The best model organisms for chromatin studies

This was written by Direncan Boyraz (Izmir Institute of Technology)

1) Saccharomyces cerevisiae 

Saccharomyces is one of easier organisms to be analysed for isoforms of histones and histone modifications chromatin structure studies than other complex eukaryotes; additionally Saccharomyces has common modifications with mostly all eukaryotes (1). There are some key points for this. Firstly, promoters of yeast as enhancers can be activated by becoming nucleosome-depleted. Second, nucleosomes on chromatins are well located. Distances and bordering regulatory of nucleosome provide convenience. Third, highly transcribed genes own low nucleosome occupancy because of RNA polymerase II activity with its associated factors (2).  

2) Drosophila melanogaster 

Fruit fly as a model organism has some advantage in chromatin structure analysis such as DNA methylation. DNA methylation in flies are manageable than other animal or models. We have available data for the system of it and also existence of less methylated genome than human genome  with less number of gene is useful for epigenetic studies. More restrainable number of chromosomes form an easier environment for observations. Fruit fly is one of most model organism for those experiments. Easy observable and high similarity to human make it a good model(3).  

3) Arabidopsis thaliana 

Histone modification and DNA methylation are to key for studying chromatin structure and natural changes Arabidopsis lives brought it to rapid modifications evolutionary.  Also epigenetic variation is proved with some ways such as modifications and the presence of repeated sequences or transposons within the promoters. First and most known plant on the earth is Arabidopsis, and this makes it a good model organism for that (4). 

References: 

[1] Rando, Oliver J., and Howard Y. Chang. “Genome-Wide Views of Chromatin Structure.” Annual review of biochemistry 78 (2009): 245–271. PMC. Web. 28 Feb. 2018.  

(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2811691/) 

[2] Rando Lab Biochemistry and Molecular Pharmacology, Umass Medical School, Chromatin Structure and Function,  

(https://www.umassmed.edu/randolab/research/chromatin-structure-and-function/) 

[3] Lyko F., Beisel C., Marhold J., Paro R. (2006) Epigenetic Regulation in Drosophila. In: Doerfler W., Böhm P. (eds) DNA Methylation: Development, Genetic Disease and Cancer. Current Topics in Microbiology and Immunology, vol 310. Springer, Berlin, Heidelberg  

[4] Turck, F. and Coupland, G. (2014), Natural Variation in Epigenetic Gene Regulation and Its Effects on Plant Developmental Traits. Evolution, 68: 620–631. doi:10.1111/evo.12286  

 16S Ribosomal RNA Sequencing and Phylogenetic Tree Construction

Aim:
The purpose of this experiment is to form phylogenetic tree between five different kinds of prokaryotic organisms by using 16S rRNA analysis.
Hypothesis:
For forming phylogenetic tree, genomic DNA of each organism should be isolated and then interested regions of DNA (rRNA sequences or ribosomal RNA sequences) are amplified by PCR. For knowing these sequences, amplified DNA fragments are sequenced and phylogenetic tree is formed by using bioinformatics tools.
Introduction:
rRNA or ribosomal RNA is essential material that joins protein production. rRNA is produced from rRNA sequences or rDNA sequences. The importance of these rRNA sequences is to have very conserved regions and also differences that species have. These sequences are used for determined place of specie in taxonomy. (1)
There are three types of rRNA in prokaryotes. These are 5S, 16S and 23S. Without eukaryotes, 16S is often used for determination of phylogenetic tree or taxonomic places of organisms. For eukaryotes, 18S is used for that. rRNA sequences include some commonplace regions. As universal, some regions are same for all organisms. These regions are much conserved because of mutual and essential sequences. These conserved regions are named as highly conserved regions. The regions 16S includes differences is called hypervariable regions or hot-spots. These differences are used for taxonomy. (1, 2)
Size of rRNA genes is suit for bioinformatics applications and tools. Also conserved regions are universal and it helps to use universal primer for amplification of the gene. After years, researchers have a huge database about rRNA genes and it makes researches easy for them. Hypervariable and conserved regions can help by different ways for constructing phylogenetic tree. (2, 3)
Before construction of phylogenetic tree, interested rRNA genes are amplified by PCR for forming better results and working easier. Ribosomal RNA sequences are used as template DNA. Universal primers also make process easier by highly conserved regions. (4)
Phylogenetic tree is a figure that is used for showing evolutionary relativeness between living organisms. Similarity of genes is related with evolutionary origins. Figures on phylogenetic tree represent different relationships or closeness. Each node is a connection between braches and it demonstrates an ancestor. Branches are formed with differences among kinds. How far two kinds are on tree can be understood by shared nodes. In a tree, if there is a common ancestor for all organisms, this tree is named rooted tree or if there is no, it is named unrooted tree. (5)
Materials and Method:
Firstly, 16S rRNA sequence of DNA that was isolated before experiment was amplified by PCR process. After PCR components were calculated, components were mixed in an Eppendorf except enzyme. Enzymes were added into mixture and then lastly sufficient amount of water was added for completing mixture 50 µl. Finally mixture was placed into PCR machine.
Required Material Stock concentration Required concentration Volume (µl)
DNA 9.4 ng 250 ng 26.6
Primer Forward 10 µM 1 µM 5
Primer Reverse 10 µM 1 µM 5
dNTP (mix) 10 µM 200 mM 1
MgCl2 25 mM 2 mM 4
Taq Pol. 5 unit/µl 2.5 units 0.5
Buffer 10X 1X 5
H20 – – 2.9
Total: 50 µl

A PCR thermal cycle:
Denaturation (30 seconds at 950C)
Annealing (60 seconds at 680C)
Extension (5minutes at 720C)
Thermal cycle of PCR was repeated for 30 times.
Products of PCR were sequenced for rRNA analysis. Sequences were used for constructing a phylogenetic tree with help of T-Coffee (6).

Discussion:
This experiment was intended to make phylogenetic tree between five different organisms according to 16S rRNA sequences of them by firstly using PCR with universal primers then sequence and bioinformatics tools (6).
After constructing phylogenetic tree between five organisms, relativeness of organisms can be seen. Any common ancestor of five organisms cannot be observed on tree. This demonstrates that this phylogenetic tree is unrooted.
The nodes that have two branches from a branch on tree utilize an ancestor kind in mutual. As seen in the Figure 1, the closeness of Acinetobacter haemolyticus and Escherichia coli creatures as affinity is the closest organisms than the other organisms. The node on the right between these organisms releases that they have a common ancestor in the past. Enterococcus faecium is also close to these kinds partially. These three species seem to originate from a common ancestor. The node on the left provides this information to us about the other two organisms too. If Bacillus subtilis and Staphylococcus aureus are observed, these two kinds don’t have a close origin to each other can be seen. Also these two kinds also aren’t close to the other three kinds.
References:
[1] Smit S, Widmann J, Knight R (2007). “Evolutionary rates vary among rRNA structural elements”. Nucleic Acids Res. 35 (10): 3339–54. Received on May 14, 2017
(https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkm101)
[2]  Woese CR, Fox GE (November 1977). “Phylogenetic structure of the prokaryotic domain: the primary kingdoms”. Proceedings of the National Academy of Sciences of the United States of America. 74 (11): 5088–90. Received on May 14, 2017
(http://www.pnas.org/content/74/11/5088)
[3] Case RJ, Boucher Y, Dahllöf I, Holmström C, Doolittle WF, Kjelleberg S (January 2007). “Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies”. Applied and Environmental Microbiology. 73 (1): 278–88. Received on May 14, 2017
(http://aem.asm.org/content/73/1/278)
[4] Schmidt TM, Relman DA (1994). “Phylogenetic identification of uncultured pathogens using ribosomal RNA sequences”. Methods in Enzymology. Methods in Enzymology. 235: 205–22. Received on May 14, 2017
(http://www.sciencedirect.com/science/article/pii/0076687994351422?via%3Dihub)
[5] Hodge T, Cope M (2000). “A myosin family tree”. Journal of Cell Science. 113 (19): 3353–4. Received on May 14, 2017
(http://jcs.biologists.org/content/113/19/3353.full)
[6] (http://www.ebi.ac.uk/Tools/msa/tcoffee/).

Nonhomologous genes on chromosome Y

ZFY(Zinc Finger Y-Chromosomal Protein)
UTY(Ubiquitously Transcribed Tetratricopeptide Repeat Containing, Y-Linked)
USP9Y(Ubiquitin Specific Peptidase 9, Y-Linked)
TSPY1(Testis Specific Protein, Y-Linked 1)
SRY(Sex Determining Region Y)
RMY1A1(RNA Binding Motif Protein, Y-Linked)
PRKY(Protein Kinase, Y-Linked)
DAZ1 and DAZ2(a member of the DAZ gene family and is a candidate for the human Y-chromosomal azoospermia factor (AZF))
BPY1(Testis-Specific Basic Protein On Y)
AZF1(Azoospermia Factor 1, a gene locus)
Reference:
http://www.genecards.org/

Staining Techniques

 

INTRODUCTION 

Microorganism is the scientific term used to describe a living organism of a very small magnitude that can be studied only by the usage of microscopes. Microbiology is the field of science that studies microorganisms, such as : bacteria, virus, fungi and protozoa. The process of staining includes the preparation of the wet mount or smear preparation which is a culture of microorganisms over the cover slip [1]. 

Bacteria are small prokaryotic cells whose length ranges from 0.2 to 1 micrometer and that are found in different environments. They are characterized by the ability to move and to reproduce each 20 minutes. Bacteria have a crucial role about the environment and all the other living organisms, because they have the ability to recycle dead plants and animals, fixate the nitrogen the plants need to function regularly, contribute to the foods by adding by giving them their characteristic scent, making the environment cleaner by converting the dangerous materials into neutral ones, by a process called bioremediation [2]. 

Bacterial staining is the procedure that enables the researchers to have better images of the microorganisms unders the microscope by coloring of the bacteria by specific dyes. There are 4 types of staining, respectively: 

  • Negative staining is the term used to describe the method that only stains the bacteria’s background and exhibiting the bacteria colorlessly. It is a very beneficial method for the determination of the size and the arrangement of the cells by the usage of the nigrosin that is an acid stain that becomes negative by providing H+ion and is repelled by the positively charged of the most bacterial cells [3]. 
  • Simple staining is the term used to descibe the method that enables a view of the cells’s shape, size and arrangement by the usage of simple basic dyes (ex:crystal violet,basic fuchsin and methylene blue). This strain realizes the colorization of the bacteria because the basicity it gains when donating OH ions or accepting H+ ions, allows it to adhere to the negative cell membrane [4]. 
  • Gram staining is the method developed by Hans Christian Gram that enables discerning between Gram-positive bacteria and Gram-negative bacteria due to the different colors they exhibit after reacting with crystal violet. This difference in reaction is caused by the presence of the peptidoglycan that is a polymer composed of several chains of polysaccharides and peptides.  The gram-negative bacteria exhibit the red color of the counterstain (safranin), because of the inability of the thin layer of the peptidoglycan to keep the crystal violet’s color bound, while the gram-positive bacteria that contain a thick layer of the peptidoglycan exhibit the purple color of the crystal violet stain [5]. 
  • Endospore staining is a differential method, because of the usage of more than one stains developed to observe the spores, that are keratin structures produced each 6-8 hours, able to survive in extreme conditions, consisting of a passive metabolism, de-hydrative, exhibit resistance to the desiccation, different chemicals used in the laboratory, radiation and the heat. The first stain used is malachite, a water-soluble stain that is encountered in the cell by the help of the heat and enables the cell exhibit a green color. The second stain is the safranin that enables the other part of the cell except the spores to develop a red color [6]. 

MATERIALS & METHODS 

Negative staining 

  • Microscopic slides                                      
  • Nigrosine solution 
  • Bacillus subtilis 

This experiment is performed near the Bunsen burner. Initially, 30 µl nigrosine dye is positioned over the microscopic slide by using a micropipette and afterwards 20 µl Bacillus subtilis is adjoined over it and mixed with it by the help of the pipetting. Finally, this preparation is scattered over the slide by using another slide and observed with a light microscope. 

Simple staining 

  • E.coli`s slant culture  
  • Methylene blue 

Initially, 20 µl E.coli is positioned over the slide by the help of a micropipette and this mixture is stirred over the slide by a heated inoculation loop. Afterwards, the smear preparation is positioned in order to get dried in the room temperature and later it is slipped over the flame in order to fix the microorganism`s sample. Later, 300µl Methylene blue is positioned over the microscopic slide for 1 minute. After that the microscopic slide is washed with water and dried so that all the drops are eliminated from the slide`s surface. At the end of the process, the slide is observed by the help of the microscope. 

Gram staining 

  • Crystal violet 
  • Safranin 
  • Iodine dye 
  • Bacterial mixture 

Initially, 20 µl bacterial mixture is positioned over the slide by using a micropipette and this sample is scattered over the slide by using a heated inoculating loop. This smear preparation is placed over the bench under the room temperature in order to get dried. Afterwards, the slide is slipped over the flame of the Bunsen burner. 300 µl Crystal Violet is positioned over the slide for one minute. Later, the stain is removed after being washed with water and 300 µl Iodine dye is positioned over the slide for 1 minute. After that, 300 µl Ethanol alcohol (95M) is used to wash the slide for 20 seconds and then water is used to wash the slide for approximately 2 seconds. 300 µl Safranin is added for 1 minute and after that washed with water for 2 seconds. The slide is dried by using a blotting paper and later a lamella is positioned over it. Finally, the sample is observed by using a light microscope.  

Endospore staining 

  • Malachite green 
  • Safranin 
  • Bacillus subtilis 
  • Spore staining kit  
  • Electric hot plate 
  • Small beaker 

Initially, 20 µl Bacillus subtilis is positioned with a micropipette over the slide and the sample is scattered around the microscopic slide by using a heated inoculating loop. Preparation smear is dried under room temperature and later, slipped across the flame of the Bunsen Burner 3 times. A toweling paper is placed over the microscopic slide and this slide is positioned over the electric hot plate. 100 µl Malachite green is placed over the toweling paper and dried for a short period of time. This step is repeated 4 times. After the last drying process, the preparation smear is washed with water for 30 seconds. Afterwards, 300 µl of Safranin is positioned over the slide for 20 seconds and the washing process with water is repeated again. After the water is removed from the slide by using blotting paper, lamella is positioned over the slide and observed under the light microscope.  

DISCUSSION 

In the negative staining, a dark background with light particles showing the presence of Bacillus was expected, but the image of our sample reflects the Bacillus bacteria in a green light color and the background as whitish. A possible reason why this has occurred can be, because of contamination of the sample during the smear preparation, such as: preparing a thick layer of smear instead of a thin one, burning of the cells or because of visualization of the sample while the sample is still wet.  

Simple staining method was successful and due to our image the E.coli‘s cells shape, size and arrangement could be visualized. The ability of E.coli cells to appear as blue is because of the ability of the positively charged dye to adjoin to the cytoplasm of the E.coli that carry a negative charge. 

Gram staining method also led to satisfactory results and a classification of bacteria as positive and negative was made. In this method, Crystal Violet contributed to give the cells a purple color, while iodine helped as a co-agent of crystal Violet to create an insoluble mass of stain. Alcohol contributed to decolorize the purple color from the Gram negative bacteria, because of their thin layer of peptidoglycan that is unable to keep the purple color bound to itself. Decolorizing does not occur in Gram positive cells, because of their thick layer of peptidoglycan to keep the purple color bound to its structure. Safranin contributes to give the pink color to the decolorized Gram negative. The samples were observed with compound light microscope assisted by the oil immersion to contribute to a clearer view.  

Endospore staining’s results were satisfactory, because our group’s image showed that the endospores reflected the green color they had gained by the Malachite green and the other cells reflected the red color of the Safranin stain.  

REFERENCES 

1. http://antranik.org/stain-uses-types-and-applications/ retrieved 09.03.2016 from 

2. http://medimoon.com/2014/04/types-of-different-staining-techniques-of-microorganisms/ retrieved 09.03.2016 from 

3. http://www.nos.org/media/documents/dmlt/Microbiology/Lesson-02.pdf retrieved 10.03.2016 from 

4. http://sudps.org/theory_project.php?id=34&type=theory&chapter=7#tab1 retrieved 10.03.2016 from 

5. http://www.bcin.ca/Interface/openbcin.cgi?submit=submit&Chinkey=36682 retrieved 10.03.2016 from 

6.https://www.researchgate.net/profile/Paul_Tak/publication/227816027_van_der_Heijden_IM_et_al_Presence_of_bacterial_DNA_and_bacterial_peptidoglycans_in_joints_of_patients_with_rheumatoid_arthritis_and_other_arthritides_Arthritis_Rheum_43_593-598/links/543bbc4e0cf24a6ddb979450.pdfn retrieved 11.03.2016 from