ANBI 470.3 Lecture Notes - Lecture 8: Hydrogen Bond, Thrombin, Plasmin

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20 Jun 2018

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Eukaryotic Transcription Regulation

bovine grow hormone gene promoter won't work in bacteria

• bring enhancer in area of......

• transcription factors: equivalent of repressor protein, binding to specific sequences in

they are used to turn up and turn down expression◦

Production of Recombinant Protein General Approach

-choose a host cell (bacteria, mammalian cells, plant, ect.)

oexpression system: term used for the system that you have chosen to manipulate to

make cells

-find/develop a promoter which drives high level, controlled expression of gene

odifferent for each expression system

-generate construct (DNA sequence) which combines promoter with coding sequence for gene of

interest

-deliver DNA to host cell

ovectors include naked DNA, viruses, plasmids

oextrachromosomal or genomic integration (need to decide whether DNA will exist in

extrachromosomal place or integrated in the genome)

-optimize host conditions for protein synthesis

-harvest and purify protein

ogetting it pure enough to inject into an animal is very hard and costly

Production of recombinant Protein Expression Systems:

Bacteria

-simplest system

oshort production timescale

ocan buy core materials easily

-typically plasmid based

obacterial plasmids are autonomous self-replicating extrachromosomal closed circular DNA

(basically viruses of bacteria)

oPlasmid features include:

Origin of replication: DNA sequence that binds DNA polymerases

plasmids take over metabolism of bacteria, one plasmid bind to host

DNA and replicate, want strong origins of replication when intentionally

using this to produce growth hormone

selection marker:

usually an antibiotic resistance gene

bacteria have mechanisms to get rid of plasmids, plasmids make

themselves indispensable, they carry antibiotic resistance

provide bacteria with medium with that it can only survive with the

plasmin in the bacteria

multi-cloning site

high density of restriction sites

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Document Summary

Eukaryotic transcription regulation bovine grow hormone gene promoter won"t work in bacteria: bring enhancer in area of, transcription factors: equivalent of repressor protein, binding to specific sequences in they are used to turn up and turn down expression. Bacteria simplest system: short production timescale, can buy core materials easily typically plasmid based, bacterial plasmids are autonomous self-replicating extrachromosomal closed circular dna (basically viruses of bacteria, plasmid features include: Origin of replication: dna sequence that binds dna polymerases plasmids take over metabolism of bacteria, one plasmid bind to host. Example plasmid-based: expression vector example: something you can buy to support any gene you wanted in plasmid bacteria three restriction enzyme sites that are unique. Ptac: steroid version of the lactose expression system, notified to be a very strong promoter, that will drive expression of down stream gene fusion protein: protein produced from rdna, fusion of genes into a protein, recombinant dna.

Related Questions

QUESTION 1

How is a bone marrow transplant able to treat someone with leukemia?

		The donor blood contains antibodies that destroy cancer cells
		Donor blood contains stem cells that regenerate cells in the immune system
		Bone marrow contains induced pluripotent stem cells that turn into red blood cells
		Bone marrow contains tumor supressor genes that slow down leukemia growth
		Bone marrow contains pluripotent human embryonic stem cells

1.2 points

QUESTION 2

What would happen if siRNAs designed to target mRNA from an oncogene were introduced into cancer cells?

		Increased genomic instability would result from adding foreign nucleic acids
		New mutations would arise in the oncogene
		The oncogene would be converted back into a proto-oncogene
		The siRNAs would compete with the oncogene's active site
		Production of the oncogene protein would decrease

1.2 points

QUESTION 3

What is the purpose of chromatin-remodeling enzymes in human cells?

		The enzymes control the rate of translation of an mRNA transcript
		The enzymes add phosphate or methyl groups to histone tails
		The enzymes slide nucleosomes farther apart or closer to each other
		The enzymes send damaged proteins to the proteasome for degradation
		The enzymes cause DNA looping to occur

1.2 points

QUESTION 4

If you wanted to resurrect the mammoth, how would you do it?

		Use somatic cell nuclear transfer to insert the mammoth genome into the egg of an elephant
		Use transcription factors to re-program adult cells from elephants into mammoth cells
		Change the gene expression patterns of elephants to increase production of mammoth genes
		Use siRNAs to decrease expression of elephant-specific genes in an elephant embryo

1.2 points

QUESTION 5

How is eukaryotic transcription initiated?

		RNA polymerase is recruited to the start codon by transcription factors
		RNA polymerase begins transcription at the enhancer sequence
		RNA polymerase is activated by binding to GTP
		An initiator tRNA binds to the ribosome
		A complex of proteins forms at the promoter of a gene

Multiple choice questions, I need the CORRECT ANSWERS, 3500points

1.The promoter is in region_____________.

a.1

b.2

c.3

d.4

e.5

2.The stop codon is identified in region __________

a.4

b.5

c.6

d.7

e.8

3.A mutation is region 8 would be expected to:

a.disrupt transcription

b.disrupt ribosome

c.disrupt DNA polymerase binding

d.disrupt splicing

e.none of the above

4.Which is responsible for the transcription of structural genesin eukaryotes?

a.RNA polymerase I

b.RNA polymerase II

c.RNA polymerase III

d.DNA polymerase

E.RNA Synthesis

5.The eukaryotic TATA box is directly bound by a basaltranscription factor known as:

a.TBF

b.TBP

c.TAF

D.TPP

E.TAT

6.mOST TRANSCRIPTIONAL ACTIVATOR proteins in eukarypotesfunction in the cell as:

a.monomers

b.dimers

c.trimers

d.tetramers

e.pentamers

7.The basal level of transcription of eukaryotic genes is quitehigh in vitro systems. This is because:

a.activation are present

b.formation of chromatin dos not occur in vitro

c.basal transcription factors are present

d.a and c

e.none of the above

8.Transcriptional repression by methylation of DNA involves:

a.methylation of adenine (A) bases of the sequence GATC

b.methylation of cytosine (C) bases of th sequence GATC

c.mthylation of cytosin (C) bases in CpG nuclotides

d.Methylation of guanosine (G) bases in CpG nucleotides

e.acetylation of hisone proteins

9.A eukaryotic DNA sequence that can increase the level oftranscription is called:

a. a transcription factor

b.a transcritpional activator

c.TATA box

d.an enhancer

e.a co-activator

10.Which is generally true of eukaryotic transcriptionalregulation?

a.activators and reprssors bind to the promoter while basalfactors bind to the enhances

b.basal factors bind to the promoter, while activators bind tothe enhancers

c.activators bind to the promoter while basal factors andrprssors bind to th enhancers

d.repressors bind to the promoter whil basal factors andactivators bind to the enhancers

e.activators and basal factors bind to the promtoer whilereprssors bind to the enhancers

11.You wish to sequence the promoters region of the mouse generab.To do this, you need to construct a library and then search forthe gene sequence.You have a DNA probe based on the sequence of theprotein coding region.Which of th follwoing strategies would youfollow in order to carry this out?

A.construct a genomic library and screen for the promoter usingPCR

b.construct a cDNA library and screen for the promotoer usingPCR

c.construct a genomic library and screen for the promoter usingPCR

d.Construct a cDNA library and screen for the promoter usingcolony hybridization

e.all of the above

12.if one were to examine th clones in a cDNA library derivedfrom th heart muscle cells and the clones in a cDNA library derivdfrom prancreatic cells, you would find that:

a.the two libraries would hav the same cloned sequences

b.the two libraries would have completely different clonesequences from one another

c.the two libraries would share some common clond sequences, butwould each have some sequences unique to that library, not to befound in the other

d.the sequences from both would also b the same as those foundin the genomic library

e.none of th above

13.In a particular population at Hardy-Weinberg equilibrium, thefrequency of recssive allele b for a particular gne is0.01.Assuming that there are only two alleles for the gene, whatwould b th expected frequency for dominant allele B in th nextgeneration?

a.0.01

b.0.02

c.0.98

d.0.99

e.none of the above

14.White wool in the sheep is controlled by a dominant allele R,and black wool by the recessive allele r.In an isolated popualtionof 6530 sheep, 514 are balck, 4981 are hterozygous, 1035 arhomozygous white. When will this population be expected to reachHardy-Weinberg equilibrium?

a.in the next generation

b.in two generations

c.it already is in equilibrium

d.impossible to tell

e.it will not reach equilibrium

15.A population has th following genotypic disturbutions:

A^M A^M=1787, A^M A^O=2011, A^OA^O=2331.Is this population atH-W equilibrium?

a.yes, with allele frequencies of p=0.45562 and q=0.54437

b.yes, with frequencies of p=0.54437 and q=0.37862

c.no, with allele frequencies of p=0.45562 and q=0.37862

d.no, with xpectd genotypic ratios of p^2=1272, 2pq=3040, andq^2=1826

e.yes, with expctd genotypic frequencies of p^2=1778, 2pq=2015,and q^2=2336

16.The result of the Luria-Delbruck fluctuation exprimentindicatd that:

a. most bacteria are naturally resistant to phage

b.a low level of bacteria become resistant to phage by mutationupon exposure to phage

c.most bacteria become resistant to phage by mutation uponexposure to phag

d.bacteria become resistant to phag by random spontaneousmutation

e.bacteria become resistant to phag by acquiring resistanttraists upon repeated exposure

17.DNA probes are used for:

a.restriction ndonuclease analysis

b.sequencing

c.PCR

d.DNA library analysis

e.all of th above

18.Colony hybridization is used to:

a.isloate a gne of interest froma DNA library

b.analyze the presence or number of copies of a particular DNAsegment in a chromosome

c.determine the size of a gene or sequencing of interest

d.replace the more tediuos method of library screening.

e.all of the above

19.For the colony hybridization procedure, which of thefollowing is true:

a.individual colonies are isolated and analyzed by DNAsequencing

b.the DNA fragments are separated by size prior to transfer ontonitrocellulose membrane

c.the plasmid DNA must be denatured before hybridization

d.the relative positions of DNA fragments on the agarose gel arepreserved after transfer to the nitrocllulose membrane

e.the cloned target sequence is detected by staining withthidium bromide

20.The following strand of DNA is to be used for sequencing3'CGACTGACTCGACGGCCTAGCTATAG.The primer is GCTGACTG.The number ofDNA fragments resulting from the ddCTP tube is expectd to be:

a.7

b.5

c.8

d.4

e.6

21.Which of the following is not true of heterozygosity for agene deletion?

a.it is often detrimental if there is a gene dosage effect.

b.as long as one wildtype copy of any deleted gene is presnt onthe homologous chromosome, no phenotypic consequnces willresult.

c.it could be detected by Southern blot analysis

d.it could be detected by PCR analysis

e.none of the above

22.Robertsonian translocation arise from:

a.breaks at or near the chromosome of two acrocentricchromosomes

b.breaks at or near th centromeres of two metacentricchromosomes

c.breaks at or near th centromeres of one acrocentric and onemetacentric chromosome

d.breaks in a large and a small chromosomes

e.transposable elements

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ANBI 470.3 Lecture Notes - Lecture 8: Hydrogen Bond, Thrombin, Plasmin

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