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lacIphenotype 

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  • LacIon Xgal = white colonies on a Xgal + IPTG=white colonies. Molecular explanation = the lac operon in the LacImutation is uninducible, it is not expressed when lactose is added to the media (when glucose is low the LacImutation abolishes the ability of the lacI repressor to bind to the inducer. In this mutation the repressor is locked in the active form. 
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what is the difference between genome, methylome, epigenome, transcriptome and the proteome 
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  • The genome: the genome is the DNA sequence of a species. it is generally the same in all the somatic cells (non-sex cells) of the species and does not change within the cell or when the cell divides into daughter cells 
  • The methylone – the methylome is the pattern of methylation on the DNA in the genome. The methylation pattern is typically different in cells from different tissues and can change within a cell in response to numerous factors including signals, environment age etc.
  • The epigenome – the epigenome includes the nucleosome, the DNA, the pattern of all methylated cysteines, the pattern of all modifications to the chromatin, transcription factors bound to the chromatin and the remodeling complexes bound to the chromatin. The epigenome is different in every tissue 
  • The transcriptome – the transcriptome is the RNA transcribed from genes in a cell or tissue. The RNA profile is used interchangeably with transcription. The transcriptome also changes in cells in response to signals, environment, age, etc. 
  • The proteome – the proteome is the full complement of proteins expressed in a cell or tissue. The proteome is closely related to the transcriptome and like the transcriptome differs between cells of different tissues. The proteome also changes in cells in response to signals, environment, age etc. 
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conversion of closed chromatin to open chromatin (30nm to 10nm fibre) 

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  • a pioneer transcription factor binds to DNA or to a nucleosome 
  • this transcription factor recruits a chromatin remodeling g complex which modifies an adjacent nucleosome
  • this increases accessibility to the DNA and allows binding of a second transcription factor 
  • the second transcription factor recruits a histone acetyl transferase (HAT) enzyme which acetylates the histones and opens the chromatin 
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lac -d phenotype 

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  • LacI-d on Xgal = blue colonies on a Xgal + IPTG = blue colonies. Molecular explanation = a single LacI-d can inactivate a lac tetramer even though the other three are wild type. The repressor activity requires all DNA sites in the tetramer to be active if one of the repressor protein in the tetramer is abnormal this affects the whole tetramer
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LacOphenotype 


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  • LacOon Xgal + blue colonies on Xgal + IPTG = blue colonies. Molecular explanation. = mutations in the DNA binding site of the repressor are constitutive because the repressor cannot bind the operator. Therefore DNA polymerase is free to express lacZ constantly 
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what is the principle of the chromatin immunoprecipitation assay 


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  • It is very difficult or not practical to carry out EMSA and DNA foot printing are typically carried out in vitro and are limited for use in vivo. Chromatin immunoprecipitation assay has been developed to study protein – DNA interaction in vivo 
  • The principle and method of ChIP assay are; the protein/transcription factors bind to DNA in the nucleus in a chromatin context. Certain agents such as formaldehyde or uv light can be used to cross-link-proteins to the DNA in a whole cell or intact nucleus. After the cross-linking the DNA with the cross-linked proteins can be purified and sheared into small fragments. An antibody that recognizes the protein of interest is then added to the mixture. The antibody binds specifically to the protein of interest which in turn has the DNA to which it binds attached to it. A secondary antibody linked to beads recognizes the first antibody is then added to the mixture. The beads are isolated by centrifugation or other means. The cross link is dissolved, and the DNA bound to the captured protein is released – the released DNA is amplified by PCR using specific primers or by ligating adapters to the DNA and then carrying out PCR using primers designed against the adapters. Once amplified the and is then sequenced to identify the exact sequence bound by the protein. The ChIP method can be adapted to investigate all protein binding sites in the genome. Over 4,000,000 binding sites have been identified using this method 
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Gal1 gene in yeast 

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  • Regulation of Gal1 gene in yeast
  • Gal1 is the first gene in the pathway induced by galactose. Gal1 encodes galactokinase which phosphorylates galactose to galactose-1-phosphate
  • Gal4 is an activator in yeast (Saccharomyces cerevisiae)
  • Gal4 activates transcription of galactose metabolizing genes in yeast 
  • Gal1 is one of these genes 
  • Gal4 binds to 4 sites 275bp upstream of Gal1 in presence of galactose and activates transcription of gal1 1000 fold 
  • Gal4 is active only in the presence of galactose
  • Even in the absence of galactose gal4 is found bound to its sites upstream of the gal1 gene 
  • But it does not under these circumstances activate that gene because the activating region is bound by a protein called Gal80
  • In the presence of galactose Gal80 undergoes a conformational change. The activating regions are revealed and the Gal1 gene is activated
  • In the presence of glucose Mig1 site between UASg and Gal1P and recruits Tup1
  • Tup1 in turn recruits a deacetylase which deacetylates local nucleosomes
  • If Tup1 is fused to a DNA binding domain and a DNA binding site for the DNA binding domain is engineered upstream of different gene, tup1 will repress expression of the gene 
  • When glucose is withdrawn or depleted the proteins Sak1, Elm1 and Tos3 phosphorylate and activate Snf1
  • Snf1-P then enters the nucleus and phosphorylates Mig1
  • The phosphorylated repressor is exported from the nucleus, allowing transcriptional initiation to occur
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how does the conversion. of the 30nm fibre two the 10nm fibre be achieved 

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  • Histone acetylation results in conversion of 30nm fiber to 10nm fibre 
  • Conversion of the 30nm fiber to the 10nm fiber 
  • Is essential for transcription and can be achieved by the coordinated and successive actions of multiple factors 
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30nm to 10nm fibres in detail 

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  • Activators recruit multiple coregulators which act in a coordinated manner to convert closed chromatin to open chromatin 
  • Recruited entities include: 
  • ATP-dependent remodeling complexes which displace the impeding nucleosomes 
  • Complexes with HAT, histone methyltransferases and histone kinases – these alter histone-histone and histone DNA interaction by acetylation, methylation, and phosphorylation of histones 
  • Coregulators can also modify activators and other coregulators, and alter their interactions 
  • Activators recruit the mediator complex which leads to formation of basal transcription/preinitiation complex with RNA pol II and GTFs. RNA pol II is modified by phosphorylation of its C-terminal tail and transcription initiates 
  • Each promoter and chromatin context is unique and as such the transcription activation of each gene is essentially unique 
  • They key players: 
  • Pioneer TF binds to DNA or a nucleosome 
  • This TF recruits a chromatin remodeling complex which modified adjacent nucleosome and increases accessibility to the DNA 
  • This allows binding of a second TF 
  • This TF recruits a histone acetyl transferase (HAT)
  • Histone acetylation results in conversion of 30nm fiber to 10nm fiber 
  • There is about 2 meters of DNA in the human nucleus. The nucleus is about 0.01mm in diameter. Therefore, major compaction of DNA is needed for DNA to fit into the nucleus. DNA in cells is wrapped around histone octamer giving a 10nm beads on a string structure. These bead structures can be further condensed to 30nm fibers. These 10nm and 30nm fibers and their associated proteins are called chromatin. Chromatin is hugely organized. Loosely compacted chromatin is 10nm in diameter and is easily accessible, the 10nm fiber is called euchromatin. Compacted chromatin is the 30nm fiber and is quite inaccessible to the enzymes and factors associated with transcribing DNA. 30nm fiber called heterochromatin. Chromatin states are dynamic and sections of 30nm chromatin are regularly remodeled to 10nm chromatin and vice versa. The main purpose of chromatin remodeling is to modulate the accessibility of the DNA chromatin to factors involved in gene transcription and DNA replication, recombination, and repair. The main aspects of chromatin remodeling are; (1) histone modification by specific enzymes including histone acetyltransferase and kinases. Nucleosomes with acetylate tails have reduced ability to form 30nm fibers (2) ATP-dependent chromatin remodeling complexes. These alter the nucleosome composition, and we use energy from ATP so the first ATPase complex described was the yeast SWI/SNF complex. The main function of chromatin remodeling ATPases is to remodel chromatin by; disrupting, sliding nucleosomes and changing the composition of the nucleosome. There are four distinct families of chromatin remodeling ATPases. They all have ATPase domains but differ with respect to their other functional domains, chromodomains, bromodomain and ATPase domain to be successful it must bind specific accessible regions (epitopes) on the nucleosome.
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TESTE DEIN WISSEN

LacIPhenotype 

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TESTE DEIN WISSEN
  • LacIphenotype on a X-gal plate = blue colonies on a Xgal + IPTG= blue colonies. Molecular explanation: in a lacI- mutation the lac repressor is not active, and the repressor will not repress the lac operon. Thus, in the presence of low glucose and absence of lactose the lac operon is expressed 
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TESTE DEIN WISSEN

what is CRISPR an abbreviation for 

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clustered regularly interspaced short palindromic repeats 

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what is the genome 

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  • the genome is the DNA sequence of a species. it is generally the same in all the somatic cells (non-sex cells) of the species and does not change within the cell or when the cell divides into daughter cells 
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Q:

lacIphenotype 

A:
  • LacIon Xgal = white colonies on a Xgal + IPTG=white colonies. Molecular explanation = the lac operon in the LacImutation is uninducible, it is not expressed when lactose is added to the media (when glucose is low the LacImutation abolishes the ability of the lacI repressor to bind to the inducer. In this mutation the repressor is locked in the active form. 
Q:


what is the difference between genome, methylome, epigenome, transcriptome and the proteome 
A:
  • The genome: the genome is the DNA sequence of a species. it is generally the same in all the somatic cells (non-sex cells) of the species and does not change within the cell or when the cell divides into daughter cells 
  • The methylone – the methylome is the pattern of methylation on the DNA in the genome. The methylation pattern is typically different in cells from different tissues and can change within a cell in response to numerous factors including signals, environment age etc.
  • The epigenome – the epigenome includes the nucleosome, the DNA, the pattern of all methylated cysteines, the pattern of all modifications to the chromatin, transcription factors bound to the chromatin and the remodeling complexes bound to the chromatin. The epigenome is different in every tissue 
  • The transcriptome – the transcriptome is the RNA transcribed from genes in a cell or tissue. The RNA profile is used interchangeably with transcription. The transcriptome also changes in cells in response to signals, environment, age, etc. 
  • The proteome – the proteome is the full complement of proteins expressed in a cell or tissue. The proteome is closely related to the transcriptome and like the transcriptome differs between cells of different tissues. The proteome also changes in cells in response to signals, environment, age etc. 
Q:

conversion of closed chromatin to open chromatin (30nm to 10nm fibre) 

A:
  • a pioneer transcription factor binds to DNA or to a nucleosome 
  • this transcription factor recruits a chromatin remodeling g complex which modifies an adjacent nucleosome
  • this increases accessibility to the DNA and allows binding of a second transcription factor 
  • the second transcription factor recruits a histone acetyl transferase (HAT) enzyme which acetylates the histones and opens the chromatin 
Q:

lac -d phenotype 

A:
  • LacI-d on Xgal = blue colonies on a Xgal + IPTG = blue colonies. Molecular explanation = a single LacI-d can inactivate a lac tetramer even though the other three are wild type. The repressor activity requires all DNA sites in the tetramer to be active if one of the repressor protein in the tetramer is abnormal this affects the whole tetramer
Q:

LacOphenotype 


A:
  • LacOon Xgal + blue colonies on Xgal + IPTG = blue colonies. Molecular explanation. = mutations in the DNA binding site of the repressor are constitutive because the repressor cannot bind the operator. Therefore DNA polymerase is free to express lacZ constantly 
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Q:

what is the principle of the chromatin immunoprecipitation assay 


A:
  • It is very difficult or not practical to carry out EMSA and DNA foot printing are typically carried out in vitro and are limited for use in vivo. Chromatin immunoprecipitation assay has been developed to study protein – DNA interaction in vivo 
  • The principle and method of ChIP assay are; the protein/transcription factors bind to DNA in the nucleus in a chromatin context. Certain agents such as formaldehyde or uv light can be used to cross-link-proteins to the DNA in a whole cell or intact nucleus. After the cross-linking the DNA with the cross-linked proteins can be purified and sheared into small fragments. An antibody that recognizes the protein of interest is then added to the mixture. The antibody binds specifically to the protein of interest which in turn has the DNA to which it binds attached to it. A secondary antibody linked to beads recognizes the first antibody is then added to the mixture. The beads are isolated by centrifugation or other means. The cross link is dissolved, and the DNA bound to the captured protein is released – the released DNA is amplified by PCR using specific primers or by ligating adapters to the DNA and then carrying out PCR using primers designed against the adapters. Once amplified the and is then sequenced to identify the exact sequence bound by the protein. The ChIP method can be adapted to investigate all protein binding sites in the genome. Over 4,000,000 binding sites have been identified using this method 
Q:

Gal1 gene in yeast 

A:
  • Regulation of Gal1 gene in yeast
  • Gal1 is the first gene in the pathway induced by galactose. Gal1 encodes galactokinase which phosphorylates galactose to galactose-1-phosphate
  • Gal4 is an activator in yeast (Saccharomyces cerevisiae)
  • Gal4 activates transcription of galactose metabolizing genes in yeast 
  • Gal1 is one of these genes 
  • Gal4 binds to 4 sites 275bp upstream of Gal1 in presence of galactose and activates transcription of gal1 1000 fold 
  • Gal4 is active only in the presence of galactose
  • Even in the absence of galactose gal4 is found bound to its sites upstream of the gal1 gene 
  • But it does not under these circumstances activate that gene because the activating region is bound by a protein called Gal80
  • In the presence of galactose Gal80 undergoes a conformational change. The activating regions are revealed and the Gal1 gene is activated
  • In the presence of glucose Mig1 site between UASg and Gal1P and recruits Tup1
  • Tup1 in turn recruits a deacetylase which deacetylates local nucleosomes
  • If Tup1 is fused to a DNA binding domain and a DNA binding site for the DNA binding domain is engineered upstream of different gene, tup1 will repress expression of the gene 
  • When glucose is withdrawn or depleted the proteins Sak1, Elm1 and Tos3 phosphorylate and activate Snf1
  • Snf1-P then enters the nucleus and phosphorylates Mig1
  • The phosphorylated repressor is exported from the nucleus, allowing transcriptional initiation to occur
Q:

how does the conversion. of the 30nm fibre two the 10nm fibre be achieved 

A:
  • Histone acetylation results in conversion of 30nm fiber to 10nm fibre 
  • Conversion of the 30nm fiber to the 10nm fiber 
  • Is essential for transcription and can be achieved by the coordinated and successive actions of multiple factors 
Q:

30nm to 10nm fibres in detail 

A:
  • Activators recruit multiple coregulators which act in a coordinated manner to convert closed chromatin to open chromatin 
  • Recruited entities include: 
  • ATP-dependent remodeling complexes which displace the impeding nucleosomes 
  • Complexes with HAT, histone methyltransferases and histone kinases – these alter histone-histone and histone DNA interaction by acetylation, methylation, and phosphorylation of histones 
  • Coregulators can also modify activators and other coregulators, and alter their interactions 
  • Activators recruit the mediator complex which leads to formation of basal transcription/preinitiation complex with RNA pol II and GTFs. RNA pol II is modified by phosphorylation of its C-terminal tail and transcription initiates 
  • Each promoter and chromatin context is unique and as such the transcription activation of each gene is essentially unique 
  • They key players: 
  • Pioneer TF binds to DNA or a nucleosome 
  • This TF recruits a chromatin remodeling complex which modified adjacent nucleosome and increases accessibility to the DNA 
  • This allows binding of a second TF 
  • This TF recruits a histone acetyl transferase (HAT)
  • Histone acetylation results in conversion of 30nm fiber to 10nm fiber 
  • There is about 2 meters of DNA in the human nucleus. The nucleus is about 0.01mm in diameter. Therefore, major compaction of DNA is needed for DNA to fit into the nucleus. DNA in cells is wrapped around histone octamer giving a 10nm beads on a string structure. These bead structures can be further condensed to 30nm fibers. These 10nm and 30nm fibers and their associated proteins are called chromatin. Chromatin is hugely organized. Loosely compacted chromatin is 10nm in diameter and is easily accessible, the 10nm fiber is called euchromatin. Compacted chromatin is the 30nm fiber and is quite inaccessible to the enzymes and factors associated with transcribing DNA. 30nm fiber called heterochromatin. Chromatin states are dynamic and sections of 30nm chromatin are regularly remodeled to 10nm chromatin and vice versa. The main purpose of chromatin remodeling is to modulate the accessibility of the DNA chromatin to factors involved in gene transcription and DNA replication, recombination, and repair. The main aspects of chromatin remodeling are; (1) histone modification by specific enzymes including histone acetyltransferase and kinases. Nucleosomes with acetylate tails have reduced ability to form 30nm fibers (2) ATP-dependent chromatin remodeling complexes. These alter the nucleosome composition, and we use energy from ATP so the first ATPase complex described was the yeast SWI/SNF complex. The main function of chromatin remodeling ATPases is to remodel chromatin by; disrupting, sliding nucleosomes and changing the composition of the nucleosome. There are four distinct families of chromatin remodeling ATPases. They all have ATPase domains but differ with respect to their other functional domains, chromodomains, bromodomain and ATPase domain to be successful it must bind specific accessible regions (epitopes) on the nucleosome.
Q:

LacIPhenotype 

A:
  • LacIphenotype on a X-gal plate = blue colonies on a Xgal + IPTG= blue colonies. Molecular explanation: in a lacI- mutation the lac repressor is not active, and the repressor will not repress the lac operon. Thus, in the presence of low glucose and absence of lactose the lac operon is expressed 
Q:

what is CRISPR an abbreviation for 

A:

clustered regularly interspaced short palindromic repeats 

Q:

what is the genome 

A:
  • the genome is the DNA sequence of a species. it is generally the same in all the somatic cells (non-sex cells) of the species and does not change within the cell or when the cell divides into daughter cells 
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