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Consurf activity- LHCII- Dmitriy, Keats, Jason

2bhw_asr_r_500LHCII- Light Harvesting Complex II is part of photosynthesis in higher plants, algae and some bacteria. It is the primary chlorophyll binding protein, andis associated with both of the Photosystems. LHCII also controls the lamellar structure of the thylakoid membrane.  It exists as a trimer its natural state composed of a varying combination of three LHCII genes (LHCb1, LHCb2, and LHCb3). Is important to note that LHCII is a transmembrane protein consisting of three thrans membrane helices per monomer.

 

Source of Structure:

Mechanisms of Photoprotection and Nonphotochemical Quenching in Pea Light-Harvesting Complex at 2.5 A Resolution.
Standfuss, J.,   Terwisscha Van Scheltinga, A.C.,   Lamborghini, M.,   Kuehlbrandt, W.
(2005) Embo J. 24: 919

Species used in MSA:

Pisum sativum
Solanum lycopersicum
Nicotiana tabacum
Zea mays
Spinacia oleracea
Petunia sp.
Nicotiana plumbaginifolia
Cucumis sativus
Pisum sativum
Glycine max
Hordeum vulgare
Pinus sylvestris
Oryza sativa subsp. japonica
Apium graveolens
Pyrus pyrifolia
Triticum aestivum
Sinapis alba
Arabidopsis thaliana
Gossypium hirsutum
Polystichum munitum
Lemna gibba
Solanum lycopersicum
Oryza sativa subsp. indica
Cucumis sativus
Pinus thunbergii
Physcomitrella patens
Malus domestica

This protein appears to be highly conserved in its internal regions as might be expected since variation in these reasons could have a large impact on protein folding. On the external surface of the protein there appear to be conserved regions intermingled with highly variable regions. It is likely that the conserved regions play a role in the functionality of the protein, whereas the variable regions probably do not. As this protein needs to interact with both chlorophyll and other LHCII proteins as well as PSI and PSII in order to perform its function, it is likely that these external conserved regions may be important for these interactions.

Alcohol Dehydrogenase

Dylan, Ellen, Stephen, and Jerreme

Alcohol Dehydrogenase

Alcohol dehydrogenase (ADH) is an enzyme that catalyzes the conversion of alcohols to aldehydes or ketones and a reduction of NAD+ to NADH.  ADH is advantageous to humans because it prevents alcohol toxicity by breaking down alcohol. ADH oxidizes only secondary and tertiary alcohols to aldehydes and ketones with the use of a coenzyme.

Reference:

Substitutions in the Flexible Loop of Horse Liver Alcohol Dehydrogenase Hinder the Conformational Change and Unmask Hydrogen Transfer
Ramaswamy, S.,   Park, D.H.,   Plapp, B.V.
(1999) Biochemistry 38: 13951-13959

A total of 190 sequences were used for the alignment including Mouse, Horse, Rat, Bovine, E.coli.

We compared side chain A of the protein and found the external regions to be highly variable with the interior regions exhibiting more conserved behavior. The ligand binding regions were also highly conserved.

Is the active site highly conserved?pbb_protein_adh5_image1

Jennifer and Ayla: GFP

Green Fluorescent Protein (GFP)

It’s a protein that glows green when exposed to blue light.  It comes from the jellyfish Aequorea victoria.  It is often used in experiments as a reporter gene.  We chose it because it GLOWS!!!

PBD ID: 2g6e

Sources:
http://www.pdb.org/pdb/explore.do?structureId=2G6E#
http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb42_1.html

Looked at chain A in jmol

Species source Aequoria victoria (jellyfish), expressed in E. coli

If we could have gotten the ribbon structure to display (it was just showing us the space-fill model for some reason), we would have expected to see a sort of barrel shape in jmol like we saw on the pbd page.

In the Consurf you can see the surface on one end is highly conserved.  We would like to know if the conserved portion has anything to do with the fluorescence function of the protein.

gfpjmol

gfppdb

Different fluorescent proteins growing in bacteria

Different fluorescent proteins growing in bacteria

NCBI Activity: Male Pattern Baldness

Group:
Ben Ernest, Joel Bucci, Letitia Olson

Goal: to find more information on male pattern baldness, how chemical imbalances and genetic variations cause hair loss, and potential treatments for this disorder

Introduction:
Male pattern baldness has several potential causes including endocrine disorders and genetic mutations, among others.  The hair loss is initiated by dysfunction in the anagen or telogen phases of the hair cycle.  Conversion of androgens to dihydrotestosterone shortens the anagen phase in hair development causing hair follicles to be miniaturized.
Mutations in androgen receptors affecting binding may also cause baldness. Recently there has been a large-scale screening of patients with male pattern baldness to determine an additional “at risk” allele that is correlated with androgenic alopecia when combined with other alleles.
Male pattern baldness in women has been shown in some cases to be caused by elevated testosterone levels resulting from an androgen-secreting tumor.  Upon the removal of the tumor the testosterone levels revert to normal and hair loss soon ceases.

References:

Alopecia: Possible Causes and Treatments, Particularly in Captive Nonhuman Primates
Comp Med. 2009 February; 59(1): 18–26.
Published online 2009 February.

Laparoscopic Adrenalectomy – A Cure for Male Pattern Baldness
Ann R Coll Surg Engl. 2007 January; 89(1): 43.
doi: 10.1308/147870807X160371.

Male-pattern baldness susceptibility locus at 20p11
Nat Genet. 2008 November; 40(11): 1282–1284.
Published online 2008 October 12. doi: 10.1038/ng.255.

Background:
For more background on Male pattern baldness see the Hair chapter in Clinical Methods. This can be found at the following link.
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cm&part=A3348#A3354

Search:
We saved a search strategy that will alert us once a month when new articles on alopecia are uploaded to the NCBI database.  So that we can stay up to date with new research being done on this disorder.

Tips:
When coming across an unfamiliar term or acronym use the find function (ctrl + f or cmd + f) to find the first mention of the term where it will be better defined.

Use the Links button on the side of the NCBI PubMed search to find more information related to the article, and subject you are searching.

NCBI : Activity Personal BLAST Database Development ; Dylan Storey

Introduction/Background:

In order to compare previously sequenced RFLP clones and EST clones used in marker development for genetic mapping in pepper to a chip developed by industry a BLAST database of proprietary sequences needs to be compiled. Using the toolkit developed by NCBI I’m in currently in the process of compiling a database of these sequences. After that database is ready, a series of sequences from RFLP clones that have been historically used for mapping will be compared to sequences on the chip. It is my hope that high sequence homology will exist between RFLP sequence and data points on the chip, if  high sequence homology does exist and linkage data also backs up the homology then the ability to call them orthologous will exist. With the declaration of these loci as orthologs comparisons between data from older southern based experiments to newer chip based experiments will be available. Because the sequence information from industry is proprietary I am unable to provide links to it.

Links to information / data repositories :

Database development

http://www.ncbi.nlm.nih.gov/staff/tao/URLAPI/unix_setup.html#2

RFLP clone sequences and linkage information:

http://sgn.cornell.edu/

EST sequence information:

Pepper EST database: comprehensive in silico tool for analyzing the chili pepper (Capsicum annuum) transcriptome,Hyun-Jin Kim et al.,BMC Plant Biology 2008, 8:101 doi:10.1186/1471-2229-8-101

Questions:

Are previously used and reported marker systems orthologous to marker systems currently in development and use.

Promoter Prediction in Genetics

Group: Jianzhuang, Xuejuan and Xiaomin.

Topic: Promoter Prediction in genetics

Introduction: A promoter is a region of DNA that facilitates the transcription of a particular gene. I am interested in this problem because this is one of the most important, difficult and popular topics in the genomics.

References:

Prediction and integration of regulatory and protein-protein interactions.

Wichadakul D, McDermott J, Samudrala R.

Methods Mol Biol. 2009;541:101-43. Review.

PMID: 19381527 [PubMed – indexed for MEDLINE

Background:

There are many books and publications available about promoter prediction. The link for some reviews are provided below: [http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=eurekah&part=A31978#A31978]

Search: I have built a search filter which contains the key word of ‘promoter prediction’. NCBI will send me the current articles about promoter prediction to my email.

Tips: This is a promoter database [http://rulai.cshl.edu/cgi-bin/TRED/tred.cgi?process=home]. You can download lots of useful information in this database.

Questions:

Seems there is no way to customize the alert functionality. Where can I create a filter similarly to search filter?

To do this research, backgrounds from Mathmatics, Engineering and Biology are needed. I need some input from others.

NCBI Activity:

Group: Brittany and Randy

Topic: Cyclic AMP

Introduction: Cyclic AMP, a secondary messenger, is invovled in numerous biological processes. We are interested in the research involving brain function and the role cAMP plays. Research is being done on cAMP and its involvement in mood disorders associated in the brain.

References:

Adenylyl cyclase-cyclicAMP signaling in mood disorders: Role of the crucial phosphorylating enzyme protein kinase A
Yogesh Dwivedi and Ghanshyam N Pandey
Neuropsychiatr Dis Treat 4(1), 161-176 (2008)

Background: Cyclic AMP is a secondary messenger involved in intracellular cascade events. No matter the mood state of a person, baseline levels of cAMP in cerebrospinal fluid under normal conditions does not fluctuate. However, patients that were diagnosed with various mood disorders showed differing levels in cAMP.

Search: We searched for full text articles on cAMP through the pubmed central.

Tips/Tricks:

The NCBI website has the option to search specifically for articles that have the full text available.

Questions:

NCBI Activity:Canine Parvovirus

Group: Yijia, Xiaoxin, Zhou

Topic: Canine Parvovirus

Intro: Canine parvovirus is a pathogen of dogs that emerged in 1970s.  It’s  a highly contagious disease that inflicts adult dogs, but mostly concentrates on puppies. The most common syndroms of this disease are depression, loss of appetite, severe diarrhea, and vomiting. Vaccines can prevent this infection, but mortality can reach 91% in untreated cases.

Reference:

Canine Parvovirus:

http://en.wikipedia.org/wiki/Canine_parvovirus

 

Canine viral vaccines at a turning point–a personal perspective

http://www.ncbi.nlm.nih.gov/pubmed/9890023?ordinalpos=23&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

Adv Vet Med. 1999;41:289-307

PMID: 9890023

 

Occurrence of canine parvovirus type 2c in the dogs with haemorrhagic enteritis in India.

http://www.ncbi.nlm.nih.gov/pubmed/19552931?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

Res Vet Sci. 2009 Jun 22

PMID: 19552931

NCBI Activity 2009: Exploring the stability of Taq Polymerase by Keats Shwab and Jason Harris

Topic:

We are invstigating a very well used enzyme, Taq polymerase. We wish to understand the structural characteristics which make it ideal for high temperature activity. We we also would like to see how related its peptide sequence is to non thermophillic polymerases.

Content:

All DNA polymerases have a similar structure that is related to their function, yet they differ in the details which comprise this uniform structure. This structure is analogous to that of a cupped right hand with the palm, fingers, and thumb being the domains of the complex (http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=polymerase,dna&rid=stryer.section.3769#3770, a NCBI Book reference 27.2.1. All DNA Polymerases Have Structural Features in Common)

If the structures of polymerases are so remarkably conserved and no mention is made of taq, a very popular DNA polymerase, then it seems safe to assume that it falls into this generalization. In order for it to maintain its DNA binding and polymerizing structure at thermophyllic temperatures, the details of its composition are expected to vary from that of other lower temperature operating polymerases.

The referenced book above was searched for in the NCBI BOOK database using the term ‘taq polymerase’. This query resulted in several books about polymerases, but none specifically on taq polymerase. It did however provide a generalized overview of DNA polymerases.

Taq polymerase is a DNA-dependent DNA polymerase derived from the themophilic bacterium Thermus aquaticus, which is found in hot springs and geothermal vents. This enzyme is widely used in polymerase chain reactions (PCR) because of its ability to catalyze DNA synthesis at high temperatures, with an optimal temparature between 72-80 C. Taq polymerase is not ideal for PCR, however, as it lacks 5′-3′ exonuclease proofreading activity that results in occasional errors in DNA synthesis. Other “High-fidelity” DNA polymerases with this proofreading activity have been isolated from other thermophilic bacteria and archaea, such as the archean Pyrococcus furiosus (Pfu polymerase).

References:

Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus.

A Chien, D B Edgar, and J M Trela

Journal of bacteriology 127 (3), 1550-7 (Sep 1976)

Molecular diversity and catalytic activity of Thermus DNA polymerases.

Moreland Gibbs et al.

Extremophiles : life under extreme conditions, (12 Jul 2009)

info:pmid/19597696 | info:doi/10.1007/s00792-009-0269-8

DNA polymerases: structural diversity and common mechanisms.

T A Steitz

The Journal of biological chemistry 274 (25), 17395-8 (18 Jun 1999)

Search: We searched for “Taq polymerase,” “Polymerase thermostability,” and “Taq polymerase structure.”

Tips:

You can find 3-dimensional protein structures in the “Structure” section of NCBI.

Questions:

There seems to be limited information of specifically what properties allow certain polymerases to be more thermostable than others.

NCBI Activity: Elizabeth & Dmitriy; Hsp 70

Group: Elizabeth & Dmitriy

Topic: Gene Conversion and GC content in Hsp 70 (Heat Shock Protein)

Introduction:

HSP70  is a highly conserved family of ubiquitous chaperon proteins. This study used several HSP70 proteins with very high similarity in the amino acid sequence but in contrast, great variance in the codons which code for the amino acids.

This paper demonstrated that the different codons for the same amino acid can up regulate or down regulate Hsp 70 expression. Specifically, greater GC content at the third position of the codon increases mRNA levels of Hsp 70. The increase in mRNA levels cannon be attributed to a change in mRNA stability because the stability of the different mRNA was virtually identical. The significance of this study is that it shows that mammals have selectively evolved different DNA sequences, without changing the protein, to have greater control of gene regulation.

Background:

Hsp 70 has a weight of 70 kilodaltons and plays a chaperoning and  housekeeping role. During periods of stress such as heat and specifically exposure to heavy metals the levels of Hsp 70 will jump in an organism.

References:

GC-Content Evolution in Mammalian Hsp70
Grzegorz Kudla*, Aleksandra Helwak{dagger},* and Leszek Lipinski{dagger},*
Mol. Biol. Evol. 21(7):1438-1444. 2004
DOI: 10.1093/molbev/msh146

The hsp70 family of molecular chaperones.Box
Molecular Biology of the Cell -> Basic Genetic Mechanisms -> How Cells Read the Genome: From DNA to Protein -> From RNA to Protein

High guanine and cytosine content increases mRNA levels in mammalian cells.
Grzegorz Kudla et al.
PLoS biology 4 (6), e180 (Jun 2006)
info:pmid/16700628 | info:doi/10.1371/journal.pbio.0040180

We used google alert to track any information or citations that appear on the internet with the title from the paper.

We learned how to set up a google alert, but we would like to figure out how to set up an alert using the actual paper not just the title.

Another good trick is using Connotea to transfer a paper between computers.

We would like to find a site that created a paper citation map.