Monday's Molecule #118: Winners

 
UPDATE: The molecule is cyclin-dependent kinase 2 (CDK2), a protein involved in signaling [PDB 1b38]. The Nobel Laureate is Paul Nurse.

This week's winners are Mike Fraser of Toronto and Alex Ling of the University of Toronto.

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This is a very famous protein but most of you won't be able to identify it from the structure alone. You'll need a hint of some sort.

Letting you know that the ligands are Mg²⁺ and adenosine-5′-triphosphate might not be enough so I'll also tell you that one of the authors on the structure paper was M.E. Noble.

There is one Nobel Laureate who is most closely identified with the function of this particular molecule, although that scientist was NOT the first to identify it. You have to identify the Nobel Laureate who got the prize for working out the function of the protein.

The first person to identify the molecule and the Nobel Laureate wins a free lunch at the Faculty Club. Previous winners are ineligible for one month from the time they first won the prize.

There are six ineligible candidates for this week's reward: Bill Chaney of the University of Nebraska, Elvis Cela from the University of Toronto, Peter Horwich from Dalhousie University, Devin Trudeau from the University of Toronto, Shumona De of Dalhousie University, and Maria Altshuler of the University of Toronto.

I note that Canadians are trouncing the rest of the world. That's as it should be.

I still have one extra free lunch donated by a previous winner to a deserving undergraduate so I'm going to continue to award an additional free lunch to the first undergraduate student who can accept it. Please indicate in your email message whether you are an undergraduate and whether you can make it for lunch.

THEME:

Nobel Laureates
Send your guess to Sandwalk (sandwalk (at) bioinfo.med.utoronto.ca) and I'll pick the first email message that correctly identifies the molecule and names the Nobel Laureate(s). Note that I'm not going to repeat Nobel Prizes so you might want to check the list of previous Sandwalk postings by clicking on the link in the theme box.

Correct responses will be posted tomorrow.

Comments will be blocked for 24 hours. Comments are now open.

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Sequenced genomes contain thousands of "unknown" genes

 
The total number of genes in the human genome has dropped from the initial estimates of 30-35,000 to about 25,000. Of these, more than 4,000 encode functional RNAs, leaving about 20,500 protein-encoding genes in the human genome [Humans Have Only 20,500 Protein-Encoding Genes].

Up to 40% of these protein-encoding genes are "unknown" in the sense that no function has been assigned to their protein products. In the jargon of genomics, the genes are "unannotated," meaning that nobody has assigned a function to the gene in the human genome database (Reichardt, 2007).

That means 8,000 unknown genes. About 1000 of these genes are "orphan" genes—genes that have no homologues in other species, including chimpanzees (Clamp, 2007).

Humans aren't unique. All sequenced eukaryotic genomes have a high percentage (~30-40%) of "unknown" protein-encoding genes.

A new paper in PLoS One looks at the "unknown" genes in the filamentous fungus Neurospora crassa (pink bread mold) (Kasuga et al. 2009). The Neurospora genome has about 9,000 protein-encoding genes and more than half of them have not been annotated. They are the "unkown" genes.

The genomes of about 40 different species of fungus have been sequenced and many of these are filamentous fungi related to Neuropsora. What this means is that it's possible to compare the Neurospora genes to those in many different genomes from closely related species; those that are part of the same family (less closelyrelated); part of the same phylum; and distantly related. You can't do such an extensive study with human genomes because there aren't very many mammalian genomes that have been sequenced and carefullyannotated. A draft sequence of the chimpanzee genome, for example, has been published but it is neither complete nor reliable enough for genomic comparisons. The only other primate genome is from macaque (Rhesus monkey) and that's far from finished. (The human and mouse genomes are the only ones listed as "complete" on the NCBI/Entrez website.)

The question is: are the unknown genes confined to Neurospora and its close relatives? If so, it would suggest that new genes have evolved within the past several million years and that's why we don't know their function.

Kasuga et al. created six sets of genes ...

1. Genes with homologs in distantly related eukaryotes and possibly prokaryotes. These are ancient genes.
2. Genes that are only found in fungi and not in plants or animals or protists (Dikarya).
3. Genes found only in Ascomycetes.
4. Genes confined to the Pezizomycotina clade to which Neurospora belongs.
5. Genes found only in Neurospora.
6. Others: genes that are found in some of the first groupings but not in all the smaller grouping.

The classification depends on the similarity cutoff. If the lowest cutoff is 25% sequence identity, then there will be more homologs in the eukarote or prokaryote class than if the cutoff is raised to 35%. The distibution of the various classes at each of three minimum sequence identify cutoffs is shown in their second figure.


Taking the 30% threshold numbers (middle group), it looks like there are 2,358 highly conserved genes with homologs in distantly related eukaryotes and prokaryotes. In contrast, there are 2,219 genes that don't have homologs in any other species. These are the orphan genes in Neurospora.

You might expect that most of the unknown/unannotated genes would be confined to Neurospora and closely related species. You might expect that highly conserved genes would be more likely to have been identified. That's partly true. Here are the numbers.


Only 16.5% of the highly conserved genes are mystery genes of unknown function. While this is much lower that the total (56%), it's still surprising that so many of the core genes remain unidentified. Presumably they are doing something very important. There are dozens of thesis projects available for talented graduate students who want to make a valuable contribution to biology.

It's not a surprise that 94% of the orphans are unannotated. These genes are likely to be new genes that have evolved recently in Neurospora and they would be expected to carry out unusual reactions that aren't found in other species. These "genes" are also the ones most likely to be artifacts (false positives) of the gene searching software. They may not be genes at all.

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[Image Credit: Neurospora-National Institute of General Medical Sciences]

Clamp, M., Fry, B., Kamal, M., Xie, X., Cuff, J., Lin, M.F., Kellis, M., Lindblad-Toh, K. and Lander, E.S. (2007) Distinguishing protein-coding and noncoding genes in the human genome. Proc. Natl. Acad. Sci. (USA) 104:19428-19433. [DOI 10.1073/pnas.0709013104]

Kasuga, T., Mannhaupt, G., and Glass, N.L. (2009) Relationship between Phylogenetic Distribution and Genomic Features in Neurospora crassa. PLoS ONE 4(4):e5286. [DOI:10.1371/journal.pone.0005286]

Reichardt, J.K.V. (2007) Quo vadis, genoma? A call to pipettes for biochemists. Trends in Biochemical Sciences (TIBS) 32:529-530. [DOI:10.1016/j.tibs.2007.10.001]

Conservative Spin

 

Canadian Cynic has built a career out of keeping an eye on The Blogging Tories. Every now and then CC comes up with something that makes you scratch your head and ask, "Can The Blogging Tories really be that stupid?"

Here's a posting from ErwinGerrits.com that will answer the question.

Funny how the current deficit budget is now universally referred to as “The Conservative Deficit”, even after this current budget was forced upon us by the Liberals, NDPers and the bloc-heads after a mid-winter stand-off on the Governour General’s front stoop. As I recall, the Conservative’s Economic Update, brought forward in December, did not make us go into a deficit at all. It was after the three stooges reared their ugly heads and blackmailed the country, that the current deficit budget was tabled.

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International team cracks mammalian gene control code

International team cracks mammalian gene control code

Stop the presses! Revise the textbooks! John Mattick and his collaborators have discovered how genes are controlled in mammals.

Anyone who knows Mattick's past history will know what's coming—Mattick overthrew the Central Dogma of Molecular Biology over six years ago (Mattick, 2003; Mattick, 2004).^1,2

An international consortium of scientists, including researchers from The University of Queensland (UQ), have probed further into the human genome than ever before.

They have discovered how genes are controlled in mammals, as well as the tiniest genetic element ever found.

Their discoveries will be published in three milestone papers in leading journal Nature Genetics.

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1. See Basic Concepts: The Central Dogma of Molecular Biology for the truth about the Central Dogma.

2. See Greg Laden Gets Suckered by John Mattick for an example of how easy it is to get fooled by John Mattick.

Mattick, J.S. (2003) Challenging the dogma: the hidden layer of non-protein-coding RNAs in complex organisms. BioEssays 25:930-939.

Mattick, J.S. (2004) The hidden genetic program of complex organisms. Sci. Am. 291:60-67.

Idiot America?

 
Oh dear, my accommodationist friends aren't gonna like this.

From the amazon.com site ....

The Culture Wars Are Over and the Idiots Have Won

A veteran journalist's acidically funny, righteously angry lament about the glorification of ignorance in the United States.

In the midst of a career-long quest to separate the smart from the pap, Charles Pierce had a defining moment at the Creation Museum in Kentucky, where he observed a dinosaur. Wearing a saddle.... But worse than this was when the proprietor exclaimed to a cheering crowd, “We are taking the dinosaurs back from the evolutionists!” He knew then and there it was time to try and salvage the Land of the Enlightened, buried somewhere in this new Home of the Uninformed.

With his razor-sharp wit and erudite reasoning, Pierce delivers a gut-wrenching, side-splitting lament about the glorification of ignorance in the United States, and how a country founded on intellectual curiosity has somehow deteriorated into a nation of simpletons more apt to vote for an American Idol contestant than a presidential candidate.

With Idiot America, Pierce's thunderous denunciation is also a secret call to action, as he hopes that somehow, being intelligent will stop being a stigma, and that pinheads will once again be pitied, not celebrated.

I can't wait for the sequel—Idiot Canada.

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[Hat Tip: Canadian Cynic]

Bob McDonald Explains why Canadian Scientists Are Upset

 
Bob McDonald is the host of Quirks & Quarks on CBC radio. He has a blog and here's part of what he posted on Friday [Another Earth Day, Canadian scientists concerned].

While people around the globe celebrate the beauty of our planet on Earth Day, April 22nd, scientists in Canada are concerned that government funding is heading in the wrong direction to provide sensible solutions to environmental problems. More than 2000 scientists from across the country have signed an open letter to Prime Minister Harper and the Leader of the Opposition, expressing concerns over cuts to basic science research. It’s basic science that takes the pulse of the planet.

The scientists are concerned that government money is overlooking vital areas. For example, the current Conservative budget allocates $2 billion for university infrastructure - in other words, renovations to aging buildings. But those funds come with a catch. They must be matched with private funding, something everyone is having trouble finding during these tough economic times. Keeping roofs on buildings is important, but if there are no scientists to work in them, what’s the point?

The Canada Foundation for Innovation, a major source of science funding, did receive $740 million, but it also comes with that match-funding hook. The other funding agencies, the Natural Sciences and Engineering Research Council and the Canadian Institutes of Health Research, have had their budgets cut back, while Genome Canada was essentially ignored.

The rest of the government’s support for science is going towards the automotive industry, carbon sequestration, biofuels and scholarships for business students. In other words, applied science is taking precedent over basic science.

While we do need both, when it comes to the environment, the two types of science are often at loggerheads.

Politicians like to support applied science because it leads to jobs and products, such as more efficient cars or new wireless devices. Basic science, on the other hand, can’t promise an immediate economic return because it simply looks at nature to understand how things work - and more importantly these days, how things are changing. As we’ve seen with climate change, basic scientists have been out in the field watching ice caps disappear before their eyes, carbon dioxide levels rise and climate patterns shift. At the same time, those dealing with the technology at the heart of the problem resist the basic science to keep the current systems in place.

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[Hat Tip: T. Ryan Gregory]

Monday's Molecule #118

 
This is a very famous protein but most of you won't be able to identify it from the structure alone. You'll need a hint of some sort.

Letting you know that the ligands are Mg²⁺ and adenosine-5′-triphosphate might not be enough so I'll also tell you that one of the authors on the structure paper was M.E. Noble.

There is one Nobel Laureate who is most closely identified with the function of this particular molecule, although that scientist was NOT the first to identify it. You have to identify the Nobel Laureate who got the prize for working out the function of the protein.

The first person to identify the molecule and the Nobel Laureate wins a free lunch at the Faculty Club. Previous winners are ineligible for one month from the time they first won the prize.

There are six ineligible candidates for this week's reward: Bill Chaney of the University of Nebraska, Elvis Cela from the University of Toronto, Peter Horwich from Dalhousie University, Devin Trudeau from the University of Toronto, Shumona De of Dalhousie University, and Maria Altshuler of the University of Toronto.

I note that Canadians are trouncing the rest of the world. That's as it should be.

I still have one extra free lunch donated by a previous winner to a deserving undergraduate so I'm going to continue to award an additional free lunch to the first undergraduate student who can accept it. Please indicate in your email message whether you are an undergraduate and whether you can make it for lunch.

THEME:

Nobel Laureates
Send your guess to Sandwalk (sandwalk (at) bioinfo.med.utoronto.ca) and I'll pick the first email message that correctly identifies the molecule and names the Nobel Laureate(s). Note that I'm not going to repeat Nobel Prizes so you might want to check the list of previous Sandwalk postings by clicking on the link in the theme box.

Correct responses will be posted tomorrow.

Comments will be blocked for 24 hours.

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Agnotology

 
A reader (David) posted a comment about my recent Denyse O'Leary quotation. He alerted me to a new word: agnotology [Comments].

The Wikipedia entry is informative [agnolology agnatology] but there's lots more to learn about this word. Everyone is agreed that the word was invented by Robert Proctor a Standford University Professor who studies the history of science. Everyone is agreed that it refers to the study of ignorance, or why we don't know certain things. But there's more to it than that ....


Here's an excerpt from an article by Clive Thompson in WIRED magazine [Clive Thompson on How More Info Leads to Less Knowledge].

Normally, we expect society to progress, amassing deeper scientific understanding and basic facts every year. Knowledge only increases, right?

Robert Proctor doesn't think so. A historian of science at Stanford, Proctor points out that when it comes to many contentious subjects, our usual relationship to information is reversed: Ignorance increases.

He has developed a word inspired by this trend: agnotology. Derived from the Greek root agnosis, it is "the study of culturally constructed ignorance."

As Proctor argues, when society doesn't know something, it's often because special interests work hard to create confusion. Anti-Obama groups likely spent millions insisting he's a Muslim; church groups have shelled out even more pushing creationism. The oil and auto industries carefully seed doubt about the causes of global warming. And when the dust settles, society knows less than it did before.

"People always assume that if someone doesn't know something, it's because they haven't paid attention or haven't yet figured it out," Proctor says. "But ignorance also comes from people literally suppressing truth—or drowning it out—or trying to make it so confusing that people stop caring about what's true and what's not."

This is an important insight. It's not something that we didn't know already but it's good to emphasize the concept and give it a name.

Creationism is an excellent example. It's not just that creationists fail to understand science, it's also that they are being actively lied to in an attempt to spread ignorance. In other words, there are people who deliberately spread misinformation in order to oppose knowledge.

If we are going to fight creationism we have to do more than just teach evolution in the schools. If we do that then we are just barely holding our own against the people who spread ignorance. At best, students will be aware of a conflict between what they learn in school and what they learn everywhere else.

In order to fight the spread of ignorance we have to take on the liars directly and show why they are lying. They need to be discredited and exposed. Unfortunately, many of the enemy are "Christians" and Christians get special protection in our society. You can criticize astrology and quackary but not religion.

That has to change. Perhaps we can lump them all under the term "agnotology" and treat them all the same?

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And Now for a Little Comic Relief

 
It's been a few weeks since my last humorous posting so at the start of a new week I thought I'd give you something to laugh about.

Here's a few words from Denyse O'Leary.

The reason so many of us have risen up against Darwinism is not that we think natural selection never occurs but that we have never accepted - without evidence - the idea that it produces a high level of information (and that was Darwin's argument) And - as Mike Behe shows in Edge of Evolution, it doesn't.

It is amazing what people who get tenure at prestigious universities are willing to support without evidence. Including "chance" as a key explanation of high levels of information, which we must all know is completely untrue.

If you doubt that, try throwing the bag of Scrabble letters around the room and reassembling them randomly, and see what happens.

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Modified Bases in DNA

 
Adenine: from the Greek adenas "gland": first isolated from pancreatic glands (1885)

Cytosine: derived from cyto- from the Greek word for "receptacle," refering to cells (1894)

Guanine: originally isolated from "guano" or bird excrement (1850)

Thymine: first isolated from thymus glands (1894)

(source Horton et al. 2006)Bacterial genomes contain a number of unusual bases in addition to the classic adenine (A), cytosine (C), guanine (G), and thymine (T). The most common of the unusual bases are 5-methylcytosine, N⁴-methylcytosine, and N⁶-methyladenine (Erlich et al. 1987).

Bacteriophage DNA, especially the DNA of bacteriophage T4 and its close relatives, can contain 5-hydroxymethylcytosine. The base is usually glycoslylated in normal phage. (A sugar group is attached to the hydroxymethyl group.)

Many of these modified bases serve to protect DNA from restriction endonucleases—enzymes that cleave foreign DNA at specific sites. The restriction endonucleases act on bacteriophage (virus) DNA preventing it from replicating inside the bacterial cell [see Restriction, Modification, and Epigenetics].

If bacteriophage modify their nucleotides at the site of cleavage, they will escape the defenses of the bacterial cell. Of course, bacteria that make restriction endonucleases have to protect their own DNA or else they will be committing suicide. That's why their genomes have modified nucleotides.

Many other modified bases have been found in DNA but they are quite rare. Examples are uracil, α-putrescinylthymine, and 5-dihydroxypentyluracil.


Eukaryotic DNA doesn't have as many modified bases. In fact, 5-methylcytosine is the only one that's common in all eukaryotes. N⁶-methyladenine is known to exist in protist and plant DNA and it is thought to exist at low levels in mammalian DNA as well (Ratel et al. 2006). The presence of hydroxymethycytosine has been reported in various animals as far back as 1972 (Penn et al. 1972).


Now Kriaucionis and Heintz (2009) have re-discovered hydroxymethylcytosine in mammalian DNA. Their paper appears in the latest issue of Science. Apparently the modified nucleotide is found in certain brain cells. Their result confirms the work done by Penn et al. (1972), a result that had not been confirmed in several other studies. This makes hydroxymethylcytosine the seventh modified base in eukaryotic DNA—unless there are some that I don't know about.

The problem with the press release is that it doesn't put the discovery in the proper context.

ScienceDaily (Apr. 17, 2009) — Anyone who studied a little genetics in high school has heard of adenine, thymine, guanine and cytosine – the A, T, G and C that make up the DNA code. But those are not the whole story. The rise of epigenetics in the past decade has drawn attention to a fifth nucleotide, 5-methylcytosine (5-mC), that sometimes replaces cytosine in the famous DNA double helix to regulate which genes are expressed. And now there's a sixth: 5-hydroxymethylcytosine.

In experiments to be published online April 16 by Science, researchers reveal an additional character in the mammalian DNA code, opening an entirely new front in epigenetic research.

We aren't told that the sixth nucleotide is actually N⁶-methhyladenine. We aren't told that many other modified bases have been discovered in bacteria, including hydroxylmethylcytosine. And we aren't told that the authors actually cite earlier work showing the presence of hydroxymethylcytosine in mammalian DNA.

That's a shame. The authors are quoted in the press release. They should have made more of an effort to ensure that it was scientifically accurate.

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Ehrlich, M., Wilson, G.G., Kuo, K.C., and Gehrke. C.W. (1987) N4-methylcytosine as a minor base in bacterial DNA. J Bacteriol. 169:939-943. [Journal of Bacteriology]

Kriaucionis, S. and Heintz, N. (2009) The Nuclear DNA Base 5-Hydroxymethylcytosine Is Present in Purkinje Neurons and the Brain. Science Published Online April 16, 2009 [DOI: 10.1126/science.1169786]

Penn, N.W., Suwalski, R., O'Riley, C., Bojanowski, K. and Yura, R. (1972) The presence of 5-hydroxymethylcytosine in animal deoxyribonucleic acid. Biochem J. :781–790. [Biochem. J.]

Ratel, D., Ravanat, J.L., Berger, F., and Wion, D. (2006) N6-methyladenine: the other methylated base of DNA. Bioessays :309-15. [PubMed

Dancing in Antwerp's Train Station

 
I have a confession to make. Ms. Sandwalk and I, along with four other couples we've known for many years, are learning how to do ballroom line dances. It's so much fun.

Perhaps that's why this video makes me smile. It's a promotion by a Belgian TV show that's looking for a "Maria" to play the lead in "The Sound of Music." I hope my daughter—who lives in Belgium— never hears about it 'cause she already knows all the words to every song in the musical.



UPDATE: Some people may not know about the T-Mobile add in the Liverpool Street Station in London [T-Mobile Dance].

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P.S. My dancing group is almost as good as these guys. Maybe we should do a performance in Union Station in downtown Toronto?

[Hat Tip: GrrlScientist]

Jerry Coyne's View of Evolutionary Psychology

 
You'll have to read Evolutionary psychology: the adaptive significance of semen flavor to find out what Coyne thinks of evolutionary psychology. I think he's captured the essence of the movement.

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P.S. I was looking for an appropriate illustration of the behavior that Coyne describes. Turns out that if you enter the appropriate term in your Google search box, you'll find quite a few photographs out there on the internet. I decided not to use any of them but the search was interesting.

Do You Want to Know the Sequence of Your Genome?

 
It's very likely that each one of us carries several recessive lethal alleles and many alleles that make us more susceptible to various diseases. You can count on it.

Do you want to know which of the many known deleterious alleles you have in your genome? That's one of the questions asked in an article published in The Economist: Getting Personal.

Though he has the world’s most advanced gene-sequencing technology at his fingertips, Dr Altshuler refuses to get his own genome sequenced: “If someone gave it to me on a CD, I’d refuse to look at the disc. The information is meaningless.” Bill Gates agrees. He has not had his genome sequenced either, nor does he plan to, though after a moment’s reflection he adds, “unless I find out I have cancer.”

Keep in mind that there's nothing much you can do if you find out that you have a predisposition for cancer or some other disease, like Alzheimer's. You can change your lifestyle but it's not clear whether that will change the probabilities significantly. Furthermore, there are so many claims out there about how to prevent disease that you'll never be sure you're doing the right thing.

And what do you do if you want to have children with someone who also knows their genome sequence? Figure out the probabilities that your children will not inherit any of your bad alleles. Is that a responsibility that you want? Why?

I'm with David Altshuler on this one and so was John Hawks until recently. Now John is changing his mind—Turning ACGT into poetry. Part of my problem is that I don't feel very comfortable seeking advice from a for-profit company when it comes to interpreting my genome sequence. But we won't have much choice; there's absolutely no way that the average person is going to be able to do the interpretation, so they will have to pay for it. And they'll have to get an update every few years as we learn more about human genes. (Whole genome interpretation is not a service that will be covered by socialized medicine.)

Do you want to know the sequence of your genome? Assuming that you can afford the sequencing cost, how do you intend to use the information and how will you interpret it?

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Sign the Open Letter

 
Are you a Canadian scientist who is upset about cuts to research funding by the current government? Read the Open letter to the Prime Minister and Leader of the opposition and add your name to the list of over 2000 scientists.

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Proportional Representation Is about to Pass in British Columbia

 
The Single-Transferable Vote (STV) is a system where each voting district (riding/constituency) has multiple members. You vote for several candidates by ranking them from most preferred to least preferred. If a candidate gets more than the minimum number of votes needed for election then the "surplus" votes are transferred to the second choice candidates.

If, after transferring "surplus" votes, there are still candidates to be elected, then the candidate with the lowest number of votes is eliminated and the votes are transferred to the voter's next choice. This process of elimination and transfer continues until the required number of candidates are elected [BC-STV].

The system was chosen by a non-partisan citizens' assembly.



A majority of voters in British Columbia voted in favor of this form of proportional representation in the last referendum but they failed to get the 60% majority required to change the voting system. It looks like they will succeed on May 12th, making British Columbia the first province to abolish the old first-past-the-post system and adopt a form of proportional representation.

I'm certain that Ontario won't be far behind. We lost the vote for proportional representation last time but that was probably because the general public didn't understand it. That, plus the fact that many prominent newspaper columnists and editors lied made untrue statements about the dangers of proportional representation. They didn't understand it either.

April 15, 2009
FOR IMMEDIATE RELEASE
Poll: 65 per cent of British Columbians support BC-STV
Younger voters overwhelming in their support

Vancouver, B.C. – The numbers are in and British Columbia voters are giving a big thumbs up to electoral reform with 65 per cent saying they will vote for BC-STV in the upcoming referendum on May 12. That is the top line result of a major survey conducted by Angus Reid Strategies. Support for a new way of electing our MLAs is particularly strong among younger voters – those 18 to 34 – at 74 per cent.

“The survey results indicate that British Columbians and particularly younger voters are ready to embrace a new electoral system in British Columbia,” said Catherine Rogers, vice president, Angus Reid Strategies. “A large majority are looking for electoral change and want an electoral system that is fair and that elects MLAs who are more accountable to them.”

When presented with the question that will appear on the ballot, 65 per cent said yes to BC-STV while only 35 per cent chose to keep the current first-past-the post system. Angus Reid Strategies conducted the online survey March 9 to 12 and polled 702 British Columbians across the province. While support for BC-STV continues to grow, awareness of the upcoming referendum is at 44 per cent.

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