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you

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[Music]

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situation is that there's only a single

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planet that we know of that actually

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harbors life so we're constrained in our

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thinking about what constitutes life by

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what we know on earth and surprisingly

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enough we know actually quite little we

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know about the three domains of life on

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Earth and that life probably originated

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at once as and a there's a single

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genetic code and all of the diversity of

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life that we see are based on this

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single genetic code we don't really know

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very much about evolution in the early

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stages and by the early stages I would

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say the first half of the history of our

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planet and particularly confounding has

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been the fact that there's been a

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tremendous amount of lateral gene

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transfer and that has confounded our

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understanding of evolutionary processes

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there's been a tremendous amount of

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evolution during this time and most of

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the metabolic processes have evolved

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during this time including oxygenic

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photosynthesis methanogenesis all sorts

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of respiration photo trophy and probably

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the most fundamental biological process

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which is chemiosmotic coupling all of

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these processes require pigments and

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there are a number of different pigments

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and the ones that I'm going to talk

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about primarily are the carotenoids

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and in particular retinol the visual

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pigment also I'll compare them to

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Flavin's and porphyrins one of the main

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questions that we have in our mind is in

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an evolutionary sense when did these

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different pigments arise because if some

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of the pigments arose earlier than

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others then we should be aware of that

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as we search for biosignatures

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elsewhere so the other hypothesis that I

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keep in mind is that generally speaking

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life has evolved

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from simplicity to complexity and the

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beta carotene and particular its

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oxidative product retinol are two of the

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simplest pigments Flavin's which are

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involved in respiration processes and

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many organisms is also relatively simple

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compared to what are the porphyrins

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which are involved in a wide variety of

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processes and are some of the most

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interesting pigments out there so if you

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look at the spectra of these different

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pigments especially the chlorophylls

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versus the retinols

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the chlorophyll pigments are there which

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there are a variety which are tuned to

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specific environments as we'll hear

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about in the next talk have to

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absorptions maxima one in the blue and

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one in the red and therefore the color

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green is observed in the case of the

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retinal pigment there's a single peak

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which is in the green region and

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therefore those pigments are purple the

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retinal pigments the prototype vector

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Dobson is the simplest biological system

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for energy transduction so again we

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think that these may have evolved very

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early it's a single protein with a

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single chromophore it is a light driven

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proton pump and it generates a proton

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motive gradient and that proton motive

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gradient can be used for akp synthesis

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and a wide variety of other by energetic

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processes so the organisms the extreme

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halophiles are yellow philic archaea are

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a very nice system in terms of by

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signatures to be thinking about retinal

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pigments and carotenoids so I think many

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of you have seen these ponds in South

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San Francisco Bay which are highly

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saline the airplane when

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lands in San Francisco generally flies

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over at least when you're coming from

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the East Coast over these ponds and

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these ponds have been the subject of

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much interest over the years the ponds

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are of different colors depending on the

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types of organisms and their salinity

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and they are some of them are red orange

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or purple the JPL a virus collection has

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actually kept ten years of data on those

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ponds and the the ponds are differing by

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the concentration of salt from about 20

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PPT to over 350 PPT very close to

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saturation so if you look at the

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reflectance spectra in these ponds you

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can see absorption for the carotenoids

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and BR you can also see absorption for

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chlorophylls however the carotenoids in

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BR are are much more prevalent in the

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highest salt concentrations and the the

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concentration of cells in these ponds is

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extremely high it's maybe ten to the

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eleventh cells per mil so we think that

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this is a pretty good system to think

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about developing a way to assay for

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biosignatures in this route in this

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range so these organisms are growing in

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extremely se line brine and I think you

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could see the the cells between the the

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salt crystals the cells can actually

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survive desiccated in the salt crystals

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and when you plate them you see highly

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pigmented colonies and one can also

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observe many mutants and that's been our

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approach okay so our approach to

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studying these halo bacterial pigments

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has been the isolation of pigment

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mutants

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then we did characterization

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biochemically and genetically and in the

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recent years we have just been able to

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sequence a whole genome to analyze them

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and then we've also done some

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spectroscopic analysis both absorption

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and reflection and characterized

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different genes and pathways so I want

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to share with you some of the data that

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we've accumulated on these so the

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carotenoid and retinol by synthetic

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pathway starts with the isoprenoid

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pathway and then ultimately gets to

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lycopene lycopene is actually the color

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of tomato then it goes on to beta

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carotene and then ultimately to retinol

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and the retinol is allows the cells to

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grow phototrophic lee the other branch

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from lycopene goes to the effector

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Ruben's and these are c50 pigments which

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have function in repair of DNA damage so

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we were able to isolate mutants that are

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white these occur some of them occur

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spontaneously and the white mutants when

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analyzed genetically have been shown to

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have a defect in life being elongate so

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that's the first step in the conversion

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of lycopene into vector Rubens another

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type of mutant that we've been able to

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isolate our retinal mutants in this case

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the phenotypes are much more subtle

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the cells on the left are the wild-type

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cells on the right are unable to make

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retinol and they have a defect in the

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enzyme for beta-carotene mono oxygenase

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and we initially knocked out the gene

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that we believed to be responsible for

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this because the VRP gene and you can

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see in number two that the retinol is

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reduced but not completely gone beta

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carotene is actually induced

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compared to the wild type there's a

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second gene it turns out that also

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encodes a beta-carotene mono oxygenase

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and when you do a double mutant then you

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can see that it has knocked out retinal

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entirely so we've gone on and isolated

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other mutants the halo vector and vector

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tops and mutants and we've isolated a br

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over producers so this is a sucrose

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gradient showing the relative proportion

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of carotenoids to vector Group B R if

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you compare that to the next two which

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has the wild-type you can see that

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there's a considerably lower amount of

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BR compared to the carotenoids and then

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we also isolated an orange mutant and

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colorless mutants and when we analyze

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these we see that the orange mutants

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have an insertions into the protein gene

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for vector Dobson and the colorless

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mutants have insertions in the bat gene

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for which is a regulatory gene for not

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just the protein but also for the

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pigment so we've done a fair amount of

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analysis of this type and we're putting

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together the pathway for these

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production of the carotenoids and this

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is the portion that I've mentioned

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before the lycopene is converted to

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retinol which binds to the vector opsin

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and produces BR the lye and another gene

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rub converse lycopene into becsher

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rubriz and then if you go backwards for

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the carotenoid pathway there are other

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genes that are involved in conversion of

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Filene and general general power

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phosphate into lycopene and then before

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that there's a complicated pathway for

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isoprenoid goes all the way from

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basically acetate up to general journal

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so just to put that

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pathway into context we think that this

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is actually a relatively simple pathway

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for production of pigments and may have

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originated fairly early in evolution and

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the carotenoid pathway is producing both

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the vector Rubens and retinols before

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the carotenoid pathways the isoprenoid

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pathway which is taking STL Co a to the

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general general power phosphate and we

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think that this is maybe one of the

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original or early by synthetic pathways

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on earth for pigments and remember these

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pigments are involved both in generating

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energy as well as protection of DNA the

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okay I'm sure this is the last slide so

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the another branch of that pathway leads

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to membrane lipids so it makes sense

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that under these circumstances you would

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be able to generate something that looks

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like a protocell and produce a protein

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with a chromophore that would be able to

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generate energy for life and this entire

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pathway of course is just direct

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offshoot from central metabolism from

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glycolysis and TCA so we think that

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there may be a temporal appearance of

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these pigments on earth the carotenoids

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might have appeared first retinal

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pigments appeared subsequently and then

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ultimately the chlorophyll and you can

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see that there is a complementarity

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between the retinal pigments and the

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chlorophyll pigments so I think I'll

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stop there and acknowledge my co-authors

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Priya Victoria and also our

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collaborators at Kenyon College and also

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thank NASA exobiology and the mirrors