Q1) How would you determine the function of PUG1 in yeast?
“A gene is a discrete genomic region […] which contains the information for the synthesis of functional proteins or non-coding RNAs” 1. Thus the found gene sequence might be encoding for a protein or a non-coding RNA since it has an unknown function. “A non-coding RNA gene sequence does not have strong statistical signals, unlike protein coding genes”2. Thus, specific motifs/specific sequences give a clue about the gene product. The ORF (="open" reading frame) ranges from the start codon (ATG) at one end to one of the three stop codons at the other end, with at least 100 bases in between. A gene encoding for a protein has normally specific additional sequences in and around the ORF, e.g. an enhancer, TATAboxes, a 5’UTR site, specific motifs like Leucine Zipper or Zinc Finger. For the following examination I assume that PUG1 encodes for a protein. With reverse genetics different approaches are feasible which can give clues about the function of PUG1.
So, to find the specific function of PUG1 there are two main approaches: One concerning the sequence itself and one concerning the function by introducing mutations.
The ORF from PUG1 was discovered through reassessing the known yeast genome sequence and it was found constantly expressed throughout all stages of the cell cycle. This implies that the protein is not involved in any specific phase of the cell cycle, or rather in its control system. But it does not mean that it is not needed in the cell cycle. It probably could be even needed for every step. Furthermore, no obvious homologous gene in yeast itself could be identified implying that the gene does not belong to a multi-family. Being homologous means that they are evolutionary linked by a common ancestor and thus, often (but not always) similar in sequence. Nevertheless, you should search for specific sequences (e.g. special motifs) giving a hint for the function. Additionally you could look on the genetic map for the surrounding genes which might indicate a linkage group functionally linked by specific sequences and consensus motifs. But the latter is probably not the case since no homologous sequences in yeast has been found. But in many cases, the structure has been highly conserved more than the amino acid sequence, since the function is more important. From the DNA sequence it is possible to predict the amino acid sequence and with that you could try to identify the 3D structure of the protein since some proteins are not similar in sequences but in structure. To accomplish this you could either do Circular Dichroism (CD) experiments/X-ray crystallography/Nuclear Magnetic Resonance or deduce the 3D structure on the basis of the amino acid sequence by structural superposition with special computer programmes. Comparing this 3D structure with other proteins from yeast could show a belonging to a family since related structures often imply related function.
In addition you could try to identify homologous gene sequences, protein sequences and 3D structures in other organisms by comparative genetics (covered by question 2-5), using sequence alignment programs like BLAST and FASTA; from their previously characterized functions you could also deduce the function of PUG1.
The found similarities could point in the correct direction, but it is still necessary to test these insights through direct experimentation.
To analyse/determine the function of PUG1 it is possible to do a knockout experiment using homologous recombination (HR) so that they lack the gene or express an altered version of it. In HR you combine PUG1 with a marker, like Kanamycin-resistance, and introduce this construct in the yeast genome, where it binds on the homologous strand and gets replicated. Then, observing the phenotype should give a hint of the PUG1’s function. I would look after obvious changes in shape and cell processes like growing and budding. In addition it is possible to introduce the yeast lacking PUG1 to al lot of chemicals or to different stress factors. If the mutant differs, it shed light on PUG1’s function.
But if the gene is essential, a knockout mutant would be lethal precluding further studies of these mutants. An alternative would be to manipulate the gene in such a way, that it is over expressing and look if the phenotype differs or if it has an effect on other proteins.
Q2) How would you identify homologous PUG1 gene(s) in one named invertebrate?
To identify homologous PUG1 gene(s) in e.g. Caenorhabditis elegans it is possible to use a sequence alignment program like BLAST or FASTA to search for homologous genes in genomes of other organism or specifically of C. elegans. The program searches cluster of residues which fall into full or partial alignment. It is possible to search for an amino sequence and a nucleotide sequence which both could allow the prediction of PUG1’s function.
But even if there is a functionally similar protein, they could be too distantly related to be identified as clearly homologous in comparison to their amino acid sequences alone. Mutations will change the sequence of genes (largely without disruption their functions). Thus, a 3D structure prediction of PUG1 from its amino acid sequence would improve the findings about its function from the sequence alignment. And since homology per definition (see page 1) does not exclusively mean that the sequences have to be similar, the structural comparisons would be a good additional way to find such dissimilar homologues. But since PUG1 is unknown, a structure determination is relatively hard to work out. So, comparison of the amino acid sequences is the preferred way. To compare PUG1 from yeast with that from C. elegans I would BLAST the sequence against the genome from C. elegans and score the number of matches. Thereby, you have to consider that the gene sequence likely has changed e.g. due to deletions, insertions, inversions and point mutations and the gene itself can be interrupted by diverse introns. Thus, you never get a complete homology and you have to allow gaps in the sequence alignments. The sequence alignment is then checked by generating randomly scrambled sequences (which also align and score) and look if the true sequence scores significantly better than the scrambled sequences. It is especially important by looking at the score alignments, that the 3’- , 5’-end regions and specific motifs giving the function are highly similar/highly conserved.
To omit the possibility that introns significantly interrupt the alignment, it would be better to compare cDNAs with each other. cDNA is made by copying the mRNA to DNA with reverse transcriptase, so that only the coding parts of the gene are in one row. I could either BLAST the cDNA of PUG1 (which first must be produced) against the C. elegans genome or against the cDNA database of C. elegans. Also a library screening can be done through hybridisation test on a cDNA library of C. elegans to look if the PUG1-cDNA binds somewhere. You could also use degenerated cDNA probes of PUG1 which do not bind too specificly, considering the changes in the sequences through evolution.
If you found a homologous gene in C. elegans by sequence alignment or with the aid of the other methods described above, you have to investigate its function through experiments, observing the mutant phenotypes and you could also try to rescue the mutant organisms with the PUG1 gene of the other organism in both directions. If they get rescued the genes are definitely homologous (or rather orthologs, which are homologues in different organisms sharing the same ancestral gene). How this can be done, is described in the following question.
Q3) How would you determine the function of PUG1 homologue(s) in this named invertebrate?
On the one hand the theoretical found sequence/protein/3D structure similarities described in Q2 give an evidence for the function of the PUG1 homologue (=PUG1c) in C. elegans. On the other hand, provided that the methods in Q2 revealed a PUG1c in C. elegans, it is possible to determine the distinct function of PUG1c in C. elegans by perturbing the gene and producing mutants for PUG1c-loss-of-function as long as the gene mutant in yeast is not lethal. The observation of the phenotypes could give a clue for the function or could prove or disprove the theoretical function found in Q2.