advanced micro exam #2

why do bacteria and viruses make good model systems for genetic studies?

• fast generation time

• short genomes

• easy to grow in high populations

• same underlying biochemistry from DNA sequence to proteins

what is a plasmid?

small loop of DNA with a few genes that aren’t essential to the organism's growth (found in prokaryotes)

• can contain virulence factors that are problematic

why does DNA with higher GC content denature at a higher temperature than DNA wih higher AT content?

GC bonds (3H) have more hydrogen bonds than TA bonds (2H)

what roles do tRNA, mRNA, and rRNA play in protein synthesis ?

tRNA: carries the correct amino acid to the polypeptide chain in the ribosome

mRNA: messenger between DNA and protein; photocopies genes from DNA to RNA

rRNA: aligns RNA to tRNA, and ribosome; catalyzes peptide bond formation between amino acids

tRNA and rRNA both more stable than mRNA*

label mRNA, rRNA, and rRNA in the figure

i. tRNA

ii. mRNA

iii. rRNA

new bacteriophage just dropped. the base composition of the phage is: A (15%), C (20%), G (30%), and T (35%). what can you conclude about the genome of the virus?

the virus has a single-stranded DNA

a certain DNA sample is found to have a makeup consisting of 22% thymine. what is the percentage of A,C, and G?

A= 22%

C = 28%

G = 28%

(add A & T, subtract from 100 and divide by 2)

what are some differences in chromosomal structures between prokaryotes and eukaryotes?

prokaryotes —> single, circular, short chromosome within the nucleoid; haploid chromosomes

eukaryotes —> multiple chromosomes arranged linearly within the nucleus; diploid chromosomes

in the chromatin of eukaryotic cells, which regions of the chromosome would you expect to be more compact: the regions that contain genes being actively copied into RNA or those that contain inactive genes?

the regions that contain inactive genes

how do prokaryotes and eukaryotes manage to fit their lengthy DNA inside of cells? what is the name of the class of enzymes that helps with this process, and what is the name of the specific enzyme that bacteria use (good antibiotic target)?

supercoiling: process where DNA is twisted and condensed to fit into the genome

• topoisomerases help with this

• DNA gyrase: topoisomerase unique to prokaryotes (antibiotic target)

what is the main challenge eukaryotes have when replicating the genome? what enzyme helps with this challenge in single-celled eukaryotes?

the ends of the linear chromosome (telomeres) remain unpaired and get progressively shorter as cells continue to divide

• telomerase: prevents degradation of telomeres

what is the main challenge prokaryotes have when replicating the genome? what enzyme helps with this challenge in prokaryotes?

after replication, DNA chromosomes must be separated from each other

• topoisomerase IV: cuts one of the circular chromosome to separate them

• dna ligase: seals the break

can two observably different cells have the same genotype? explain.

yes! because the genotype is the full collection of genes contained in a cell, and they’re not expressed at all times

different phenotypes arise based on the environment

what is the purpose of RNA processing in eukaryotes? Why don’t prokaryotes require similar processing?

protects RNA from degradation and initiates translation by ribosomes (adding 5’ cap and 3’ poly A tail)

• prokaryotes don’t have a nucleus or non-coding introns that require splicing

what are the steps involved in RNA processing?

• 5’ cap is added

• 3’ poly-A tail is cleaved

• splicing (removal of introns)

below is a DNA sequence. envision that this is a section of a DNA molecule that has separated in preparation for replication, so you are only seeing one DNA strand. construct the complementary DNA sequence (indicating 5’ and 3’ ends).

 

DNA Sequence: 3’- T A C T G A C T G A C G A T C -5’

complementary sequence: 5’ ATGACTCACTGCTAG 3’

below is a DNA sequence. Envision that this is a section of a DNA molecule that has separated in preparation for transcription, so you are only seeing the antisense strand. Construct the mRNA sequence transcribed from this template.

 

Antisense DNA strand: 3’ – T A C T G A C T G A C G A T C- 5’

sequence: 5’ AUGACUGACUGCUGCUAG 3’

below is an antisense DNA sequence. translate the mRNA molecule synthesized using the genetic code, recording the resulting amino acid sequence, indicating the N and C termini.

 

Antisense DNA strand: 3’ – T A C T G A C T C A C G A T C- 5’

sequence: 5’ AUGACUGAGUGCUAG 3’

Met-Thr-Glu-Cys

why does translation terminate when the ribosome reaches a stop codon? what happens?

because the sequences don’t code for any amino acid

previous example: a nonsense codon reaches A site on the ribosome and release factors that cause the last amino acid at the P site to detach

how does the process of translation differ between prokaryotes and eukaryotes?

prokaryotes —> 70s ribosome; amino acid is formylated into methionine; Shine Dalgarno (sequence upstream of start codon AUG); occurs simultaneously

eukaryotes —> 80s ribosome; methionine isn’t formulated; methionine isn’t formulated; occurs temporarily and physically separate

why is it more likely that insertions or deletions will be more detrimental to a cell than point mutations?

insertions/deletions frequently cause frameshift mutations

what are the 3 main forms of horizontal gene transfer (HGT)? briefly define all 3.

1. transformation: occurs when a prokaryote takes up naked DNA found in its environment (from dead/lysed cells)

2. transduction: occurs when a bacteriophage transfers bacterial DNA from one host to another during sequential infections

3. conjugation: process of transferring DNA directly from one prokaryote to another by means of a conjugation pilus

describe two ways in which chromosomal DNA from a donor cell may be transferred to a recipient cell during the process of conjugation.

1. F-plasmid occasionally integrates into bacterial chromosome through recombination, forming an Hfr cell; it can be excised imprecisely and bring chromosomal genes with it

2. Hfr cells sometimes treat the entire chromosome like an F-plasmid and attempt to transfer using rolling circle replication

what happens when a nonsense mutation is introduced into the gene encoding transposase within a transposon?

synthesis of the transpose would end early, resulting in a nonfunctional version of the enzyme

transpoon would become permanently integrated and unable to jump/move

what’s an operon? what’s the difference between an inducible operon and a repressible operon (suggestion: practice using correct vocabulary/terminology as you explain this)?

genes controlled by the same promoter and transcribed together into a single mRNA molecule in prokaryotes (some needed some not!)

inducible operon: inducer molecule removes a repressor from the operator immediately upstream of the operon

repressible operon: repressor isn’t attached to the operator until an inducer attaches to the repressor which blocks RNA polymerase from moving foward with transcription

what are two ways that bacteria can influence the transcription of multiple different operons simultaneously in response to a particular environmental condition?

1. production of alarmones (small intracellular nucleotide derivatives) in response to enviromental stress

2. changing of sigma factor in RNA polymerase for transcription; changes its recognization of new promoters

label the following in the figure: ribosomal E, P, and A sites; mRNA; codons; anticodons; direction of translocation; small ribosomal unit; large ribosomal unit

a. growing polypeptide

b. incoming amino acid

c. ribosomal E, P, and A sites (L to R)

d. codons

e. small ribosomal unit

f. direction of translocation

g. mRNA

h. anticodons

prior to the elucidation of the genetic code, prominent scientists had predicted that each mRNA codon, coding for one of the 20 amino acids, needed to be at least 3 nucleotides long. why is it not possible for codons to be any shorter?

because shorter combinations (less than 3) don’t have enough unique codons for each amino acid

what makes an organism transgenic? what forms of horizontal gene transfer do humans use to intentionally make an organism transgenic?

if it contains recombinant DNA from a different organism

humans use —> transformation, transduction, conjugation

why was the discovery of Thermus aquaticus important for the field of genetics?

produces a heat-stable version of DNA polymerase that survives high temperatures in PCR

in chain-termination sequencing, how is the identity of each nucleotide in a sequence determined?

fluorescently labeled ddNTPs that mark the location of DNA fragments at different lengths

how are proteins separated in SDS-PAGE?

by size

what are restriction enzymes used for, broadly speaking?

to cut DNA at palindromic sites

which is more efficient: blunt-end cloning or sticky-end cloning? why?

sticky end cloning —> because they can easily anneal to complementary nucleotides on inserted DNA fragment

what’s the difference between reverse-transcriptase PCR and real-time quantitative PCR (qPCR)?

reverse transcriptase PCR: used to obtain DNA copies of a specific mRNA molecule; detects whether a certain gene has been expressed

real-time quantitative PCR (qPCR): uses fluorescence to monitor the increase in a double-stranded template during the PCR reaction as it occurs

what are some advantages of cloning human genes into bacteria to treat human diseases caused by specific protein deficiencies?

• lowering immune reactions in proteins (eliminating pathogens)

• continuous expression of human bacteria

what is a reporter gene and in what situation(s) might a reporter gene be useful?

encode for easily observable characteristics that can be attached to genes of interest

• gives reseacher info about the gene of interest’s location and expression

how do fluorescent DNA probes work, in general? what are some applications where you might want a fluorescent probe?

short single-stranded pieces of DNA that bind to a specific target sequence; if the sequence is present, it’s highlighted by light

southern blotting, flurouscent in situ hybridization (fish), quantitative PCR, microarrays

when would a scientist want to generate a cDNA library instead of a genomic library? what virus-derived enzyme is used to create a cDNA library?

in research about genes being expressed by a particular organism

• reverse-transcriptase from HIV

what is the Ames test used for? Describe how the Ames test works.

used to identify carcinogens and mutagens.

• isolates auxotrophic bacteria for a particular amino acid based on a recent mutation

• bacteria are cultured in liquid media with carcinogen/rat liver and plated onto agar that doesn’t have the missing amino acid

• compare growth to the same strain with rat liver, no carcinogen, and place on the same media

result: if there’s more growth on mising amino acid plate its considered mutagenic/carcinogenic

draw and label one complete PCR cycle (include temperatures)

a viral genome was sequenced and the composition of the genome was A (24%), U (32%), C (21%), G (23%)? what type of genome does the virus have?

single stranded RNA

why are TA bonds often found in this region rather than several GC bonds?

fewer H bonds and easier to denature (break)

what class of enzymes is responsible for assisting in supercoiling and preventing overwinding in DNA?

topoisomerase

during what process is mRNA synthesized?

transcription

in additon to physically holding tRNA and mRNA close together, what role does rRNA play in protein synthesis?

catalyzing the formation of peptide bonds

which type of RNA is least stable, especially in prokaryotes?

mRNA

what are the 3 main steps involved in DNA replication, mRNA, transcription, and translation?

1. initiation

2. elongation

3. termination

which is faster, DNA replication in Salmon or Salmonella?

salmonella

what enzyme is used to separate complementary DNA strands during DNA replication?

helicase

fragments of DNA synthesized on lagging strand during replication are referred to as?

Okazaki fragments

many eukaryotic organisms contain the enzyme ____ to remedy the fact that DNA replication results in unpaired nucleotides at the end of linear chromosomes

telemorase

at the termination step of DNA replication in prokaryotes, the enzyme topoisomerase IV is used to solve what problem?

link circular chromosomes

what is the name of the strand mRNA uses as a template during transcription?

anti sense strand

what modifications are made to mRNA after transcription in eukaryotes?

5’ cap, 3’ poly-A tail, splicing

translate the mRNA code into amino acid sequence: AUGUUAGGC

Met Glu Gly

there are 3 important sites on 50S ribosomal subunit during translation what are they in the order a tRNA molecule would experience them?

A, P, E

what is the main signal that initiates termination of translation?

stop codon

what type of organism can perform transcription and translation simultaneously?

prokaryotes

what type of mutation results in early termination of the polypeptide chain?

nonesense mutations

what type of mutagen results in the formation of thymine dimers?

UV radition

what is the purpose of the Ames test?

identifying carcinogens and mutagens

what mechanisms of horizontal gene transfer results in the transfer of the F-plasmid from 1 cell to another?

conjugation

what type of transcription factor promotes transcription of an operon?

repressor

how does the presence of lactose allow for transcription of the lac operon?

removes repressor

what mechanisms of prokaryotic horizontal gene transfer are used by humans to create transgenic organisms?

transformation, transduction, conjugation

what type of enzyme is used to cut DNA into fragments based on location of palindromic sequences known as recognition sites?

restriction enzymes

a ____ is a collection of an organisms genes stored as a recombinant DNA in either bacterial cells or bacteriophages

genomic library

what are the 3 steps (in correct order) in one PCR cycle?

denature (95°), anneal (55°), extension (72°)

how can we visualize a specific DNA sequence? (especially useful for microarrays and southern blotting)

fluorescence probes

what molecule derived from certain viruses is used to create cDNA using only expressed genes?

reverse transcriptase

where was Thermas aquaticus(the organism used to produce heat-stable DNA polymerase) first isolated?

yellowstone national park