Genetic Nomenclature

[Beata]

Hello,

I am confused by some genetic nomenclature and concepts which I’ve come across in the literature. Could someone please tell me if my interpretations below are correct and answer questions? (I apologize that this is kind of lengthy - if you only want to take a stab at one or a few questions - I’d still appreciate it!) Many thanks!

1. Gene fusions vs. transcriptional reporters - example: is there a difference between unc-17::GFP and Punc-17GFP, or do they mean the same thing? It seems weird to me to have a gene fusion without a promoter being expressed in the worms? What would be the purpose of such a strain unless you also inserted a promoter upstream of the gene fusion which would drive the expression of the genes? Moreover, could you have the sequence of unc-17 in front of GFP? (I suppose you could, if it was a translational fusion?). Lastly, Wormbook says “promoter fusions may not give a complete representation of the real expression pattern of a gene, both spatially and temporally.” Why?

2. Genetic designations - I have seen two different designations in the literature, i.e.: a) Punc-119YFP and b) unc-119p::YFP. In a) there is no semicolon, while in b) there is and yet they both appear to be used interchangeably?! In a) the semicolon seems to be only used for a translational reporter, whereas in b) it is used even for a transcriptional one? Is this correct?! According to wormbase, a semicolon designates linkage groups, so I am not sure why it would also not be used in a)?

3. In translational fusions - is the entire genomic sequence of GFP fused to the gene under investigation, or is it some functional portion, like the exons, etc? And, do I understand correctly that the GFP sequence can be inserted anywhere, in the beginning or end of the ORF? And, “translational reporters that exhibit subcellular localization can make cell type identification more difficult because the shape of the cell may not be visible (especially for neurons).” Why would this be the case?

4. In Fig. 1 (link below), what is the difference between black lines and black, dashed lines?
   http://www.wormbook.org/chapters/www_reportergenefusions/reportergenefusions.html#figure3

5. I have been told that “back in the day,” before fluorescent reporters, people used other types of reporters like lin-15(+) by crossing the translational reporter strain into the lin-15 mutant background. If the lin-15 defect was rescued, you knew that your new strain carried the transgene of interest. I am confused about how this works. How do you know that [i]lin-15/i is always linked to your transgene of interest? Does [i]lin-15/i mean that the strain is WT for the lin-15 gene, or does it mean that it’s one of the isoforms of lin-15? Could someone elaborate on what the lin-15 mutation does and what phenotype it gives? I see that wormbase lists 3 different versions of it. Lastly, if you were using lin-15(+) as a reporter, why would it make sense to have it NOT in the lin-15 mutant background? I would think if you were using lin-15 as a reporter, you would always want to have it in a translational fusion strain AND in a lin-15 mutant background?

Many thanks!

[lmu1]

1. Gene fusions vs. transcriptional reporters - example: is there a difference between unc-17::GFP and Punc-17GFP, or do they mean the same thing? It seems weird to me to have a gene fusion without a promoter being expressed in the worms? What would be the purpose of such a strain unless you also inserted a promoter upstream of the gene fusion which would drive the expression of the genes? Moreover, could you have the sequence of unc-17 in front of GFP? (I suppose you could, if it was a translational fusion?). Lastly, Wormbook says “promoter fusions may not give a complete representation of the real expression pattern of a gene, both spatially and temporally.” Why?

transc reg sequence outside of the “promoter” used. posttranscriptional regulation (splicing, miRNAs, phosphorylation etc etc)

2. Genetic designations - I have seen two different designations in the literature, i.e.: a) Punc-119YFP and b) unc-119p::YFP. In a) there is no semicolon, while in b) there is and yet they both appear to be used interchangeably?! In a) the semicolon seems to be only used for a translational reporter, whereas in b) it is used even for a transcriptional one? Is this correct?! According to wormbase, a semicolon designates linkage groups, so I am not sure why it would also not be used in a)?

use of 2 colons (not semicolons) indicates a junction between two separate elements of a construct.

3. In translational fusions - is the entire genomic sequence of GFP fused to the gene under investigation, or is it some functional portion, like the exons, etc?

i don’t think anyone uses the jellyfish genomic version of gfp. it’s always a cDNA or construct with synthetic introns.

And, do I understand correctly that the GFP sequence can be inserted anywhere, in the beginning or end of the ORF?

wherever you want. it might not work though.

And, “translational reporters that exhibit subcellular localization can make cell type identification more difficult because the shape of the cell may not be visible (especially for neurons).” Why would this be the case?

GFP with a nuclear localization signal just looks like a circle when expressed in a neuron, but if you can see cytoplasmic axonal processes you could figure out which neuron it is.

4. In Fig. 1 (link below), what is the difference between black lines and black, dashed lines?
   http://www.wormbook.org/chapters/www_reportergenefusions/reportergenefusions.html#figure3

didn’t look this up

5. I have been told that “back in the day,” before fluorescent reporters, people used other types of reporters like lin-15(+) by crossing the translational reporter strain into the lin-15 mutant background. If the lin-15 defect was rescued, you knew that your new strain carried the transgene of interest. I am confused about how this works. How do you know that lin-15(+) is always linked to your transgene of interest?

lin-15(+) was used as a coinjection marker, so you had to have the lin-15 mutation in the strain you injected (this is still very useful, not some random old method). you know lin-15(+) is on your array since you select for it. you don’t know for sure that your other thing is on the array, but it probably is since all the injected dna recombines into a big amalgam.

Does lin-15(+) mean that the strain is WT for the lin-15 gene, or does it mean that it’s one of the isoforms of lin-15? Could someone elaborate on what the lin-15 mutation does and what phenotype it gives?

it’s a complex locus AB synMuv. look up some lin-15 papers

I see that wormbase lists 3 different versions of it. Lastly, if you were using lin-15(+) as a reporter, why would it make sense to have it NOT in the lin-15 mutant background?

you’re right. it would not make sense.

I would think if you were using lin-15 as a reporter, you would always want to have it in a translational fusion strain AND in a lin-15 mutant background?

[Snug]

@Beata post:1

2. Genetic designations - I have seen two different designations in the literature, i.e.: a) Punc-119YFP and b) unc-119p::YFP. In a) there is no semicolon, while in b) there is and yet they both appear to be used interchangeably?! In a) the semicolon seems to be only used for a translational reporter, whereas in b) it is used even for a transcriptional one? Is this correct?! According to wormbase, a semicolon designates linkage groups, so I am not sure why it would also not be used in a)?

From the WormBase Nomenclature section:

Gene fusions incorporated in transgenes that consist of a C. elegans gene or part thereof fused to a reporter such as lacZ or GFP are indicated by the C. elegans gene name followed by two colons and the reporter, all italicized: pes-1::lacZ, mab-9::GFP. [i]No specific recommendations have been made for distinguishing between transcriptional and translational fusions[/i]

@Beata post:1

3. In translational fusions - is the entire genomic sequence of GFP fused to the gene under investigation, or is it some functional portion, like the exons, etc? And, do I understand correctly that the GFP sequence can be inserted anywhere, in the beginning or end of the ORF? And, “translational reporters that exhibit subcellular localization can make cell type identification more difficult because the shape of the cell may not be visible (especially for neurons).” Why would this be the case?

Technically you can insert it anywhere in the ORF, but as lmu1 says, it may not work. It can make it more difficult because your reporter is not lighting up the whole cell body.

@Beata post:1

4. In Fig. 1 (link below), what is the difference between black lines and black, dashed lines?
   Reporter gene fusions

From how I interpret the figure, the solid black lines indicate introns and the dotted black lines suggest the presence of possible exons. This is an example of inserting the GFP ‘anywhere in the ORF’. For example, in B), you can have the first exon of your gene of interest followed by GFP and then the rest of the exons of your gene.

[Beata]

Thank you, Imu1 and Snug for your answers, they were very helpful! If you or anyone else wants to take another stab at the remaining questions, I’ve reorganized them to make it easier to read and reply. Thanks!

1. Does lin-15(+) mean that the strain is WT for the lin-15 gene, or does it mean that it’s one of the isoforms of lin-15?

2. Is there a difference between unc-17::GFP and Punc-17GFP, or do they mean the same thing?

3. What would be the purpose of a strain with two genes fused and no promoter to drive the expression of those genes? Snug, I did read that explanation from Wormbase, but it still makes no sense to me.

4. Lastly, if you cross two strains, each containing a translational reporter that has been integrated, is there any way of knowing how their DNA recombined in the new strain? i.e. would you write the new strain name separated with a “;” or some other way?

[Sperm_or_Egg]

Be aware that not all of these conventions are standardized, but you should be able to sort out the meaning from the reference (particularly the methods section).

1) Does lin-15(+) mean that the strain is WT for the lin-15 gene, or does it mean that it's one of the isoforms of lin-15?

It should mean WT; isoform-specific versions would be indicated, e.g., lin-15A(-) or A(-)B(+).

2) Is there a difference between unc-17::GFP and Punc-17GFP, or do they mean the same thing?

The first contains both the promoter and some portion of unc-17; the second indicates only the promoter.

3) What would be the purpose of a strain with two genes fused and no promoter to drive the expression of those genes? Snug, I did read that explanation from Wormbase, but it still makes no sense to me.

If there is no promoter, then how would the genes be expressed? Do you mean that the nomenclature does not include ‘P’ before the gene name? Then see answer 2.

4) Lastly, if you cross two strains, each containing a translational reporter that has been integrated, is there any way of knowing how their DNA recombined in the new strain? i.e. would you write the new strain name separated with a ";" or some other way?

If the reporters are integrated, recombination is unlikely. The reporters would segregate as single genes. If the two are on different chromosomes(e.g., I and II), that would be designated ‘reporter-1 I; reporter-2 II’. If they are on the same chromosome, that would be designated ‘reporter-1 / reporter-2 I’.

[Beata]

Thank you for your reply, Sperm or Egg?. Things are definitely making more sense after reading everyone’s answers. Thanks to all who replied!