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Nima uchun erkak Drosofilada rekombinatsiya sodir bo'lmaydi?

Nima uchun erkak Drosofilada rekombinatsiya sodir bo'lmaydi?



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"Erkaklar genetik tadqiqotlarni osonlashtiradigan meiotik rekombinatsiyani ko'rsatmaydi."

Bir muncha vaqt davomida men bu hodisaning sodir bo'lishini bilaman, bu iqtibos Vikipediya sahifasidan olingan. Drosophila melanogasterva men bu xususiyatni tez -tez ishlataman Drosofila miqdoriy genetika bo'yicha o'z tadqiqotlarim uchun gemiklonlar qurishda.

Ammo shu paytgacha men hech qachon so'ramaganman nima uchun rekombinatsiya sodir bo'lmaydimi? Nima uchun bu xususiyat rivojlanadi?

Bundan tashqari, nima uchun rekombinatsiyaning yo'qligi erkaklar uchun, ayollar nega rekombinatsiyaga ega?


Bu hurmatli haqiqat. Istisnolar D. melanogaster Y-007 shtammi kuzatilgan va D. ananassae izchil erkak krossoverlari bor, lekin bu ish 1970-yillarga to'g'ri keladi.

Ushbu Current Biology 2002 hujjati mutlaqo yangi emas, lekin ko'rib chiqilayotgan masalaga biroz oydinlik kiritadi.

Erkaklarda Drosophila melanogaster, meioz rekombinatsiya yoki taniqli sinaptonemal kompleks (SC) bo'lmaganda sodir bo'ladi.

Mualliflar GFP -lac termoyadroviyidan foydalanib, erkak chivinining mayozda biriktirilgan lak o'ziga xos bog'lanish joylari bo'lgan bir qator bo'limlarini kuzatishdi. Ular, asosan, DNKning GFP bilan bog'langan segmentlari hujayra bo'linishi bilan bog'liq emasligini ko'rishadi. Ya'ni, homolog xromosomalar meyoz paytida (SC orqali) va erkak drozofilada fizik jihatdan bog'lanadi, ular yo'q.

Yaqinda o'tkazilgan ishlardan ko'rinib turibdiki, bu ishning tafsilotlari hali ishlab chiqilmoqda, biroq bir necha gen mintaqalari va omillari aniqlangan. Turli gomologik xromosomalarni uyg'unlashtirishga yordam beradigan shubhali omillar o'rganilmoqda. Batafsil ma'lumot uchun P.I.bibliosiga qarang.

Bu mening asosiy soham emas, lekin men tushunganimdek, meiozning ko'p jihatlari hali ham xaritada.

Degan savolga kelsak bu qanday rivojlangan va nima uchun; albatta g'alati. Ko'rinib turibdiki, erkaklarda SC shakllanmaydigan jins tanlangan xususiyat bor. Erkaklarda faqat rekombinatsiyani yo'qotgan mutatsiyani tasavvur qilish oson - jarayon shu qadar murakkabki, bunday xususiyatni ko'rsatmasligini tasavvur qilish qiyin.

Shunga qaramay, bu xususiyat nafaqat saqlanib qoldi, balki dominant bo'lib qoldi deb o'ylash ajablanarli Drosofila butun dunyo bo'ylab. Ko'pchilik uchun umumiy xususiyat bo'lish Drosofila turlar, bu 10 million yil oldin sodir bo'lgan bo'lishi kerak. Jinsiy ko'payishning afzalliklarini tushuntirish ko'pincha rekombinatsiyani o'z ichiga oladi. Hatto inson Y xromosomasi kodlash hududlarida xatolikni kamaytiradigan o'z-o'zini rekombinatsiya qilish mexanizmiga ega. Shunday qilib, bu chivinlar uchun katta kamchilik emasligiga ishonish qiyin. Shunday bo'lsa-da, agar bu juda katta bo'lsa, siz Y-007 ustunlik qilganini va bu xususiyatni qayta kiritgan bo'lardi, deb o'ylar edingiz.

Men o'ylay oladigan yagona nazariyani "dangasa erkaklar" deb atash mumkin; Erkaklar urg'ochilarga nasl uchun yana bir narsani qilishga majbur qilishdi va bu qandaydir tarzda erkaklarning spermatozoidlaridan ustun turadi. Ko'p sperma harakatsiz; Bu odamlar uchun haqiqat, men birlashadigan chivinlarga qanday o'xshashligini bilmayman. Morfologiyadagi tafovutni ham ko'rib chiqing - spermatozoidlar ba'zida juda xunuk bo'ladi va juftlashish jarayonining asosiy qismi nuqsonlilarini yo'q qilishga yordam beradi. Rekombinatsiya bo'lmasa, erkaklar sperma raqobatida ko'proq yashovchi sperma ishlab chiqarishi mumkinmi, menimcha, bu pashsha juftlashuviga mos keladi - Drosophila ham monogam emas, deb o'ylayman? Yaxshiyamki, bu tadqiqot qilingan fikr emas, faqat men taxmin qilaman.


Bilishimcha, buning sababini tasdiqlovchi kuchli dalillar yo'q. Ko'rinib turibdiki, bu erkak jinsi xromosomasining rekombinatsiyasini oldini olishning qo'pol mexanizmi sifatida paydo bo'lgan.

Siz keyin so'rashingiz mumkinki, nima uchun jinsiy xromosomaga maxsus mexanizm (odamlarda bo'lgani kabi) rivojlanmagan, men evolyutsiyani tez-tez "etarlicha yaxshi" echimlar bilan davom ettirishni taklif qilaman, bu erda, ehtimol, yaxshiroq echimlarning foydasi unchalik katta emas. t qo'shimcha daromadlarni o'z ichiga oladi va shuni esda tutingki, Drozofilada rekombinatsiyaning past darajasi foydali ekanligi haqida ba'zi dalillar mavjud.


Bu hodisa axiazmiya deb nomlanadi, bunda turning bir jinsida rekombinatsiya bo'lmaydi. Haldane Xuxli qoidasi, achiazma turlarida, rekombinatsiyasiz jins har doim heterogametik jins (XY yoki ZW) bo'lishini bildiradi. Bu, asosan, rekombinatsiyaga oid yagona izchil qoida. Ko'rinib turibdiki, kuzatilgan har bir nazariya va naqshdan istisnolar bor. Haldane Xuxli qoidasi achiyazmiya X va Y o'rtasida gomologik bo'lmagan rekombinatsiyani oldini olish uchun rivojlangan degan nazariyani qo'llab-quvvatlaydi. Ammo X va Y rekombinatsiyasini boshdan kechiradigan turlar (ko'pchilik sutemizuvchilar) bor, lekin rekombinatsiya xromosomalarning kichik bir qismi bilan cheklangan. psudo-autosomal mintaqa.


"Xromosoma xaritasi"

50% rekombinatsiya tezligiga turli xil xromosomalarda joylashgan genlar, faqat mustaqil assortiment orqali erishish mumkin.
Ammo bir -biridan uzoq, lekin bir xil xromosomada bo'lgan genlar shunga o'xshash natijalarga olib kelishi mumkin (agar rekombinatsiya sodir bo'lsa).
E'tibor bering, Drosophila erkaklarda kesishish sodir bo'lmagani uchun, hatto katta xromosomaning qarama -qarshi uchlaridagi genlar ham erkak gametalarini ishlab chiqarishda to'liq bog'langan.

O'tish orqali rekombinatsiya
Rekombinant chastotasi 50% dan sezilarli darajada kam bo'lsa, genlar bog'langanligini ko'rsatadi.
50% rekombinant chastotasi odatda genlarning alohida xromosomalarda uzilganligini anglatadi.


Biologiya 15-bob

I. uning to'rt juft xromosomasi
II. juda ko'p sonli ko'rinadigan, shuningdek biokimyoviy mutant fenotiplar
III. oson va arzon texnik xizmat ko'rsatish
IV. qisqa avlod vaqti va ko'p sonli nasl

I. anafazada xato
II. anafaza II xatosi
III. birinchi postfertilizatsiya mitozining xatosi
IV. juftlashtirishda xatolik

55) O'g'illarining qaysi qismi rang ko'r va bo'yi normal bo'lar edi?
A) yo'q
B) yarim
C) har to'rtdan biri
D) to'rtdan uchtasi
E) hammasi
Old
Oddiy ko'rish qobiliyatiga ega bo'lgan akondroplastik mitti erkak, bo'yi ko'r bo'lgan, bo'yi ko'r ayolga uylanadi. Erkakning otasining bo'yi 6 fut, ayolning ikkala ota-onasi ham o'rtacha bo'yli edi. Akondroplast mitti-autosomal dominant, qizil-yashil rang ko'rligi X bilan bog'liq retsessiv.

56) Ularning oddiy rangli ko'rish qobiliyatiga ega mitti qizi bor. Uning ikkala gen uchun ham heterozigot bo'lish ehtimoli qanday?
A) 0%
B) 25%
C) 50%
D) 75%
E) 100%
Old

Pandora sayyorasidagi o'simliksimon organizm uchta retsessiv genetik xususiyatga ega bo'lishi mumkin: mavimsi barglari, A genining allelidan (tukli), tukli poyasidan, B genining allelidan (b) kelib chiqishi va allel (c) tufayli bo'sh ildizlardan Uch gen bir -biriga bog'langan va quyidagicha rekombinatsiyalanadi:

56) Ularning oddiy rangli ko'rish qobiliyatiga ega mitti qizi bor. Uning ikkala gen uchun ham heterozigot bo'lish ehtimoli qanday?
A) 0%
B) 25%
C) 50%
D) 75%
E) 100%
Old

Pandora sayyorasidagi o'simliksimon organizm uchta retsessiv genetik xususiyatga ega bo'lishi mumkin: mavimsi barglari, A genining allelidan (tukli), tukli poyasidan, B genining allelidan (b) kelib chiqishi va allel (c) tufayli bo'sh ildizlardan Uch gen bir -biriga bog'langan va quyidagicha rekombinatsiyalanadi:

Genetika mutaxassisi uchta retsessiv xususiyat uchun heterozigotli bo'lgan organizm bilan test o'tkazdi va u quyidagi fenotipik tarqalishning avlodlarini (+ = yovvoyi turi) aniqlay oldi: (Rasmga qarang)

57) Bu xochda ota -onalarning fenotiplari qaysi?
A) 2 va 5
B) 1 va 6
C) 4 va 8
D) 3 va 7
E) 1 va 2
Old

Pandora sayyorasidagi o'simliksimon organizm uchta retsessiv genetik xususiyatga ega bo'lishi mumkin: mavimsi barglari, A genining allelidan (tukli), tukli poyasidan, B genining allelidan (b) kelib chiqishi va allel (c) tufayli bo'sh ildizlardan ) gen C. Uch gen bir-biriga bog'langan va quyidagi tarzda rekombinatsiyalanadi:


Asosiy natijalar

Unionidea va Mytilidae oilalaridagi mitoxondriyalarning g'ayrioddiy ikki ota-onalik merosi taxminan o'n yil davomida ma'lum bo'lgan va hayvonlarning mtDNKsi uchun onalik merosining boshqa universal qoidasidan qiziqarli istisno hisoblanadi.. Odatda, ayol (F) va erkak (M) mitoxondrial ketma-ketliklar 20% ga farq qiladi - bu gomologik rekombinatsiyani kuzatish uchun juda katta miqdor. Yaxshiyamki, bir g'alati Mytilus tizimi mtDNK rekombinatsiyasini amalda kuzatish uchun noyob imkoniyatni beradi. Vaqti -vaqti bilan F genomlari sperma orqali M uzatish yo'lini bosib o'tib, "erkaklashgan" bo'lib qoladi (1 -rasmga qarang). Ushbu M F genomlari endi ajdodlar F shaklidan ajralishi mumkin, shuning uchun biz bitta hujayradagi mitoxondrial genomlarni topishimiz mumkin, ular gomologik rekombinatsiyani aniqlashga imkon beradigan etarli ketma-ketlik farqiga ega, ammo rekombinatsiya bostirilmaydigan darajada emas.

Mualliflar sitoxrom oksidaza III subbirligi uchun genning bir qator ketma-ketliklari haqida xabar berishadi, bu erda DNKning qisqa qismlari F va M F mtDNK o'rtasida almashinishi aniq ko'rinadi. O'n uch xil ketma -ketlikdan oltitasi populyatsiyada eng ko'p uchraydigan ikkita allel o'rtasida rekombinant bo'lib ko'rinadi, rekombinant bo'laklari uzunligi 24 asosdan 200 dan oshadi.


Natijalar

Yuqori aniqlikdagi CO xaritasi D. melanogaster

Barcha xoch natijalarini birlashtirib, biz yuqori aniqlikdagi CO xaritalarini yaratish uchun ishlatilgan jami 32,511 CO hodisalarini aniqladik. D. melanogaster (1-rasm). Markerlarning yuqori zichligi va har bir xromosoma va genotipli pashshada CO hodisalarining kamligi tufayli har bir CO har ikki tomonda ko'plab qo'shni markerlar tomonidan qo'llab-quvvatlanadi va biz barcha CO ni aniqladik deb kutmoqdamiz. Umumiy genetik xarita uzunligi D. melanogaster Bizning xochlarimizda olingan 287,3 sm, klassik o'lchovlarga chambarchas mos keladi (282 sm [26]). CO stavkalarining taqsimlanishiga past aniqlikdagi yaqinlashish (c) bizning ma'lumotlarimizga asoslanib, xromosoma qo'llari bo'ylab (S2-rasm) ko'rinadigan belgilarga asoslangan oldingi xaritalar kabi umumiy, keng miqyosli taqsimotni tiklaydi [11]–[13], [26], [49]–[53]. Kutilganidek, c telomerlar va sentromeralar yaqinida keskin kamayadi va biz kichik to'rtinchi (nuqta) xromosomada xiazmatasiz meiotik segregatsiyaga o'tadigan CO hodisalarini aniqlamaymiz [54].

O'tish tezligi (c) barcha xochlardan olingan ma'lumotlarga asoslangan va har bir ayol meioziga (qizil chiziq) megabaza (Mb) boshiga santimorganlarda (cM) ko'rsatilgan. c 100 kb oynalar uchun xromosomalar bo'ylab va 50 kb qo'shni oynalar orasidagi harakat ko'rsatilgan. Moviy chiziqlar 90% ishonch oralig'ini ko'rsatadi c har bir oynada.

Bizning batafsil xaritalarimiz so'nggi paytlarda CO stavkalarining xromosoma ichidagi o'zgarishiga bo'lgan minnatdorchilikni kuchaytiradi Drosofila [29], [55], [56] va bu heterojenlikni butun genom bo'yicha ancha nozik miqyosda tasvirlab bering. Har bir xromosoma bo'yicha CO stavkalarining heterojenligi 100 kb dan 10 Mb gacha bo'lgan tahlil qilingan barcha jismoniy shkalalarda, hatto sezilarli darajada pasaygan sentromerik va telomerik hududlarni olib tashlaganidan keyin ham muhim (P& lt0.0001 barcha holatlarda qarang: Materiallar va usullar). Barcha xromosoma qo'llari (to'rtinchi xromosomadan tashqari) an'anaviy ravishda past aniqlikdagi xaritalar asosida kamaytirilmagan rekombinatsiya stavkalari hududlari sifatida belgilangan hududlarda 15 dan 20 barobargacha o'zgaruvchanlikni ko'rsatadi. CO stavkalaridagi bu heterojenlik juda qisqa nuqtalarda aniqlanadi, qisqa masofadagi keskin o'zgaruvchanlik va bir nechta qo'shni 100 kblik derazalar 15-20 barobar farq qiladi (masalan, X xromosomadagi 15.9-16.1 Mb). issiq- va sovuq joylar CO uchun D. melanogaster. Ko'pgina sovuq nuqtalar rekombinatsiyasi kamaygan kattaroq hududlarga o'rnatilgan 100 kb-lik mintaqalardir, ammo biz sentromerikdan tashqari doimiy ravishda past CO kontsentratsiyasini (masalan, 2R xromosoma qo'li bo'ylab 15,8 Mb atrofidagi hudud) ko'rsatadigan bir nechta kattaroq hududlarni aniqlaymiz. telomerik ketma -ketliklar.

CO landshaftlarining o'ziga xos o'zgarishi

Tabiiy xochlarni o'rganish D. melanogaster shtammlar CO stavkalarini o'zgartirishi mumkin bo'lgan omillar bilan bog'liq o'zgarishlarni nazorat qilgandan so'ng, sakkizta CO xaritalarini yaratish va solishtirish imkonini berdi. Drosofila masalan, yosh, harorat, juftliklar soni yoki oziq-ovqat [57]–[60]. Statistik quvvatni oshirish uchun biz xromosomalar bo'ylab 250 kb shkaladagi xochlar orasidagi farqlarga e'tibor qaratdik. Sakkizta CO xaritalari yuqori darajadagi o'ziga xos o'zgarishlarni ochib beradi, xususan, xochlar qo'shni mintaqalarga yoki boshqa xochlarga nisbatan juda yuqori tezlikka ega (>40 baravar) mintaqalarga ega (2-rasm). Kutilganidek, bitta ota-ona shtammiga ega bo'lgan xochlar ota-ona shtammi bo'lmagan xochlarga qaraganda ko'proq o'xshash xaritalarga ega, ammo bu xochlar o'rtasidagi korrelyatsiyaning umumiy kattaligi, garchi muhim bo'lsa ham, juda kichik (Spirmanning R = +0.451). Bu kuzatish CO ning xromosomalar bo'ylab tarqalishi uchun juda poligenik va polimorfik asos tushunchasini mustahkamlaydi.

c sakkiz xil xoch (har xil rang) uchun (har bir ayol meioziga cM/Mb) va qo'shni 250 kb derazalar uchun ko'rsatilgan.

Sakkizta CO xaritasida CO stavkalari o'zgarishini aniqlash uchun biz dispersiyani o'rtacha nisbatga qarab baholadik. RCO) va ma'lum bir mintaqadagi har xil miqdordagi CO hodisalarini Puasson jarayoni bilan izohlash mumkinmi yoki yo'qligini tekshirdi. Bundan tashqari, biz o'zgaruvchanlikka e'tibor qaratdik tarqatish xromosomalar bo'ylab CO ning tezligi va shuning uchun biz har bir xromosoma uchun umumiy hodisalar sonini hisobga oldik (batafsil ma'lumot uchun Materiallar va usullarga qarang). Bizning tadqiqotimiz RCO xromosomalar bo'ylab kutilganidan kattaroq xochlar oralig'ida (haddan tashqari tarqalish) ko'p mintaqalar (genom bo'ylab bir-biriga to'g'ri kelmaydigan 250 kb bo'lgan barcha viloyatlarning 107 yoki 22%) aniqlanadi va bu xromosomalarda kuzatiladi (3-rasm). Ushbu ortiqcha dispersiyaning kattaligi xromosoma qo'li 2L uchun eng yuqori, xromosoma qo'li 3L uchun esa sezilarli darajada kamayadi. Xochlar o'rtasida CO stavkalarining sezilarli darajada haddan tashqari ko'payishi, biz katta genomik hududlarni o'rganganimizda ham aniqlanadi. 1 Mb jismoniy miqyosda genomik hududlarning yarmidan ko'pi ortiqcha dispersiyani ko'rsatadi, bu esa CO ning o'zgaruvchan stavkalari bo'lgan hududlarda etarlicha tez-tez bo'lishini ko'rsatadi. D. melanogaster genom bu uzunroq ketma-ketliklarning katta qismida aniqlangan rol o'ynaydi.

RCO sakkizta xochdan o'tish stavkalarini taqqoslash yo'li bilan olingan (tafsilotlar uchun Materiallar va usullarga qarang) va qo'shni 250 kb oynalar uchun ko'rsatilgan (ko'k chiziq). Nuqtali qizil chiziq belgini ko'rsatadi P = 0.0005 ishonch chegarasi (teng P (= 0,05)/butun genom tahlilidagi derazalar soni).

Ushbu natijalar dastlabki tadqiqotlar bilan mos keladi Drosofila sun'iy tanlash tajribalari ([61] va ularga havolalar) asosida CO stavkalarining tabiiy o'zgarishi haqida xabar bergan. Bizning genom bo'yicha tadqiqotimiz ushbu o'zgarishning genomik joylashuvi va kattaligini batafsil bayon qiladi va CO stavkalarining birinchi yuqori aniqlikdagi polimorfik landshaftini tasvirlaydi. D. melanogaster. Bir nechta genomik hududlar barcha xochlarda past ko'rsatkichlarga ega, shuning uchun CO ning monomorf (yoki yuqori chastotali) sovuq nuqtalarini ifodalaydi. D. melanogaster. Birlashtirilgan xaritalar asosida CO stavkalarining eng yuqori nuqtasi sifatida tayinlangan boshqa hududlar, ammo bizning namunamizdagi past chastotali polimorfik nuqtalar kuchli ta'sir ko'rsatadi. Darhaqiqat, xochlar orasidagi CO stavkalari haddan tashqari xilma-xil bo'lgan hududlarning ko'pchiligi past chastotali sovuq nuqtalar bilan emas, balki past chastotali ulanish nuqtalari bilan bog'liq bo'lib, bu nuqtalar vaqtinchalik (qisqa muddatli) xususiyatlar ekanligini ko'rsatadi. D. melanogaster populyatsiyalar.

Bizning natijalarimiz shuni ko'rsatadiki, "turlar" rekombinatsiya landshaftining vakillik tasvirini olish uchun bir nechta xoch va genotiplarga asoslangan CO stavkalari zarur. Bundan tashqari, issiq nuqtalarning past chastotasi barcha xaritalarning arifmetik o'rtacha qiymatiga asoslangan rekombinatsiya o'lchovlariga kuchli ta'sir ko'rsatadi, bu harmonik o'rtacha yoki median o'lchovlariga qaraganda yuqori tezlikni taklif qiladi (o'rtacha va o'rtacha CO qiymatlari o'rtasidagi taqqoslash uchun S3-rasmga qarang). Ta'kidlash joizki, biz o'rtacha CO stavkalari juda past (yoki nol) bo'lgan genomik hududlarni kuzatamiz, namunaviy o'rtacha ko'rsatkich o'rtacha ko'rsatkichlarni ko'rsatadi.

Geni konvertatsiya qilish xaritalari D. melanogaster

Biz jami 74 453 GC hodisasini aniqladik. Shunga qaramay, qo'shni markerlar orasida joylashgan GC traktlari o'tkazib yuborilishi kutilmoqda. Bundan tashqari, SNP va marker zichligi farqlari tufayli, bu kam baholanish, ehtimol, genom bo'ylab o'zgarib turadi. Shuning uchun biz GC traktlarining uzunligini baholash uchun taklif qilingan maksimal ehtimollik algoritmini [62] kengaytirdik (LGC) bir vaqtning o'zida taxmin qilish LGC va GC boshlanishining tezligi (g) va markerlarning ixtiyoriy taqsimlanishi va zichligi har qanday hududiga qo'llanilishi mumkin (batafsil ma'lumot uchun Materiallar va usullarga qarang).

Bizning genom bo'yicha γ va o'rtacha baholarimiz LGC mos ravishda 1,25 × 10 -7 / bp / ayol meiosis va 518 bp. Har bir xromosoma qo'lini alohida o'rganish (4 -rasm) shuni ko'rsatadiki, CO (2L, 2R, 3L, 3R va X) dalillari bo'lgan qurollar similar (1.13-1.49 × 10-7 /bp /ayol meioz) va LGC (456–632 bp). Shunisi e'tiborga loyiqki, biz to'rtinchi kichik aixazmatik xromosomada bir nechta GC hodisalarini kuzatamiz, bu erda CO butunlay yo'q. Γ va bizning taxminlarimiz LGC to'rtinchi xromosoma uchun mos ravishda 0,46 × 10-7 /bp /ayol meiozi va 1062 bp.

Gen konversiyasini boshlash tezligi (g) va gen konvertatsiya qilish traktining o'rtacha uzunligi (MLE) bo'yicha qo'shma maksimal ehtimollik taxminlari (MLE)LGC) ichida D. melanogaster. γ birliklar bp va urg'ochi mayozga to'g'ri keladi va LGC bp da. Qizil/sariq konturlar confidence va uchun 95 ishonch oralig'ini bildiradi LGC Har bir xromosoma qo'li uchun mustaqil ravishda. Ko'k nuqta g va uchun o'rtacha genomni ifodalaydi LGC jami 74 453 kuzatilgan GC hodisalariga asoslangan.

The qizg'ish joylashish D. melanogaster intragen rekombinatsiyasi uchun yuqori metazoa bilan tavsiflanganlardan biridir [63], [64]. Ushbu tadqiqotlar shuni ko'rsatdiki, GC hodisalari CO ga qaraganda tez-tez uchraydi, har bir CO uchun o'zaro bog'liq bo'lmagan to'rtta GC hodisalari [63]-[65]. Mutlaq tezligi bo'yicha, intragenik CO hodisalarining tiklanishi qizg'ish ochib beradi c∼3.0 × 10 −8 /bp /ayol meioz [66] shunday qilib, bu lokalda γ∼1.2 × 10-7 /bp /ayol meiozini bashorat qiladi. Biz 100 kb genomik mintaqaga e'tibor qaratganimizda pushti lokus bizning taxminimiz $ pi $ - 1,17 × 10 -7 /bp /ayol meioz. Butun genom miqyosida bizning ma'lumotlarimiz g (1,25 × 10 -7 / bp / ayol meiozi) va GC∶CO nisbati (voqealarning ~83% GC ga olib keladi) ga yaqin, lekin yuqoriroq bo'lsa-da, taxmin qiladi. qizg'ish. Bizning natijalarimiz va natijalarimiz o'rtasidagi asosiy farq qizg'ish lokus gen konversiya yo'llarining o'rtacha uzunligi, bizning o'rtacha hisobimiz bilan LGC (518 bp) 352 bp ga teng pushti [62].

GC∶CO nisbatlarini baholashning yana bir yondashuvi DSB hosil bo'lishi uchun molekulyar marker sifatida g-His2Av ga antikordan foydalanishga asoslangan [67] va DSB tuzatuvchi nuqsonli mutantlarda g-His2Av o'choqlari sonini kuzatish [68]. Hisoblangan DSB soni D. melanogaster Bu metodologiyadan foydalanish genom boshiga 24,2 [68] ni tashkil qiladi, bu shuni ko'rsatadiki, biz har bir ayol mayozida kuzatilgan CO hodisalari sonidan foydalansak, barcha DSBning 76,2 foizi GC sifatida hal qilinadi. Bizning tadqiqotimizda kuzatilgan GK ning o'rtacha darajada yuqori ulushi, agar barcha DSBlar (yoki DSB-ni ta'mirlash yo'llari) g-His2Av bo'yalishi [68] bilan belgilanmagan bo'lsa yoki DSB-tuzatish uchun nuqsonli mutantlar ruxsat etilgan bo'lsa, ishlatilgan shtammlar orasidagi farqlar bilan izohlanishi mumkin. qoldiq ta'mirlash uchun, shuning uchun ba'zi DSB'larni aniqlashni qiyinlashtiradi. To'rtinchi xromosoma va boshqa genomik mintaqalarda CO mavjud bo'lmagan, ammo GC aniqlangan DSBlarni eksperimental ravishda lokalizatsiya qilishga urinishga qaratilgan kelajakdagi tadqiqotlar alohida qiziqish uyg'otadi.

100 kb shkalada x ning xromosomalar bo'ylab taqsimlanishini tahlil qilish CO ga nisbatan bir xil taqsimotni ko'rsatadi.c) stavkalari, telomerlar yoki sentromeralar yaqinida pasayishsiz (5 -rasm). 100 kb hajmli derazalarning 80% dan ko'prog'i 2 marta diapazonda show ni ko'rsatadi, bu CO ning taqsimlanishidan farq qiladi, bu erda xromosomalar bo'ylab 100 kbli derazalarning atigi 26,3 foizi ko'rinadi. c o'rtacha xromosomaning 2 barobar oralig'ida. CO hodisalarining taqsimlanishi GC yoki GC va CO hodisalarining kombinatsiyasi (ya'ni, DSBs soni) genomida ko'proq o'zgaruvchanligini aniqlash uchun biz har uchtasi uchun xromosomalar bo'yicha o'zgarish koeffitsientini (CV) baholadik. har xil oyna o'lchamlari va xromosoma qo'llari uchun parametrlar. Barcha holatlarda (deraza o'lchami va xromosoma qo'li) CO uchun rezyume GC yoki DSB (CO+GC) ga qaraganda ancha katta (2 baravar ko'p), DSB uchun CV esa faqat bir oz kattaroqdir. GC: 100 kb oynalar uchun CO, GC va DSB uchun xromosoma qo'li uchun o'rtacha CV mos ravishda 0,90, 0,37 va 0,38 ni tashkil qiladi. Shunga qaramay, biz GC hodisalari yoki DSBlarning tarqalishi mutlaqo tasodifiy, har bir xromosoma bo'ylab sezilarli heterojenlik bilan bo'lishini istisno qilishimiz mumkin.P<0.0001 tahlil qilingan barcha jismoniy shkalalarda, 100 kb dan 10 Mb gacha, batafsil ma'lumot uchun Materiallar va usullarga qarang). GC ning CO hodisalaridan oshib ketishi ajablanarli emas, chunki GC genlar bo'yicha umumiy DSBs yoki umumiy rekombinatsiya hodisalarining CO stavkalariga qaraganda ancha yaxshi bashoratchisi bo'lib, yarim qisman korrelyatsiyalari GC uchun 0,96 va CO uchun 0,38 ni tushuntiradi. DSBlardagi umumiy dispersiya (to'rtinchi xromosomani hisobga olmaganda).

γ (/bp/ayol meiozi) barcha xochlarga asoslangan va qo'shni 100 kb derazalarda ko'rsatilgan.

G/C nukleotidlarini qo'llab-quvvatlaydigan noto'g'ri gen konversiyasini tiklashning yo'qligi D. melanogaster

DSB o'lchamlari heterodupleks ketma -ketlikni shakllantirishni o'z ichiga oladi (CO yoki GC hodisalari uchun ham, S1 -rasm). Ushbu heterodupleks ketma-ketliklar tasodifiy tuzatiladigan yoki o'ziga xos nukleotidlarni qo'llab-quvvatlaydigan A (T): C (G) nomuvofiqliklarini o'z ichiga olishi mumkin. Bir qator turlarda, G va C nukleotidlari [69] - [71] ni qo'llab -quvvatlaydigan va rekombinatsiya tezligi o'rtasidagi ijobiy munosabatni bashorat qiladigan, gen konversiyasi mos kelmasligini tuzatish bir tomonlama bo'lishi taklif qilingan.sezgi heterodupleks hosil bo'lish chastotasi) va kodlanmagan DNKning G+C tarkibi [72], [73]. Yilda Drosofila, G + C tarafkash gen konversiyasini ta'mirlashni qo'llab-quvvatlovchi to'g'ridan-to'g'ri eksperimental dalillar yo'q va evolyutsion tahlillar CO stavkalarini heterodupleks shakllanishi uchun proksi sifatida ishlatganda qarama-qarshi natijalar berdi ([73]-[75], lekin qarang [13], [76]) . Shuni yodda tutingki, GC hodisalari CO hodisalariga qaraganda tez-tez uchraydi Drosofila shuningdek, boshqa organizmlarda [32], [65], [77], [78] va shuning uchun GC (g) stavkalari CO ga qaraganda ko'proq mos kelishi kerak (c) heterodupleksni ta'mirlashning mumkin bo'lgan oqibatlarini tekshirishda stavkalar.

Bizning ma'lumotimiz γ ning G +C nukleotidlar tarkibi bilan intergenik ketma -ketlikda (R = +0.036, P & gt0.20) yoki intron (R = −0.041, P>0.16). G+C nukleotid tarkibi bilan solishtirganda, assotsiatsiyaning ekvivalentligi kuzatiladi c (P& gt0.25 intergenik ketma -ketliklar va intronlar uchun). Shunday qilib, bizda G va C nukleotidlarini qo'llab -quvvatlaydigan genlarning konversiyasi tarafdorligi haqida hech qanday dalil topilmadi D. melanogaster nukleotidlar tarkibiga asoslanadi. G va C nukleotidlariga genlarning konversiyasi tarafkashlik qilgan ba'zi oldingi natijalarning sabablari Drosofila ko'p bo'lishi mumkin va siyrak CO xaritalaridan foydalanishni, shuningdek to'liq bo'lmagan genom izohini o'z ichiga oladi. Chunki gen zichligi D. melanogaster kamaytirilmagan CO [13], [79] bo'lgan hududlarda yuqoriroqdir, ko'plab yaqinda izohlangan transkripsiyalangan hududlar va G+C ga boy ekzonlar [31], [80], [81] avval neytral ketma-ketliklar sifatida tahlil qilingan bo'lishi mumkin, ayniqsa kamaytirilmagan CO bilan bu genomik hududlarda.

Rekombinatsiya motivlari Drosofila

Rekombinatsiya hodisalari (CO yoki GC) bilan bog'liq DNK motiflarini kashf qilish uchun biz 500 BP yoki undan kam chegaralangan 1.909 CO va 3.701 GC hodisalariga e'tibor qaratdik.500 va GC500mos ravishda). Bizning D. melanogaster ma'lumotlar rekombinatsiya hodisalari atrofidagi ketma-ketlikda sezilarli darajada boyitilgan ko'plab motivlarni ochib beradi (CO va GC uchun mos ravishda 18 va 10 motiflar) (6-rasm va 7-rasm). Alohida, CO hodisalari atrofidagi motiflar (M.CO) 6,8 dan 43,2% gacha CO tarkibida mavjud500 ketma-ketliklar, GC voqealari atrofidagi motivlar (MGC) GK ning 7,8 dan 27,6% gacha mavjud500 ketma-ketliklar. E'tibor bering, barcha CO ning 97,7%500 ketma -ketlikda kamida bitta M mavjudCO motif va GCning 85,0%500 ketma-ketlikda bir yoki bir nechta M mavjudGC motiv (S4-rasm).

Biz 500 bp yoki undan kamroq bilan chegaralangan 1909 CO hodisasiga e'tibor qaratdik (CO500 ketma -ketlik). Faqat E-vale va lt1 × 10 -10 bo'lgan motiflar ko'rsatiladi va E-qiymati bo'yicha tartiblanadi. Mavjudlik 100 CO uchun motiflarning umumiy sonini ko'rsatadi500 ketma -ketliklar, shu jumladan bitta ketma -ketlikda mumkin bo'lgan bir nechta mavjudlik. Motif M.CO4-rasmda 7-nukleotidli CCTCCCT motifi birinchi bo'lib odamlarda hotspotni aniqlash bilan bog'liq [87], motif esa MCO16 10-mer ketma-ketligini o'z ichiga oladi (CCNTCGCCGC), bu odamlarning issiq nuqtalarida o'zaro faoliyat faoliyati bilan bog'liq bo'lgan 13-mer uzun CCNCCNTNNCCNC bilan bir-biriga to'g'ri keladi [86]. Ko'rsatish maqsadida A va/yoki nukleotidlarning mavjudligini maksimal darajada oshirish uchun oldinga va orqaga ketma -ketlik motiflari tanlanadi.

Biz 500 bp yoki undan kam chegaralangan GC 3,701 hodisalariga e'tibor qaratdik (GC500 ketma -ketlik). Faqat E-qiymati<1×10 −10 boʻlgan motivlar koʻrsatilgan va E-qiymati boʻyicha tartiblangan. Mavjudlik 100 GC uchun motiflarning umumiy sonini ko'rsatadi500 ketma -ketliklar, shu jumladan bitta ketma -ketlikda mumkin bo'lgan bir nechta mavjudlik. Ko'rsatish maqsadida A va/yoki nukleotidlarning mavjudligini maksimal darajada oshirish uchun oldinga va orqaga ketma -ketlik motiflari tanlanadi.

Oldingi tahlillar D. pseuddobscura rekombinatsiya tezligi va oddiy takrorlashlar, shuningdek CACAC [55], CCTCCCT va CCCCACCCC [29] motiflari o'rtasida sezilarli ijobiy korrelyatsiyani aniqladi. Ekvivalent tadqiqot D. persimilis odamlarning rekombinatsiya tezligi bilan bog'liq bo'lgan CCNCCNTNNCCNC ketma -ketligi bilan CO tezligining ijobiy aloqasini topdi [82]. Yaqinda o'tkazilgan tadqiqot shuni ko'rsatdiki, GTGGAAA motifi CO tadbirlari yaqinida bo'lishi kerak D. melanogaster [83]. Bizning tadqiqotimiz ushbu ketma -ketliklarning bir qismini (CACAC va CCTCCCT) sezilarli darajada boyitilishini tasdiqlaydi va yangilarini aniqlaydi, shu bilan birga CCCCACACCCC yoki GTGGAAA -ni rekombinatsiyaning kuchayishi bilan bog'liq motif sifatida qo'llab -quvvatlamaydi. D. melanogaster.

Sutemizuvchilarda metontransferaza gistoni PRDM9 yuqorida tasvirlangan 13-darajali CCNCCNTNNCCNC motifini [84], [85] rux-barmoq qatori orqali nishonga oladi va bu motif odamlarning 41% nuqtalarida o'zaro faoliyat faoliyati bilan bog'liq [86]. Bizning tadqiqotimizda CO yoki GC uchun aniqlangan motiflarning hech biri to'liq 13-mer PRDM9 motifini o'z ichiga olmaydi. Biz ushbu 13-merning qisqaroq versiyalarini CO ning ikki xil motivlarida kuzatamiz (MCO4 va M.CO6-rasmdagi 16). Motif M.CO4da 7 ta nukleotidli CCTCCCT motifi mavjud bo'lib, u birinchi navbatda odamlarda nuqta nuqtasini aniqlash bilan bog'liq [87], motifi MCO16 uzunroq PRDM9 motivi bilan bir-biriga mos keladigan 10-merli ketma-ketlikni (CCNTCGCCGC) o'z ichiga oladi.

Aniqlangan boshqa motiflar orasida biz bir qator qisqa takrorlarni topamiz, shu jumladan [CA]n, [CAG]n va [CCN]n, shuningdek, ikkala CO bilan boyitilgan poli(A) cho'zilgan500 va GC500 ketma -ketliklar. Biz [A] to'plamimizga ta'sir etuvchi omil sifatida LTR bo'lmagan transpozitsiyali elementlarning va ularning poli-A dumli 3 'UTR ning mumkin bo'lgan ta'sirini istisno qildik.n-boyitilgan motivlar, genomik skanerlash shuni ko'rsatadiki, rekombinatsiya motivlarini tekshirish uchun ishlatilgan 500-bp uzunlikdagi ketma-ketliklarning atigi 0,8 foizi izohli bo'lmagan LTRlar bilan bir-biriga mos keladi. E'tibor bering, juda boyitilgan CA dinukleotidi (M.CO1 va M.GC2) ko'pincha transkripsiya boshlanadigan saytlar (TSS) bilan bog'liq Drosofila odamlarda bo'lgani kabi [88] va madaniyatda inson hujayralarida homolog rekombinatsiyani rag'batlantirish taklif qilingan [89]. Ushbu tadqiqotda aniqlangan boshqa motivlar ham ilgari rekombinatsiya bilan bog'langan, xamirturushdagi CO yoki GC bilan bog'liq qisqa poli(A) cho'zilgan [32].

Garchi barcha motiflar bizning CO va GC hodisalari o'rtasida taqsimlanmagan bo'lsa -da, ushbu tadqiqotdan olingan umumiy rasm motiflar ketma -ketligidagi jiddiy heterojenlik va rekombinatsiya hodisalarining har ikkala turidagi motiflar o'rtasidagi o'zaro bog'liqlikni tasvirlaydi. CO va GC hodisalari atrofidagi ketma -ketlikda sezilarli darajada boyitilgan motiflarning sonini (yoki populyatsiyasini) aniqlash sutemizuvchilar va hayvonlar o'rtasidagi tub farqni ko'rsatadi. Drosofila DSB ulanish nuqtalari. Yilda DrosofilaBizning natijalarimiz shuni ko'rsatadiki, ko'p sonli turli xil ketma -ketlik motiflarini o'z ichiga olgan (yoki yaratgan) yuqori xromatinli genomik mintaqalarda uchraydigan DSBlar.

CO va GC landshaftlari genom bo'ylab

Biz CO juda kam uchraydigan yoki to'rtinchi xromosomada bo'lgani kabi, butunlay yo'q bo'lgan genomik hududlarda GC hodisalarini aniqladik. Bu natija ushbu mintaqalarda rekombinatsiya hodisalarini aniqlagan va nolga teng bo'lmagan population populyatsiyani genetik tahlilini o'tkazishga yordam beradi. D. melanogaster genom [46], [90] - [95]. Bizning tadqiqotimiz shuni ko'rsatadiki, CO aniq bo'lmagan genomik hududlarda ham, GC bilan bog'liq rekombinatsiya, CO kamaymagan mintaqalarda (masalan, gen uzunligi va kodondan foydalanish tendentsiyasi yoki oqsillar evolyutsiyasi tezligi bo'yicha genlarning ifoda darajasi [12], [13], [17], [21], [96] - [99]). Bizning hisob-kitoblarimizga asoslanib, biz shuni taxmin qilishimiz mumkinki, to'rtinchi xromosoma CO aniqlanmagan boshqa eukromatik mintaqalarga qaraganda kuchli bog'lanish bilan bog'liq naqshlarni namoyish qilishi kerak.

Ta'kidlash joizki, GC va CO stavkalari mustaqil emas. 100 kb shkalada biz γ va o'rtasidagi salbiy korrelyatsiyani kuzatamiz c Bu butun xromosomalarni tahlil qilganda aniq bo'ladi (Spearman R = −0.1246, P = 1,6 × 10 -5,) va telomerik/sentromerik hududlarni olib tashlaganidan keyin (R = −0.1191, P = 1,2 × 10 -4) (8 -rasm). Ushbu jismoniy shkalada g/c nisbati >100 qiymatlariga yetganda c≤0,1 cM/Mb, g/ populyatsiya genetik baholariga mos keladic X xromosomasining telomerik hududlarida D. melanogaster [94].

c va γ 100 kb qo'shni derazalarga asoslangan bo'lib, ular teng toifadagi 6 toifaga bo'lingan c [CO1, CO2,. Ortib borayotgan tezlikni ko'rsatuvchi CO6 c] O'rtacha c Olti toifadagi (cM/Mb/ayol meioz) qiymatlari: 0.078 (CO1), 0.727 (CO2), 1.439 (CO3), 2.294 (CO4), 3.299 (CO5) va 5.964 (CO6). Moviy ustunlar butun xromosomalar tahlil qilinganida natijalarni ko'rsatadi. To'q rangli ustunlar CO stavkalari sezilarli darajada kamaygan sentromerik va telomerik hududlarni olib tashlaganidan keyin natijalarni ko'rsatadi. O'rtasida jiddiy salbiy munosabatlar mavjud c va g mustaqil (bir-biriga to'g'ri kelmaydigan) 100 kb oynalar yordamida: Spearman's R = −0.1246 (P = 1,6 × 10 -5) butun xromosomalar uchun va R = −0.1191 (P = 1,2 × 10 -4 ) telomerik / sentromerik hududlarni olib tashlaganingizdan so'ng.

CO va GC uchun turli xil landshaftlarni tushuntirish uchun bir nechta farazlarni ilgari surish mumkin D. melanogaster genom Turli xil DSB tuzatish yo'llari taklif qilingan Drosofila xamirturushda bo'lgani kabi [100], [101], faqat GC hodisalari bilan bog'liq bo'lgan sintezga bog'liq bo'lgan paychalarining tavlanishi (SDSA) yo'li bilan, Xolliday qo'shilishining (DHJ) erishi CO yoki GC hosil qilishi mumkin (S1-rasm) ). To'rtinchi xromosomada GC hodisalarining aniqlanishi, hech bo'lmaganda CO to'liq bo'lmagan xromosoma uchun SDSA ta'sirini ko'rsatadi va SDSA butun genom bo'ylab harakat qilishi mumkinligini ko'rsatadi. Qachon g ning ortishi kuzatilishi c to'rtinchi xromosoma va telomerik/sentromerik hududlarni olib tashlaganidan keyin ham past bo'ladi (8 -rasmga qarang), boshqa yo'l bilan tuzatilgan DSBli juda katta xromosoma domenlari variantiga qarshi chiqadi. Γ va o'rtasidagi xuddi shunday salbiy munosabatlar cCO va GC hodisalari bilan bog'liq bo'lgan motiflarning bir -biriga o'xshashligi (yuqoriga qarang) umumiy kelib chiqishni taklif qiladi va shu bilan GC va CO stavkalari bo'yicha landshaftlarning nomutanosibligini ko'rsatadi. D. melanogaster genomga DSB ta'mirlash yo'llarining nisbiy qo'llanilishidagi farq (masalan, DHJ SDSAga nisbatan) yoki DHJ oraliq moddalari CO yoki GC mahsulotlarini hosil qilish uchun hal qilinganda tuzatish qaroridagi o'zgaruvchan tarafkashlik ta'sir qilishi mumkin.

Xamirturushda, mos kelmasliklarning mavjudligi, DSBni mitotik va meiotik tuzatish paytida heterodupleks qidiruv vositalarining shakllanishi va/yoki kengayishiga to'sqinlik qiladi [102] va ketma -ketlikdagi divergensiya mitoz CO ni GClarga qaraganda ko'proq darajada inhibe qiladi [103]. Yaqinda xamirturushdagi meiotik rekombinatsiya oraliq moddalarining genom bo'yicha tahlili shuni ko'rsatadiki, mos kelmaslikni tuzatish CO∶GC nisbatini oshiradi [104]. Kelishuvga ko'ra, tahlillar pushti joy D. melanogaster show a small increase in the ratio CO∶GC in the presence of sequence polymorphisms (27 CO and 5 GC) when compared to a case where polymorphisms are virtually absent (23 CO and 8 GC events) [66]. If this tendency is confirmed across the Drosofila genome and if the differences in mismatch presence across the genome are of sufficient magnitude to alter either DHJ/SDSA relative use or the resolution of DHJ into CO or GC, then genomic regions with reduced heterozygosity could favor a DSB repair favoring GC over CO events [105].

At a whole-genome level, nucleotide differentiation between parental strains (ranging between 0.005 and 0.007 for total pairwise differences per bp) shows no association with overall γ/c, c or γ (P>0.4 in all cases). To test the possible influence of mismatch presence across the genome we investigated the correlation between levels of total nucleotide polymorphism (π) and the γ/c ratio based on adjacent 100-kb regions (Figure 9 see Materials and Methods). Congruent with the hypothesis that the choice to repair DBS into either GC or CO is heterozygosity-dependent, we observe a strong negative correlation between total π and γ/c across the whole genome (R = −0.56, P<1×10 −12 ) and after removing telomeric/centromeric regions (R = −0.499, P<1×10 −12 ). We also observe a negative relationship between total π and γ [R = −0.197 (P = 8×10 −12 ) and R = −0.175 (P = 1.2×10 −8 ) across the whole genome and after removing telomeric/centromeric regions, respectively]. The negative relationship between π and γ remains significant after controlling for the influence of CO rates [semi-partial correlation r = −0.163 (P = 1.2×10 −10 ) across the whole genome and r = −0.109 (P = 9×10 −5 ) after removing telomeric/centromeric regions].

π indicates total pairwise nucleotide variation (/bp) based on 100-kb adjacent windows. π values for X-linked are adjusted to be comparable to autosomal regions. γ/c shown in log-2 scale. There is a significant negative correlation between π and γ/c (Spearman's R = −0.56, P<1×10 −12 ) also detectable after removing telomeric/centromeric regions (R = −0.499, P<1×10 −12 ).

It is also interesting to note that the observed patterns of CO and GC distribution along chromosomes can inform us about models proposed to explain chiasma interference. The “counting model” assumes that double-strand breaks occur independently, and that a fixed and organism-specific number (m) of noncrossovers (GC events) occur between neighboring crossovers [106], [107]. A later extension of the model included the possibility of a fraction of meiotic crossovers associated with a second pathway that is not subject to interference [108]. The extreme variation in the ratio of CO and GC events observed along chromosomes together with the negative relationship between CO and GC rates therefore seem to be inconsistent with the “counting model” while supporting a more dynamic one involving a variable DSB repair pathway or DHJ resolution across genomes.

Local distribution of CO and GC events

At a 100-kb scale, we have shown that CO, and to a much lesser degree GC, are not randomly distributed across chromosomes. To study the distribution of CO and GC events at a more local scale (the level of single genes) we again focused on the 5,610 CO and GC events delimited by 500 bp or less (CO500 and GC500 see above). We found that the distribution of CO and GC events is not random in terms of intergenic/genic sequences, with a significant tendency to be located within genic sequences (P<0.00001, Figure 10A see Materials and Methods for details). This excess is mostly due to GC500, with a highly significant preference for genic regions (P<0.00001) while CO500 show no preference or avoidance (P>0.40). The differential distribution of GC and CO when looking at genic and intergenic sequences is consistent with the heterozygosity-dependent GC∶CO repair of DSB proposed above, given that intergenic sequences have higher levels of heterozygosity than genic sequences. Overall, our data suggest a higher probability of DSBs within annotated transcriptional units.

Analyses based on 1,909 and 3,701 CO and GC events delimited by 500 bp or less (CO500 and GC500). (A) Frequency of recombination events (CO or GC) within genic sequences. Probability [P (Freq. Observed<Freq. Expected) based on 100,000 replicates of the observed number of recombination events distributed across the D. melanogaster reference genome taking into account differences in marker density between genic and intergenic regions. The expected frequency of a recombination event to be located within a genic sequence is 0.607 when taking into account the influence that maker density plays in detecting CO or GC events delimited by markers separated by 500 bp or less. The genomic location of genic sequences was obtained from the D. melanogaster genome annotation (release 5.3) (B) Relative position of 2,627 GC500 events along transcripts, shown in 10 intervals from 0 at the transcription start site (TSS) to 1 at the end of the transcript. The frequency of GC500 along the transcript is shown with 95% confidence intervals.

In yeast, some DSBs do not require transcriptional activity but depend on the binding of transcription factors, thus predicting an accumulation of recombination events near promoter regions. Alternatively, transcription may alter local chromatin structure, increasing the likelihood of DSB formation along the transcript unit ([109] and references therein). We therefore investigated the distribution of GC events along these sequences. We observe that the median position of GC500 is +910 from the transcription start site (TSS), close to the median midpoint of all D. melanogaster transcripts (+1,058). A split of transcripts into short (<2.5 kb) and long (>2.5 kb) shows the median GC500 position shifting significantly relative to the TSS (from +556 in short transcripts to +3588 in long transcripts Mann-Whitney test U = 51,192, P<1×10 −12 ). Moreover, the relative position of GC500 events along transcript sequences is uniform (Figure 10B), indicating that DSBs are not strongly associated with the binding of transcription factors. This latter result is also consistent with analyses of recombination at the rosy locus, where recombination is initiated throughout the gene [65]. Altogether, our results favor a model where increased chromatin accessibility contributes to the definition of DSB sites in Drosofila, probably associated with transcriptional processes. Note that the preponderance of GC over CO events in many species, and the difference in their physical location across the genome, may limit analyses trying to assess the role of chromatin accessibility on DSB formation and their genomic distribution when using only data associated with COs.


Speciation

Inversions are implicated in speciation in several ways. An intriguing pattern is that rates of chromosome evolution and speciation seem to be correlated [20], but that pattern alone does not tell us which factor causes the other, or whether both are driven by a third variable. Some workers, most famously M.J.D. White [21], have argued that fixed inversion differences between species are important for postzygotic isolation because of their underdominant fitness effects. A difficulty with this idea is that drift is unlikely to fix inversions that are strongly underdominant, while those that are more likely to spread because they are only weakly selected will produce little isolation [22]. With favorable demographic conditions (e.g., frequent colonisations and extinction) and life histories (e.g., self-fertilization or close inbreeding), however, models show that populations can fix underdominant chromosomal rearrangements that contribute appreciably to hybrid fitness loss [8].

No matter how fixed differences for inversions between populations get established, we expect them to become hotspots for accumulating positively selected differences and genes that cause incompatibility between species [23]. This is one explanation for an intriguing pattern seen in sunflowers [24] and flies [25]: loci involved in both pre- and postzygotic isolation map to inversions that distinguish closely related species.

An alternative hypothesis is that the inversions in fact became fixed because of adaptive differences that pre-existed at some of those very loci. The idea depends on local adaptation, to which we now turn.


Genetics 16: 'Introduction to Drosophila: genotypes, recombination and balancer chromosomes'

5 days in prepupa and pupa stages. Then the adult emerges from the pupal case, an event called eclosure, and they become reproductively active within 8 hours after. They can live 45-60 days under good conditions.

A few classic phenotypes are Curly wings (Cy dominant), white eyes (w), yellow body (y), Stubble (Sb), and irregular facets in the eye (If). Flies are often used for studying development, chromosome structure, neurobiology, and behavior. Morgan and Muller did the earliest famous work on Drosofila. Drosofila have polytene chromosomes in some tissues, which made the chromosomes large enough to see even with the microscopes of yesteryear. Lewis, Nüsslein-Volhard & Wieschaus won the 1995 Nobel Prize in Physiology or Medicine for studying embryonic development in Drosofila and discovering Hox genes. Anntennapedia is a gain-of-function mutation where legs, instead of antennae, grow out of the head.

Comparison to other organisms

In contrast to model organisms we’ve recently studied in this class, Drosofila come in male (♂) and female (♀) only (no hermaphrodites ⚥) and are always diploid. Here are some other comparisons:

organizm hujayralar neurons genome size protein-coding genes
xamirturush 1 0 12 Mb 6,600
C. elegans 1031 (males), 959 (hermaphrodites) 302 100 Mb 20,000
Drosofila ? 100,000 165 Mb 13,000

Drosofila have chromosomes X, Y, 2, 3 and 4. Females are X/X and males are X/Y. Chromosome 4 is small and contains hardly any genes. Like most organisms, Drosofila have the annoying property that most genes are named after their loss-of-function phenotype, i.e. the opposite of what they do. For instance, the white gene makes eyes red. However, you can’t count on this always being true. For instance, cinnabar (cn) and brown (bw) are two genes which encode cinnabar -colored and brown -colored pigments, such that a cn bw / cn bw double mutant is white.

Nomenklatura

A singed (sn crooked bristles, recessive, on chrom 2), cinnabar (cn) and brown (bw) (both recessive, both on chrom 3, this combination gives white eyes), Serrate[d] wing (Ser dominant, hence Initcaps, located on chrom 4) fly would be written:

Avvalboshdan, sn means the mutant gene, while wild-type genotypes are simply omitted. If you need to emphasize for clarity, you can write the wild-type allele as sn+ and the mutant as sn-. If you have multiple singed mutants you could write them sn 1 , sn 2 , etc. Genes known to be on the same chromosome are written in one fraction. Reference is Flybase.org

Chromosomal position and phase are represented in this fraction nomenclature, such that cn bw / + + has the cinnabar and brown mutations in cis, while cn + / + bw has them in trans.

Protein products are written in Initcaps, no italics. Flybase has full nomenclature rules.

Balancer chromosomes

A challenge of Drosofila is they cannot be readily frozen or archived - mutants have to be continuously propagated. In addition, many of the most interesting genes in Drosofila are essential for life, and have to be propagated as heterozygotes which have no phenotype. This gives you two problems: first, it leaves you blind as to whether you’re still propagating the mutation. Second, as you cross your heterozygotes to one another, you lose all the mut1/mut1 offspring, and this selection reduces the allele frequency of your mutation of interest in every generation. This is a problem in all constitutively diploid organisms, including mice. Yilda Drosofila, the solution for studying mutations that are recessive lethal or recessive sterile is as follows:

  1. “Balance” the mutation with another lethal/sterile marker, so for instance all your flies are of a mut1 + / + mut2 genotype, such that only hets for both mutations can survive.
  2. Add a visible dominant marker in cis with one of the two balancing mutations: mut1 + + / + mut2 Curly
  3. To prevent recombination from short-circuiting your system, you additionally introduce multiple inversions into the trans chromosome. This gives you a multiply inverted balancer chromosome which contains mut2 va Curly. A downside is that this doesn’t butunlay prevent recombination - in fact, when recombination does still occur, it results in crazy copy number variations because parts of the multiply inverted chromosome will be gained or lost.

Misol

DTS is a dominant temperature sensitive mutation that causes flies not to develop at 29°C (their preferred temperature is around 22°C but wild-type flies can go from 19°C to 29°C). DTS is recessive lethal at any temperature. CyO (“Curly O”) is a balancer chromosome which is wild-type at the DTS locus and also has a recessive lethal mutation. Implicit in the nomenclature DTS/CyO is that each chromosome is wild-type for the other chromosome’s mutation. In a cross:

DTS CyO
DTS</b> DTS/DTS DTS/CyO
CyO</b> DTS/CyO CyO/CyO

The DTS/DTS va CyO/CyO are both recessive lethal, so you have successfully propagated only the parental genotype, DTS/CyO.

Chunki Drosofila have only three chromosomes of appreciable length, there are only three balancer chromosomes in common use.

Chromosome Name of balancer chromosome Dominant phenotype
X (First) FM7 Bar eye
2 (Second) SM5 or CyO Curly wings
3 (Tird) TM3 or TM6 Stubble bristles, Ser wings or Tb (Tubby) body

How to get mutants

Usually you mutagenize males with EMS or X-rays. Males are used because they don’t need to be virgins. Also they are more resistant to DNA damage, which means they can tolerate enough damage to get useful mutations. Note that you only care about mutations in the germline, not soma. Then you cross to females. Each F1 will have a unique heterozygous mutation inherited from the father’s germline. At the F1 stage, you can only identify viable dominant mutations. (An exception is if you mutagenize females instead of males, you can find X-linked recessive mutations in the male F1s). If your viable dominant mutation is sterile, you’re out of luck, because you can’t propagate it beyond that one male. This motivates a need for more complicated screen designs, which will be covered later this week.

Erik Vallabx Minikel haqida

Erik Vallabx Minikel prion kasalligining oldini olish uchun umr bo'yi izlanmoqda. U MIT va Garvardning keng institutida joylashgan olim.


The Scientific Importance of Drosophila Melanogaster

Drosophila melanogaster, or the fruit fly as it is more commonly called, has played an important part in science. It has aided scientists in the discovery of many different principles. Its importance continues today.

The fruit fly has been used for approximately a century in scientific research, according to the University of Michigan Museum of Zoology. It has played an especially large role in the study of genetics. Thomas H. Morgan utilized the fruit fly to prove the chromosomal theory of inheritance. This showed that chromosomes carry genetic information. The fruit fly was vital in discoveries regarding gene mapping, multiple alleles, spistatis and sex-linked inheritance. Research continued on Drosophila melanogaster by H. Sturtevant. He created genetic maps of the fruit fly.

There are various reasons why Drosophila melanogaster has been such an ideal subject for studies in genetics and biology. First, it can reproduce very easily in captivity. It is very easy to breed many different subjects for use in studies.

The fruit fly has a very short lifespan. In seven days it matures into an adult. Because of this, researchers could study many generations in a short span of time. This is especially useful for genetic research.

It is simple to care for fruit flies. It is also inexpensive. Fruit flies reproduce very quickly, with one female creating a hundred eggs every single day. It is easy to tell the difference between males and females, as well as identify virgin females. There are only four pairs of chromosomes, and this makes it very easy to study them. Meiotic recombination does not occur in males. Also, because they have been studied so extensively, their entire genome was sequenced, allowing for easy research. All of these traits make Drosophila melanogaster the ideal subject to use in scientific research.

Despite the simplicity of Drosophila melanogaster, scientists can learn a great deal about genetics and biology from it. Many of the same principles about genetics are the same for fruit flies and other animals, including humans.

Drosophila melanogaster is even used to teach children about the importance of science. The same benefits that make it a great subject for established research scientists make it easy to use for high school and college students, according to the biology department at the University of Arizona. Thus they can help teach the next generation of scientists.

Although Drosophila melanogaster are simple creatures, their contribution to science through the years has been nothing short of amazing. They continue to be studied all over the world.


San'at aloqalari

[link] In a test cross for two characteristics such as the one shown here, can the predicted frequency of recombinant offspring be 60 percent? Nima uchun yoki nima uchun?

[link] No. The predicted frequency of recombinant offspring ranges from 0% (for linked traits) to 50% (for unlinked traits).

[link] Which of the following statements is true?

  1. Recombination of the body color and red/cinnabar eye alleles will occur more frequently than recombination of the alleles for wing length and aristae length.
  2. Recombination of the body color and aristae length alleles will occur more frequently than recombination of red/brown eye alleles and the aristae length alleles.
  3. Recombination of the gray/black body color and long/short aristae alleles will not occur.
  4. Recombination of the red/brown eye and long/short aristae alleles will occur more frequently than recombination of the alleles for wing length and body color.

Why doesn't recombination occur in male Drosophila? - Biologiya

In his autobiography, Darwin mused with regret at his failure to learn more mathematics, observing that those with an understanding of the “great leading principles of mathematics” “seem to have an extra sense”. This extra sense is beautifully exemplified in a subject that was near to Darwin’s heart, namely, the origins of heredity, the study of which gave rise to modern genetics. Gregor Mendel was intrigued by the same question that has perplexed naturalists as well as parents for countless generations, namely, what are the rules governing the similarities and differences of parents and their offspring? His approach required the painstaking and meticulous act of counting frequencies of various traits such as pea shape from carefully constructed plant crosses, where he found that out of a total of 7,324 garden peas, 5,474 of them were round and 1,850 were wrinkled. The subsequent analysis of the data showed for this case a ratio of these traits in the second generation of crosses of 2.96 to 1, providing a critical clue permitting Mendel to posit the existence of the abstract particles of inheritance we now call genes.

Figure 1: Schematic of the first genetic map of the X chromosome of Drosophila redrawn with modern symbols. Sturtevant’s map included five genes on the X chromosome of Drosophila. Adapted from: http://www.nature.com/scitable/topicpage/thomas-hunt-morgan-genetic-recombination-and-gene-496. Locations updated from Green & Piergentili, PNAS 2000. Based on: Pierce, Benjamin. Genetics: A Conceptual Approach, 2nd ed. (New York: W. H. Freeman and Company), 161. From:

To cause a sea change in biological research required going beyond phenomenological observations to a situation where genetic manipulations could be more easily performed and more detailed predictions made. This came about when Morgan, head of a lab already overflowing with studies of pigeons and starfish, undertook with his students an object of study with minimal space requirements and faster generation times. So came to the scene one of the great protagonists of modern genetics, the fruit fly Drosophila Melanogaster. As Morgan’s lab transformed to what became known as the “fly room” (first at Columbia University, then at Caltech), it harbored flies with several distinct morphological properties akin to Mendel’s mottled and different colored peas. Systematic crosses of these mutant flies showed deviations from the predictions of Mendelian genetics on the relative fractions of different progeny. An inquisitive Columbia University undergraduate student in Morgan’s lab decided to analyze the frequencies of linkage, that is of pairs of co-inherited traits. During a long night that was supposed to be devoted to homework for his undergrad studies, the young Alfred Sturtevant instead made a conceptual leap that was to become textbook material and a cherished story from the history of science. He found that the tendency of the traits they studied to be inherited together such as white eyes instead of red eyes or a more yellow body color could be quantitatively explained if one assumes that the genes for these traits are ordered along a line (chromosome) and the tendency not to be inherited together is then reasonably predicted as increasing linearly with their distance. Using this logic, that night Sturtevant created the first genetic map reproduced in Figure 1.

Table 1: Recombination rates in various mammals and marsupials of similar genome sizes. Genetic map length is the sum of genetic map lengths summing in units of cM over all chromosomes in each genome. The right most column, recombination events per chromosome, is calculated by dividing the genetic map length (cM/100) by the number of chromosomes. Note how this genetic map length per chromosome is close to one over the range of organisms. (BNID 107023, adapted from Dumont BL, Payseur BA. Evolution of the genomic rate of recombination in mammals. Evolution. 62:276, 2008. Choromosme numbers are from: http://www.genomesize.com.)

The mechanism explaining the frequency with which characteristics are inherited together is that of recombination. This is an act of two chromosomes of similar composition coming together and performing a molecular crossover, thereby exchanging genetic content. Two genes on the chromosome that have a 1% chance of crossover per generation are defined to be at a distance of one centimorgan, or cM for short. In humans, the average rate of recombination is about 1cM per 1Mbp (BNID 107023), that is, for every million base pairs there is a one in a hundred chance of crossover on average per generation. The variation in the rate of recombination is shown in Table 1. It tends to scale inversely with genomic length. This interesting scaling property can be simply understood by noting that in most species there are one to two crossover events per chromosome per replication. This results in an organism-wide rule of thumb of one recombination event per chromosome as demonstrated in the right-most column of Table 1, or equivalently as 100 cM (i.e. one Morgan or one crossover) per chromosome per replication. Beyond general rules of thumb, we now also know that some locations along chromosomes are hotspots that are more labile for crossovers. Finally, human females have ≈50% higher recombination rates than males (42 versus 28 on average in one recent study, BNID 109268). So even though you tend to get more of your single base mutations from your father as discussed in the vignette on “How many chromosome replications occur per generation?”, your crossovers are mostly thanks to your mother.

Figure 2: Detection of recombination events based on sequencing of a single sperm cell. The two columns in each chromosome represent the two homologous chromosomes carried by the subject. The source of the sperm single chromosome copy can be traced to one or the other homologous chromosome based on single nucleotide polymorphisms that appear in one chromosome but not the other. Blue lines show the association of the sperm sequence to the two chromosome sets based on those single nucleotide polymorphisms. Each switch (haplotype block) indicates a recombination event. (Adapted from J. Wang, et al., Cell 150:402,2012.)

Recent breakthroughs in genotyping have made it possible to perform a single-cell analysis of recombination activity. Single nucleotide polymorphisms (SNPs) are locations in the human genome where there is variation between people such that say more than 1% of the population has a nucleotide different than the majority of the population. For the human population there are on the order of 10 6 such locations on the genome. Here is how this can be used to infer the number and location of recombination events. The chromosomes of a male were separated in a microfluidic device (arbitrarily marked as left and right for each of the 22 pairs) and then each chromosome was separately analyzed for the variant of nucleotide it carries by a microarray technology. The same process was repeated for a sperm cell leading to maps such as that shown in Figure 2. At the locations where it is known that there is polymorphism in the genome it was checked if the variant in the sperm cells is the one that appears in one chromosome but not the other, and if so its location was marked as a blue stripe on the relevant chromosome. The events of recombination are clearly seen as switches of those polymorphism locations from one arm to the other. On average, 23 recombination events were found for a human sperm cell (BNID 108035). Short stretches consisting of a single SNP switching chromosome, as highlighted in chromosome 8, are cases of what has been termed gene conversion where one allele (gene copy) has performed homologous recombination that makes it replace the other copy (its heterozygous allele). Such analysis at the single-cell level, in contrast to inference at the population level or from studying progeny in a family, makes it possible to see the rates of events such as recombination and mutation in the gametes including for those gametes that will not lead to viable progeny. This is relevant as the human monthly fecundity rate, that is the chance of a menstrual cycle leading to pregnancy, is only about 25% (BNID 108080) even at the peak ages of 20-30. Aberrations in the genome content are often detected naturally early in development, within the first few weeks following conception, and lead to natural termination of the pregnancy even before the woman is aware she is pregnant.

Today recombination also serves researchers as a key tool in genetic engineering for creating designer genomes. Homologous recombination enables incorporation of a DNA sequence at a prescribed location within the genome. Its use has transformed our ability to tag genes of interest and resulted in genome-wide libraries enabling high-throughput analysis of key cellular properties ranging from localization of proteins to different cellular locations to genome-wide assessments of protein levels and variability. Though recombineering, as it is called, is incredibly powerful, unfortunately it can only be used in some organisms and not others, giving those lucky organisms a strong selective pressure in labs around the world as attractive model systems. Outstanding examples are the budding yeast and the moss physcomitrella patens. The method of homologous recombination requires a sequence of homology flanking the integrated sequence. The length of this sequence varies depending on the organism, the gene of interest and the specific technique and protocol employed. Some characteristic values are ≈30-50 bp in budding yeast (BNID 101986) whereas in mouse it is ≈3-5 kbp (BNID 101987). With the longer stretches also comes a much lower efficiency of performing the act of homologous recombination complicating the lives of molecular biologists, though modern CRISPR techniques have effected a new revolution in genome editing that may largely supersede recombineering methods.