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The chicken and the ink - bi3mw - 26-12-2021
In a thread about Biocodicology, the following idea occurred to me:
The Mc Crone Institute studied the ink in the VMS in 2009. It was found that Glair (eggwhite) was used for clear or the color white. It was used in the headdress of bather on folio 78r ( clear, not white ) and a face on folio 70v ( see table in the report ). Thus, inks containing protein were used which are hardly contaminated, i.e. they were only mixed with calcium carbonate but not processed in any other way (in contrast to the parchment).
Samples of these inks could help determine the origin of the chicken and thus the ink itself if the ovalbumin (OVA) is sufficient to determine the exact species ( that would be the first and most important question, does it differ from sub species to sub species ?). The protein sequence of chicken ovalbumin was fully elucidated in 1981 [2].
The Red junglefowl ( gallus gallus ) is the ancestor of all domestic chickens existing today ( Wikipedia ). The variations in late medieval times were locally different. The DNA of chicken bones from archaeological excavations could be used for comparison. These are found worldwide ( for example also in my neighboring town ) in large numbers.
From the 15th century onwards breeding of chickens has been demonstrably started, which facilitates the differentiation according to local sites.
Quote:Benecke (1994: 370-372)[1] proves that chickens definitely became larger as well as more different in shape and color from the late Middle Ages on. From about the 15th century, there are first indications of incipient breeding. In the Middle Ages, the animals can be divided according to the construction of the skeleton into small chickens similar to today's bantams and heavier chickens such as Leghorn or Italian. Chicken breeds or different types of chickens that emerged in the late Middle Ages are the origin of some breeds still living today ....
[1] Benecke, N. Der Mensch und seine Haustiere, Theiss, 1994
[2] A. D. Nisbet, R. H. Saundry u. a.: The complete amino-acid sequence of hen ovalbumin. In: European Journal of Biochemistry / FEBS. Band 115, Nummer 2, April 1981, S. 335–345, doi:10.1111/j.1432-1033.1981.tb05243.x (free Fulltext). PMID 7016535.
My question now is whether this idea is plausible and considered feasible or ( justified ) why not.
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RE: The chicken and the ink - Linda - 26-12-2021
Sure, why not? Just need the chicken database to be in place to see where ours falls on the dna timeline.
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I found this study on Thai chickens so it is possible to see where chickens come from through dna study
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So it appears chicken dna databases do exist.
As to which came first, the chicken or the egg, the egg did, it just wasn't a chicken egg per se.
RE: The chicken and the ink - Bernd - 27-12-2021
I'm no expert in biocodicology but that's probably not going to work.
The albumen of an egg does not contain cell nuclei and thus only traces of DNA from the hen's oviduct. Also the paint is an extremely thin and transparent layer that has exposed any genetic material to the environment for centuries, thus massively degrading it. Ideally you'd like to have samples with lots of DNA copies protected by thick walled cells like in bones or teeth. Therefore I think obtaining usable chicken DNA from the paint is extremely unlikely.
The protein sequence, even if it could be determined from the tiny amount of paint, doesn't nearly have the same resolving power as SNPs and STRs from DNA and cannot be correlated with archaeological DNA findings from chicken bones. At least not in any straightforward way.
It is possible to identify some albumen traits associated with chicken breeds though but you'd likely need a proteome database from other contemporary paintings containing egg white of known origin.
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I'll leave that to the biocodicologists, maybe it is indeed possible, at least some day.
The only material theoretically containing ample DNA is the vellum, but it is most likely severely damaged and contaminated due to processing. Similarly the vellum proteome could be extracted and compared to other manuscripts to assess a shared origin. Again we'll have to see what's possible, but I'd still bet my money there and not on any paint.
We have seen spectacular developments regarding ancient DNA in the last years in palaeontology (even from cave soil!) so extracting the desired DNA while removing contaminants is certainly possible if you hire the right specialists.It's all about developing the correct sample processing method. And then interpreting the vast data from Next Gen Sequencing of course. But it's certaily feasible.
Yet, forensic palynology would be much more promising in my opinion, basically extracting individual pollen grains that were trapped in or under the paint during creation of the manuscript and analyzing them in an electron microscope. It's only minimally destructive and the plant species and thus rough geographic region should be identifiable. Unfortunately Middle Europe isn't exactly diverse and rather uniform regarding plant species but it might be possible to determine a location north or south of the alps. The flora of Tuscany is different from South Germany or an alpine valley.
Or maybe we'd find some exotic pollen proving the VM was indeed made in [enter fringe VM origin theory of your choice]
RE: The chicken and the ink - bi3mw - 27-12-2021
(27-12-2021, 02:44 AM)Bernd Wrote: You are not allowed to view links. Register or Login to view.The albumen of an egg does not contain cell nuclei and thus only traces of DNA from the hen's oviduct.
Thank you @Bernd for your conclusive argumentation. The thought that the oviduct of the chicken could directly provide DNA has not occurred to me. However, as you said, these would be only traces of it and probably very difficult to extract.
(27-12-2021, 02:44 AM)Bernd Wrote: You are not allowed to view links. Register or Login to view.It is possible to identify some albumen traits associated with chicken breeds though but you'd likely need a proteome database from other contemporary paintings containing egg white of known origin.
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Unfortunately, the effect of being able to get comparison material (bones) quickly and easily is eliminated. Building such a database is notoriously difficult and lengthy.
As for the durability of egg white, it should be noted that it was used, among other things, for gilding with real gold leaf. Quote: "These gildings are characterized by special luster and durability, their durability can be calculated after centuries." ( Wikipedia, section "Use in bookbinding" )
RE: The chicken and the ink - MichelleL11 - 27-12-2021
(27-12-2021, 02:05 PM)bi3mw Wrote: You are not allowed to view links. Register or Login to view.Unfortunately, the effect of being able to get comparison material (bones) quickly and easily is eliminated. Building such a database is notoriously difficult and lengthy.
As for the durability of egg white, it should be noted that it was used, among other things, for gilding with real gold leaf. Quote: "These gildings are characterized by special luster and durability, their durability can be calculated after centuries." ( Wikipedia, section "Use in bookbinding" )
Hi, bi3mw:
I agree that building the database for ovalbumin (protein) for particular strains of chickens is going to take some time.
I did find this publication concerning cross-species comparison:
You are not allowed to view links. Register or Login to view., Li et al.
On second thought -- this is just too complicated -- here's one that focuses on species differentiation in just the yolk -- more focused on the questions we're trying to answer:
You are not allowed to view links. Register or Login to view., Liu et al.
And a general discussion of the protein's presence in chicken products (eggs):
You are not allowed to view links. Register or Login to view., Kanaka et al. (I guess there is a whole family of serpin proteins?)
I was actually initially concerned that albumen could be so evolutionarily constrained that it would not differ significantly between chicken breeds -- and that could still be the case. But at least we know that it does differ enough between species to be used for some sort of identification -- I wasn't sure that would be possible.
Also, a very clear initial complicating factor is that there appears to be three very, very similar genes in chickens (resulting from gene duplication in the far distant evolutionary past). The result is there may be a complex relationship between these sequences that could be tough to tease out (e.g. three similar genes in each specific species -- how would SNPs in the albumin be differentiated from maybe being protein sequence from one or the other of the very similar proteins?). However, I do note that actual production of these two further proteins is still being figured out -- so if these ovalbumen-like genes (X and Y) are used to make proteins very, very infrequently, then maybe that issue would go away.
You are not allowed to view links. Register or Login to view., DaSilva et al.
So -- don't mean to be a "nay-sayer" but there are definite issues that would have to be figured out to get a confident identification using ovalbumen.
It is worth thinking about these things so the complications of structuring these types of experiments can be understood -- so that the answer to -- "Why don't they do X DNA sequence or Y protein sequence?" may be it just isn't possible right now (or may never be possible given the structure of that DNA or protein -- it may just not be a good choice in order to differentiate).
But I do appreciate the board being willing to think about these things in a sophisticated fashion!
RE: The chicken and the ink - bi3mw - 27-12-2021
Thanks @Michelle for your informed comment. I will work my way through the papers you presented first.
I also think that sometimes it can be worthwhile to "think outside the box". The direct way ( examination of the parchment ) is not necessarily without alternative. Of course it always needs actors who have the will and the resources to implement alternative ideas.
RE: The chicken and the ink - Aga Tentakulus - 27-12-2021
Just thrown into the room.
Today's farm animals are crossbreeds of different breeds.
How can an exact pedigree be recorded here.
It is here not a normal evolution, but pure intention.
RE: The chicken and the ink - bi3mw - 27-12-2021
(27-12-2021, 08:55 PM)Aga Tentakulus Wrote: You are not allowed to view links. Register or Login to view.Today's farm animals are crossbreeds of different breeds.
How can an exact pedigree be recorded here.
(27-12-2021, 02:44 AM)Bernd Wrote: You are not allowed to view links. Register or Login to view.It is possible to identify some albumen traits associated with chicken breeds though but you'd likely need a proteome database from other contemporary paintings containing egg white of known origin.
(27-12-2021, 05:05 PM)MichelleL11 Wrote: You are not allowed to view links. Register or Login to view.I agree that building the database for ovalbumin (protein) for particular strains of chickens is going to take some time.
According to the current state of discussion, one would build a database with comparative material ( tempera / glair ) from Middle Ages Manuscripts and with known origin. The samples from the VMS would then be compared directly with this database ( ovalbumin / protein ). So a complete pedigree is not necessary. Of course, this is all associated with the problems described by Bernd and Michelle.
In addition, one could take over today still regionally existing, old chicken races into the database (e.g. Leghorn or Italian). The today standardized turbo chicken can be safely left out.
It should also be noted that tempera / glair was usually mixed on site. So, it was not (possibly) bought from far away, like other colors.
RE: The chicken and the ink - bi3mw - 28-12-2021
I was wondering what exactly of the egg is actually used to make glair. It is not the beaten egg white but the liquid egg white that forms on the bottom of the container after a few hours. As I understand it, beating the egg white breaks down the proteins ( denaturation ). That probably has a negative impact on any analysis although the primary biochemical structure remains unchanged.
Quote:.... until the later Middle Ages manuscript miniatures were mainly painted using glair. Glair was made from the egg white, presumably the ones that were left over from painting wood panels with egg yolk. The proteins of egg white must be broken down by beating to make a runny substance for mixing paint.
Here is a short video on how glair is made:
Quote:If egg whites are beaten until they are stiff, they are fully denatured and have no elasticity; they lose their original properties and aren't able to return to their former state. If egg whites are beaten only until they form soft peaks, the proteins are only partially denatured and retain some of their elasticity.
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RE: The chicken and the ink - MichelleL11 - 28-12-2021
(28-12-2021, 03:26 PM)bi3mw Wrote: You are not allowed to view links. Register or Login to view.I was wondering what exactly of the egg is actually used to make glair. It is not the beaten egg white but the liquid egg white that forms on the bottom of the container after a few hours. As I understand it, beating the egg white breaks down the proteins ( denaturation ). That probably has a negative impact on any analysis although the primary biochemical structure remains unchanged.
Focusing on egg white proteins, here are some possibilities:
You are not allowed to view links. Register or Login to view., Mann and Mann
You are not allowed to view links. Register or Login to view., Arena et al.
and -- a possible candidate gene:
You are not allowed to view links. Register or Login to view., Kinoshita et al.
So -- what you want is a gene with high genetic variability but can also be distinguished from other similar gene sequences. Please note that "high genetic variability" will very likely result in high protein sequence variability -- but not necessarily. Thank you for understanding that I'm not trying to be discouraging, just want to bring a realistic approach:
The reason why high genetic variability doesn't necessarily translate to high protein variability is because the way that DNA (or RNA) is translated into amino acids is through nucleotide triplets. The third position of the triplet is particularly "flexible" (often termed "wobble" or degenerate codons) -- thus, triplets of different nucleotide sequence results in the same amino acid being added to the protein. Note there are 64 different triplet codons, but only 20 amino acids -- so you can change the triplet codon and end up with the same amino acid. Also, some of the codons are "metadata" -- like the "stop" codons.
Here is a chart that illustrates this principle. In this chart, U, C, A, and G are the four nucleotides found in the mRNA (uracil, cytosine, adenine, and guanine -- (uracil takes the place of the thymine (T) in DNA structures).
But, that being said, the authors do identify 11 "nonsynonymous" variations (a substitution that doesn't change the amino acid would be synonymous) in the 29 chicken breeds that they tested. So I do think that the TENP gene (ovoglobulin G2 protein) could be a good candidate for differentiation. I'll take a closer look at this paper sometime this weekend and will come back with further thoughts -- but for now, you do have a candidate that could be used focusing only on an egg white product.
Finally, please note that the variations in proteins aren't necessarily all due to changes in the primary sequence. Proteins undergo a high amount of post-translational (mRNA --> amino acid step) modification. Thus, you can see proteins that are distinguished from each other when isolated and run on a gel (movement through the gel is dependent on a combination of size and total charge) -- but the differences aren't at the amino acid levels but instead are differences in sugar molecules that "decorate" a finished protein sequence. Different sugar molecules means changes in both size and charge -- so you see the difference on the gel migration. But, a quick look shows they did find primary sequence differences (it's not just post-translational modifications) so that is positive for your purposes. Again, I'll look more closely when I have more time. But, in my opinion, this is a good development for your proposed approach.