Tetrad analysis. Life cycle and meiosis in yeast. Fig.1. Life cycle of yeast

Transcription

1 Tetrd nysis Tetrd nysis in genetics refers to nysis of four products formed from meiosis. In orgnisms like yest the tetrd contins four spores while in cse of Neurospor the scus in which products of meiosis re held, there re eight scospores. Although meiosis occurs in l sexuly reproducing orgnism, in most of them, the products of meiosis re mixed such s in cse of pollen grins. However, in few cses the products of single meiosis re held in seprte ody. It is possile to isolte ech such spore contining ody which cn e further dissected to isolte nd culture ech spore seprtely. Thus, it is possile to study the phenotype nd the genotype of ech isolted spore. This nysis is known s tetrd nysis nd some orgnisms in which it is crried out re yest, Chlmydomons nd Neurospor. Tetrds in which the spores re rrnged ccording to the order in meiotic division re cled ordered tetrds s in Neurospor, while where the order or sequence is not specific re cled unordered tetrds s in yest or Chlmydomons. Higher orgnisms which undergo meiosis re diploids (or polyploids). Some lower orgnisms which undergo meiosis spend considere prt of life cycle in hploid stte. The hploid orgnisms provide suite system since there is only one lele in hploid stte nd there is no dominnce phenomenon. Life cycle nd meiosis in yest Fig.1. Life cycle of yest Fig.1. shows life cycle of yest nd Fig.2. shows meiosis in yest nd sci contining scospores.

2 Diploid cells strved for nitrogen or cron source undergo meiosis. After nucler division, ech prospore memrne closes on itself to enclose hploid nucleus within two distinct memrnes. Spore wl Fig.2. Meiosis in yest nd sci with spores. synthesis then egins in the lumen etween the two prospore memrne -derived memrnes. After spore wl synthesis is complete, the mother cell collpses to form the scus. Since the spores in the scus re not rrnged in ny prticulr order this is cled s unordered tetrd. Normly spores form tetrhedr shpe, some times l spores re in one plne. Life cycle nd meiosis in Neurospor Neurospor spends most of its life cycles in the hploid stte Fig.3. N. crss which is most commonly studied is heterothlic which mens g two different mting types re required to undergo sexu reproduction. In the sexu phse, hyphe of different mting types come into contct nd nuclei fuse resulting in mny trnsient diploid nuclei inside fruiting odies cled peritheci. Ech diploid nucleus undergoes meiosis. The four hploid products of one meiosis sty together in sc cled n scus. In N. crss ech of the four products of meiosis undergoes further mitotic division, resulting in n octd of eight scospores within ech scus. Ascospores germinte nd produce hyphe resulting in colonies. Ascospores from single scus cn e individuly picked up nd inoculted to produce colonies.

3 Fig.3. Life cycle, perithecium nd sci contining scospores. Segregtion of leles From pir of homologous chromosomes the homologues segregte in the 1 st meiotic division. If there is no crossover, the two leles on the homologous chromosomes thus segregte in the first meiotic division. Even when there is crossover, the leles etween the centromere nd the crossover segregte in the 1 st Homologous with two leles Alleles segregte in 1 st division Alleles segregte in 2nd division Zygote No crossover Single crossover Chromosome dupliction Mitosis A Fig. 4. Segregtion of leles in (A) 1 st division or () in 2 nd division.

4 meiotic division Fig.4A. When there is crossover etween the centromere nd the locus, the leles of the locus segregte only in the second division of meiosis Fig.4. In Neurospor fter meiosis there is mitotic division which rings the numer of spores in ech scus to eight. Auxotrophic mutnts Microorgnisms cn mutte to produce different types of mutnts. Mutnts which hve lost the ility to produce prticulr metolite nd cn e mde to grow y supplying the sme s nutrient re cled uxotrophs. The wild type is prototroph which mens it does not require ddition of the metolite in the medium s supplement. Auxotrophs for prticulr mino cid or vitmin re commonly used mutnts in genetic studies. Tetrd nysis in Neurospor Neurospor crss produces sci with eight spores which re rrnged in n order fter meiosis. Hence ordered tetrd nysis is possile. This is prticulrly useful in studying recomintion, estimting linkge nd mpping of genes. Crosses re mde etween strins of known muttions nd from the fruiting odies sci re dissected out, spores re isolted nd their phenotypes re determined. The question if crossover occurrs t two strnd stge or four strnd stge (i.e. if crossover took plce nd then chromosome douling occurred or first chromosome douling occurred nd then crossover took 1. If crossover occurs t two strnd stge X 2. If no crossover occurs t two strnd stge 3. If crossover occurs t four strnd stge Since spore rrngement seen in 3 is oserved crossover occurs t four strnd stge. Fig. 5. Spore rrngement shows tht crossover tkes plce t four strnd stge.

5 plce) is nswered y looking t the spore rrngement (Fig.5). The sequence of spore rrngement indictes if crossing over hs occurred etween the genes under study or the centromere nd gene. Assume cross etween wild type (+) nd ino mutnt (). If there is no cross over etween the centromere nd the gene, the spore rrngement oserved is 4 + :4 nd if there is crossover etween the centromere nd the gene involving two of the four strnds, the spore rrngement is 2+:2:2+:2 (Fig.6) No crossover etween centromere nd the gene results in 4:4 rrngement of spores. Crossover etween centromere nd the gene involving two of the four strnds results in 2:2:2:2 rrngement of spores. Fig.6. No crossover etween centromere nd the gene or crossover involving two of the four strnds results in chnge in the rrngement of the spores. In such cross if 129 sci showed 4:4 rrngement nd 141 sci showed 2:2:2:2 rrngement, the tot numer of sci scores is =270. In the 141 sci which showed recomintion, only two out of the four strnds took prt in crossing over. Hence, recomintion% is hf of the numer of sci showing segregtion. The recomintion is cculted s ½(141/270) = 0.26 or 26%. If two genes (P nd Q) re on the sme side of the centromere, nd if t four strnd stge two strnds re involved in crossover etween the centromere nd gene nd the sme two strnds re involved in crossover etween two genes, the consequence is shown in Fig.7A. However if the first crossover etween centromere nd gene P is etween two strnds the second crossover etween P nd Q the other two strnds the consequences re shown in Fig. 7. P Q P q A P Q P Q P q P q P P p p Q Q q q Pq Pq P P p p Q Q q q PQ PQ Pq PQ Two crossovers, one etween centromere nd gene, the other etween two genes, involving the sme two strnds. Two crossovers, one etween centromere nd gene, the other etween two genes, involving the sme two strnds. Fig.7. Consequences of two crossovers (A) etween the sme two strnds () etween different two strnds.

6 Two genes on two seprte chromosomes, two rms of the sme chromosome or on the sme rm of the sme chromosome re different possiilities of loction of genes nd, no crossover, single crossover etween centromere nd gene or single crossover etween the two genes, two crossovers one etween centromere nd the other etween the two genes involving the sme two strnds or involving two different strnds ech time, two crossovers involving three strnds re the different possiilities nd correspondingly there re sequence rrngements of the spores. Detection of linkge Consider dihyrid cross etween n denine requiring strin (d) with A mting type nd wild type strin with mting type. The cross cn e written s d A X + on oserving the sci there my e prent comintions (prent ditypes or PD) which re formed s result of no recomintion nd nonprent ditypes (NPD) formed s result of recomintion. In ddition, recomintion so results in tetrtypes (T) in which four types of spores re formed. Te1. Types of tetrds in cross etween two strins of Neurospor. Prent ditypes Nonprent ditypes Tetrtypes d A d d d A d d A + A + + A + A Numer oserved: 10 Numer oserved: 9 Numer oserved :1 The difference etween prent ditypes nd nonprent ditypes is not significnt which indictes independent ssortment etween the mting type gene nd the denine requiring locus. A significntly higher numer of prent ditypes would men tendency to e inherited together or genetic linkge. In Neurospor strin uxotrophic for pyridoxine (pdx) nd strin uxotrophic for pntothenic cid (pn) were crossed. Results of tetrd nysis re shown in Te2. Te2. Types of tetrds nd numers oserved in crosses etween strin pdx + nd + pn pdx + pdx pn pdx + pdx + pdx + pdx + pdx + pdx pn pdx pn pn + pn + pn pdx pn pdx + pdx pn + pn pn + pn + pn Recomintion t pdx is seen in 4, 5 nd 6 (1+13+2=16), nd recomintion t pn is seen in 3, 5 nd 6 ( =32). This recomintion is due to crossover etween the centromere nd gene. As recomintion types re less for pdx, this locus is nerer to the centromere nd recomintion types re more for pn, the locus is frther.

7 The recomintion t pdx nd pn loci cn e used to cculte recomintion % nd drw linkge mp on which the two loci cn e locized with respect to the centromere. In cculting recomintion percentge, the frequency of tetrds with single crossover is multiplied y ½ since in these cses two of the four strnds re involved in crossover while the frequency of doule crossovers is used s it is since in doule crossover four out of the four strnds prticipte in crossover. The distnce etween centromere nd pn is otined s follows The tot numer of tetrds scored is =49, 32 showed recomintion t pn due to single crossover nd four due to doule cross over, therefore Recomintion = ½(32/49) + (4/49) =0.387 or 38.7%. Recomintion t pdx is cculted s ½ (16/49) =0.163 or 16.3% (there re no doule crossovers etween centromere nd pn). Distnce etween pn nd pdx is otined y sutrcting from which is Distnce etween pn nd pdx cn so e otined s; ½ (17+1+2)/49 + 1/49 = or 22.4%. The reltive positions of the centromere, pdx nd pn re shown in Fig pdx pn Fig.8. Schemtic representtion of reltive positions of centromere, pdx nd pn in linkge mp. Unordered tetrds These occur in mny four spored yests, some eight-spored euscomycetes nd in sidiomycetes. Here the spores resulting from single meiosis event re held together, however, the rrngement of the spores hs no correltion with the order during meiosis. As result of this the centromere cnnot e used s

8 mrker in genetic studies, linkge distnces etween two genes hve to e determined through their recomintion vue. Three types of tetrds result fter meiosis prent ditype, nonprent ditype nd the tetrtype. In cse of unlinked mrkers prent nd nonprent ditypes occur in equ proportions. A sttisticly significnt devition from equ proportion indictes linkge. Informtion out linkge nd the loction of the genes cn e otined y compring the distnces etween ech of the two genes. However, three fctor crosses re necessry to estish the order of loci with certinty. If the previous dt in Te2 re pertining to unordered tetrds, the crossovers etween centromere nd pdx will not e considered. Only crossovers etween pdx nd pn loci will e considered for ccultion. The dt re then interpreted s shown in Te3. Te3. Types of tetrds nd numers oserved in crosses etween strin pdx + nd + pn pdx + pdx Pn pdx + pdx + pdx + pdx + pdx + pdx Pn pdx Pn pn + pn + pn pdx pn pdx + pdx pn + pn Pn + pn + pn No crossover only prent ditypes Two crossovers etween pdx nd pn, l four strnd involved Single crossover etween pdx nd pn, two strnds involved Single crossover etween pdx nd pn, two strnds involved No crossover etween pdx nd pn Single crossover etween pdx nd pn, two strnds involved Prent types (no recomintion etween pdx nd pn)= 15+13=28. Recominnts with one crossover =17+1+2=20. Recominnts with two crossovers=1. Tot numer scored=49. Recomintion % will e [½(20)+1]/49=11/49=0.224 or 22.4%. The vue is sme s in previous ccultion.

9 Addition informtion G.W. edle ( ) nd E.L. Ttum ( ) Americn geneticists edle nd Ttum's experiments involved exposing N. crss to x- rys, to crete muttions. They showed tht muttions cused chnges in specific enzymes involved in metolic pthwys. This led them to propose link etween genes nd enzymtic rections, which ecme known s the one gene, one enzyme hypothesis. They were wrded Noel Prize in Physiology or Medicine in 1958 ong with J. Ledererg. Micromnipultion technique in yest A sporulted culture is used for isoltion of scospores. The culture contining sci is digested with zymolyse in controlled mnner. Seprtion of spores requires use of micromnipultion technique. The treted sci re plced on petridish with medium or on thin gr sls. The four spored scus is picked up using the microneedle of the micromnipultor. Ech spore is then seprted using micromnipultor nd the spores re plced on the medium surfce for growth s colony.