Linkage testers having markers near centromere

Linkage testers having markers near centromere Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This contribution on genetic stocks is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol19/iss1/21 Perkins, D. D. Linkage testers having markers near the centromere. Strains have been constructed that contain readily rcorable muton+ morken near the centromere in six linkage groups. MDting type morkr the remoinino wow. Although the workers are not ideal for efficiencv of scoring in .I: ccmbinotionr;experience her shown these multicentromere terterr to be quite useful, erpeciolly for situations where olcoy is not effective, os with temperature-sensitive mutonh ( because cot-l is a marker in olcoy), with mutonb where olcoy foils to detect linkage (because olcoy markers ore for distal in two linkage g= and VII is unmarked), and with chromosome reorrangementr (becoure three tronrlocations are olreody present in olcoy ). The following genes ore present os markers: Markers: A/a bol au-2 pcfx at m&g--type baTcon .JiCi”* pyridoxine ottezoted yl0-l WC yellow whithollor Irolotion No. : 856 KH5 37803 Ml11 Y30539y PB29 Linkage group: I II 1 1 1 IV V VI VII Four stocks hove been deposited with the Fungal Genetics Stock Center: Multicent (oil markers) A and o FGSCff 2014 and 2015, respectively Multicent (without&l) A ond o FGSC# 1085 and 1086, respectively. Our normal procedure with multicent is os follows: Use multicent os fertilizing parent. Suspend mycelial fmgmentr in I ml water in o 10 x 75 mm tube, using a pipette to homogenize by grinding agoinrt the wall. Multicent con with some persistence be used as Pmtoperitheciol parent, but perithecia ore slow to develop. Isolate 100-150 orcorporer to minimal + pyridoxine IO doyr after spores rtort shooting. Germinate at 34’C. Sort for bol, ot and W C ot 3 ond 4 days. Set up scoring sheets and number tuber ot 4 doyr. Because balloon grows os o restricted colony, it zozrt towork only among the bal+ half of the progeny, even though this requires thot more spores be isolated originally. ot is readily scoroble on minimal (with or without supplenenh) ot 2 or 3 days (34’C), but conidioter more profusely on a complete meTurn, creating some scoring difficulties in older cultures. Growth is flat on the rurfoce, with scottered specks of conidiotion. W C is cleorert ot tempemturer above 25’C, and is rcoroble by the obrence of corotenoidr in mycelio, though not in conidio. Germiznts ore best kept cl+ 34’C for 3 or 4 doyr under illumination till W C scoring is accomplished (uruolly in two readings 24 hours apart; ovoid reading just after cultures ore brought from dark into light). Germinmts ore then moved to 25’C, where increased development of pigment in W C facilitates the scoring of&. Unlike W C, ~10-1 scoring improves with age, and is likely to be unreliable in young cultures. Corotenoid i n yl0-l l o o k orange at first, then become yellow. yl0-l scoring at 3 or 4 days should be considered preliminary, and should be checked Ioter. au-2 is scored clearly by tronrfer to min + IO pg pyridoxine/ml + 50 pg ocriflovine/ml. pdx is most easily scored by transfer to min + 100 pg deroxypyridcxine~HCl/ml. It con be scored sotirfoctorily on minimal withoutthe antagonist if sufficiently small inoculo are used. If linkage is no+ shown to markers in II VII , mating type is scored on flPA and flp a testers, either by spotting onto ‘I-doy old SC plater, SC plater, or by fertilizing 75 mm fl tuber. (Tubes rother than plater ar~olwoys u?ed for chromosome reormngementr, so that isolates con be scored as Normal or &r&ion sequence according to the presence of white deficiency oscosparer among those shot to the wall of the tube. ) Effort is minimized by the stepwise scoring procedure. If on unmapped point mutont is scored early in the sequence, growth tests for& and au-2 ore required only if linkage to the visible mo&ers is not opporent. Mating-type tests ore then required only if no linkogeirpparent to pdx or ocr-2. With tronrlototionr, the normolly independent multicent markers ore examined for linkages to one another. 6epaGt of Biological Sciences, Stanford University, Stanford, Colifornio 94305. Letter to the Editor: Over the port few years, I hove been orked several times about the alleles of crisp, of osmotic and of crisp, osmotic that were used in the work by Trevithick and Metzenberg (1966 Molecular sieving by Neurosporo cell walls during secretion of invertore irozymes. J.Bocteriol. PZ:.?OlO.) crisp was allele 8123, and osmotic was allele E IIZCXJ. Unforhmotely, we do not have o record of the allele numberrofthe double mutant. It might hove been 8123, Ell200, or it might hove been 8122, 8135, since we once obtained this double mutant from the Fungal Genetics Stock Center; this detail is now lost in antiquity. Forfunotely, the single mufonts, from which most of the experimental information was obtained, con be identified with certainty. R. L. Metzenberg, Deportment of Physiologic.1 Chemistry, University of Wisconsin, Modiron, Wisconsin 53706.

Perkins, D. D. Linkage testers having markers near the centromere.
Strains have been constructed that contain readily rcorable muton+ morken near the centromere in six linkage groups. MDting type morkr the remoinino wow.
Although the workers are not ideal for efficiencv of scoring in .I: ccmbinotionr;experience her shown these multicentromere terterr to be quite useful, erpeciolly for situations where olcoy is not effective, os with temperature-sensitive mutonh ( because cot-l is a marker in olcoy), with mutonb where olcoy foils to detect linkage (because olcoy markers ore for distal in two linkage g= and VII is unmarked), and with chromosome reorrangementr (becoure three tronrlocations are olreody present in olcoy ). Our normal procedure with multicent is os follows: Use multicent os fertilizing parent. Suspend mycelial fmgmentr in I ml water in o 10 x 75 mm tube, using a pipette to homogenize by grinding agoinrt the wall.
Multicent con with some persistence be used as Pmtoperitheciol parent, but perithecia ore slow to develop.

Sort for bol, ot and WC ot 3 ond 4 days.
Set up scoring sheets and number tuber ot 4 doyr. Because balloon grows os o restricted colony, it zozrt towork only among the bal+ half of the progeny, even though this requires thot more spores be isolated originally.
ot is readily scoroble on minimal (with or without supplenenh) ot 2 or 3 days (34'C), but conidioter more profusely on a complete meTurn, creating some scoring difficulties in older cultures. Growth is flat on the rurfoce, with scottered specks of conidiotion. WC is cleorert ot tempemturer above 25'C, and is rcoroble by the obrence of corotenoidr in mycelio, though not in conidio. Germiznts ore best kept cl+ 34'C for 3 or 4 doyr under illumination till WC scoring is accomplished (uruolly in two readings 24 hours apart; ovoid reading just after cultures ore brought from dark into light). Germinmts ore then moved to 25'C, where increased development of pigment in WC facilitates the scoring of&.

Unlike WC, ~10-1 scoring improves with age, and is likely to be unreliable in young cultures.
Corotenoids in yl0-l look ora at first, then become yellow. yl0-l scoring at 3 or 4 days should be considered preliminary, and should be checked Ioter. au-2 is scored clearly by tronrfer to min + IO pg pyridoxine/ml + 50 pg ocriflovine/ml. pdx is most easily scored by transfer to min + 100 pg deroxypyridcxine~HCl/ml. It con be scored sotirfoctorily on minimal withoutthe antagonist if sufficiently small inoculo are used.
If linkage is no+ shown to markers in II -VII , mating type is scored on flPA and flp a testers, either by spotting onto 'I-doy old SC plater, SC plater, or by fertilizing 75 mm fl tuber.
(Tubes rother than plater ar~olwoys u?ed for chromosome reormngementr, so that isolates con be scored as Normal or &r&ion sequence according to the presence of white deficiency oscosparer among those shot to the wall of the tube. ) Effort is minimized by the stepwise scoring procedure. If on unmapped point mutont is scored early in the sequence, growth tests for& and au-2 ore required only if linkage to the visible mo&ers is not opporent.
Mating-type tests ore then required only if no linkogeirpparent to pdx or ocr-2. With tronrlototionr, the normolly independent multicent markers ore examined for linkages to one another. ----6epaGt of Biological Sciences, Stanford University, Stanford, Colifornio 94305.

Letter to the Editor:
Over the port few years, I hove been orked several times about the alleles of crisp, of osmotic and of crisp, osmotic that were used in the work by Trevithick and Metzenberg (1966 Molecular sieving by Neurosporo cell walls during secretion of invertore irozymes. J.Bocteriol. PZ:.?OlO.) crisp was allele 8123, and osmotic was allele E IIZCXJ.
Unforhmotely, we do not have o record of the allele numberrofthe double mutant. It might hove been 8123, Ell200, or it might hove been 8122, 8135, since we once obtained this double mutant from the Fungal Genetics Stock Center; this detail is now lost in antiquity.
Forfunotely, the single mufonts, from which most of the experimental information was obtained, con be identified with certainty.