Western Blot - overview

Control of Gene Expression - part 1

Attenutation = Premature termination of transcription. 

Tat-Tar gene regulation in HIV:

Normally, in host cell, transcription of HIV virus will be terminated by host cell to prevent trascription occur. To avoid this, HIV transcribes a Tat protein that binds to the stem loop Tar on RNA sequence of HIV,  allows for elongation of mRNA to be made.

Riboswitch :

It is a part of mRNA that can "sense" small molecules in which binding of these small molecules will affect the gene activity. Riboswitch often can be divided into 2 parts: one is aptamer which binds the small molecules that regulates gene expression, and the other is platform that responses upon the conformation change based on the interaction of small molecules with aptamers.





Alternative RNA splicing:

This is one form of control transcription based on alternative of splicing. There are 4 patterns of alternative RNA splicing: option exon, optional intron, mutually exclusive exons and internal splice site. Because of this mechanism, even with a small amount of genes in cell can express million different proteins based on the stimulation under some certain conditions. This alternative splicing can be controlled in many different ways. Many distinct ways to control and alternate RNA splicing includes the intron sequence ambiguity, regulated splicing ( involve both repressor and activator), change in the site of RNA trasnscript cleavage.

Intron sequence ambiguity: 

the standard spliceosome mechanism for removing intron sequences is unable to distinguish clearly between two or more alternative pairing of 5' and 3' splice site, so that different choices are made by chance on different transcripts that occurs on transcriptional level.

Regulated splicing:

Splicing is controlled by both repressor and activator at the trancriptional level.

A change in the site of RNA transcript cleavage:

While transcribing to synthesize new RNA, for somehow, the elongation can be controlled by RNA cleavage reaction that is catalyzed by additional factors and results different size of RNA strand and alternate the C-terminus of the resultant protein. During the 3' end cleavage for additional pol A tail, the factor CPSF will recognize the cleavage site, allowing for pol A tails to be added. However, in some certain circumstances, CPSF might recognize different cleavage site, and results different size of mature mRNA.

For example: B-cell switches from anchored Ab to secreted Ab. Note that anchored Ab has hydrophobic sequence at C-terminal that allows it to anchor into cell membrane while secreted Ab does not have hydrophobic sequence. Also, that anchored Ab has a longer sequence than secreted Ab due to the cleavage process.

RNA editing:

this mechanism alters the nucleotide sequences of RNA transcripts once the are synthesized and thereby changes the coded message. This process involves guide RNA ( that is complementary in sequence to one end of the region of the transcript to be edited) or deamination of nucleotides ( change from A to I or C to U).

If the edit occurs in a coding region, it can change the amino acid sequence of the protein or produce a truncated protein.

If edits occur outside the coding sequences, it can affect the pattern of pre-mRNA splicing, the transport of mRNA from the nucleus to cytosol, or efficiency of RNA  being translated.

Indirect ELISA

Wash solution: is used to wash the unbound materials
Stop solution: as for the second antibody that coupled with the enzyme-labelled conjugation. This enzyme will be bound with reagent ( normally added in the end) to cause color development. The color is directly proportional to the amount of bound antibody-antigen interactions. Thereby, the stop solution is used to stop the enzyme-substrate reaction and allows for color to develop.

The relationship between bacteriophage and host polymerase for the transcription of its own gene

Since this process is very complex, I will simplify this mechanism

- Normally, bacteriophage does not encode its own RNA pol, so it must rely on the host RNA pol to transcribe its gene.

- Transcription from early bacteriophage promoter is initiated by the host holoenzyme containing the host sigma factor.

- There will be a competition for host sigma factor to bind with between bacteriophage promoter and host promoter. In this case, if bacteriophage wants host holoenzyme binds it promoter, it must make sure that its promoter must be strong ( meaning that it must have high affinity for host holoenzyme).

- When this process is accomplished, this transcription produces viral sigma factor. This viral sigma factor will steal RNA pol from host to synthesize its own gene and a new sigma factor ( let just denote as sigma *.

- The second sigma factor ( sigma*) will again bind to host RNA pol for its transcription to generate other part of viral structure like capsid.

- Note: always, there will be a competition for the binding of host holoenzyme between viral promoters and host promoter, as well as viral sigma factor with the new viral sigma factor*. This process totally is dependent on affinity of the molecular interactions.

Holliday junction

-First, the homologous chromosomes are both nicked at identical locations.

-Then, the strand from on side of the nicks invade and base-pairs with the other homologous complementary strand.

-The invading strand is covalently linked to the original strand at the nick site, forming Holiday junctions.

- The Holiday junction migrates away from the original nick sites in which this is called branch-migration.

- As it does so, the DNA strands are swapped between the chromosomes. This creates the heteroduplex regions on both chromosomes where minor base sequence differences between homologous chromosomes result a region of DNA with low percent of mismatches base-pairs.

Now, what happens if cleavage happens? To answer this, there are 2 ways that they can happen

1/
- At some point of the branch migration process,  breaks are made in the DNA that end the migration and resolves the entangles DNA into two separate chromosomes. This results both non-recombinant chromosomes and recombinant chromosomes. If cleavage takes place at the cross-strands by endonuclease, then after the ligation within chromosomes, there will be two non-recombinant chromosomes with short heteroduplex regions.

2/
- Alternatively, if one rotates one DNA helix 180o ( a process called isomerization), and if the cleavage takes place between uncross strands by endonuclease, ligation can produce recombinant chromosomes with short heteroduplex regions.


  

Nadia

Nadia ! The legend !

A big guy in Virology ! ( Source from actual Jack Johnson lab)

Oh I can say .... WOW 

NAME	POSITION TITLE
John E. Johnson	Professor, Dept. of Molecular Biology
EDUCATION/TRAINING
Carthage College, Kenosha, WI	B.A.	1967	Chemistry
Iowa State University, Ames, IA	Ph.D.	1972	Physical Chemistry
Purdue University, W. Lafayette, IN	Post Ph.D.	1972-1975	Virus Crystallography

Positions and Employment
1972-1975	Postdoctoral Research Associate, Dept. of Biological Sciences, Purdue University, W.Lafayette, IN.
1975-1977	Visiting Assistant Professor, Department of Biological Sciences, Purdue University, W.Lafayette, IN.
1978-1981	Assistant Professor, Department of Biological Sciences, Purdue University, W.Lafayette, IN.
1981-1985	Associate Professor, Department of Biological Sciences, Purdue University, W.Lafayette, IN.
1985-06/30/1995	Professor, Department of Biological Sciences, Purdue University, W.Lafayette, IN.
07/01/1995-Present	Professor, Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA.
01/01/1986-07/31/1986	Visiting Professor, Institute Biologie Moleculaire et Cellulaire, Strasbourg, France.
01/01/1993-07/31/1993	Visiting Member, The Scripps Research Institute, La Jolla, CA.
07/01/1998-Present	Adjunct Professor, University of California, San Diego, La Jolla, CA.
12/2010-Present	Eldon R. Strahm endowed chair in Structural Virology
Other Experience and Professional Memberships
1985-1989	Member, National Institutes of Health Biophysical and Biochemical Study Section.
1989	Purdue Chapter Sigma Xi Faculty Research Award Recipient
1991-Present	Board of Governors, Consortium for Advanced Radiation Sources, University of Chicago.
1994-1998	Board of Scientific Councilors, National Cancer Institute, Frederick, MD.
1996-2002	Board of Scientific Councilors: NIAMS (Arthritis, Muscoloskeletal and Skin Diseases).
1999-Present	Scientific Advisory Board (Chairman) NIGMS/NCI Synchrotron Beamline Development.
2000-2005	Scientific Advisory Board Finnish National Science Academy.
1998-Present	Scientific Advisory Board Donald Danforth Plant Science Center, St. Louis, Missouri.
2004-Present	Scientific Advisory Board International Society for Nanoscale Science, Computation and Engineering.
2007-Present	Scientific Advisory Board NIH National Resource for Automated Molecular Microscopy (NRAMM)
2007-Present	Scientific Advisory Board Institute for Protein Research, Osaka University, Osaka, Japan
2008-2010	Scientific Advisory Board European Structural Biology Consortium (SPINE II)
2010-2013	Scientific Advisory Board National Biomedical Computation Resource UCSD
2010-2013	Proposal Review Panel Linac Coherent Light Source (LCLS) Stanford
1994-1998	International Committee on Taxonomy of Viruses.
1998-Present	Editorial Board, Virology.
1992-1998	Editorial Board, Biophysical Journal.
1998-2003	Editorial Board, Journal of General Virology.
1998-Present	Editorial Board, Structure.
1998-Present	Editorial Board, Journal of Molecular Recognition.
2002-2005	Editorial Board, Journal of Structural Biology.
2002-2012	MERIT award NIGMS GM34220-19.
6/2002	Co Organizer (with Margaret Killian) FASEB Virus Assembly Meeting, Saxtons River VT.
1/5/2000-1/20/2000	Travel fellowship/lectureship Japanese Society for the Promotion of Science.
1/7/2001-1/23/2001	Travel fellowship/lectureship Taiwan National Science Committee.
10/1/2003-10/30/2003	Visiting Lecturer National University of Singapore.
1996	Kaesberg Lecturer, American Society for Virology Annual Meeting, London, Ontario.
3/2004	Mathers Lecturer, Department of Chemistry, University of Indiana, Bloomington.
4/2004	Colter Lecture, Department of Biochemistry, University of Alberta, Edmondton, Alberta.
7/2004	Keynote (Harold S. Ginsberg) Lecture, American Society for Virology Annual Meeting, Montreal, Quebec.
3/2006	Frank Nelson Distinguished Lecturer, Montana State University, Bozeman.
2007	Carthage College Distinguished Alumnus
2008	American Chemical Society (San Diego Section) Distinguished Scientist
2009-2012	Appointed to the Council of The National Institute of General Medical Sciences NIH
Patents
11/1996	Modified Plant Viruses as Vectors European Patent No. 92 907 583.6.
2/1998	Modified Plant Viruses as Vectors US Patent, approved/No.: pending.
2007	Dow Chemical Co.Global Innovator Award (US patent 7,208,655)