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Web sites: The RNA World Web site: http://www.imb-jena.de/RNA.html Tree of life: http://www.tolweb.org/tree/ ARB (downloadable program): http://www.arb-home.de/ Ribosomal Database Project (RDP): http://rdp.cme.msu.edu/index.jsp
Exercise 1 A. Sequence Match 1. Which of the sequences (>1200) in the RDP most resemble the sequence given below? 1 gacgaacgct ggcggcatgc ctaatacatg caagtcgaac gcttttgttt caccgggtgc 2. Check the information (GenBank flat file accessed via S00 number)
for your best match. What habitat was the organism
isolated from? B. Use the Hierarchy Browser (search) to find Methylomonas scandinavica. a. Does this organism belong to the domain Archaea or Bacteria? b. M. scandinavica is found in which phylum? c. M. scandinavica is found in which class? d. M. scandinavica is found in which order? e. M. scandinavica is found in which family? f. Where was this organism isolated from? g. Download the sequence to your on-line files. This will be one of the sequences used for labs 2.2 and 2.3
Probe Match (set size to "both") C. For each of the following sequences, determine: a. Which genus, family, or order of organisms the signature sequence or probe is specific for? [That is, which of these headings encompasses (contains) the bulk of the matches given.] How many "hits" (query is complementary to sequence found in this organism) fall into this category out of the total hits. Be sure the settings are correct for your entry - they will differ depending on if you are entering a signature sequence or a probe sequence. b. For each of the following, how many sequences within the genus (or family or order) aren't complementary to the signature sequence/probe?
Possible Signature sequences (- strand, 5'-3'): 1. TAGAGTGCAGCAGAGGGG 2. GATGTGGAGCGAACCTGAGA Possible Probes (+ strand, 5'-3'): 3. CTTCCATACTCTAGGTAC 4. ACCGAGGTACATGTACCCCGACAT
D. Which of the 4 sequences above would work the best for a genus-specific probe (or signature sequence)? Defend your answer. Provide at least 2 reasons for your choice. E. When you run Probe Match the size default is "both". Why do you suppose you'd want both instead of just >1200 or <1200? When set to "both", if the results show your probe hits 23 of the 52 sequences, does this mean that 29 organisms have sequence with mismatches to the probe in the region of the 16S rRNA where the probe is targeted? Defend your answer. F. Look back at your results for signature sequence 2 above (i.e. matched it in Probe Match). Was the probe complementary to sequence from all members in this group (with no mismatches)? Probe match allows you to restrict your search by entering a region of the sequence that should contain your signature sequence/probe target. This narrows the search to only include the sequences in the database that contain the target region complementary to your probe. Note: it still checks the entire sequence though and not just the region entered. Run Probe Match again setting the region to 1240 to 1290. How do these results compare to those you had before? Why are they different? What do these results tell you about the utility of the probe compared to what you knew before?
(Do not hand in this next part. These sequences are needed for Lab 2.2 and so you need to have found these sequences before you can do this next lab. Be sure to use appropriate nucleic acid databases and not protein databases - otherwise you can use any database you prefer. ) Find the following rRNA sequences (or the rRNA gene sequences) and download or copy the sequences into Biology Workbench, a FASTA file or a text file (do not use a word processing program like Microsoft Word). Note most of these aren't in the RDP currently as they are still preparing the Eukarya section and so you should use either Workbench or Entrez to retrieve the sequences. For some of these there will be more than one option to choose from. Please select complete or large partial sequences rather than small partial sequences if possible (about 1500 nt for 16S, over 1400 to preferably close to 1600 for the protozoa and at least 1700 nt for the other 18S, although 1900 is better ) and do NOT use sequences which also contain other genes or spacers. The protozoa files may refer to these as 16S or 16S-like as they are much smaller than typical 18S rRNA. We will be using these sequences to perform multiple alignments and create phylogenetic trees and the sequences you select can affect your results. Mouse 18S rRNA Methylomonas scandinavica 16S rRNA Human (Homo sapiens) 18S rRNA Xenopus laevis 18S rRNA Actaea japonica 18S rRNA Chlamydomonas nivalis 18S rRNA Trichomonas tenax 18S rRNA Giardia intestinalis 18S rRNA Methanocaldococcus jannaschii 16S rRNA Escherichia coli 16S rRNA Pyrodictium occultum 16S rRNA
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