1. Locating genes in Plasmodium falciparum You have seen how artemis is used to view, analyse and annotate bacterial genomes, but now we are going to move on to looking at predicting genes in a eukaryotic genome. The eukaryote is Plasmodium falciparum, a parasitic protozoan which causes malaria. The gene prediction process and the use of Artemis are fundamentally the same for eukaryotes and prokaryotes. One key difference is that eukaryotic genes can in some cases, contain regions within them which do not encode the protein product as they are removed before protein synthesis (translation), by enzymatic processing. These removed regions are called introns (blue). The regions which flank the introns and are translated are called exons (orange). This process is summarised in Figure 1 which shows a theorectical eukaryotic gene with two introns. transcription gene mRNA prior to processing mRNA processing Processed mRNA exon intron translation protein Around 53 % of Plasmodium falciparum genes have one or more introns, and so this must be considered when predicting genes. The nucleotide composition of the genome of Plasmodium falciparum is such that it is very rich in adenine (A) and thymidine (T) and poor in cytosine (C) and guanine (G). If we take the genome as a whole, G + C comprises about 19.5 %, but if we look only at genes the percentage G + C is higher, at around 23 %. So coding regions often appear as distinguishable peaks in a plot of G + C composition over a sliding window. We can use this as a line of evidence to identify the location of genes (and their component exons if they are multiexon genes) Figure 1

2. Gene prediction in eukaryotic genomes: location of exon/intron boundaries. The exact locations of exon/intron boundaries (also referred to as splice sites) is helped by the fast that they have particular sequence patterns (see Figure 2, see also Appendix 4). These patterns have a biological function as recognition signals to the cellular machinery that removes introns during RNA processing. We can identify these sequences by eye, or train computers to identify them, and so locate introns. Note: The first two bases of the intron are always GT and the last two bases always AG. The others may vary...AAGGTAAAGA.....TTTAGNNN....TTCCATTTCT.....AAATCNNN.. exon intron 5’ 3’ Figure 2 Sequence patterns at exon/intron boundaries in P. falciparum. Exercise 3 The aim is to examine a region of chromosome 6 of P. falciparum, find several areas where genes may be missing by examining GC and Correlation Score graphs. Your gene predictions will then be refined by comparing them to genes in the database. The second part of the exercise is to design PCR primers to genes in the areas flanking a therorectical region where the sequence quality is poor. It is useful to have an internet browser open in the background for viewing search results Firstly open the file MAL6.region in artemis. Use the slider on right hand side to get a view of the whole region as below. From the Graph drop down menu select ‘GC Content %’.Use the slider on the GC graph to a adjust the GC content window size. Do the genes that are marked have corresponding peaks in the CG trace?

3. Display the ‘Correlation Scores’ graph by selecting it from the the same Graph drop down menu. What do you notice about the coloured traces in regions where genes are located? If your curiuos ask a demonstrator what correlation scores are based on. We are now going to look for missing genes in this region on the basis of GC content, correlation scores and BLAST or FASTA search results. Look for peaks in the GC trace which correspond with separation of the Correlation Score traces. Are there also corresponding gaps in the stop codons in any of the six frames? Once you have identified such a region mark up a CDS in the gap in the stop codons. You do this by rapidly double clicking the central (scrolling) mouse button when the cursor arrow is pointing at the gap between stop codons. Then select ‘Create New Feature From Base Range’ from the create drop down menu. Then select OK. A light-blue CDS will then be marked up in that frame and also listed in the feature list (lower panel). We can now ready to run a search on the nucleotide or amino acid sequence of the selected feature against the databases. An example is shown left, but note that a gene is not present at this location, If you have any problems ask a demonstrator. Note this possible CDS has quite extended stretches of K and F. Unlikely to be a protein. Compare this with the amino acid sequence of one of the marked CDS’s.

4. Go through and mark up CDS’s in places where you think a gene may be present. Before moving on to searching we’ ll look at how genes with introns are represented in Artemis. Either look at PFF0520w or PFF0550w. Note that the exons are linked by a kinked line and may be present on the same frame, as in the case for PFF0520w, or be located in different frames as in PFF0550w. PFF0550w is shown in the screenshot below. The sequence of the exon/intron boundaries is visible in the lower panel. The ‘GT’ at the 5’ end of the intron and the ‘AG’ at the 3’ end are marked and arrowed for clarity. If you’re curious, quickly check how the sequence at the boundaries compare with the sequences described in Appendix 4. Now we are ready to run searches on predicted CDS’s. This will allow us to compare predictions against genes already in the database and give us information to decide if the predicted CDS is really a gene and also give us information which will help in the location of exon/intron boundaries if any introns are present. To search your predicted CDS features against the database: Select it by clicking in it, this should result in a black line outlining the CDS. Select the ‘Run’ dropdown menu, and from it ‘Run fasta on selected features against’ and ‘% uniprot’. These searches may take a few minutes so be patient. You can run the searches simultaneously by selecting all your predicted features and selecting the above option. When the search is complete a box appears. Access the results by pressing ‘r’or by selecting ‘View’ then ‘Search results’ then ‘fasta results’ from the drop down menu (for both the CDS feature must be selected). These results can be sent to a internet browser by choosing ‘Send to browser’ which gives you a convenient way to click between the hit and the alignment as they are bookmarked. Your search give you a list of similar proteins in the database as well as alignments against your query protein (ask a demonstrator if you want more details).

5. Repeat this process of integrating GC data, correlation scores and FASTA searches to define the remaining upstream exon or exons of this gene. Remember that it could be in the same or a different frame, and that the first bases of the intron should be ‘GT’. To show the start codons (vertical magenta bars) right click in the middle panel and click the start codons box. If your gene prediction contains an intron it is necessary to merge them to show that they are linked. Do this by first clicking to select adjacent exons. Then select the ‘Edit’ drop down menu, then click OK and OK again when prompted ‘delete old features’. The merging process may cause the exon to shift frame as the exact exon/intron boundary may be within a codon. Go back and check that the intron starts with a ‘GT’ and ends with ‘AG. If necessary you adjust the boundaries by clicking and dragging the end of the exons in the lower frarme panel. To check your predictions reveal the missing genes by selecting ‘File’ then ‘Read an entry’ and choose the file ‘excercise3.tab’ Now we are in a position to define the CDS’s precisely. Compare information from the alignment with the region that you have marked up (it is convenient to have the results window open in front of artemis). Alignments can tell you whether the CDS region you have defined is internal to the gene, extends beyond the start or end (both), or whether it is likely to contain an intron. In the example below the CDS matches a protein in the database from position 53, ‘VAVIA.. to the end of the gene, but the start of the gene is missing. We have found an exon of the gene. Move the CDS from position 1 (red arrow) back to position 2 (red arrow). Note that an ‘AG’ sequence (red arrow 3 and boxed white) is present just before the start of the exon (starts with) ‘VAVIA’ giving confidence that this is a real intron – exon boundary. If you have any questions on this ask a demonstator. 1 2 3

6. The second part of the exercise is to design PCR primers to two genes in this region. For the sake of this exercise we’ll assume that more sequence information is needed between PFF0520w and PFF0530w and so we want to PCR amplify this region. Designing PCR primers in exons rather than intergenic regions (introns and regions between genes) is a recommendable approach to obtain a PCR primer that is specific. As intergenic regions often have regions of low complexity, i.e. where there are long tracts of A and T. Such regions of low complexity make PCR primers design difficult. Select regions that you think are appropriate for PCR primers. Mark them up as genes by selecting ‘Create new feature from base range’ then in the upper left corned select ‘gene’ in the drop down menu. Remember that one primer must be designed on the coding strand and the other on the complementary strand. Ask a demonstrator if you have any questions. Then estimate the approximate size of the PCR product.