 regl genes that metabolize gal by binding to enhancer, when no gal, no transcription, when gal, transcription activated

How is gene expression regl at mRNA processing level?

Alternative splicing; T antigen gene encodes for both large and small T antigens, SF2 enhances mRNA splicing for small T antigen

Explain RNA interference differences between microRNA (miRNA) and small interfering RNA (siRNA)

siRNA is from mRNA, transposon, or virus and targets the genes from which they were transcribed; miRNA is from RNA transcribed from distinct gene and targets genes other than the ones it was transcribed

Explain RNA interference mRNA cleavage process (RISC, RNA induced silencing complex protein involved)

ssmiRNA or sssiRNA pairs with prot to form RISC and cleaves mRNA, which is degraded

Explain RNA interference translation inhibition process (RISC, RNA induced silencing complex protein involved)

miRNA pairs w prot to form RISC to imperfectly pair w mRNA, which prevents translation

Explain RNA interference transcription silencing process (RITS, RNA induced transcriptional silencing protein involved)

siRNA pairs with prot to form RITS, binds to DNA and causes methylation, restricting transcription

Somatic vs. germline mutations Fig. 18.1

most cases (of cancer), randomly acquired and not passed to child, used in vegetative propagation; present in all cells of body and passed to child (familial cancer)

Transition vs. transversion mutations Fig. 18.3

Most common, same type of nitrogenous base, ex purine to purine; pur to pyr or vice versa

Insertion vs. deletion mutations, compare to sub Fig. 18.2

Adds or delete one or more nuc; affects all downstream coding seq, and may lead to frame- shift mutation, more severe than sub

Expanding tri-nucleotide repeats –Huntington’s disease (mutations caused how) Fig. 18.5

Tri nuc repeats separate and replicate, forming a hairpin, causing part of the template to be replicated twice in the new strand

Nonsense, missense, and silent mutations as defined by effect on translation Fig. 18.6

Premature termination; encodes for new AA; new codon encodes same AA

causes of mutations Biological (4):

Mismatch: wobble sub error and unconventional bp; Strand slippage: if new strand loops, additional nuc, if template loops, nuc deletion; Expanding trinucleotide repeats :special case of strand slippage; transposition: mvmt of TE/transposon to diff location in genome, facilitated by transposase

3 common features during transposition; what kind of effects?

Staggered breaks made by transposase in target DNA with 2 sticky ends, TE joined to ss sticky ends of DNA, DNA repl at ss gaps; 50% spont mutations

causes of mutations Chemical (2):

alkylating agents: donate groups to nuc bases; base analogs: chm with similar structures as nuc that DNA mistakes and uses (5 bromouracil to thymine)

causes of mutations Physical:

UV-radiation causes bonds to form btwn adjacent pyr (thymine) causing distortion, block repl, and inhibit cell div

Define and Explain DNA repair mechanisms Mismatch (Fig 18.36)

Repairs repl errors, including mispaired bases and strand slippage by cutting the mismatch seq after repl complete

Define and Explain DNA repair mechanisms Direct (Fig 18.37)

Repairs pyr dimers and demethylation by returning damaged bases to original state and photolyase which breaks thymine dimer bond

Define and Explain DNA repair mechanisms Base excision (Fig 18.38)

(DNA glycosylases) recognize and remove mod or incorrect bases and pyr dimers

Define and Explain DNA repair mechanisms Nucleotide excision (Fig 18.39)

Repairs lesions like dimers, mod/abnormal bases, and damaged DNA that distort the double helix

List the common properties shared by cancer cells.

Failure to respond to normal controls, cause enhanced cell proliferation, enhanced survival, and reduced diff

Define oncogenes and tumor suppressor genes and understand how they contribute to cancer Fig. 23.5

90% of cancer genes, and promote proliferation and survival of cells from proto oncogenes (responsible for basic cell funct); encode prot that restrict cell growth and div, inact causes loss of funct and cells can grow out of control

Explain different causes for the inactivation of tumor suppressor genes: mutation, epigenetic modification, Fig. 23.14

only nonfunct exp, tsg not exp, DNA methylation

Explain different causes for the activation of oncogenes: gene mutation, retrovirus, gene amplification, and chromosome translocation Figs 23.11, 23.12

Point mut in H Ras for bladder cancer; virus insert RNA into cell, reverse transcription allows the gene to be inserted in host DNA and viral promoters stimulate over exp of proto oncogene; extra chromosomes; chronic myeloid leukemia and burkitt’s lymphoma

Explain the roles of tumor suppressor genes in cell cycle checkpoints, what happens if mutated? Fig. 23.9

RB: binds E2F in G1/S checkpoint to control progression; uncontrolled transcription; p53 halts cell growth and division at G2/M, allowing DNA repair, apoptosis if not repaired; accumulation of damaged cells

How does HPV induce cancer?

Inact TSG p53 and RB

Define the roles of the defective DNA repair genes in cancer

Cells w mutation in DNA repair genes tend to develop additional mutations in other genes

Role of DNA repair genes

Identify and correct DNA to control rate of repl error and efficiency of DNA repair

Describe the principle and procedure of PCR and gel electrophoresis. Figs. 19.4, 19.5 What are the different components and steps of the process?

Components: template DNA, 2 primers, DNA polymerase, and dNTP; steps: heated denaturation of dsDNA into ssDNA, cooling to anneal primers to ssDNA, heat for DNA pol to extend new strands

Illustrate the function of restriction enzymes and its application in gene cloning Fig.19.8

Recognize and cut DNA at specific nuc seq, resulting in sticky ends that can pair with other fragments and create recombinant DNA; need unique restriction sites

Describe the plasmid features (4) and explain its function and application for gene cloning

Have their own origin of repl, repl is indep from chromosomal DNA, have genes that provide antibiotic res, don’t carry essential genes for survival; origin of repl needed for cloning

Describe the procedure of gene cloning

Construct vector by inserting foreign DNA into commercial vector, transfer construct into bacterial cell through electroporation for repl, screen for antibiotic res by observing colonies that remain white (don’t syn B gal)

Cloning plasmid vectors characteristics Fig. 19.6

Origin of repl, selectable marker, unique restriction enz site

Describe Ti (tumor inducing) plasmid structure, function, and application in plant transformation Fig. 19.11

Has transferable DNA (T-DNA) that infects plants and integrates DNA into plant chromosome; was modified as a vector to remove Ti plasmid and replace with gene of interest to created plants with selected traits

Describe the Sanger sequencing procedures and principles (4) Figs. 19.16, 19.17, 19.18

Long time standard, 1st gen, dideoxy seq method, relies on DNA repl; products separated on polyacrylamide gel and band image capture with fluorescent dyes

Function of dNTPs and ddNTPs in sanger seq

dNTP has OH at 3’ to continue seq, ddNTP has H at 3’ to prevent new dNTP from attaching and terminate syn

how is Sanger seq gel read?

Complementary strand obtained starting from bottom, 5’ to 3’

Describe the general differences in capabilities between Sanger sequencing and NGS

NGS largely inc throughput (number of seq rxn that can be simultaneously run)

Define genomics

Studies content, origin, funct, and evolution of whole genetic info of an organism

define and describe methods of genome annotation

process to assign funct to all genes in genome; Ab initio prod: intrinsic method based on detection of gene struc signals (splice sites, poly A, start/stop codon) using statistical algorithms; seq similarity search: alignment of unannotated genome to prot seq in genome of related species to predict gene struc and funct

how to seq DNA? (2)

map based seq: phys mapping; whole genome shotgun seq: simpler, fragmentation of genome into small pieces, seq each fragment, assembly of overlapping seq using computer