S1B4 - Regulation of Gene Expression Flashcards Preview

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Flashcards in S1B4 - Regulation of Gene Expression Deck (27):
1

What is the cDNA problem and what is the way around it?

cDNA Library Problem: If one wants to express a eukaryotic protein, in E. coli most eukaryotic genes will not work because the genes contain introns. In that case a way around the problem is to make a cDNA or complementary DNA library using reverse transcriptase.

2

How could one isolate a gene for a particular protein with unknown nucleotide sequence?

If one has a purified protein and amino acid sequence from even a small portion of the protein then one can synthesize an oligodeoxynucleotide probe (oligo, a nucleotide probe) which would be complimentary to one of the strands of the cloned (DNA) gene. The probe is made radioactive by attachment of  32P to its 5' terminal.

Example: Screening for the cloned cDNA for the  chain of fibrinogen. Since the amino acid sequence of the lambda chain of the fibrinogen is known, we can use a synthetic oligonucleotide that would be expected to hybridize with a portion of lambda chain cDNA. But which oligonucleotide to use? The pentapeptide  sequence 334-338 contains amino acids that have least degenerate codons.

3

Which amino acides are coded for by only one codon?

Which amino acids have minimum woblle?

Trp and Met are coded for by only one codon.

Asn and Lys have minimum wobble.

4

Describe immunoscreening.

Immunoscreening:

  • Antibodies to the protein of the cloned DNA can be produced; for example, to human insulin.
  • Colony proteins can then be transferred to a filter and  exposed to an antibody to the protein.
  • The antigen-antibody complex is then detected by either radioactive or colorimetric methods.

5

What is the basic concept of the Polymerase Chain Reaction (PCR)?

Basic concept: 

  • Limitations in the amount of genetic material from a patient can be a major obstacle to performing DNA analysis for genetic disease.
  • The polymerase chain reaction (PCR) provides a method for the enormous amplification of a selected segment of DNA.
  • The method permits as high as 1010 fold increase in the concentration of a single DNA molecule.
  • PCR has broad application in molecular biology and can be used to amplify a gene from even a single cell for analysis.

6

What is the basic concept of RADP analysis?

RAPD analysis (random amplified polymorphic DNA):

  • A PCR technique which produces a fingerprint of an organism.
  • That would allow identification of disease causing organisms for example.
  • In principle amplification of DNA is carried out with a mixture (10 or more different) random, short (about 10 bases) oligonucleotide primers.
  • With this set of primers different organisms will produce unique PCR products visualized as a pattern of bands on agarose gel electrophoresis. 

 

7

What's the difference between constitutive and inducible proteins?

Constitutive proteins are those proteins which are found in relatively constant amounts in cells. Their rates of synthesis and degradation are relatively constant in a given cell type regardless of the metabolic state of the cell.

Inducible proteins are those proteins which are usually found in low concentrations in a given cell type but whose concentration can be increased markedly in response to the proper stimulus.

8

What must happen to a gene in order for it to be "expressed"?

A gene is said to be expressed when it is transcribed and also translated.

9

Which is more important for regulation of gene expression, gene amplification and gene rearrangements, or transcriptional activators?

Regulation of gene expression can occur at any of a number of points.

Note: Gene amplification and gene rearrangements appear to be exceptions rather than the rule. Transcriptional activators are most important for gene regulation in eukaryotes.

10

During oogenesis in the fruit fly, the DNA (genes) which codes for the chorion (eggshell protein) increase in number apparently due to a process of repeated initiations during DNA synthesis. 

What is this process called?
 

Gene Amplification: The replication of a gene responsible for forming a certain protein is greatly increased; in other words the cell makes extra copies of the gene.

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11

Methotrexate is an inhibitor of dihydrofolate reductase (DHFR), an enzyme indirectly important to the synthesis of thymidine and thus DNA.

How can cancer cells develop resistance to methotrexate?

Gene amplification.

Cancer cells can develop resistance to methotrexate by increasing the number of genes for DHFR to 40 to 400copies per cell. These extra copies recombine to somehow form double minute chromosomes. These extrachromasomal elements are inherited in an unstable fashion.

12

What is a gene called that moves from site to site within a genome?

What mechanism of gene expression are these involved in?

What is the significance of these genes in bacteria?

Transposons:  “Jumping Genes.”

  • Most genes are immobile but certain sequences of DNA can move from site to site within the genome.
  • These “jumping” fragments of DNA are usually short (< 2 kilobases in length) and contain within them coding sequences for the enzyme transposase which carries out the actual “jumping”.
  • Although humans have many transposons in their genomes there is little to fear because many are inactive and they jump infrequently.
  • Transposons have been found to cause human gene mutations, although infrequently. (i.e. 1/100 hemophilia patients).
  • Much more important, transposons in bacteria sometimes contain antibiotic resistance genes and can “jump” onto plasmids which can be taken up by sensitive strains as one mechanism for resistance transfer.

 

13

The three regions in the gene for the light chain in  immunoglobulins (V, J and C) are involved in what specific mechanism of control of gene expression?

Immunoglobulins:The millions of potential antibodies that an animal can possess are generated through gene rearrangement. For example, the genes for the variable regions of light chains of immunoglobulins are composed  of 3 regions (V, J and C). There are over 500 VL(light) genes, 5 or 6 JL and 10 to 20 CL. One VL plus one JL plus one CL would code for one variable region of one light chain; for example, VL376, JL3CL17.

14

What is the general description of transcriptional activators?

Where on a gene are transcriptional activators usually located?

Transcriptional activators:

  • Most eukaryotic genes are regulated by activators and transcription is very low in the absence of an activator.
  • The site of activation of the gene is often located some distances from the transcription start site; i.e., usually upstream (5'), but could also be located downstream (3') side.

15

What are enhancers in the context of transcriptional activation?

Enhancers (What are they).

  • Enhancers are cis acting DNA sequences.
  • They activate transcription only in the chromosome (DNA molecule) where they reside.
  • Enhancers are often effective in either orientation.
  • Enhancers can affect transcription even when they are located thousands of base pairs away from the transcription start site. They are sometimes found within an intron or at the 3' end of a gene.
  • Enhancers affect the transcription of any gene in their vicinity.

16

What's the difference between basal expression of a gene and regulated expression of a gene?

 Basal expression of the gene is due to TATA and CAAT boxes

Regulated expression is due to enhancer elements.

17

How do transcription activating enhancers work?

Enhancers (How they work)   

  • Transcriptional activators bind to the enhancer and the enhancer stimulates RNA polymerase transcription of specific genes.   
  • Multiple activators used for most genes

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18

If SV40 is cloned together with the promoter and structural gene for beta globulin, it will markedly increase transcription of the beta globulin gene. And, it works whether cloned in a 5' --> 3' or 3' --> 5' direction.

How many base pairs is SV40?

What does the above experiment show?

Examples of Enhancers

  • SV40
    • The prototype enhancer was isolated from the animal virus SV40.
    • It is a 72bp sequence which can enhance the transcription of many genes.
    • For example, if SV40 is cloned together with the promoter and structural gene for beta globulin, it will markedly increase transcription of the beta globulin gene. And, it works whether cloned in a 5' --> 3' or 3' --> 5' direction.
    • This experiment shows that permissive enhancers can stimulate any promoter in the vicinity.

19

Metallothionein (mt) enhancers: Zinc by stimulating the mt enhancer increases the transcription of the gene for the metal-binding my protein. If mt enhancer is cloned with the promoter for thymidine kinase (tk) followed by the gene for human growth hormone.

What does this experiment show?

 

Metallothionein (mt) enhancers: Zinc by stimulating the mt enhancer increases the transcription of the gene for the metal-binding my protein. If mt enhancer is cloned with the promoter for thymidine kinase (tk) followed by the gene for human growth hormone. This experiment shows that certain enhancers are not specific for a given promoter.

20

Pancreatic beta-cell tissue enhancer: Cloning this enhancer (fron the insulin gene) with the polyoma large T antigen (cancer causing gene) caused pancreatic tumors in transgenic mice.

What does this experiment show?

Pancreatic beta-cell tissue enhancer: Cloning this enhancer (fron the insulin gene) with the polyoma large T antigen (cancer causing gene) caused pancreatic tumors in transgenic mice. This experiment showed that enhancers can be tissue-specific. Presumably a factor in the pancreas activated by beta-cell tissue enhancer.

21

Liver specific enhancers: Albumin has an enhancer (named PE) which binds a liver specific transcription factor.

What does this explain?

Liver specific enhancers: Albumin has an enhancer (named PE) which binds a liver specific transcription factor. Explains why albumin is synthesized exclusively by the liver.

22

What are four types of binding between transcription factor proteins and DNA?

  1. Helix-Turn-Helix: These proteins consist of at least 2 connected coils of a-helix with one of the coils lying flush in a major groove (covers 5 base pairs, bp) and the other “turned” away about 90 degrees C.
  2. Zinc fingers:  Zinc coordinates loop domains in the protein with each domain binding to the major groove of DNA.  Multiple “fingers” are found in these type of proteins.  (Estrogen receptor contains several zinc fingers, for example.)
  3. Leucine Zipper: Actually this is a modified or smaller region of a much larger DNA binding protein.
    1. A leucine at every 7th position in the “zipper” alpha helix. The leucine zipper holds the DNA binding α-helical domains in the correct position for interaction with the DNA.
  4. Loop Binding: The transcription factor NF-Kappa β binds to the major groove of DNA through multiple interactions which bonds or loops in the protein structure. Amazingly this transcription factor shows similarities to Ig light chain antigen binding domains (see below, NfKappa B left(DNA center),  IgG right). 
     

23

What is the importance of the JAK-STAT family of transcription factors?

JAK-STAT is a signaling pathway vital for the function of cytokines, soluble mediators of inflammatory and immune responses.

Examples of cytokines are the interleukins and interferons which bind to receptors on T-cell and activate the JAK-STAT pathway.

JAKs are [Janus Kinases] tyrosine kinases which phosphorylate specific tyrosines on STATs (Signal Transduction Activators of Transcription) upon cell surface receptor ligand binding.

STATs are a family of transcription factors which migrate to the nucleus upon tyrosine phosphorylation, bind to DNA specific sites and stimulate the transcription of genes which mediate cytokine function.  

24

What are the three important STATs and what is associated with loss of each of their genes?

  1. Loss of STAT 1 -- loss of response to viral or bacterial infection. 
  2. Loss of STAT 4 no T helper 1 cell function. 
  3. Loss of STAT 5No breast development or lactation.

25

What do post-transcriptional nuclear controls play a role in?

Post-transcriptional Nuclear Controls

  • Post-transcriptional controls appear to play a role in regulating which mRNAs actually reach the cytoplasm.
  • Messenger RNA stability is important for regulating message levels post-transcriptionally.

26

What can cause an rna message to be unstable?

Message stability. Certain messages are unstable (targeted by nucleases) because the message contains an extra A-U rich sequence near the 3 end of the message (in the untranslated region).

27

What do you need to know about micro-RNA (mi-RNA)?

  • Micro-RNAs are small RNA transcripts (20-30 nucleotides) which regulate the levels and translation of messages. 
  • They bind to complementary sequences on messenger RNA to form double-stranded segments which signal nuclease degradation or block translation on the ribosome.

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