Our first part we will be going through some of the eukaryotic protein coding gene sequences
and the corresponding GenBank records. RAG1 is our example. This is showing the Takifugu
gene and there are four exons for this gene in the puffer fish. The first exon is a non-coding
exon that has the 5-prime UTR untranslated region. It is followed by an intron and additional
exons for the rest of the gene. The second and fourth exons are both coding and non-coding.
I once had a submitter who thought exons were only coding. It depends on your organism and
on the gene. In the puffer fish, of the four exons for this gene, only one exon is ONLY
coding.
If we continue to look at RAG1 sequences, the puffer fish gene is 5146 bases long. Showing
on the screen are other GenBank accessions. Most people who use RAG1 as a molecular marker,
they only determine the sequence for part of exon 4. The sequences they obtain from
the sequencing technology are 5-prime and 3-prime partial and they are approximately
1400 bases long. Please recognize 5-prime and 3-prime partial say nothing about whether
it is untranslated. You want to make sure you keep straight the 5-prime and 3-prime
nature of your sequence data.
Let's look at the GenBank record. There is a link on the page that will give you more
information about the individual fields in a GenBank display format as shown on the screen.
Primarily there are three parts. There is the top part shown in pink. The next part
is outlined in red, and the blue part, and I will go through that in a moment. The top
part is the part that describes the study, the organism, the taxonomic information, the
references and it gives you the definition and the accession number. The bottom section
which we have a small part shown here at the bottom of the screen is the actual sequence,
whether it's DNA or RNA, and it has numbered rows and it is in groups of 60 nucleotides
for each row.
If we look at the FEATURES section, there is source information. For this particular
GenBank record, we only have the organism name, but this is where you would include
the culture collection or the strain or other isolate information. Then we have the annotated
gene and other features.
Sometimes people look at a GenBank record and they see other information that is included
in the CDS section of the GenBank display record. They add this information in their
data submission. You should not do this. This information is provided by GenBank and NCBI
staff. The translation is what will be done by the submission tool when you add the CDS
feature. You only need to worry about the specific features, namely the gene, messenger
RNA and CDS feature when you do your data submission.
If we look at the FEATURES section in a little more detail, it's important you indicate with
the correct partial indicator, whether it is 5-prime or 3-prime partial, and when you
look at the graphic representation of the RAG1 gene, this record again, the first base,
and then it is 1439 bases long and it is 5-prime and 3-prime incomplete. Sometimes submitters
think they need to give coordinates that are related to the gene itself or to the chromosome,
the genomic piece of DNA, but you should always provide your information for the features
as they are on your sequence, whatever length your sequence is, that should be the total
number of bases included in the features you are adding at the time of your submission.
Let's look at beta-tubulin as another example, from this fungus. This is a member of the
tubulin family. It has seven exons. In this organism, none of the exons include untranslated
regions. All of the exons in this particular organism for this gene are coding.
I want to focus in on one specific splice site. This is the crux of the matter of eukaryotic
nuclear genes. In the DNA sequence, and this gene is on the plus strand, introns start
with a GT, the splice donor site or splice donor pair. And introns end with an AG, that
is called the splice acceptor pair. 5-prime is usually GT, 3-prime is AG. This is not
the only donor-acceptor pair. There are some others, but predominately in eukaryotic genes
it is GT-AG. If you indicate your coding region, such that it includes this G, you will get
an error in some of the submission tools, but it will be biologically incorrect and
GenBank staff will ask you to correct it because it's not biologically valid. The GT-AG pairs
are something you want to pay special attention to when you are submitting data that is protein
coding and that has exons and introns.
Here are more examples of beta-tubulin sequences. Most of the beta-tubulin sequences submitted
again are 5-prime and 3-prime partial sequences. Here we have some GenBank accessions shown
on the screen and they cover several different exons. We are going to have several pieces
or several parts of our coding regions that we need to include in our CDS feature.
This is a genomic scaffold. This is a specific GenBank accession number I'm showing again
for the exons, but this KP189336 accession number starts at base 1, it's 5-prime partial
so it has the less-than-sign, and it is 675 nucleotides long so it has the greater-than-sign
to indicate it is a 3-prime partial. We determined the individual nucleotide numbers for this
sequence for each part of the coding region and that is what we will need to add in our
CDS feature.
We have a specific submitter who has written to us. They have two different data sets.
One for TEF1a and one for beta-tubulin or TUB2, and they have already identified some
GenBank records that are similar to their data sets so they are asking for help. And
the important thing to recognize is to understand whether the person doesn't know how to determine
the CDS locations or she may know them, but she doesn't know how to add the information
in the submission tool.