Ma `lumot

Organizmlar orasidagi ketma -ketlik o'zgarganda, GPD genini qanday aniqlash mumkin?


Men zamburug'larning genetik transformatsiyasi haqidagi maqolani o'qiyapman va qog'ozda ishlatiladigan plazmid GFP genini boshqarish uchun bir xil GPD (gliseraldegid3-fosfat dehidrogenaza) promouterining ikkita shaklidan foydalanadi, biri Agaricus bisporus va ikkinchisi Lentinula edodes (GenBank). : GQ457137.1).

Biroq, men yuqorida aytib o'tilgan GPD promouterlari ketma -ketligi boshqa organizmdan olingan GenBank (NC_007251.2) mos yozuvlar ketma -ketligiga mos kelmasligini payqadim.

Nima uchun bitta promouter uchun turli xil ketma -ketliklar mavjud? Bundan tashqari, agar men uni ma'lum ketma-ketlik bilan taqqoslay olmasam, boshqa organizmdagi GPD genini qanday aniqlashim mumkin?

Men o'zgartirmoqchi bo'lgan organizmda to'liq genom ketma -ketligi bor edi va agar men GPD kabi mahalliy promouterdan foydalansam, transformatsiyam ancha samarali bo'ladi.


Bu yerda sizni noto‘g‘ri tushunayotgan bo‘lishim mumkin, lekin yuqoridagi havolalaringizdan ikkita (uzoqdan bog‘liq) zamburug‘larning GPD promouter mintaqalariga mos kelishni xohlayotganingizni tushunaman. Agaricus bisporus va Lentinula edodlari, GPD promouteriga Katta leyshmaniya, bu butunlay boshqa Shohlikka tegishli!

Targ'ibotchi hududlar, hatto yaqin turlar uchun ham, oqsillarni kodlovchi hududlar bilan solishtirganda, turlar orasida nisbatan yomon saqlanib qolgan. Siz aytib o'tayotgan turlar orasidagi evolyutsion masofani hisobga olsak, ularning promouterlari o'rtasida har qanday gomologiyani topish ehtimoli nolga teng.

Bundan tashqari, agar men uni ma'lum ketma-ketlik bilan taqqoslay olmasam, boshqa organizmdagi GPD genini qanday aniqlashim mumkin?

Men nima qilardim, tarjima qilingan GPD protein ketma-ketligini olishdir, buning uchun Lentinula edodes GenBank BAA83550.1 bo'ladi. Keyin men undan protein moslamalarini qidirish uchun ishlatardim portlash, ayniqsa uchun subsetting Katta leyshmaniya; va natijadan foydalanib, genomda kodlovchi genni toping. Buni bir qadamda ham qilishingiz mumkin tblastn, tarjima qilingan nukleotidlar bazasida gugurtlarni qidiradi (bu tblastn misol so'roviga qarang).

Keyin siz GPD promouteringizni ifodalash uchun kodlash hududining 1000 bp yoki undan yuqori oqimini olishingiz mumkin.


9: Proteinlarni saqlash

  • Clare M. O & rsquoConnor hissasi
  • Boston kollejining faxriy professori (biologiya).

Ushbu laboratoriya yakunida talabalar quyidagilarni bilishlari kerak:

  • aminokislotalarni 1 harfli kod bilan aniqlang.
  • BLOSUM 62 matritsasi bo'yicha yuqori va past ballar orasidagi farqni tushuntiring.
  • oqsil ketma -ketligini solishtirish uchun BLASTP algoritmidan foydalaning.
  • bir nechta ketma -ketlikda saqlangan hududlarni aniqlash.

Turlarning rivojlanishi bilan ularning oqsillari o'zgaradi. Alohida oqsil ketma-ketligini o'zgartirish tezligi organizmlar boshdan kechiradigan evolyutsion bosimni va oqsilning fiziologik rolini aks ettiruvchi juda katta farq qiladi. Bizning maqsadimiz - bu semestrda Met va Cys biosintezida ishtirok etadigan oqsillar funktsional jihatdan saqlanib qolganligini aniqlash. S. pombe vaS. cerevisiae, evolyutsiyada milliard yilga yaqin bo'lgan turlar. Ushbu laboratoriyada siz homologlar uchun ma'lumotlar bazalarini qidirasiz S. cerevisiae bir nechta turlarning ketma -ketligi, shu jumladan S. pombe. Gomologlar o'xshash genlardan kelib chiqqan o'xshash DNK ketma -ketligi. Gomologlar har xil turlarda topilsa, ular deyiladi ortologlar.

Xuddi shu genomdagi homologlar paraloglar deb ataladi. Analoglar genlarning ko'payishi natijasida paydo bo'ladi, lekin vaqt o'tishi bilan diversifikatsiya qilinadi va alohida funktsiyalarni oladi. Evolyutsiya davrida genomning to'liq takrorlanishi sodir bo'lgan S. cerevisiae (Kellis va boshqalar., 2004), metionin super yo'lidagi faqat bir nechta genlarda paraloglar mavjud. Qizig'i shundaki, MET17 Oltingugurtni o'tkazishda ishtirok etadigan uchta genga parallel: STR1 (CYS3), STR2 va STR4, bir nechta genlarning takrorlanishini aks ettiradi. Bu to'rtta alohida fermentning mavjudligi o'ziga xos moslashuvchanlikni beradi S. cerevisiae oltingugurt manbalaridan foydalanishda. The SAM1 va SAM2 genlar ham bir -biriga o'xshashdir, lekin ularning ketma -ketligi deyarli bir xil bo'lib qoladi, agar bitta gen inaktiv bo'lsa, funktsional ko'paytirishni ta'minlaydi (6 -bob).

Bu semestrdagi tajribalarimiz evolyutsion tafovut paytida Met va Cys sintezida ishtirok etadigan genlar saqlanib qolganligini tekshiradi. S. cerevisiae va S. pombe . Turli xil algoritmlar tadqiqotchilarga oqsil ketma -ketligi evolyutsiyasini o'rganish vositalarini taklif etadi. To'qqizta divergent model organizmlardan olingan Sam2p ketma-ketliklarining ushbu grafik tasvirida harfning balandligi ma'lum bir aminokislotalarning ushbu pozitsiyadagi chastotasini aks ettiradi.

Protein funktsiyasi uning tuzilishi bilan chambarchas bog'liq. Esingizda bo'lsa, oqsilning oxirgi katlangan shakli uning birlamchi ketma-ketligi, aminokislotalarning ketma-ketligi bilan belgilanadi. Aminokislotalarni almashtirish konservativ bo'lsa, evolyutsiya jarayonida oqsil funksionalligi kamroq tez o'zgaradi. Konservativ almashtirishlar yangi aminokislotalar yon zanjirining o'lchami va kimyosi almashtiriladigan zanjirga o'xshash bo'lganda sodir bo'ladi. Ushbu laboratoriyada biz aminokislotalarning yon zanjirlarini muhokama qilishni boshlaymiz. Keyin BLASTP algoritmidan foydalanib, bir nechta model organizmlardagi ortologlarni aniqlaysiz. Siz boshqalarga qaraganda yuqori darajada himoyalangan hududlarni ajratib turadigan bir nechta ketma -ketlikni tekislashni amalga oshirasiz.

Mashqlar ustida ishlayotganda, ma'lumotlar bazalaridagi oqsillar ketma-ketligi 1 harfli kodda yozilganligini sezasiz. 1 harfli kod bilan tanishish bugungi molekulyar biologlar uchun zaruriy mahoratdir.


Fon

Klasterli muntazam intervalgacha qisqa palindromik takrorlashlar (CRISPRs) Bakteriyalar va Arxeyadagi takrorlanuvchi tuzilmalar bo'lib, uzunligi 24 dan 48 tagacha bo'lgan aniq takrorlash ketma-ketliklaridan tashkil topgan (bu erda takrorlashlar deb ataladi) o'xshash uzunlikdagi noyob ajratgichlar (bu erda oraliqlar deb ataladi) [1, 2]. CRISPR ketma-ketligi genomning eng tez rivojlanayotgan elementlaridan biri bo'lib ko'rinadi, shuning uchun bir-biriga yaqin turlar va shtammlar, ba'zan DNK darajasida 99% dan ortiq bir xil bo'lib, CRISPR tarkibida bir-biridan farq qiladi [3, 4].

CRISPR bilan bog'liq ketma-ketliklar (CAS) deb nomlangan 45 tagacha genlar oilasi bu takrorlanishlar bilan birgalikda paydo bo'ladi va ular CRISPRning tarqalishi va ishlashi uchun javobgar deb faraz qilinadi [2, 5, 6]. CASlarni operon tashkil etilishi va gen filogeniyasiga ko'ra etti yoki sakkizta kichik tipga bo'lish mumkinligi taklif qilingan [5, 6]. Filogenetik tahlil qo'shimcha ravishda CAS -larning keng gorizontal gen o'tkazilishini boshdan kechirganligini ko'rsatadi, chunki juda o'xshash CAS genlari uzoq qarindosh organizmlarda uchraydi [6, 7]. CRISPR va CAS plazmidlar kabi mobil genetik elementlarda topilgan. teri mobil elementlar va hatto profaglar tizim uchun mumkin bo'lgan tarqatish mexanizmini taklif qiladi [7-9].

CRISPRlar replikon bo'linishi [1], DNKni ta'mirlash [10], tartibga solish [5] va xromosomalarni qayta tashkil etishda [11] rol o'ynashi taklif qilingan. Yaqinda ma'lum bo'lishicha, ajratgichlar ko'pincha fag yoki plazmid DNK kabi ekstraxromosoma DNK fragmentlariga juda o'xshashdir [3, 12]. CRISPR/CAS tizimi antiviral javobda, ehtimol RNK aralashuviga o'xshash mexanizm bilan ishtirok etishi taklif qilindi. Ushbu CRISPR funktsiyasining taklif qilinadigan mexanizmi DNKning invaziv elementlarining namunalarini olish va rekordini saqlashni, shuningdek, invaziya uchun zarur bo'lgan gen funktsiyalarini inhibe qilishni o'z ichiga oladi [12]. Darhaqiqat, yaqinda CRISPRlar prokaryotlarda viruslarga qarshi orttirilgan qarshilik ko'rsatishi ko'rsatildi [13].

CAS-larni chuqur tahlil qilishiga qaramay, takroriy ketma-ketlikning tabiati yaqindan o'rganilmagan. Bu, ehtimol, qisqa DNK ketma-ketligi kabi takrorlanishlar oqsil kodlovchi genlarga qaraganda solishtirma salohiyatga ega emas. Oldingi tadqiqotlar shuni ko'rsatdiki, takrorlanishlar juda o'zgaruvchan va ular organizmlar o'rtasida o'xshash emas [2, 7]. Biroq, biz shuni ko'rsatadiki, turli xil organizmlarning takrorlanishlari ketma -ketlik o'xshashligiga qarab klasterlarga birlashtirilishi mumkin va ba'zi klasterlar kompensatsion bazaviy o'zgarishlarga ega bo'lgan ikkinchi darajali tuzilmalarga ega. Biz yana shuni ko'rsatamizki, CAS pastki turlari va takroriy klasterlar o'rtasida aniq yozishmalar mavjud. Bizning topilmalarimiz CRISPR funktsiyasi va xilma -xilligiga muhim ta'sir ko'rsatadi.


Maxsus fikrlar

Bir nechta yig'ilishlarning izohi

Agar ma'lum bir organizm uchun yaxshi sifatli bir nechta yig'ilishlar mavjud bo'lsa, barchasini izohlash muvofiqlashtirilgan holda amalga oshiriladi. Assambleyalar bo'ylab mos keladigan hududlar bir xil tarzda izohlanishini ta'minlash uchun yig'ilishlar izohdan oldin bir -biriga moslashtiriladi.

  • O'rnatish-yig'ish natijalari transkript va tuzilgan genomli hizalamalarni saralash uchun ishlatiladi: berilgan so'rovlar ketma-ketligi uchun ikkita yig'ilishning tegishli hududlariga hizalanish bir xil darajani oladi.
  • Bir nechta yig'ilishlarning mos keladigan joylari bir xil GeneID va joylashuv turiga ega.

Yig'ish-yig'ish moslamalari NCBI Genom Remapping Service orqali mavjud.

Qayta izoh

Yangi dalillar mavjud bo'lganda (masalan, RNK-Seq) yoki yangi yig'ilish chiqarilganda, organizmlar vaqti-vaqti bilan qayta izohlanadi. Izohning bir chiqarilishidan ikkinchisiga modellar va genlarni kuzatishga alohida e'tibor qaratiladi. Bir -biriga o'xshash genomik joylarga izoh berilgan oldingi va hozirgi modellar aniqlanadi va yangi modellarga GeneID -larni tayinlashda oldingi modellarning lokus turi va GeneID hisobga olinadi. Agar yig'ilish izohning ikki bosqichi o'rtasida yangilangan bo'lsa, yig'ilishlar bir -biriga moslashtiriladi va xaritali hududlardagi oldingi va joriy modellarga moslashtirish uchun ishlatiladi.


Natijalar

GM guruch hodisalarining molekulyar tavsifi uchun bioinformatik ish oqimi

Ko'pgina tadqiqotchilar katta miqdordagi bioinformatik ma'lumotlar bilan ishlashda qiyinchiliklarga duch kelishadi. Biz an'anaviy aniqlash usullari o'rniga NGS ma'lumotlari yordamida kiritilgan D-DNK birikmalarini aniqlash uchun foydalanuvchilar uchun qulay usulni ishlab chiqdik. Bioinformatik ish oqimining diagrammasi 1-rasmda ko'rsatilgan. Birinchi bosqichda malakali xom juftlangan o'qishlar Burrows-Wheeler Aligner dasturi yordamida maksimal aniq mosliklarga ega (BWA-MEM) transformatsiya plazmid vektoriga moslashtirildi [22] . Transformatsiya plazmid vektorining tuzilishi dumaloq bo'lgani uchun biz chiziqli vektor mos yozuvlar ketma -ketligini (pPZP200) tuzdik, bu erda chap va o'ng chegara ketma -ketligi plazmid ketma -ketligining qarama -qarshi uchidan 150 bp ni o'z ichiga oladi. Ulanishlarni qamrab oluvchi o'qishlarni tanlash uchun, T-DNK joylashuvi (6392 dan 10,291 bp gacha) asosida xaritalangan o'qishlar ularning xaritalangan pozitsiyalariga ko'ra ayirildi. Ushbu to'plangan o'qishlar mos yozuvlar guruch genomiga qarshi noto'g'ri-ijobiy o'qishlarni tasniflash uchun BLASTN tahlili uchun so'rovlar sifatida ishlatilgan (O. sativa versiya 7.0) [23]. Kiritilgan T-DNK endogen elementlarni o'z ichiga olishi uchun mo'ljallanganligi sababli, endogen promotorlar ketma-ketligini o'z ichiga oladi. RbcS3 noaniq hizalamayı kamaytirish uchun ketma -ketlik o'xshashlik ballari (mahalliy guruch ketma -ketligi) asosida ehtiyotkorlik bilan olib tashlandi. Qolgan o'qishlar transgenik vektorga moslashtirilib, IGV yordamida juft o'qish bilan tasvirlangan. Natijalardan biz T-DNK ning ikkala uchiga qisman mos keladigan (ya'ni, T-DNK va guruch genomini qamrab olgan o'qishlar) birlashma o'qishlarini tanladik va FASTA ketma-ketligini ajratib oldik, bu T-DNKning birlashma mintaqasiga kiritilganligini aniqlash uchun. genom (1 -rasm).

T-DNK joylashuvi va nusxa raqami

Taxminan 28 Gb ketma -ketlik ma'lumoti, 72 × ketma -ketlik chuqurligiga mos keladi, "Illmi" nazorat qiluvchi asosiy navidan olingan. Bundan tashqari, SNU-Bt9–5, SNU-Bt9–30 va SNU-Bt9–109 dan taxminan 78 ×, 54 × va 68 × ni tashkil etuvchi 30 GB, 21 GB va 26 GB xom ma'lumotlar olingan. mos ravishda genom qamrovi (1-jadval).

Bizning ulanishlarni aniqlash tahlilida qo'llaniladigan ketma-ket qadamlardan (usullarning "T-DNK qo'shilish joyini tahlil qilish" bo'limida ta'riflanganidek) GM guruchidan SNU-Bt9-5 dan 11539 o'qish, shu jumladan 2790 juft xaritali o'qish olingan. Bundan tashqari, SNU-Bt9–30 va SNU-Bt9–109 navli GM guruchlaridan 8371 va 9767 o'qishlar, mos ravishda 1792 va 2336 to'g'ri o'qish juftlari xaritasi tuzilgan (2-jadval). Kutilmaganda, yovvoyi "Illmi" dan olingan 8125 o'qish transgenik vektor ketma-ketligi bilan xaritaga kiritildi, shu jumladan atigi 648 juft o'qish. Qolgan juftlanmagan juft o'qishlar Illumina ketma-ketligining qisqa ketma-ketligi tufayli yuzaga kelishi mumkin bo'lgan xususiyatga bog'liq deb taxmin qilingan. Shuni ham ta'kidlash kerakki, bizning T-DNK konstruksiyamiz ushbu tadqiqotda guruchning endogen promotor genini o'z ichiga olishi uchun yaratilgan. rbcS3 (Os12g0291100), u 1824 bp T-DNKni oladi va guruch xromosomasi 12 da ifodalanadi [24]. Mahalliy genomdan (ya'ni T-DNKdan emas) kelib chiqadigan yolg'on-pozitiv o'qishni yo'q qilish uchun har bir xaritali ketma-ketlik BLASTN yordamida guruch mos yozuvlar ketma-ketligi bilan solishtirildi. Illmi guruchiga mos keladigan jami 915, 1019, 729 va 899 o'qishlar mos ravishda SNU-Bt9-5, SNU-Bt9-30 va SNU-Bt9-109, barchasi 12-xromosomaga to'g'ri keldi va noto'g'ri musbat deb tasniflandi.

Transgen chegara mintaqasining ikkala uchi bilan qisman hizalangan o'qishlar, ularning xaritalash pozitsiyasiga qarab yig'ilgan (2a va b -rasm). Keyin tanlangan o'qishlar T-DNKning butun ketma-ketligi bilan yonma-yon joylashgan joyni aniqlash uchun moslashtirildi. Olingan natijalar guruch xromosomalarida qo'shimchalar birikmalarini ko'rsatdi (2c -rasm). SNU-Bt9-5 guruchidan olingan mezbon genom va transgen o'rtasidagi kesishgan hududlarni o'qiydi, guruch xromosomasi 10 ga 22,498,218 dan 22,498,279 bp gacha 79-soniyali o'chiriladi. SNU-Bt9-30 guruch hodisasi 51-bp o'chirilishi bilan 22,473,585 dan 22,473,636 bp gacha bo'lgan guruch xromosomasi 11 bilan to'g'ri ko'rsatilgan (3-jadval va 3-rasm). Ikkala transgenik hodisa ham guruch genomida bitta nusxa va bitta lokusni muvaffaqiyatli aniqladi va ikkala natija ham Southern blotga asoslangan aniqlash usuli bilan olingan natijalar bilan bir xil edi [21].

NGS o'qish moslamalari yordamida transgen guruchning molekulyar tavsifi. a T-DNK o'z ichiga olgan pPZP200 plazmid transformatsiyasi tasviri Agrobakteriyalar-SNU-Bt9–5, SNU-Bt9–30 va SNU-Bt9–109-ni yaratish uchun vositachi transformatsiya. MCS, bir nechta klonlash sayti. b IGV natijalarining batafsil namunasi. Navbat chizig'idagi gorizontal chiziqlar (panelning yuqori qismi) mos yozuvlar ketma-ketligini ko'rsatadi (ya'ni, T-DNK kiritilgan plazmid vektor ketma-ketligi). Tanlangan treklar juftlashgan yo'nalishni ko'rsatadi (yuqori panel = o'qish 1, pastki panel = o'qish 2). Rangli qutilar T-DNK chegarasi va genomik yonma-yon ketma-ketlikni o'z ichiga olgan birlashma hududini ko'rsatadi. v Birlashma oralig'idagi o'qishlarning ketma-ketligi (yuqori = chap chegara yonboshi ketma-ketligi, pastki = o'ng chegara yonboshi ketma-ketligi). Qizil va qora nukleotidlar mos ravishda guruch xromosomasini va T-DNKni ko'rsatadi

Guruch xromosomasida T-DNK qo'shilishining aniqlangan lokuslarining ifodalanishi

SNU-Bt9-109 guruchining integratsiya joylari bu erda tasvirlangan usul yordamida aniqlanmagan bo'lsa-da (3-jadval va 3-rasm), o'ng chegara (RB) yaqinidagi integratsiya joyi 3-xromosomada 14,707,459 dan 14,707,391 bp gacha topilgan. Chap chegara (LB) yaqinidagi yonboshlab ketma -ketliklar aniqlanmagan. BLASTN tahlili (NCBI nr ma'lumotlar bazasidan foydalangan holda) LB mintaqasi va mezbon genomining birlashishi "Ds/T-DNK vektorining pDsG8 tutilishi (e-qiymati: 4e-28)" ga juda o'xshashligini ko'rsatdi. Solanum tuberosum proteinaz inhibitori geni (e-qiymat: 6e-28). Biroq, S. tuberosum Qisqa so'rov va o'ziga xosligi pastligi sababli, gen artefakt sifatida qaraldi.

Yuqoridagi natijalarni tasdiqlash uchun biz olingan birikmalar ketma -ketligi bo'yicha primerlarni ishlab chiqdik (Qo'shimcha fayl 1: Jadval S1). Bizning PCR natijalari shuni ko'rsatdiki, ikkita transgenli guruch hodisasini kiritish NGS yordamida muvaffaqiyatli tavsiflangan. Bundan tashqari, SNU-Bt-109 ning birlashma ketma-ketligi yaqin atrofdagi LB ketma-ketliklari yordamida PCR yonboshlab aniqlandi (Qo'shimcha fayl 1: S2-rasm).

T-DNKning qayta joylashishini aniqlash

T-DNK ketma-ketligini aniqlash uchun biz transgen plazmid DNKga nisbatan xaritalangan juftlarni o'qishdan foydalanib, qo'shimcha o'lchamdagi taqsimotlarni hisoblab chiqdik (Qo'shimcha fayl 1: S3-rasm). Qo'shish hajmini hisoblash orqali kiritilgan DNKning qayta tashkil etilganligini aniqlash mumkin. SNU-Bt9–5, SNU-Bt9–30 va SNU-Bt9–109 uchun moslamalarning o'rtacha o'lchami mos ravishda 479, 469 va 535 bp bo'lib, ular kutubxona qurilishida tayyorlangan o'lchamlarga to'g'ri keldi (Qo'shimcha fayl 1: S4-rasm) ). T-DNK ichida hech qanday ichki o'zgarishlar yoki dublikatsiyalar mavjud emas deb taxmin qilingan. Natijalar bizning oldingi maqolamizda genomik DNK PCR va ketma-ket tahlil orqali T-DNKni to'liq olish natijalariga to'g'ri keladi [21].

Transgenli o'simliklarda umurtqa pog'onasi ketma -ketligi bo'lishi mumkin

Yangi GM zavodlarini ishlab chiqish jarayonida kutilmagan genomik o'zgarishlar yuz berishi mumkin. Plazmidli orqa miya ketma -ketligi mezbon genomiga qo'shilishi mumkin Agrobakteriyalar-vositalangan transformatsiya [10]. Shuning uchun, plazmid magistrallarining mumkin bo'lgan ifloslanishini aniqlash uchun IGV bilan ketma-ketlik hizalamalari vizualizatsiya qilindi. Plazmid magistral tuzilishiga hech qanday o'qishlar kiritilmagan (Qo'shimcha fayl 1: S5 va S6-rasm). Bu topilma shuni ko'rsatadiki, bu transgen genomlarga umurtqa pog'onasidan olingan ketma-ketliklar kiritilmagan.


Kanareyka genomidan mavsumiy qo'shiqchi qushlarda gormonga sezgir gen regulyatsiyasi evolyutsiyasini ochish uchun foydalanish

Fon: Hamma qo'shiqlarning qo'shiqlari bir xil neyron zanjiri tomonidan boshqarilsa -da, qo'shiqchilikning gormonlarga bog'liqligi turlar orasida katta farq qiladi. Shu sababli, qo'shiqchi qushlar gormonlarga bog'liq xatti-harakatlar va neyronlarning egiluvchanligi mexanizmlarini o'rganish uchun ideal organizmlardir.

Natijalar: Biz 1,2-Gbp ayol kanareyka genomining yuqori sifatli yig'ilishi va izohini taqdim etamiz. Qush taksilaridagi kanareykalar va 13 genom o'rtasidagi genomlarning to'liq mosligi juda yaxshi saqlanib qolganligini ko'rsatadi, bir martalik o'lchamda esa turlarning sezilarli farqlari bor. Bu farqlar kichik ketma -ketlik motiflariga ta'sir qiladi, masalan, estrogen javob elementlari va androgen javob elementlari kabi transkripsiya faktorlarini bog'lash joylari. Ushbu turga xos bo'lgan javob elementlarini kanareykalar qo'shig'ining gormonlarga sezgirligi bilan bog'lash uchun biz qo'shiq bilan bog'liq bo'lgan asosiy miya hududlari, HVC va RA ning mavsumiy testosteronga sezgir transkriptomalarini aniqlaymiz va neyronlarning differentsiatsiyasi bilan bog'liq mavsumiy gen tarmoqlarini faqat quyidagi joylarda topamiz. HVC. Testosteronga sezgir, qo'shiqchi erkaklarning HVC gen tarmoqlari neyronlarning differentsiatsiyasi bilan bog'liq. Testosteron bilan boshqariladigan kanareykali HVC genlari orasida, zebra finchidagi ortolog promotorlarda 20% estrogen va 4-8% androgen javob elementlari yo'q.

Xulosa: Kanar genomlari ketma-ketligi va qo'shimcha ekspressiv tahlillar mavsumiy qo'shiqchilik xatti-harakatlarini boshqaruvchi ko'p mintaqaviy neyron davridagi intra-mintaqaviy evolyutsion o'zgarishlarni ochib beradi va bu mavsumiy qo'shiqchilik xatti-harakatlarining gormonlarga sezgirligi bilan bog'liq genlar evolyutsiyasini aniqlaydi. Kanareykalarda testosteron va estrogenga sezgir bo'lgan va neyronlarning qayta ulanishida ishtirok etadigan bunday genlar mavsumiy qo'shiq naqshlari asosidagi HVC ning mavsumiy qayta differensiatsiyasi uchun juda muhim bo'lishi mumkin.


Serologiya: umumiy ma'lumot

Boshqa tana suyuqliklari

DNK profilini olish keng tarqalgan testlar bo'lmagan tana suyuqliklari va to'qimalarida muvaffaqiyatli amalga oshirildi. Misollarga teri (shu jumladan kepek), terlash, burunning shilliq pardasi, yiring, ona suti va quloq mumi kiradi. Ko'pincha, bu holatlarda biologik kelib chiqishi materialning ko'rinishi yoki uning tekshirilgan buyumda joylashishi, masalan, bosh kiyimdagi terlash, to'qimalarda burun mukusi va boshqalar bilan bog'liq. Ushbu materiallarning uyali identifikatorini aniqlash uchun maxsus testlarni talab qilish juda kam, ammo ularning har biri o'ziga xos biokimyoga ega, agar kerak bo'lsa, identifikatsiya testini ishlab chiqish uchun ishlatilishi mumkin.


Natijalar va muhokama

Biz yaxshi o'rganilgan 5 ′ -UTRni tanladik S. cerevisiae CYC1 promouter [15, 16]. Biz pCYC1minni (-143 pozitsiyasidan boshlab) xamirturush bilan boyitilgan yashil lyuminestsent oqsilga (yEGFP) [17] va CYC1 terminator. To'liq bilan solishtirganda CYC1 rag'batlantiruvchi, pCYC1min uchta TATA qutisidan ikkitasini o'z ichiga oladi va oqimni faollashtiruvchi ketma -ketlik yo'q. pCYC1min - mo''tadil zaif targ'ibotchi va shu sababdan, oqim mutaxassisi oqimi ifodalovchi etakchisidagi nuqta mutatsiyalarining ijobiy va salbiy ta'sirini aniqlash uchun ideal nomzod bo'lib ko'rinadi. The CYC1 5 ′ -UTR promotori 71 nukleotid uzunligidan iborat.

Quyidagi tahlilda biz qismiga murojaat qilamiz CYC1 5 ′ -UTR −1 dan −8 gacha pozitsiyada kengaytirilgan Kozak ketma-ketligi va bu -9 dan -15 gacha bo'lgan yuqori oqim mintaqasi. Kengaytirilgan Kozak ketma-ketligida adenin beshta holatda kuchli saqlanadi, yuqori oqimda esa hech qanday nukleotid kuchli saqlanib qolmaydi. Biroq, adenin deyarli har bir joyda eng ko'p uchraydi (qarang. Fon).

Kengaytirilgan Kozak ketma-ketligi

Asl CYC1 -15 dan -1 gacha bo'lgan pozitsiyalar ketma -ketligi CACACTAAATTAATA (bundan keyin - deb nomlanadi) k 0). Dvir va boshqalarga ko'ra. [9], adenin -1, -3 va -4 -pozitsiyalarda bo'lishi va -2 -pozitsiyada guaninning yo'qligi, bu etakchi ketma -ketlikni yuqori ifodalash uchun deyarli maqbul holga keltirishi kerak. Biroq, -2 pozitsiyasida timin va -13 pozitsiyasida sitozin 20 dan past chastotaga ega. % va 10 %, o'z navbatida, yuqori ifodalanganlar orasida S. cerevisiae genlar [8]. Biz birinchi sintetikani yaratdik CYC1 yetakchilar ketma-ketligi (k 1) -1 dan -15 gacha bo'lgan har bir pozitsiyaga adenin qo'yish orqali.

Bilan bog'liq bo'lgan floresan darajasi k 1 6,5 edi % bilan o'lchanganidan yuqori k 0. Biroq, bu ikki etakchi ketma -ketlikda to'plangan ma'lumotlardan statistik jihatdan muhim farq yo'q edi (p-qiymati = 0.13). Biz ushlab turdik k 1 (optimallashtirilgan etakchi ketma -ketligi) bizning keyingi sintetik konstruktsiyalarimiz uchun shablon sifatida va bitta yoki bir nechta nukleotidlarni mutatsiyalash orqali yana 57 sintetik 5 '-UTRni yaratdi. k 1.

Sintetik etakchi ketma -ketliklarning birinchi guruhi -1 pozitsiyadan 8 pozitsiyagacha bo'lgan bitta nuqta mutatsiyasi orqali amalga oshirildi (1 -jadvalga qarang). Shunday qilib, biz faqat kengaytirilgan Kozak ketma-ketligini o'zgartirdik, yuqori oqim esa -9 dan -15 gacha bo'lgan pozitsiyalarda adeninlar bilan yuqori gen ifodasi uchun optimallashtirilgan konfiguratsiyada saqlangan.

Uchun eng yuqori floresans yozilgan k 16 (bu erda guanin adeninni -5 pozitsiyasida almashtirgan) va eng pasti bilan k 9 (-3 -pozitsiyada adenin o'rnini timin egallagan). Bundan tashqari, floresans darajasi k 16 dan statistik jihatdan sezilarli darajada farq qilgan k 0 va k 1. Guanin -5 pozitsiyasida floresansning kuchayishi hayratlanarli natija bo'ldi, chunki guanin achitqida eng kam uchraydigan nukleotiddir. S. cerevisiae etakchi ketma-ketliklar. Bundan tashqari, bu pozitsiyada hech qanday guanin aniqlanmagan genlar [8] orasida aniqlanmagan yoki Dvir va boshq. [9].

Dan statistik jihatdan muhim farq yo'qligiga qaramay k 1, dan boshqa yagona konstruktsiyalar k 16 natijada & gt5 ga oshdi % ning floresans darajasida k 1 edi k 3, k 10, va k 24. Xususan, ichida k 3, timin -1 -holatida adenin o'rnini bosdi k 10 -3 holatidagi adenin guaninga mutatsiyaga uchradi. Yuqorida aytib o'tilganidek, adenin -1 va -3 pozitsiyalarda yuqori gen ifodasini kafolatlashi kerak. Shunga qaramay, bunday adenin fonida, gen ekspresiyasini yanada kuchaytirish uchun -1 yoki -3 pozitsiyalarda kam uchraydigan nukleotidlar kerak bo'ladi. Bundan farqli o'laroq, adenin o'rniga -3 -pozitsiyada timin (k 9) a & gt5 ni keltirib chiqargan yagona mutatsiya edi % ning qisqarishi k 1 floresan darajasi. Bu natija [9] dagi kuzatuvga mos keladi -3 pozitsiyasida timin kam ifodalangan genlarda ko'p (1-rasm a).

Kengaytirilgan Kozak ketma-ketligidagi nuqta mutatsiyalarining floresans ifodasiga ta'siri. Floresan sathi nisbatan chiziladi k 1 (a) va k 0 (b). Nazorat yEGFP genisiz xamirturush turiga to'g'ri keladi. Adenin o'rnini bosadigan nukleotid k 1 va mutatsiya sodir bo'lgan pozitsiya har bir sintetik lider ketma -ketligi nomi ostida berilgan. Yulduzcha, p-qimmat & lt0,05 vs. k 1 (a) yoki k 0 (b)

Ga kelganda; .. ga kelsak; .. ning haqida k 0, barcha 25 ta yangi sintetik yetakchi ketma-ketligi olti va sakkiz mutatsiyani o'z ichiga olgan. Dan tashqari k 9, barcha sintetik 5 ′ -UTRlar flüoresanlik darajasini undan yuqori ko'rsatdi k 0, ulardan beshtasi sezilarli darajada yuqori edi. Bularga -1, -4 va -5 pozitsiyalar kiradi. Taqqoslashda allaqachon ta'kidlanganidek k 1, START kodonidan yuqorida joylashgan adenin gen ekspressiyasi uchun alohida afzalliklarga ega emasdek tuyuldi. Bu erda sitozin va timin (k 2 va k 3, mos ravishda) adeninga qaraganda ancha yaxshi ishladi. Biroq, nisbatan k 0, yuqori oqimda yana etti nuqta mutatsiyasi bor edi. −4 holatida timin (k 12) eng yuqori lyuminestsent o'sishiga olib keldi, -5 -pozitsiyada esa har ikkala sitozin (k 14) va guanin (k 16) & gt10 ga kuchaytirilgan floresans % ning ustida k 0. beri k 0 -2, -5 va -6 pozitsiyalarida timin mavjud bo'lib, ularning har biri sintetik 5 '-UTR ning har biridan statistik jihatdan sezilarli farqlarni ko'rsatdi. k 0 ikki yoki undan ortiq qo'shni joylardagi nuqta mutatsiyasidan ta'sirlangan. Yana uchta sintetik lider ketma -ketligi (k 10,k 17, va k 24) >10 ni keltirib chiqardi % nisbatan floresansning ortishi k 0Garchi bu farqlar ahamiyatli bo'lmasa ham (p-qimmat va gt0.05). k 10 va k 17 qo'shni joylarda ham ikki nuqtali mutatsiyalar mavjud edi (1-b-rasm).

Guaninga bir nechta mutatsiyalar

Bizning dastlabki 25 ta sintetik 5 ′-UTR ketma-ketligimizning tahlili hayratlanarli natijani berdi: guaninga bitta nuqta mutatsiyasi - bu yuqori darajada ifodalangan kengaytirilgan Kozak ketma-ketligida umuman yo'q. S. cerevisiae genlar - lyuminestsentlik darajasini oshirishi mumkin k 1, genlar ifodasi uchun optimallashtirilgan etakchi ketma -ketlik. Bundan tashqari, bizning sintetik 5 ′ -UTR -larimizdan beshtasi aniq (& gt9 %) pCYC1min bilan bog'liq bo'lgan floresans darajasini oshirdi.

Bizning ma'lumotlarimizga ko'ra, guaninga bitta mutatsiya gen ekspressiyasini kuchaytirishi mumkin. Biroq, oldingi ikkita maqola [18, 19] START kodoni oldiga joylashtirilgan bir nechta guaninlar oqsil sintezini sezilarli darajada kamaytirishi haqida xabar berdi. Shuning uchun, biz guaninga bo'lgan bir nechta nuqta mutatsiyalari pCYC1min tarjima samaradorligiga qanday ta'sir qilganini, ular gen ekspresiyasini modulyatsiya qilish uchun ishlatilishi mumkinligini aniqlash uchun baholadik.

[8] ga ko'ra, yuqori darajada ifodalangan S. cerevisiae genlar, guanin -1 va -15 pozitsiyalar orasida eng kam uchraydigan nukleotiddir, -7 pozitsiyasidan tashqari, eng kam uchraydigan nukleotid sitozin. Biz bu ketma -ketlikni aks ettiruvchi sintetik 5 ′ -UTR qurdik (k 26 2-jadval). Tegishli floresans darajasi sezilarli darajada farq qilmasligi bilan ko'rsatilgandek, bu gen ifodasini o'chirib qo'yadi (psalbiy qiymatimizdan (qiymat = 0,21) S. cerevisiae yEGFP genini o'z ichiga olmagan shtamm).

Guaninga bir nechta mutatsiyalar (-7-pozitsiyadagi sitozin) butun kengaytirilgan Kozak ketma-ketligini qamrab olganida, gen ekspressiyasiga boshqacha ta'sir qiladimi yoki yo'qligini sinab ko'rdik.k 27) yoki yuqori oqim mintaqasi (k 28). Nisbatan mutatsiyalar qilingan k 1, barcha mutatsiyaga uchramagan saytlarda adenin mavjud edi. Ajablanarlisi shundaki, biz ikkita konfiguratsiya gen ifodasi uchun ekvivalent ekanligini aniqladik (p-value & gt0.40) va kamaytirilgan k 1 floresans darajasi taxminan yarmi.

Dan boshlab k 27, biz guaninni -1 pozitsiyalariga almashtirdik (k 29), −2 (k 30) va -3 (k 31STAREN kodonining yuqorisida joylashgan uchta pozitsiyadagi bitta adenin, kengaytirilgan Kozak ketma -ketligining boshqa joylari guanin yoki sitozin bilan ishg'ol qilinganida, floresans ifodasini kuchaytiradimi yoki yo'qligini aniqlash uchun adenin yordamida. -1 -pozitsiyada adenin floresansining yaxshilanishini ko'rsatmadi k 27. Qizig'i shundaki, -2 va -3 pozitsiyalarida adenin gen ifodasini taxminan 7 ga pasayishiga olib keldi. % ning k 1 floresans darajasi. Bu natijalar adenin ekanligini ko'rsatadi o'z -o'zidan -3 yoki -1 -pozitsiyani egallagan taqdirda ham gen ifodasini yaxshilay olmaydi. Umuman olganda, etakchi ketma-ketlikda bitta nuqta mutatsiyasining gen ifodasiga ta'siri kuchli kontekstga bog'liq degan xulosaga kelishimiz mumkin.

Nihoyat, yuqori oqim gen ekspressiyasi uchun qanchalik muhimligini yaxshiroq tushunish uchun biz asta -sekin guaninlar sonini 7 tadan kamaytirdik.k 28) biriga (k 38). -9 pozitsiyasidan boshlab, biz har bir qadamda guaninni adenin bilan almashtirdik va ko'rdikki, floresans darajasi adeninlar soni bilan deyarli chiziqli ravishda oshgan (2 -rasm va Qo'shimcha fayl 1). Floresan sathi statistik jihatdan undan farqli bo'lgan oxirgi ketma -ketlik k 1 edi k 36, unda guaninlar -13 dan -15 gacha bo'lgan pozitsiyalarda mavjud edi. Guaninning o'zi -15 pozitsiyasida yoki boshqasi bilan birga -14 pozitsiyasida floresans darajasida sezilarli farqga olib kelmadi. k 1. Shuning uchun, yuqori gen ifodasi uchun optimallashtirilgan kengaytirilgan Kozak ketma -ketligi mavjud bo'lganda ham, yuqori oqimdagi ko'p mutatsiyalar oqsil sinteziga aniq ta'sir ko'rsatadi va ularni oqsillar ko'pligini sozlash vositasi sifatida ishlatish mumkin. Bu natija uchun tushuntirish quyida "Hisoblash tahlili" bo'limida keltirilgan. Qizig'i shundaki, adeninlar bilan aralashgan to'rtta guanin (k 33) yuqori oqim mintaqasida kamayadi k 1 ketma-ket to'rtta guanindan kichikroq darajada floresans (k 32), "5" -UTR ichidagi nuqta mutatsiyalarining gen ekspressiyasiga ta'siri nukleotid kontekstga juda bog'liqligini yana bir tasdiqlash bilan ta'minlash mumkin (2 -rasm bilan solishtirish uchun 1 -faylga qarang. k 0 floresans).

Guaninga ko'p nuqtali mutatsiyalar. Dan sintetik 5 ′ -UTR ning lyuminestsent darajasi o'rtasidagi nisbat k 26 ga k 38 va bu k 1 xabar qilinadi. Yuqori oqimdagi adeninlar yoki guaninlar soni yetakchi ketma-ketlik nomi ostida berilgan (dan k 27 ga k 38). −1, −2 va −3 pastki belgisi kengaytirilgan Kozak ketma-ketligida adeninning faqat tegishli pozitsiyada mavjudligini bildiradi. Pastki indeks i ifodalaydi aralashgan (asosiy matnga qarang). Yulduzcha, p-qimmat & lt0,05 vs. k 1

Yuqori oqim mintaqasi

Oldingi tahlil shuni ko'rsatdiki, 5 ′ -UTRda bitta va bir nechta mutatsiyalar tufayli gen ekspresiyasiga ta'siri kuchli kontekstga bog'liq. Bundan tashqari, bizning ma'lumotlarimiz aniq ko'rsatdiki, nafaqat Kozak ketma -ketligidagi, balki yuqori oqim ichidagi o'zgarishlar ham gen ekspresiyasiga sezilarli ta'sir ko'rsatadi. Shuning uchun biz nuqta mutatsiyalarini amalga oshirdik k 1 -9 va -15 pozitsiyalari o'rtasida (3-jadval) adenindan farq qiladigan bitta nukleotid yuqori oqim mintaqasiga joylashtirilganida tarjima tezligini o'zgartirishi mumkinmi yoki yo'qligini baholash uchun.

Barcha nuqta mutatsiyalari (kiritilganidan tashqari) k 38) bilan bog'liq bo'lganidan yuqori floresans darajasiga olib keldi k 1. Ta'kidlash joizki, sakkizta holatda floresansning o'sishi statistik jihatdan ahamiyatli edi (>10). % dan yuqori k 1 floresans). Bu sakkizta mutatsiyalar -11 dan -14gacha bo'lgan to'rtta bir -biriga yaqin pozitsiyalarni o'z ichiga olgan. Dvir va boshqalarning ma'lumotnomasida ularning hech biri hisobga olinmagan. [9].

-11 -pozitsiyada adenin o'rniga guanin (k 47) >15 tomonidan kuchaytirilgan floresans ifodasi %, holbuki sitozin va timin sezilarli ta'sir ko'rsatmadi. −12 holatidagi har bir mutatsiya floresansini oshirdi k 1. Eng katta o'zgarish (& gt15 %) guanin tufayli edi (k 50). -13 pozitsiyasidagi mutatsiyalar ham kuchli darajada kuchaygan k 1 floresan darajasi. Ikki nuqtali mutatsiyalar - sitozin (k 51) va guanin (k 53) - dan floresansning statistik jihatdan muhim farqlari natijasida k 1, holbuki timin (k 52) kengaytirilgan k 1 taxminan 14 ga floresans % lekin bu statistik ahamiyatga ega emas edi. Shuni ta'kidlash kerakki, bizda 58 sintetik 5 - -UTR mavjud. k 51 eng yuqori floresans darajasiga ega edi - deyarli 17 % dan yuqori k 1.

Nihoyat, -14 pozitsiyasida ikkita turli nuqta mutatsiyalari floresansning oshishiga olib keldi: sitozin (k 54) va timin (k 55) (3 -rasm bilan solishtirish uchun 1 -qo'shimcha faylga qarang k 0).

Yuqori oqimdagi nuqta mutatsiyalarining floresansga ta'siri k 1. The nucleotide that replaced an adenine in k 1 and the position at which the mutation took place are given below the name of each synthetic leader sequence. Asterisks, p-value <0.05 vs. k 1

Together, the results of this last analysis of the upstream region underline another surprising result: single point mutations upstream of the Kozak sequence, in particular at positions −12 and −13, were those that most enhanced gene expression from a context rich in adenines.

Computational analysis

We carried out simulations with RNAfold to investigate possible correlations between computed mRNA secondary structures, together with their corresponding minimum free energies (MFEs), and measured fluorescence levels. Our analysis provides an explanation for the drop in fluorescence due to multiple mutations from adenine to guanine (and cytosine) in the −15…−1 region. In contrast, no plausible justification for the effects of single point mutations on translational efficiency emerged from simulations with RNAfold.

As an input for RNAfold, we used mRNA sequences starting at the transcription start site of pCYC1min [16] and ending at the poly-A site of the CYC1 terminator [20]. Each sequence was 937 nucleotides long. From preliminary simulations, we observed that a poly-A chain with a variable length of 150–200 nucleotides had no significant effect on mRNA folding. All mRNA secondary structures were calculated at 30 °C (the temperature at which we grew S. cerevisiae cells for the FACS experiments).

k 0 va k 1 have the same MFE: −241.21 kcal/mol. This is the highest—and the most common—within the collection of 59 sequences analyzed in this work (see Additional file 1). The mRNA secondary structure corresponding to this MFE is characterized by the presence of a giant hairpin between positions −40 and +10. The hairpin loop goes from position −31 to position +1 and contains the whole 5 ′ -UTR portion we have targeted here. The hairpin stem is made of nine base-pairs, of which only one gave a “mismatch” because of an adenine at position −38 and +8 (see Fig. 4 a).

mRNA secondary structures. a A giant hairpin is present in the mRNA secondary structure corresponding to the MFE of both k 0 va k 1. The hairpin loop contains the −15…−1 region. The portion of the 5 ′ -UTR in our analysis is free from any pairing interactions in its wild-type configuration (k 0) and in that theoretically optimized for high protein expression (k 1). The loop of the giant hairpin is reduced in k 4 owing to the base-pairing interaction between the guanine at position −1 and the cytosine at position −31. In every mRNA structure presented, a green arrow indicates position +1, and a red arrow indicates position −15. b The disruption of the giant hairpin induces a decrease in the MFE of the mRNA secondary structure. k 26 va k 31 are associated with the lowest MFEs computed in our analysis. The two sequences contain multiple guanines in the extended Kozak sequence involved in pairing interactions with the CDS. A similar pattern is also present in k 30. Here, however, a second mini-loop around the START codon provokes an increase in MFE. The MFE of k 26 is substantially lower than those of k 30 va k 31 because of the presence of another stem due to pairing interactions between the upstream region and the CYC1 terminator. Nevertheless, the fluorescence levels of k 30 va k 31 are only approximately 1.2-fold higher than that of k 26

Multiple mutations to guanines either in the upstream region or the extended Kozak sequence originate base-pairing interactions between, at least, a portion of the −15…−1 region and the CDS (yEGFP) or the CYC1 terminator. As a consequence, the giant hairpin is destroyed and replaced by one or two stems that lower the MFE of the mRNA secondary structure (Table 2). Most of the MFE values smaller than −241.21 kcal/mol were associated with fluorescence levels lower than that of k 1 (Fig. 5). This result is in agreement with the notion, supported also by [8, 9], that stable mRNA secondary structures in the 5 ′ -UTR reduce protein expression. However, the fluorescence levels we measured did not increase proportionally to increments in the MFE. Moreover, in two cases (k 32 va k 36) RNAfold predicted a giant hairpin in the mRNA structure, whereas the fluorescence levels from our experiments were significantly lower than that of k 1 (Fig. 5 and Additional file 1).

Low MFE values are associated with reduced fluorescence expression. Red bars, difference between MFEs of the corresponding 5 ′ -UTR and k 1 (DMFE). Blue bars, 10-fold magnified ratio between the fluorescence level of the indicated 5 ′ -UTR and that of k 1. Dan tashqari k 1, sequences are sorted by increasing DMFE. All sequences except k 4 contain multiple point mutations with respect to k 1. Asterisks above blue bars, p-value <0.05 vs. k 1

k 26 was designed by choosing the least frequent nucleotides between positions −15 and −1 among a set of highly expressed S. cerevisiae genlar. The corresponding MFE (−261.39 kcal/mol) was the lowest within the ensemble of transcription units considered in this work. No giant hairpin was present in the MFE mRNA secondary structure as the −15…−1 region was sequestered into two different stems. The guanines between positions −1 and −6 were part of a long stem and paired with a hexamer at the beginning of the yEGFP sequence (positions +33 to +38). In contrast, positions −9 to −15 paired with a region of the CYC1 terminator, at positions +750 to +758 (Fig. 4 b).

A fluorescence level just above that of k 26 was registered for k 30 va k 31. Both differed from k 26 for the upstream region (made of seven adenines) and the presence of an adenine in the extended Kozak region (at positions −2 and −3, respectively). Xuddi shunday k 26, the first five nucleotides of the extended Kozak region of k 30 and the first six of k 31 were sequestered into a stem with the CDS. However, differently from k 26, the upstream regions of k 30 va k 31 were entirely free from any pairing interactions (see Fig. 4 b). Their MFEs (−244.28 and −247.26 kcal/mol, respectively) were also significantly higher than that of k 26. These three sequences suggest that a condition for markedly lowering protein expression is to enclose the nucleotides at positions −1 to −5 in an mRNA secondary structure. Moreover, not all of these nucleotides have to participate in base-pairing interactions. Indeed, a guanine at position −1 (k 30) or −2 (k 26 va k 31) is “free” and responsible for the presence of a mini-loop in the mRNA structure.

However, this hypothesis is contradicted by k 29. The MFE of this sequence (−245.97 kcal/mol) is comparable to that of k 30 va k 31, and the corresponding mRNA secondary structure is very similar to that of k 31 (Fig. 6 a). Nevertheless, the fluorescence level associated with k 29 was more than 6-fold higher than that of k 31 and amounted to 45% of that of k 1.

mRNA secondary structures. a k 27 dan farq qiladi k 29 only by a guanine instead of an adenine at position −1. However, their mRNA secondary structures are dissimilar. Yilda k 27, the extended Kozak sequence is involved in base-pairing interactions with the CYC1 terminator, whereas in k 29 the extended Kozak sequence is locked into a stem with the CDS. The MFE associated with k 27 is lower than that of k 29, but there is no difference between the fluorescence levels of the two sequences (p-value =0.20). b Multiple guanines in the upstream region give rise to mRNA structures characterized by base-pairing interactions between the 5 ′ -UTR and the CYC1 terminator. k 28 va k 34 have six guanines in a stem with the CYC1 terminator, whereas k 35 has only 5 guanines in an analogous structure. This causes an increase in MFE and consequently a higher fluorescence

k 27 shared with k 29k 31 an upstream region made only of adenines. However, unlike in these three sequences, the extended Kozak sequence of k 27 did not contain any adenine. The MFE of k 27 (−247.04 kcal/mol) was comparable to that of k 29k 31, but its corresponding mRNA secondary structure had a different configuration. Indeed, all nucleotides of the extended Kozak sequence (with the exception of the cytosine at position −7) were involved in base-pairing interaction not with the CDS but with the CYC1 terminator (positions +755 to +762 Fig. 6 a). The fluorescence level of k 27 was slightly higher than that of k 29, i.e. almost 7-fold greater than that of k 31.

The five sequences considered so far (k 26, k 27, k 29k 31) have in common an extended Kozak region rich in guanine that was sequestered into a stem in the MFE mRNA secondary structure. In four cases, the extended Kozak sequence paired (partially) with the CDS, and in one case (k 27) with the CYC1 terminator. The MFE of k 26 was the lowest, as its upstream region was also sequestered into a stem. The other four sequences showed very similar MFE values but rather different fluorescence levels.

The other group of sequences affected by multiple mutations with respect to k 1 had only adenines in the extended Kozak sequence and a variable number of guanines in the upstream region.

k 28, k 34, va k 35 had, respectively, 7, 6, and 5 guanines in a row from position −15 downstream. Although the MFE of k 35 was clearly higher than that of k 28 va k 34 (Table 2), the three sequences gave rise to similar mRNA structures where at least five guanines of the upstream region (plus the first adenine downstream) were locked into a stem due to base-pairing interactions with the CYC1 terminator (see Fig. 6 b).

Interestingly, both the MFE and fluorescence level of k 28 were comparable to those of k 27 va k 29. Hence, even if the Kozak sequence was free of pairing interactions, the sequestering of the upstream region into a stem was enough to guarantee a clear drop in protein expression. This is further confirmation of the role played by the nucleotides upstream of the Kozak sequence in tuning protein expression.

A different MFE mRNA secondary structure was obtained for k 33 (four guanines, intermixed with adenines), in which half of the extended Kozak sequence and almost the whole upstream region were involved in base-pairing interactions with the CDS, giving rise to a long stem. However, compared to k 35, where only five nucleotides of the upstream region were locked into a stem with the CYC1 terminator, k 33 showed a higher MFE as well as a higher fluorescence level (Fig. 5 and Additional file 1).

Finally, for k 32, k 36, va k 37 (with four, three, and two guanines in the upstream region, respectively) RNAfold returned the same MFE as for k 1. The corresponding mRNA secondary structures were all characterized by the presence of the the giant hairpin (see Additional file 1). Compared to our experimental data, this result was plausible only for k 37 but in apparent disagreement with the measurements for k 32 va k 36, whose fluorescence levels were significantly lower than that of k 1 (Fig. 5). In particular, the fluorescence of k 32 only corresponded to about 69% of that of k 1. Therefore, it can be argued that in vivo k 32 va k 1 share the same MFE and mRNA secondary structure, as suggested by the in silico simulations.

In contrast to the multiple point mutations, of the single point mutations on k 1, only k 4 caused a modification in the structure of the giant hairpin and a consequent decrease in the MFE. k 4 carries a guanine at position −1 that pairs with the cytosine at position −31 such that the length of the loop is reduced from 32 to 29 nucleotides and the MFE is lowered to −241.42 kcal/mol (Fig. 4 a). According to our data, this minimal change has no effect on fluorescence expression. All the other point mutations that induced a fluorescence level significantly higher than that of k 1 (namely, k 16, k 47k 51, va k 53k 55) were characterized by the same MFE and corresponding mRNA secondary structure as k 1, according to the RNAfold simulations.


The next steps: making new DNA

One of the original DNA strands is used as a template for the synthesis of new DNA. The primers anneal to the template strand, and the DNA polymerase enzyme makes a new strand of DNA by creating a complementary sequence of nucleotides drawn from the reaction mixture.

The new DNA strand is made by complementary base pairing with the original DNA template. Because all four ordinary DNA nucleotides are present in large amounts, the chain elongation continues normally – until by chance a dideoxynucleotide (terminator) is added in the place of a normal DNA nucleotide.

The dideoxynucleotides are just like ordinary DNA nucleotides except that one hydroxyl (OH) group has been chemically changed to a hydrogen (H). With normal DNA nucleotides, one nucleotide can be attached to another and so on, forming a chain. The chemical change in a dideoxynucleotide, however, means that no additional nucleotides can be added, hence the name ‘terminator nucleotides’.

The synthesis of new DNA is terminated when one of the dideoxynucleotides is added to the strand. Because there are many more ordinary nucleotides than dideoxynucleotides, some chains will be several hundred nucleotides long before a dideoxynucleotide is added. The end result is a whole lot of new DNA fragments, of varying length, all ending with a dideoxynucleotide.


How to identify the GPD gene when the sequence varies between organisms? - Biologiya

Proteomics is the study of the entire set of proteins produced by a cell type in order to understand its structure and function.

O'quv maqsadlari

Explain how the field of genomics led to the development of proteomics

Asosiy xulosalar

Asosiy nuqtalar

  • Proteomics investigates how proteins affect and are affected by cell processes or the external environment.
  • Within an individual organism, the genome is constant, but the proteome varies and is dynamic.
  • Every cell in an individual organism has the same set of genes, but the set of proteins produced in different tissues differ from one another and are dependent on gene expression.

Asosiy shartlar

  • proteomika: the branch of molecular biology that studies the set of proteins expressed by the genome of an organism
  • proteome: the complete set of proteins encoded by a particular genome
  • genomika: the study of the complete genome of an organism

Proteomics is a relatively-recent field the term was coined in 1994 while the science itself had its origins in electrophoresis techniques of the 1970’s and 1980’s. The study of proteins, however, has been a scientific focus for a much longer time. Studying proteins generates insight into how they affect cell processes. Conversely, this study also investigates how proteins themselves are affected by cell processes or the external environment. Proteins provide intricate control of cellular machinery they are, in many cases, components of that same machinery. They serve a variety of functions within the cell there are thousands of distinct proteins and peptides in almost every organism. The goal of proteomics is to analyze the varying proteomes of an organism at different times in order to highlight differences between them. Put more simply, proteomics analyzes the structure and function of biological systems. For example, the protein content of a cancerous cell is often different from that of a healthy cell. Certain proteins in the cancerous cell may not be present in the healthy cell, making these unique proteins good targets for anti-cancer drugs. The realization of this goal is difficult both purification and identification of proteins in any organism can be hindered by a multitude of biological and environmental factors.

Proteomlarning funktsiyasini o'rganish proteomika deb ataladi. Proteoma - bu hujayra turi tomonidan ishlab chiqarilgan oqsillarning butun to'plami. Genomics led to proteomics (via transcriptomics) as a logical step. Proteomes can be studied using the knowledge of genomes because genes code for mRNAs and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins. Proteomika genomikani to'ldiradi va olimlar o'zlarining genlarga asoslangan gipotezalarini sinab ko'rishni xohlaganlarida foydalidir. Even though all cells of a multicellular organism have the same set of genes, the set of proteins produced in different tissues is different and dependent on gene expression. Shunday qilib, genom doimiy, lekin proteoma o'zgaradi va organizm ichida dinamikdir. In addition, RNAs can be alternately spliced (cut and pasted to create novel combinations and novel proteins) and many proteins are modified after translation by processes such as proteolytic cleavage, phosphorylation, glycosylation, and ubiquitination. There are also protein-protein interactions, which complicate the study of proteomes. Garchi genom reja tuzsa -da, yakuniy arxitektura proteom hosil qiluvchi hodisalarning rivojlanishini o'zgartirishi mumkin bo'lgan bir qancha omillarga bog'liq.

Large-scale proteomics machinery: This machine is preparing to do a proteomic pattern analysis to identify specific cancers so that an accurate cancer prognosis can be made.


Few steps to find amino acid sequence

STEP 1 – Know which DNA strand is given. There are two strands: Coding strand or non-coding strand.

One can either read the coding strand from 3’ to 5’ or read the template strand from 5’ to 3’ when making the corresponding m-RNA strand.

STEP 2 – Write the corresponding m-RNA strand.

Using Coding strand: (A= U, T= A, G=C, C=G) Read from left to right

Using template strand: (T=U)Read from left to right

We can see that we achieve the same sequence irrespective of the strand used.

STEP 3 – Convert m-RNA as a sequence of codons. ALWAYS start from the codon AUG and NEVER count the same nucleotide twice!

STEP 4 – Use the below table to find the relevant amino acid sequence.

Also remember,
a. Start codon AUG stands for Methionine.
b. If you come across a stop codon UAA, UGA, UAG you should stop sequencing.


Videoni tomosha qiling: Ketma (Dekabr 2021).