Thanks to Henry Brzeski for holding this meeting.
This transcript can be found on the tape 'Synthesis_tape' in the PPS Base, but it will eventually have to be deleted to save disk space.
Participants:
Jzt says, "testing synthesis_tape 20th March 1996"
jzt turns the C-recorder off.
jzt turns the C-recorder on.
Hb [guest] says, "no I'm just trying to get my typing together"
Hb [guest] says, "let us go. shall we work our way throught the agenda?"
Jzt [to hb]: Sounds good
Rob finds its way in.
Ahotz finds her way in.
Hb [guest] says, "let's start on familiar territory (for me) DNA and genetic code"
Rob says, "Morning all"
Ahotz waves
Paolo waves
Hb [guest] says, "hi"
CrisCan waves
Jim Pitts stands and waves
JosteinA finds its way in.
Jzt [to hb]: Will you ask for questions or do you have subjects you want to expound on?
Hb [guest] says, "lets start with qeuestions"
Iddo will be right back...
Jim Pitts says, "Hi Raj"
Jzt says, "OK would people like to give Henry a list of questions/issues for discussion?"
TRex says, "Henry, strictly not genetic code, but why T in DNA and U in RNA?"
Hb [guest] says, "T can be C[C[C[C[C[C[C[C methylated in DNA and give rise to U if it i deaminated"
Hb [guest] says, "that shopuld have been C can be methylated"
Raj [guest] materializes out of thin air.
Hb [guest] says, "this causes mutation problems because a CG base pair cabn give rise to a AT base pair"
Hb [guest] says, "so the cell has to know that U in DNA is an error and it is ecsxcised and replaced"
TRex says, "Does this imply that the cell "evolved" the use of T instead of U?"
Hb [guest] says, "I suppose so"
Hb [guest] says, "Evolutionasry biolohgists think that life started with RNA and DNA followed"
Hb [guest] says, "so it would seem reasonabl e that this correction mechanism came with DNA"
TRex says, "Yes, I'd read of that. I'd also read that a base called inosine can be used by some of the polymerases - any info?"
Gustavo1 [guest] finds its way out.
Hb [guest] says, "you can do this in the lab but in vivo it does not occur"
Hb [guest] says, "inosine is found in tRNAs but this comes about through modification afetr synthesis"
SteveTate finds his way in.
JohnW says, "and what about the hypoxanthine base- where does that occur?"
KarlS finds his way in.
KarlS waves to everybody
TRex says, "That's interesting - there must be some very discerning correcting enzymes in vivo?"
Hb [guest] says, "As far as I know it is just used as a metabolic precursoe to the four bawses found in DNA"
JohnW nods
Hb [guest] says, "Correcting enzym,es must obviously be very clever if tehy have to look afeter 3000000000 bases wityhout making a mistake"
TRex says, "Yes Hypoxanthine - must also occur by modification after synthesis?"
TRex grins
Iddo points out that a discerning enzyme is the rule rather than the exception. The only polymerase (that I know of) which is DNA and RNA based occurs in retroviruses
Iddo says, "...For example"
Hb [guest] says, "I don't knw how Taq DNA polymerase works in vivo but in vitro it isnot very discerning"
Iddo says, "Depends on the species you obtain the Taq from. There are major improvements with Taqs obtained from deep-sea hotwater spring bacteria. Those critters live in 120C... very resilient polymerases"
Hb [guest] says, "if the talk is now about reverese transcriptase then we can use this as a means of mioving t poinyt 2 on the Agenda ie cell biology (which I don't nkow much about)."
TRex says, "I thought that some of the polymerases had a proof-reading capacity i.e check and correct? True or not?"
Hb [guest] [to Iddo]: how is DNA stable at 120?
Iddo says, "Most polymerases do. Exception: reverse transcriptases"
JosteinA has disconnected.
Jzt says, "In view of time perhaps we should move on to questions on Item 2, i.e. Cell Biology"
Raj [guest] says, ""is this the same as vent polymerase ?""
Iddo [to hb]: Good question... let me know when you find the answer. Maybe the bacteria od not proliferate in those temperatures
Hb [guest] [to TRex]: in vivo Taq polymerse does not but presumable in vivo its a different story
Jim Pitts [to Iddo]: strictly speaking Taq is the species, Thermus aquaticus
SteveTate says, "say is the specificity of these enzymes governed by sigma factors etc???"
Jim Pitts [to Iddo]: but I know what you mean
Hb [guest] [to Jim Pitts]: pitte
Raj [guest] says, ""thermophillus aquaticus actually""
Hb [guest] [to SteveTate]: sigma factors are for initiation and so will probably be used here accuracy is a consequence of proof reading by the polymerase
TRex says, "In the cell, what are the relative contributions of regulator sequences and secondary structure to control? Or are these relateditems?"
Jim Pitts [to raj]: Thanks
Iddo says, "reverse transcriptases have no error-correction facility. Hence the high mutation rate among retroviruses"
Hb [guest] [to ]: Trxex
TRex says, "I mean like promoters - they have to be "visible" to be effective."
Jim Pitts [to raj]: but it is Thermus aquaticus
Hb [guest] [to TRex]: primary control is at the sequence level but obviously the DNA has to be reachable so secondary structure plays a role
Jzt has disconnected.
SteveTate has disconnected.
Jim Pitts [to raj]: vent is a trade name
Raj [guest] says, ""in eukaryotic cells, expression of genes is very likned to unwinding of the DNA on histones""
Raj [guest] says, ""[to Jim] I know I was just having some fun with you as I know you probably delve into textbooks while biomooing""
SteveTate has connected.
Jim Pitts [to raj]: you have always kept me on the hop
TRex says, "From what Raj says, it seems control is at the level of structure first, then sequence?"
Hb [guest] [to raj]: what comes first is it DNA unwinds and RNA is transcribed or RNA is transcribed so DNA unwinds?
TRex says, "I suppose the argument is that sequence determines structure (for NAs as well as proteins??)"
The housekeeper arrives to cart JosteinA off to bed.
Raj [guest] says, ""but obviously unwinding of dna fo transcription is governed by sequences which mark out the unwinding region !""
Iddo says, "in the case of steroid receptors, those proteins recognize a very specific sequence (termed half-site). There are some good crystal structures of those in the PDB"
Hb [guest] says, "dna must be unwould, the promoter must be rfound, rna must be synthesised, nucleosomes must be uncoupled but the irder is not defined yet"
TRex says, "DNA is double strande. Do messages always get read off the same strand? Is the same strand always the "sense" strand?"
Jzt has connected.
Raj [guest] says, ""the DNA must unwind and let RNA pol attach and then chug along... maybe it is still unwinding further upstream at the same time, but my understanding is that certain genes will be unwound and looping out of the chromosomes e.g. insulin
gene in pancreatic B-cells, brain enzyme genes in the brain etc..""
Hb [guest] [to TRex]: no any strand can carry the sequnce for a protein
Hb [guest] [to raj]: expressed sequences are not complexed with histones but it is difficult to decide which comes first ie transcription or removal odf histones
TRex says, "Within one chromosome, sometimes strand A is the active one and sometimes its stramd B, its mate, is read??"
Hb [guest] [to TRex]: yes
Chandra [guest] materializes out of thin air.
Hb [guest] [to TRex]: look at Science cvol 269 p496 to see how heaemoopjhilus uinfluenzae does it!
TRex says, "In prokaryotes, I'm unclear about the difference between represiion and attentuation? Is the latter partial repression?"
Hb [guest] [to TRex]: in operationa; l terms yes. But attenuation works by a totally different mechanism from repressors which simply bind ro DNA
Hb [guest] [to TRex]: repressors are either on or off attenuation can be variably controlled
TRex says, "I'll check that reference"
Jzt says, "Is it time we moved on to the next item Henry? i.e. item 3 transcription - or maybe we could skip that in view of your last BioMOO and move to Translation?"
Hb [guest] says, "we seem to be moving down the agenda without any interference from me . I was going to suggest item 5 "
JohnW says, "Did anyone from a non-biology background have any questions about the relevant cell biology with which they are unfamiliar?"
TRex says, "Are we straying too far from the topic here if we talk about how proteins are "sent" to different destinations?"
Iddo . o O ( have splicing and alternative splicing been dealt with yet (now or ever)? )
Jzt [to Henry]: OK, a self-assembled agenda then...
Raj [guest] says, ""what does trex want to know about targetting and transport""
SteveTate says, "how is the degrree of attenuation contrlolled?"
Rob says, "Im afraid Im not following this conversation at all. Maybe we need a separate remedial MOO for those unfamiliar with the terms you all keep using."
Hb [guest] says, "total chaos"
JohnW laughs
Hb [guest] says, "lets backtrack to transcription and alternative splicing"
JohnW nods
CrisCan says, "is there any hypothesis about the rule of such a complex gene organization in eucariote cell, that is introns and so on"
Raj [guest] says, ""[to criscan] what sort of rule?""
Hb [guest] says, "splicing signals exist and we have a consensus esequence for htis"
Hb [guest] says, "but the problem is if you have twenty splices to perfirm and all the consensus sugnals look the same how do you know what to slice with what?"
Hb [guest] says, "the rule appears to be splice pne site with the next on te mRNA "
CrisCan says, "it seems so complicate and it needs a very complex regulation. I ask why it is organized in this way?"
Iddo adds especially that the same gene can be alternatively spliced
Hb [guest] says, "But the cell does not always follow rthisd rule"
TRex [to raj]: Do the proteins get sent along actin or microtubule cables?
Raj [guest] says, ""is there any evidence that splicing must take place in a certain order ? if not iguess it doesn't matter if one splice site looks like any other""
Hb [guest] [to raj]: muscular dystrophy is ifetn a disease whic is due to sdplicing errors
TRex says, "There are certain mechanisms which the cell can use to put different exons in the same splice site to create variant forms of a protein"
Raj [guest] says, ""[to trex] I only know of the well defined sorting and transport pathway of the ER, but who really knows what might be going on in the cell ?""
Hb [guest] [to TRex]: yes. But how does it know which one to choose?
Raj [guest] says, ""does the muscular dystrophy mutation fall on a mrna slice site ?""
Iddo [to answer]: Cris's question (incomplete, I admit): in eukaryotes you have a lot of 'junk' - non-encoding DNA. Current hypothesis has that it mainly serves a structural function: histone wrapping, etc.
TRex [to raj]: Yes, but it intrigues me how the machinery gets to the membranes
TRex [to hb]: I have heard of "organelles" (?) called spliceosomes, but that doesn't relly answer the question
Raj [guest] says, ""so called junk dna may be of importance in genome evolution, acting as material for translocations and transpositions""
Hb [guest] [to raj]: definitively I don't know but I do know that globin diosorders fall into this category. If they can't find the right sute they use another close by (a cryptic site)
Iddo says, "Junk DNA may also contained incorporated viruses. Some oncogenes nad the Testis-Determining Factor are hypothesised to be incorporated retroviruses"
TRex says, "Men are a viral infection??!!"
Hb [guest] [to Iddo]: but the primitive organism e coli gets along fine without intorns so they can't be that important
Raj [guest] says, ""many receptors have alternatively spliced exons e.g. insulin receptor in some cells has a domain encoded by exon 11, in others an exon 12 domain - this gives the receptor different response characteristics""
Iddo laughs "it cracked up the entire Human-genetics class I took when this info was presented"
KarlS has disconnected.
Iddo says, "To Hb E. coli doesn't wrap itself around histones"
TRex says, "Although e cli goes well without inrons, it's not very co,plex"
TRex says, "Maybe introns have a real role in complexity of geneomes"
Hb [guest] [to TRex]: no because it has organised its geniome. Maybe this is outr problem
Raj [guest] says, ""but e.coli doesnt have to package so much DNA , nor does it have different specialised cell types. Also a limited repertoire, basic repressor mechanisms - is it different from a machine ? Humans and higher organisms adapt to environmen
t, seemingly making choices - maybe the complexity of adaptability is linked to genomic structure in some way""
SteveTate says, "if introns were only for histone wrapping then why is it so differnet surely there would be a common repeating DNA etc for a histone wrap and not so much junk - or other usefuf coding DNA in the interon spaces?"
Raj [guest] says, ""are introns parasitic, just hitching a free ride ?""
TRex says, "Do plants have large amounts of junk DNA?"
Malma finds its way in.
Iddo [to TRex]: I'm not sure... but the rule-of -thumb is that eukaryotes have large amounts of junk
Hb [guest] [to raj]: could be? To quote a saying from the bible "to him that have the lord givetrh"
TRex says, "Plants hang around in the sun so much - maybe junk DNA has a protective role in radiation"
Ahotz says, "I thought also the probability that an error would have consequences is lower if some of the DNA is junk"
Iddo . o O ( 'junk' that is: the fact that we don't know of a function doesn't imply there isn't one )
Raj [guest] says, ""I guess fast reproducing species are genomically streamlined., if a retrovirus got in it would be rapidly selected against by competing e.coli etc. In higher organisms there is time for a host of parasitic DNA to hitch a ride, get repl
icated and perhaps subsume a beneficioal role to the host - i.e. from parasitism to symbiosis""
Ahotz says, "beside that there are copies of funcional genes in the junk, where evolution can play with mutations"
Iddo [to Raj...]: the "Selfish Gene" paradigm...
Malma says, "Hallo everybody, sorry to be late"
TRex says, "A lot of the precision of our understanding in the test tube gets lpst when we put it in the cell :("
TRex says, "Hi Malma"
Jim Pitts says, "There are of course pseudo-genes present in higher organisms"
CrisCan says, "Sorry I have to go. Hi everyone and thanks"
Rob has disconnected.
TRex says, "Pseudo genes - are these partially replicated genes?"
The housekeeper arrives to cart KarlS off to bed.
Chandra [guest] has disconnected.
CrisCan has disconnected.
The housekeeper arrives to remove chandra [guest].
Jim Pitts says, "but usually only one or perhaps two that are functional at any one time in evolution"
SteveTate has disconnected.
Jim Pitts says, "pseudo genes appear to be "normal" to some extent but do not become transcribed and translated"
Hb [guest] says, "one of the nicest examples of pseudogens past and present is the globin locus . You can see pseudogens which have evolved different functions and som eqwhich are strilllooking for the niche inl ilife"
TRex says, "Missing essential promoters??"
Iddo says, "Oncogenes are not transcribed or translated... excepting pathological conditions"
Jim Pitts says, "this degeneracity may explain some of the usefulness of "JUNK" DNA"
Hb [guest] says, "rpomoter mutated, stop dcodonc in mRNA base uinsertions etc"
Hb [guest] says, "the best explanation I heard of for "junk" DNA is tio sepratae exons which are protein coding domains. You can then evolve new functions bty mixing and matchiong fully fucfionla units after crossing over and splicing."
Hb [guest] [to Iddo]: oncogenes occur in towo forms cellular and transforming. The cellular gene functons normally and is expresssed as a protien. The mutated form is overexpresserd and corrupts a cells signalling machinery
Raj [guest] has disconnected.
The housekeeper arrives to remove raj [guest].
Hb [guest] says, "junk DNA discussions can go on forever. shall we move to translation?"
Iddo [to hb]: There are oncogenes which are constitutively repressed: until the repressor mutates and is there no longer.
JohnW says, "(Raj had trouble with his connection, but had to leave in any case...)"
JohnW [to hb]: : Ok
The housekeeper arrives to cart rob off to bed.
TRex says, "I think translation, if everyone wants to keep going"
Iddo . o O ( Junk DNA is God's way of commenting out bad lines from the source code.. my final word on that )
The housekeeper arrives to cart CrisCan off to bed.
JohnW smiles
Hb [guest] says, "translation should be less problematical"
The housekeeper arrives to cart SteveTate off to bed.
JohnW asks, ""Does anyone have any specific questions on this subject?"
JohnW . o O ( I've got one )
Hb [guest] says, "Oh dear"
Iddo smiles broadly
TRex says, "I feel fairly safe with translation, but maybe I'm kidding myself"
Hb [guest] says, "so lets hear it"
Zerwan finds his way in.
JohnW says, "Well, actually my question relates to the genetic code really, but translation is the process implicated. Its about the 'Universality' (or not) of the genetic code:"
Zerwan says, "hello everybody"
Hb [guest] says, "hello wzerwan"
TRex waves
Ahotz waves
Hb [guest] says, "yes is it unibversal?"
Iddo says, "no"
JohnW says, "So, just how universal is this code? I think I'm right in saying that certain bacteria have a slightly different set of codons, as do some mitochondrial genomes?"
TRex says, "I think in the sense that it is triplet code, its universal"
JohnW says, "- and this depends on there being 'novel' amino acyl-tRNA molecules, right?"
Zerwan [to Johnw]: Yes I think you are right
TRex says, "I mean there are no duplex or quad codes are there?"
Iddo says, "There is the case of selenocysteine, which is translated to such only of a stem-loop structure occurs in the mRNA"
Hb [guest] says, "I don't know where the differences lie. I would guess that both tRNA and aatRNA must be different"
Gayle schulte says, "maybe getting off the topic but in certain bacterial cell walls,"
JohnW says, "For example, a novel Seryl-tRNA was found in the fungus Candida albicans, which translates the CUG codon, instead of leucine"
Gayle schulte says, "maybe getting off the topic but in certain bacterial cell walls, d-alanine is incorporated by d-ala-d-ala ligase, an enzyme whose crystal structure was solved by Jim Knox last year, how is this done? D-alanine being the enantiomer of
the usual l-ala"
Iddo says, "Yes. How are D-aas synthesized, anyway?"
Hb [guest] says, "I can't cope with proein synthesis by mRNA let alone protein synthesis by proteins"
TRex says, "There are several instances of d-amino acids in antibiotics and cell walls, but these are specially synthesised by enzymes, not on ribosomes"
Iddo says, "Yes. Also, the bacterial cell-wall is not a protein in the strict sence: it is more of a grid made up form sugars & amino-acids"
Iddo says, "N-peptidoglycan, if memory serves"
Hb [guest] says, "proetin syntheisi by proeins is not one of my areas of expertise. Unless anybody is prepared to answer these wquaestion then I suggest we revert to norma translation"
TRex says, "I stiil think the code can be described as universal"
Gayle schulte says, "sorry to digress :)"
TRex says, "Sure some of the triplets are ambiguous, and used differently in different organisms and organelles"
JohnW [to TRex]: But surely not if the same triplet codon codes for different amino acids in different organisms?
JohnW . o O ( beat me to it )
Iddo [to TRex]: the triplets, maybe. But there are quite a few species which use different aas to codons
TRex says, " I think I mean the concept of a triplet is universal"
Hb [guest] says, "is this not a semantic arument? iyt is universal in that three bases code for one amino acid its just that in the odd case he amino avcids differ"
TRex says, "Yes. Language is universal in humans, but we don't al speak the same language :)"
JohnW says, "Could someone remind me just how many tRNAs exist in a given organism- is there one for each of the 64 triplets (minus the 2 stop codons), or does the tRNA-mRNA pairing always rely on 'wobble'?"
JohnW [to TRex]: ok, in that sense yes, I see your point.
Hb [guest] says, "if I remember rightl;y it is 20 aatRNA synthetases, about 45 -50 tRNAs and 61 codons"
TRex says, "I think the minimum nimber of tRNAs needed is something like 22 or 23"
JohnW is looking up the triplet code
Zerwan says, "several "
TRex says, "One for each of the amino acids, one to start and one to stop. Mitochondria come closest to this minimum"
JohnW says, "Ah, I see I should have said '3 stop codons'"
JohnW says, "Is the Met codon the start codon then?"
TRex says, "I think!"
Hb [guest] [to TRex]: yes start is the same as met
Iddo [to JohnW]: yep, same as Met
Iddo says, "AUG"
TRex says, "Yes synthesis begins with a met"
Zerwan finds his way out.
Hb [guest] says, "but not all proteins, mature that uis, syart with met"
Iddo says, "could someone remind me about the amber and ochre codons?"
Jim Pitts says, "The level of tRNAs vary in different organisms and so there is usually seen a codon preference normally one or two codons used most of the time."
Jim Pitts says, "for a particualr aas"
TRex says, "A lot of mets are cut off after synthesis is complete"
TRex says, "amber and ochre are historic terms for two of the stop codons"
Hb [guest] [to Iddo]: the amber name was a joke (somebody'd am name?) then it was continued with ochre and opal
Iddo nods
Jim Pitts says, "The amber mutation was found before DNA sequencing so not assigned to a specific codon"
TRex [to Iddo]: I think that amber is not as efficient a stop codon, and ochre was discovered last - so they called it a olour too. I think it was a lab thing
Jim Pitts says, "Normal E. coli genes often have two different stops in a row"
JohnW asks, "so are the '3 stop codons' actually 'stop', 'amber' and 'ochre'?"
TRex says, "There also has to be a way of distinguishing a start met form an internal met"
Hb [guest] [to JohnW]:
Iddo [to trexx]: a consensus sequence -10 downstream to the initial met
Iddo says, "e.g. tataat in bacteria"
Iddo . o O ( I meant upstream, sorry) )
Jim Pitts says, "UAA = Ochre, UAG = Amber, UGA = Opal "
Hb [guest] [to TRex]: this consensus sequence S-D sequence has ho,ology to the 18S RNA in ribosomes and is situated 7 bases preceding the start AUG
TRex says, "Is it true that one of the stop codons is more effective than the others?"
Hb [guest] says, "yes its easy to get amber mutations cos of double stops spoken about earlier but difficult to get opal mutants"
Iddo 's got to go now :-( Great talking to you all
JohnW waves to Iddo
TRex says, "I seem to remember selenocysteine is read on a stop codon. I that correct, and which is it?"
Hb [guest] says, "bye iddo"
Iddo waves to all
Iddo has disconnected.
Hb [guest] says, "I think you are right about stop codons and selenocyystein but I don't know whic"
Malma says, "UAG is the selenocystein codon"
Hb [guest] says, "I' sorry but I must go to. Its been nice talking to you all. Maybe we should continue this discussion some time"
Ahotz says, "I have to go, too, thanks,bye"
TRex says, "Thanks Malma"
Ahotz has disconnected.
Hb [guest] has disconnected.
The housekeeper arrives to remove hb [guest].
TRex says, "Henry thanks for your time and help. It would be good to revisit some items later"
JohnW . o O ( another Mammoth MOO session... )
TRex smiles
TRex says, "Let's call it a night!"
JohnW says, "Yes, thanks very much Henry. This has been very interesting."
Gayle schulte says, "thank you!"
Gayle schulte has disconnected.
JohnW says, "By the way, I'll just add, have a look at Leos Kral's translation movie if you havent done so..very impressive!"
TRex says, "Bye to all - look forward t the next"
JohnW [to TRex]: one of these days (nights) I will have to make it to a Guanine group meeting.
TRex says, "John is that movie url in the PPS pages?"
TRex says, "We'd enjoy that John"
TRex says, "Guanine's are usually 2400 GMT Tuesdays at baseG"
JohnW says, "Yes- try http://www.cryst.bbk.ac.uk/mirror/prtsynth/ which is the mirror I set up, but the original is at Georgia State Univ, U.S. - movie, slide show, quiz, the lot."
JohnW says, "its also pointed to from the PPS Course Material, Section 4"
TRex says, "Thanks John. Have a good evening"
Paolo says, "Thanks a lot and bye all! "
TRex waves goodbye
Paolo has disconnected.
JohnW says, "And you, Trex"
TRex has disconnected.
The housekeeper arrives to cart Iddo off to bed.
JohnW asks, "Shall we pull the plug on the recorder?"
Malma says, "bye everybody"
jzt turns the C-recorder off.