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Chapter 2
Discovery Questions
Discovery Questions
This activity contains 97 questions.
Read the sequence from the real X-ray film in Figure 2.2. Record the sequence for both strands of DNA, with the top strand containing the sequence on the X-ray film. Be sure to keep track of 5' and 3' ends for both strands. Perform a BLASTn (nucleotide sequence) search with the top strand of DNA. BLASTn searches allow you to query the constantly updated database of all DNA sequences to find the best matches from the database for your query sequence (see Math Minute 1.1). Read the top "hit" from the BLAST results. What gene did you sequence? Now try a BLASTn search with the bottom strand (remember to enter it 5' to 3'). Do you retrieve the same gene?
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To get a complete understanding of the sequencing process, join two students who tour the Genome Sequencing Center at Washington University in St. Louis.
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Go to the Chromat 1 web page and examine the entire sequence. Don't bother trying to read the letters yet. Can you tell which end is the 5' end?
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Beginning at base 80, read 50 bases of the sequence and write down both strands of the DNA, with the top strand being the one on the chromat.
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Perform a BLASTn search of the DNA in Chromat 1, but use only the first 30 bases of the 50. What was your best match? Record the
E-value
(measures quality of BLAST hits) presented in the right column. Now BLASTn all 50 bases and compare the new results with the search that used only 30 bases. Explain what happened to the E-value and why. You can read Math Minute 1.1 to understand why the E-value changed for the two BLAST results.
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Go to Ensembl (European version of NCBI) and click on "Genomic Data" under the "Help & Documentation" menu on the left side. Click on "Downloads" and then click on the FTP link under the DNA column for your favorite species. Are the genome sequences submitted as one single file? What level of organization has been used to post the DNA sequences?
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Do mammals or amphibians have larger genomes, as revealed on the
less expensive
web site? Why does the answer seem counterintuitive?
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Go to the Assembly Archive (chromat database) to view some chromats from an anthrax sequencing effort. Click on
Bacillus anthracis
str. (strain) Kruger B. You will see a list of many assemblies; click on Contig ID 607. When the new window opens, you will see three frames. The top frame shows the coverage of this 15.3 kb segment of anthrax genome. The middle frame shows the individual sequences used to assemble the 15.3 kb contig. What does the graph in the top frame summarize from the middle frame? The bottom frame shows you the tiling path of the individual clones that span the entire 15.3 kb contig. The small red dashes indicate marker sequences used to help create the overlapping tiling path of clones. Mouse over the large blue segment that is centered on the 2 kb tick mark with trace ID ti494464459. When you see this trace ID number, click on it. A new window will open to show you the sequence, but click on the box next to "in color" and hit the "Show" button. This will produce a color graph that indicates the quality assessment score produced by PHRED. Where does the quality tend to be best? Scroll to the first two regions with quality scores between 0 and 20. Now change the menu from "
FASTA
" (plain text format) to "Trace" and hit the "Show" button. You should see the chromat for this sequencing read. Next to the "in color" button should be a new option for the Applet size. Change "normal" to "Big" and hit the "Show" button. Right above the chromat is a "confidence" option; turn that on. On the far left is a scroll bar; move it down and up to see more and less of the chromat, respectively. The confidence is indicated as bar graphs for each base, with higher-confidence bases having longer bars; bar colors match the base colors, not quality assessment values. Find the regions of low quality scores and determine why the scores were so low.
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Go to the Human Genome Browser and locate section chr19:8,584,715-8,606,616 by typing it into the search window. Click on the large black box in the gap row and read how gaps are depicted. Click the "back" button once on your browser, and scroll down below the image. Click on "hide all," except modify these individual options: base position = full; chromosome band = dense; gap = pack; Ensembl genes = dense. Be sure to click the "refresh" button at the top of the display options to implement your modifications; these settings will speed up subsequent navigation. Click on the "base" button to the right of the 10X zoom-in button. This will show you the consensus sequence where known, and an x where there is no sequence information. Below the DNA sequence, all three reading frames are translated with red boxes marking stop codons and green boxes marking start codons. Zoom out 10X three times. Is this gap near a gene? Do you think this gap affected the nearest neighboring gene's annotation? Continue zooming out until you see a second gap. Now hit the >>> button until you find a third gap. Continue to move >>> through the third gap to define the extent of this gap. Which gap is bigger, the first one you looked at or this third one? What chromosomal structure(s) are in the area of the bigger gap?
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Go to the Finishing web page and determine the order of DNA fragments needed to build the largest possible contig. How many gaps remain after you have created the largest possible contigs?
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Imagine you're a finisher working on the DNA you assembled in Discovery Question 10. How might you have isolated the gapped DNA if you knew the entire region of DNA was 20 kb long?
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Go to the genomic DNA #1 (gDNA1) web site, where you will see three pieces of DNA sequence. Copy and paste one of the sequences and then click on the "TestCode" button. One at a time, submit the three segments of gDNA to TestCode to find which one harbors the ORF.
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Copy and paste the real ORF from Discovery Question 12 into the scramble web site to have a Perl script generate a scrambled version of the same DNA. Take the scrambled version and resubmit it to TestCode. Does the randomized version of the coding DNA look like coding DNA? Would you expect it to?
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Calculate the average percent nucleotide identity for the three COX gene regions from your BLAST2 alignment.
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Go back to BLAST2 Sequences and enter the two protein accession numbers for COX2, "NP_000954," and COX1, "NP_000953." Be sure to change the search from BLASTn to BLASTp. Verify that the top blank contains NP_000954, so COX2 will be the query in the resulting page. a. What is the overall amino acid identity? Is this higher or lower than the overall nucleotide identity? b. Notice that a separate percentage is calculated for similarities (called "Positives"), which takes into account the similar structures of some amino acids. What is the percent similarity? c. Which parts of the proteins appear to be poorly conserved? Look at the sequence alignment that uses the single amino acid code and find where one protein has several Xs in a row, to mark areas of low complexity (see Math Minute 2.3). d. Use your browser's "Find" function to locate the amino acid sequence GAPFS. Serine (S) is the amino acid modified by aspirin. Is GAPFS in a region of high sequence identity or similarity? (See pages 3-3 for details.)
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Go to HGNC (Human Genome Nomenclature Committee) and perform a gene search for cyclooxygenase to see how many cyclooxygenase genes there are in the human genome. HGNC is a good, quick way to perform a gene search with links to many other databases. However, compare the HGNC results with an OMIM search using the gene name COX3. Do you find any surprises?
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Mouse over the
domain
boxes to determine the number of different CDs from your search. Don't just count the number of boxes, but determine the types of domains revealed when you mouse over each box. Notice that the E-values are provided when you mouse over each box.
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Click on the "Show" Domain Relatives button and see what hits you get. At what protein have you been looking?
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Go back and click on "gnl/CDD/7333," to the left of "smart00291,ZnF_ZZ, . . ." Read the text at the top of your screen. Does this domain have an important function? Explain your answer.
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Copy and paste this uncharacterized protein (as of spring 2005) amino acid sequence into web sites of your choosing to characterize the protein's possible functions. Determine as much as you can about its structure and function.
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How can a single protein yield more than one answer to the why, what, and where questions to describe its roles in cells?
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Go to WormBase (
C. elegans
gene database), search for the gene "
pmr-1,
" and learn its biological process, molecular function, and cellular components (about halfway down, next to Gene Ontology listing). Does
pmr-1
have more than one biological process, molecular function, and cellular component?
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Search NCBI's Human Map using the term "obesity." You will get hits for every locus that has obesity associated with it. How many loci do you see? Are they clustered or distributed throughout the genome?
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Click on the blue number "10" below the cartoon of chromosome 10. What gene did you identify?
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Click on the "Maps & Options" button to modify the view. From the new window, you can choose from the list in the left window; your choices are displayed in the right window. Modify the display until only "Gene," "Morbid/Disease," and "Ideogram" are displayed. Click on "Morbid/Disease" and then on the "Make Master" button followed by "Apply." The ideogram on the far left shows how much of the chromosome you are viewing. You can zoom in or out as needed. This database allows you to search for your favorite disease or condition and track down all this information. You could place an order for this DNA or amplify it yourself using PCR.
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Go to Electronic PCR and enter the accession number "M18533" in the big open box to determine if there are any STSs in this sequence. What gene have you located, and how many STS markers are there? Click on one of the blue links and see how much information is there. Do you have all the information you need to amplify this STS? What else did you learn, other than sequences of the primers?
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BLASTn this mystery sequence and select "est_mouse" from the "Choose database" menu. What can you learn based on the hits you obtained? For example, what gene have you identified? Scroll down and see how many tissues are described in this search. Imagine you were studying obesity in mice; how might this help your efforts? (See Chapter 5 for details.)
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Did the EST database provide you with more than just sequence identification information? With the completion of many genomes, is there any utility for the EST databases? Support your answer with specific examples.
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Go to the UniGene Statistics page to read the latest information on human ESTs. Based on this information alone, calculate the average number of ESTs for each human gene (assuming 23,000 genes).
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What does GeneCards say about
Nox1
? Is it an NADPH oxidase? Go to the Human MapViewer for the human genome, enter "Nox1," and hit "Find." You will see a red dash next to the X chromosome. Click on the blue "X" under the ideogram and notice the other genes in the area. Next to the gene "NOX1," click on the link "sv." You should see a graphical version of this gene. a. How many different mRNAs (listed as
CDS
for
c
o
d
ing
s
equences) are produced by this one gene? The color code is just below the expanded view of this gene. Notice that only the first exon is shown in the sequence (as denoted by the red bracket in the cartoon above). b. Use the navigation button icon to zoom out by clicking once on the "-" sign. How many genes are in this region of the X chromosome? Do any other genes produce more than one mRNA?
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Take a few minutes to draw a flowchart illustrating the steps you would take to annotate a newly sequenced genome (define the genes; describe each protein's biological process, molecular function, and cellular component; summarize the major metabolic pathways the organism needs to survive and evolve).
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Analyze the dystrophin gene with the Genome Browser. Enter the name "dystrophin" and click the "Submit" button. You will get a list of hits. Click on the first "dystrophin" option to see a graphic view of the human dystrophin gene. Use the 3X zoom-out button until you can see the entire gene. You may have to modify the view using the options below the display to answer the following questions. a. Are there any STS markers in this gene? b. Look at the Gap and Coverage lines. Has the public HGP sequenced all of the chromosome in this region? c. Change the "Coverage" option to "full" and then count how many BACs span the DMD gene. Gray BACs are draft-quality sequencing and black BACs are finished. Can you determine the minimum number of BACs required to span DMD based on coverage? d. How many DMD mRNAs use more than one exon? What can you infer from the number of alternatively spliced mRNAs?
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In the "position" box, enter "7p15.2" and then hit the "jump" button. At the bottom of the page, hide all features, except set to full "Known Genes" and then set to dense every species "Net" (e.g., Fugu Net) under "Comparative Genomics." Then hit refresh. You are looking at 12
Hox
genes, which are critical to body development. Center the
Hox
genes and zoom in to see the near-universal conservation of DNA, especially the
Hox
exons. a. Which species highlight the conserved exons the best, closely related species or more distant? b. Set repeat masker to "dense" and refresh this diagram. Do
Hox
genes contain a lot of repeats (black indicates repeat sequences) compared to portions outside the
Hox
genes? Do you think this is significant? Explain your answer. c. Set the SNPs (single nucleotide polymorphisms, which can be thought of as point mutations) option to dense. Do
Hox
genes have more or fewer SNPs than the surrounding area? (See Color Key.) Do you think the
Hox
frequency of SNPs is significant? Explain your answer. For a comparison, click on the "Move >>>" button. In these three questions, we are working with only one strain of mice, not two.
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If the
Igf2
gene were deleted from the sperm, predict the phenotype in the offspring. What would the phenotype be if the egg's
Igf2
gene were deleted?
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If the maternally expressed gene
Igf2r
were deleted from eggs, predict the phenotype in the offspring. What would the phenotype be if the sperm lacked an
Igf2r
gene?
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What would you predict for the offspring if the sperm's
Igf2
gene and the egg's
Igf2r
gene were deleted?
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Do you think methylation is an ancient mechanism or one limited to vertebrates? How could you answer this question?
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Go to GenBank and search "methyltransferase." Can you find any DNA methyltransferases in organisms other than vertebrates?
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Jackson-Grusby et al. found that most of the genes with altered expression increased their expression levels when the methyltransferase was deleted, as you might expect since methylation normally silences genes. Explain how some genes could be repressed when hypomethylated.
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Perform an NCBI Gene search for PPA1880 (use the pull-down menu on the NCBI main page to select "protein"). Click on the first link, then click on the "CDS" link to see the coding DNA. Copy the DNA sequence into the GC calculator to determine the %GC.
P. acnes
has an average of 60% GC and human has an average of 41%. Which genome does PPA1880 more closely match?
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Find AAH14236.1 from NCBI's protein pull-down menu. Copy the DNA sequence and determine the %GC. Does this sequence look more human-like or more
P. acnes
-like? What interesting annotation did you uncover? How could this cDNA get into a human cDNA library?
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Now determine the GC content for one rRNA gene and one tRNA gene. How do they compare to the genome average of 60% GC?
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Go to the P. acnes genome view, enter 1 into the "Start from" box, and hit go. Notice that the first gene is
DnaA
(accession number YP_054724). Click the blue arrow pointing to the left. Do you notice anything peculiar about this region upstream of
DnaA
? Compare this region to any other by clicking somewhere on the genome map to see any other region.
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Find the Conserved Domain of LPXTG. What does this domain help proteins do?
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Perform an NCBI Medical Subject Heading (MeSH) search for "CAMP Factor." You should see a hit called "CAMP protein, Streptococcus [Substance Name]." On the far right side, click on the "link" link and choose "NLM [National Library of Medicine] MeSH Browser." What can CAMP factors do to our blood cells?
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Search Google Scholar for autoinducer-2. Do you see any evidence that autoinducer-2 plays a role in communication? Is this protein expressed in many species?
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Search for "biofilm" in OMIM and click on the one hit. Perform a find function with your browser for "biofilm" and see what this protein has to do with preventing biofilm formation.
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Conduct PubMed and Google searches for "Blue Light Acne" and see what you can learn about a novel method to combat acne. Do you think genome sequences can help us understand this method better? Explain your answer.
Bonus Material
: A study of the tetanus genome is available on this book's web site.
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Go to the Microbial List web page and click on "
Bacteroides thetaiotaomicron
VPI-5482." Then click on the link "4778" to produce a list of all the proteins in the proteome, ordered the way the genes appear on the chromosome. Find "mannosidase" as many times as you can (you can stop when you get tired). This basic search illustrates the high level of polysaccharide utilization enzymes in the proteome--and you only searched for a single sugar.
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Go back to the list of 4,778 proteins and do a find function for "one-component" (the sensor and signal-transduction proteins are fused). Each time you find one, look to see if a sugar-metabolizing protein is nearby. Perhaps the placement of a sensor gene and a metabolizing gene is not random. Propose a reason why evolution might have selected for these two types of genes to be neighbors.
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Look at mannose metabolism, then find
Bacterioides
in the pull-down menu and click on the "Go" button. (The fastest way to do this is to open the menu and start typing
Bacterioides
fairly quickly. The species will be highlighted as you spell out the genus.) Next, click on the oval labeled "Galactose metabolism." Can you verify that our symbiont is well suited to help us digest sugars?
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Search
Scientific American
for "pylori" to read a surprising proposal that even the ulcer-causing bacterium
H. pylori
might produce beneficial consequences by living in our stomachs. Are we harming ourselves when we take antibiotics unnecessarily?
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Go to the TIGR CMR web site, click on "Comparative Tools" then choose "Align Whole Genomes." Choose from the two menus
Haemophilus influenzae KW20 Rd
and
Mycoplasm genitalium G-37
with a minimum alignment of 20 nt (this display looks best if viewed in Netscape or Firefox browsers). Do these two genomes look like they evolved from a common ancestor in the recent past? You can increase the sensitivity by changing the minimum alignment to 15 nt; see if this helps. For a comparison, compare two strains of
H. influenzae
to each other at 20 nt minimum alignment.
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Go to the
M. genitalium
genome page and "Gene Search" by "Locus" for the genes MG064 and MG101 (note they are "unknown genes" in figure 2.15). Do we know their functions now?
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Go to DEG and search for entry number 1169 (DEG10060038 or
clpB
) from
M. genitalium
. Does the name of this gene lead you to believe it would an essential gene? Copy the ORF sequence from the DEG link and perform a BLASTx search (submit DNA sequence and search for protein matches) search against all DEG entries using DEG's BLASTx program. Then perform a BLASTx search at NCBI. Which search is more informative and why?
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Go to the
M. genitalium
Genome Browser at Genome.Net to see an interactive genome map. At the bottom is a genome comparison tool that will show you genes in your query species aligned with the species of your choice (choose
E. coli K-12 MG1655
, "Gene cluster search", then click on "Exec"[ute]). You will see colored bands for orthologs of the two species and their location in the query genome. Mouse over the genome, and the green bar will show you on which portion it will zoom; click on the section with the most color (red). On the new page, click on the "ORF Color" button to understand the colors, and "View Genome map" to see the position of the conserved genes. What category of genes is in this area? Are the genes clustered or widely distributed?
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Perform an NCBI Entrez search for
Nanoarchaeum equitans
. Click on the "MeSH" link at the bottom to learn about this species. Go back to the full Entrez results, click on the "Genome" link, and follow this until you see the circular map of the smallest cellular genome in the world (as of September 2005). Click on "GenePlot" to align
N. equitans
and
M. genitalium
by changing the default species for the pull-down menu on the right, then click on "compare selected pair." How many protein-coding genes are conserved between these two smallest genomes (number of "bets" [best hits] listed below the 2D alignment maps)? You can navigate around by clicking and changing the zoom button. Indentify the gene they have in common that is located nearest to the 0 - 0 origin of the graph.
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In Discovery Question 57, you determined that both species have histidyl-tRNA synthetase, but if you studied the whole genome of
N. equitans
, you would discover it lacks a tRNA
His
for the codon GUG. Go to the tRNA
His
web page and BLASTn each of the three sequences. Note the first hits when you submit each half and then the difference when you submit the full sequence. Do any of these BLAST results indicate they might be a tRNA gene? Why doesn't the full sequence give a better score than just half of the tRNA gene?
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Perform a Genome NCBI search for "Mimivirus" and click on the link. Change the view of the genome to show only tRNAs and hit "Refresh." How many are in this genome?
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Return to the Mimivirus main page from above and click on the number next to "Protein coding" under the "Features" heading to get a list of Mimivirus protein-coding genes as they appear on the genome. In the "Start from" field, enter these nucleotide numbers one at a time to find three genes and then click on "Refresh": (1) 234000, R194; (2) 267000, L221; (3) 633000, R480 (L and R refer to genes pointing to the left or right, respectively). Click on each gene name to learn about this family of proteins. Which one looks most like a eukaryotic protein?
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If you were going to construct a minimum genome, would you choose a virus or a bacterium? Explain why.
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Go to the Oak Ridge National Laboratory (ORNL) Microbial Genome web site, choose the
Prochlorococcus marinus
sp. MED4 genome from the "Finished Eubacteria" pull-down menu, and note the %GC. Compare this percentage with the genomes of
P. marinus
MIT9313 and
Synechococcus
sp. WH8102. Do the two
P. marinus
genomes look like two ecotypes of the same species to you?
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Go back to the
P. marinus
sp MED4 genome and click on the "View genome in Web-Artemis" link. It will take a while to load this Java applet, but it is worth the wait. Warning: Do not close the web page that simply says "loading entry - done" or you will lose the applet. You will see the full, annotated genome in three frames. The top and middle frames are duplicates but the vertical slider bars allow you to adjust magnification, with the default showing the top frame in medium scale and the middle frame in high-resolution scale. Thin vertical lines in the top frames represent stop codons. The bottom frame is the complete list of every gene and annotated feature (e.g., transmembrane domain, signal peptide, etc.). A "c" in the gene list indicates "crick" strand of DNA. Your browser should display a few new menus that add extensively to the Artemis viewing and analysis. Under the "View" menu, choose "Show CDS Genes and Products" and under the "GoTo" menu, choose "Navigator... ." Check the "Goto Feature with This Qualifier Value:" and search for "
cobA
" (see Figure 2.19), telling the navigator to pay attention to case. Double-click on the highlighted gene in the list of "Genes and Products," and you will see the ORF displayed in the main graphic window, complete with DNA and protein sequences. What does
cobA
do, and is it on the Crick or Watson strand? Now click on the main graphic window to make sure it is the active one (a Java requirement). Under the "Graph" menu, choose "GC content (%)" to see how the GC content shifts with different genes. In the list of features in the bottom frame, scroll down until you see a tRNA gene (light green box). Double-click on the box and look at the %GC. Try a few more genes and describe the pattern you observe. Artemus is very powerful, so feel free to explore and make new discoveries on your own.
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Go back to the ORNL Microbial Genome page, select
Synechococcus
and note its %GC again. Now launch the Artemis viewer (wait...) and view the following regions with attention to the GC graph and how many genes are in these AT-rich sections: (1) 427233;(2) 622199; (3) 912098; (4) 2379778. Did you notice one of the world's longest prokaryote ORFs in one of these sections? Compare this long ORF to the average gene on the
Synechococcus
statistics page. You have just examined four areas with different codon bias and GC content. Propose an explanation for these four apparent anomalies.
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Perform a PubMed search for the term "selenocysteine" and find out what this is. Does it matter functionally whether a protein incorporates a cysteine or a selenocysteine?
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Search Google for "isoprenoid Wiki" and select the link for Wikipedia to read what isoprenoids are. Explain why loss of the apicoplast would be lethal given it's the source of isoprenoids.
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Go to the KEGG Pathway web site and click on the "ATP synthesis" link to see a model of ATP synthase. Change the pull-down menu from "Reference pathway" to "Plasmodium falciparum" located near the top of this long list of species, then click on "Go." Does
Plasmodium
have all the parts necessary to synthesize ATP from an H
+
-ion gradient? Explain your answer.
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Return to the KEGG Pathway, choose "glycolysis," and see which enzymes
Plasmodium
has. Follow the pathway from b-D-Glucose to pyruvate and see if any steps are missing. Compare
Plasmodium
to
Saccharomyces cerevisiae
(baker's yeast) to see which one has the more robust metabolic capacity. Finally, look at Aminoacyl-tRNA biosynthesis on the list of maps and see if
Plasmodium
is missing any enzymes that are needed to synthesize tRNAs coupled with their amino acids (aminoacyl-tRNA). Would you predict that a parasite might depend on the host for any of these enzymes? Explain your prediction and then test it by searching the database.
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Go to PlasmoDB, click on "Genome browser" next to "Quick Tools" to view bases 400,000-450,000 on chromosome 1 (enter MAL1:400000-450000) and click "Search". Below the busy graphics, Click on "Configure tracks..." choose to hide all options except "Gene Density," "Annotated Genes," "BLASTX Alignments," "DNA/GC Content," all of which should be set to "show one line." Hit the "Accept Changes and Return...". Do the BLASTx (DNA query against protein database) hits align with the annotated genes? Do any of the hypothetical proteins show hits from the BLASTx search?
Now go back to PlasmoDB and choose to view the mitochondrial genome (may have to use the older version 4.4 if not available yet in the newer release) at the bottom of the page. Change the display so that all RNAs and genes are displayed as "show-expanded." How many genes and how many RNAs are encoded on this organellar genome? Explain why the two numbers are different.
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Perform a search at the
Saccharomyces
Genome Database (SGD) web site and perform a Quick Search for maltose-metabolizing genes
Mal1, Mal2, Mal3, Mal4, Mal6, Mal10, Mal12,
and
Mal13
. Determine which of these genes are true paralogs or phenotypes with uncertain genomic information. Which gene or genes have the most detailed information?
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Near the bottom of the
Mal13
page, click on the "MIPS" (German genomics database) link to see a different source of information about Mal13p (p for protein). Are the two databases identical in content, or do they present different information?
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Go to SGD's Advanced Search to get an up-to-date count on the number of ORFs, ncRNA (nonprotein-coding), pseudogenes, rRNAs, and tRNAs. The search takes a couple of minutes.
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Compare the yeast gene
Sir2
in all the Model Organisms and determine if this gene is widely conserved. Compare this result with Mfa1. Why might you get such different results with two genes?
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Go to the yeast metabolism map, click on the citric acid cycle, then the "more detail" button until you cannot zoom in any more. Move around the circle until you see the two isocitrate dehydrogenase genes (
Idh1
and
Idh2
). Mouse over the enzymes to see the chemical reaction, then click on the EC number 1.1.1.41. On the resulting page, go down and click on
Idh1
and
Idh2
to find their chromosomal locations. Are these redundant genes located next to each other in the genome?
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Enter the BruinFly database created through the original research of many undergraduates at UCLA. First, search for the term "misshapen." Click on the link in the first column and view the eyes. Read the description and then zoom in by clicking on the images. Compare the eye phenotype of
misshapen
to the
patched
eye phenotype. Click on the name "
patched
" to see information about this gene from FlyBase. Go back and click on the "P-element insertion site in the genome" to see where the transposable element landed to cause the
patched
phenotype. Is the insertion in a coding or noncoding portion of this gene? Explain how this insertion could lead to a mutant phenotype. If you look at other genes, notice some of the quirky names used by fly biologists (e.g.
Ken and Barbie
,
Sunday driver
, and
deadpan
).
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Search Entrez for the largest fly gene, called
Kakapo
, then click on the "gene" database option. Notice the polytene band location on the results page, and then click on the link. What name was given to this gene based on its mutant phenotype? How many different mRNAs are produced from this one gene? While on the gene page, click on the "link" link in the top right corner and choose the map viewer option. You should see the gene highlighted with its location shown on the drawing of the polytene chromosome on the left side. Notice how long this gene is. Click on the "hm" (homology) link to the right of the gene name. Is this gene found in many different species?
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Go to Ensembl and click on the fruit fly link to access the fly database. Enter "P450" in the top text box, use the pulldown menu to choose "gene" and search "e!FruitFly". Only a few hits will be displayed out of a large number possible; click on CG10093, which may be the first one listed. The gene
Cyp313a3
should be displayed with other genes nearby. Do you see any clustered paralogs? Scroll down to determine how many exons are used in the mRNA, as shown in a diagram.
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Go to BDGP and click on the "Expression Patterns" link to see where a particular mRNA is produced. Search for
bicoid
(abbreviated
bcd
) and follow the links until you see a bar graph and images of the blue-stained mRNA. When and where is
bcd
transcribed? Compare the
bcd
expression pattern to the pattern for
Mkp3
to see how differently some genes are transcribed.
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Search OMIM for "transferrin" to see what this human protein does. On the page, do a find function for the word "Alzheimer" to see one possible medical role for this gene. Now perform a search on FlyBase for "
Tsf1
" to see if transferrin could be studied in the fly as a possible model for this protein's influence on human Alzheimer's disease.
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Go to the IRGSP web page and click on the status tab to see where the project is currently. "PLN" means that the finished sequence has been submitted to the PLaNt database. Click on the finished bar graph for chromosome 5 to see a clone-by-clone list of all the DNA sequenced for this chromosome. Click on the P0668H12 clone link called "INE" (for INtegrated rice genome Explorer) to see an interactive version of the rice genome (this launches a Java applet, so be patient). Move down to about 5.5 cM on the chromosome to locate the marker "S14158"; mouse over this text to see how many different maps it is on, or not on. Click on the text to launch a new window containing information about this marker. Now click on the image file name link "S14158.JPG" to see a DNA fingerprint of the DNA insert that contained this locus. How were these data used by the sequencers during the assembly phase of the project?
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On the clone-by-clone list page, click on "RiceGAAS:Rice Genome Automated Annotation System" for the same P0668H12 clone you explored by INE. This view gives you a very different insight into the same DNA. For example, does this portion of chromosome 5 contain any tRNA genes? Does the repeat masker identify highly repetitive DNA within genes or outside of genes? Many genes have been predicted by the various computer programs, but how many of these failed to yield BLASTx, cDNA, or EST hits and therefore could not be verified by biological evidence? You can change the view by altering the default preferences in the bottom frame. If you find a segment you really like, you can select the "MAP Download" link and print out a copy to hang on your wall.
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Go to the Rice Annotation Database (RAD) and click on the link to gene length distribution; then choose, in this order, chromosomes 1, 4, 10, 3. What characteristic changes the most" Does this characteristic correlate with chromosome length? Return to the RAD main page and determine the source of the trend you detected for these four chromosomes by studying the
ideogram
of the chromosomes.
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Go to the Chromosome 8 link and you will get basically a blank page. Click on one of the black areas in the ideogram at the top, then scroll to the right and left until you see some content. What area have you landed in, based on what you do not see and the physical location within the chromosome? Explain why you did not see anything when you first explored the chromosome.
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Go to BGI's Rice Information System (RIS), click on the "ComView Tab" at the top, and then click on the "Refresh" button on the right side if the settings indicate the base organism is 9331 (
indica
), chromosome 1, the first 1 Mb. Explore the two syntenic chromosome sections until you find two cDNAs that have drastically different locations between the two cultivars; click on the
japonica
cDNA with the higher nucleotide numbers. By how much do the two cultivars vary in this encoded protein? (Look for the single nucleotide polymorphism or
SNP
.) Click on the "Mapviewer" link to the right of the SNP information and determine if this variation altered the protein sequence. Do you consider this to be a conservative amino acid substitution? Now BLAST the amino acid sequence to determine what is known about this protein.
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Many academics are concerned about free access to genome information. Go to Syngenta's web page and read the second paragraph. Follow the links to see how quickly you can access the data. Compare this effort with the BGI databases in the preceding Discovery Questions and draw your own conclusions about the availability of data.
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Look at Figure 2.39 and consider chromosome 13. Was it duplicated, or was it unlucky enough never to have been duplicated? If 13 was duplicated, describe what happened to the duplicated version. Which chromosome pairs have been duplicated and retained nearly intact?
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Find the location of the human genes oligophrenin and arrestin, using MapViewer. Now go to the Tetraodon Genome Browser and search for oligophrenin and arrestin. Can you detect the interleaving genes shown in Figure 2.40 and the genome duplication in Figure 2.39 using these genes? Do they have paralogs near each other in the puffer fish?
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Go to the Human Genome Browser and search for "sarcospan." Is sarcospan highly conserved in the diverse species shown in the browser? You should see the Tetraodon Net line; click on the
Tetraodon
exons until you get a tabular report showing the gene's summary statistics. What is the size difference between the human and fish genes?
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Go back to Tetraodon Genome Browser and examine the ideograms. Is the genome finished yet? Click on a gap and show the resolution at 50 kb, then change the viewing options so that all are hidden, except turn on "DNA/GC content," "Gap: All Sequence Gap," "Genescan," "Hox Genes," "
Takifugu
ecotigs," and "
Tetraodon
cDNAs." What effect do gaps have on the number of genes predicted by Genescan? Compare the predicted genes to the number of cDNAs to see if any validating sequences are available. If so, how well did Genescan predict the genes' correct sizes? Now search for
HoxA
, zoom out to 200 kb, and change the settings to highlight mouse, human,
Takifugu,
and
Tetraodon
gene conservation. Which
HoxA
gene is in
Tetraodon
but not the mammals? Move your cursor over human, Fugu, and mouse
HoxA5
genes and note in which chromosomes each resides.
Bonus Material
: A study of the chicken genome is available on this book's web site. 90. Let's do some quick estimations about our DNA using these numbers: haploid genome of 3,289,000,000 bp; 35,000 genes; and the numbers from Table 2.7. a. What percentage of your genome is spent on genes? Exons? Introns? b. What percentage of your genes is spent on exons? Introns?
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Let's do some quick estimations about our DNA using these numbers: haploid genome of 3,289,000,000 bp; 23,000 genes; and the numbers from Table 2.7.
What percentage of your genome is spent on genes? Exons? Introns?
What percentage of your genes is spent on exons? Introns?
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Are any chromosomes missing from Figure 2.43b?
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Given the information in Figure 2.45, name one aspect that makes humans different from other species.
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Go to the BLAST2 web page and perform a protein alignment with human sarcoglycan delta (NP_758447) and sarcoglycan gamma (NP_000222) by pasting their protein accession numbers into the smaller accession boxes and choosing "blastp" from the program menu. What are the percent identity and percent similarity between these two proteins? Now align the
C. elegans
sarcoglycan ortholog
sgn-1
(CAA92663) with both of the human sarcoglycan genes and determine which one is more similar (compare identity, similarity, and gaps).
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Go to UCSC's Genome Browser and search for "syntrophin." How many syntrophin genes do humans have? Click on the link for "(NM_009228) syntrophin, acidic 1." Change the view to hide all except "Known genes" in full view, and all the different species "Net" views in dense, then refresh. Is this gene's structure (combination of exons and introns) highly conserved? Click on the dog alignment twice until you have a non-graphic report for this locus of the dog genome, then click on the "Open Dog browser" link. Set the species "Blat" or "Net" views to full, and "Conservation" to full, then refresh. Is the conservation confined to the exons only? Explain the significance of the conservation in the noncoding regions.
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Go to the Comparative Maps web page and click on "Chromosome 9" under the "Rat and Mouse compared to Human" column. Using the "Region Shown" box in the left frame, enter "124M" in the top box and "125M" in the bottom box. Locate the human gene called
PTGS1
, which is the
cyclooxygenase
1 gene and the target of painkillers such as aspirin (see Chapter 9 for details). Use the "Maps & Options" button, activate the "Show Connections" option, and hit "Apply." Do all 3 species have
PTGS1
? Are these regions syntenic, or are the human genes not linearly related to those in the two rodents? What pattern in the alignment of genes is evident just below
PTGS1
? Click on the Rat ortholog and choose the "ev" (evidence) link. Do you think there are sufficient data to support the annotation that this is a true ortholog?
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Go to UCSC's ENCODE web site and chick on the most recent “Regions” (e.g. hg17 or higher). "Alpha Globin" from the menu in the left frame. Alpha Globin ENm008 is abbreviated HBA, but you can see several genes that begin with HB. How many can you identify? (You may want to modify the view and zoom in to help you focus on the HB genes.) Considering the conservation in other species, how many of these HB genes are conserved in vertebrates? Do all these genes produce functional protein? Click on "HBA1" to find the answers in text. In the “Sequence and Links to Tools and Databases” table, click on “GeneLynx” and then the "AceView" for HBA1 to see more information, including graphic depictions of alternative splicing for these genes.
Return to the list of ENCODE genes in the left frame and choose "CFTR". Modify the “Comparative Genomics” settings below to show “Conservation” as full, and as many species as you want (choose only the “Net” option for each species) and set these to “dense”. On what chromosomes are CFTR orthologs located for each species (you can double click on the colored lines to open a report page)? Is CFTR conserved in chicken, or is the gene truncated? Pay special attention to the degree of conservation within introns. You can recenter and zoom in to see the genes in better detail.
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Perform a Gene search for the human
ITGB3
(integrin beta 3) gene which is illustrated in Figure 2.47c. Is the correct version of the gene in the database, or is the number of exons incorrect? Click on the KEGG pathway for regulation of actin cytoskeleton 04810. Where is integrin located in cells (its cellular component, in GO terminology)? Click on the highlighted box labeled "ITG" and use your browser's find function to locate the amino acids "RNRD." Does this database have the old or new acid sequence?
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