Antigenic screening method for genetic studies

Antigenic screening method for genetic studies Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This technical note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol19/iss1/11 A protoperithecia-less (ff-1 ) mutant (Tan and Ha 1970 Mol.Gen. Genet. 107: 158) also doer not develop protoperithecio under these conditions. Severolxer mutants defective in protoperithecial development hove also been cultured. For several of these, the aerial hyphae growing on the vertical walls of the dish are shorter than those of wild type. ( This work was carried out under tl Gorney Fellowship at the laboratory of N. H. Horowitz, Division of Biology, California Institute of Technology) Division of Genetics, School of Biological Sciences, University of Malaya, Kuolo Lumper, Moloysia. Garre+t,M. K. An antigenic screening method of potential application in genetic studier. It is frequently Ioboriour to examine the segregation of isozymer which are recognized by electmphoretic criteerio. The problem is aggmvoted when recombinontr of &rely linked genes are being sought, as is the case with the genes for treholore’in N. crc+ss(~. In this example recombinontr were being sought between the z gene (coding for the presence of treholare ) and the Ggxcoding for the electrophoretic mobility of the enzyme, viz. fast or slow) which mopped less than one unit apart (Sussman et al. 1971 J. Bocteriol. 108:59). A n enrichment of recombina~wor achieved in the usual way through the ure of flanking mwkerr, and the identification of “fast” and “slow” forms of trehalare in +re+ recombinonts would normally have involved electrophorerir in polyacrylamide gel. However, the use of on immunological technique made it possible to screen up to 100 arcorpore isolates Per day with a high degree of rpecificity. The technique is described here since it may have general application in Neurorpora studier. Numerous methods have been employed for the production of antibodies and generally require up to IO mg of pure antigen (II starting material. In the case of treholase, Z-IO mg of plre enzyme in 0.05 M phosphate buffer (pH 5.6) was emulsified with an equal volume of Freund complete adjuvont and injected intra-murcularly into rabbits over a period of 2 weeks. Booster injections contained approximately I mg of antigen in incomplete odjuvant. A maximum tihe was obtained in the third week and the animals were then bled by cardiac Puncture or from an ear vein. The 20-30 ml of blood of blood obtained was oIlwed to clot in a Petri dish and, after standing overnight cl+ 4’C. the serum which had separated was decanted, centrifuged for 20 min at 3,000 x g to remove cells, and stored at -2O’C. The Ouchterlony dwble diffusion method was used to screen extracts. Degreased gloss slides (2 in x 2 in ) were first dipped in 0, .~%Ionagar”Z (in deionized water), drained and permitted to dry. 2 ml of 1% lonag& 12 (in 0.04 M veronol/HCI buffer, pH 7 .S, + 0.003% methyl orange) war then poured to form an even foyer on one side of each slide: After storage at 4-C for I2 hr, one central and four peripheral wells were cut in each slide wing o template and cork-borer. The ontisewm was placed in the central well and peripheral wells contained extracts of undesignated phenotypes alternoting with sampler of the electrophoretially identified irozymes “fart” or “slow”. The extracts were prepared from acetone powdered mycelium os described previously for electropharerir (Yu et 01. 1971 Genetics 68:473) and IO pl sampler of ontirero and ontigeos were used throughout. Clear-cut differences between extracts of known “fast” and “slow” strains were obtained using antiserum prepared clgainrt trehalcrre from the wall fmction of “fast” strains, and such differences were onnulled by obrorption of the antiserum with purified “fort” trehalore. A total of 574 tre+recombinonts were screened by this method and only one yielded on ambiguous result on Ouchterlony plates. Electrophor~wor used to check 10% of the samples selected ot random and in alI cares the expected result WOI obtained. This work wal carried w t whilst the author was o Research Associate in the Deportment of Botany a+ the University of Michigan, and was supported by NSF grant GB 6811X to A. S. Surrmon, to whoni I om indebted for support and focilitier. The cooperation of S. Liu and T. Jepron in the preporotion of antirero is gratefully acknowledged. Deportment of Botany, The Queen’s University, Belfast, Northern Ireland. Johnron,T. E. and D. R. Stodler. A method for In this method, protoperithecio ore grown for seven days a+ room temisolating protoperithecio. perature on petri plater containing I5 ml of Westergoord’s crossing medium with 1.5% agor. After this time, large numbers of protoperithecia are visible. The agar from one plate is cut into squares, and is then immersed in 50 ml of sterile water and ground in o Sorvoll Omnimixer ot setting nine for one minute. This suspension is diluted into 2 liters of wdeer and stirred continually at room temperature during +he rest of the procedure. It is important rho+ this suspension be dilute and that clumps of mycelia not be allowed to form. The suspension is siphoned a+ a rate of 300 ml/hr into a race tube filled with distilled water. Both ends of this U-shaped tube hove been bent about 30’ from the horizontal. The bent portion o+ one end is slightly longer 50 that the suspension will flow through the tube and out the lower end without pumping. Most of the myceliol materiol either flows completely through the tube or collects near the for end, while the protoperithecio tend to collect near the mouth of the tube. We collect the material found in the first ten cm of the tube and find it to be relatively free of mycelio. A second possage of this material giver protoperithecia almost free of any contominoting mycelia. We have been able to collect as much 0s one gram (wet wt.) of protoperithecia from four petri plates. The pratoperithecia seem no+ to have suffered too badly from the isolation procedure, 01 most of them con still serve as foci for vegetative growth and form a distinctive colony type when plated on so&se media. We hove been wobble to fertilize these is&ted protoperithecia, possibly because the trichogynes ore removed during the initial process of shearing. We are currently characterizing some of the proteins which we contained in the protoperithecio. _ _ _ Deportment of Genetics, University of Washington, Seattle, Washington 98105.

It is frequently Ioboriour to examine the segregation of isozymer which are recognized by electmphoretic criteerio.
The problem is aggmvoted when recombinontr of &rely linked genes are being sought, as is the case with the genes for treholore'in N. crc+ss(~. In this example recombinontr were being sought between the z gene (coding for the presence of treholare ) and the Ggxcoding for the electrophoretic mobility of the enzyme, viz. fast or slow) which mopped less than one unit apart (Sussman et al. 1971 J. Bocteriol. 108:59).
An -enrichment of recombina~wor achieved in the usual way through the ure of flanking mwkerr, and the identification of "fast" and "slow" forms of trehalare in +re+ recombinonts would normally have involved electrophorerir in polyacrylamide gel. However, the use of on immunological technique made it possible to screen up to 100 arcorpore isolates Per day with a high degree of rpecificity. The technique is described here since it may have general application in Neurorpora studier.
Numerous methods have been employed for the production of antibodies and generally require up to IO mg of pure antigen (II starting material. In the case of treholase, Z-IO mg of plre enzyme in 0.05 M phosphate buffer (pH 5.6) was emulsified with an equal volume of Freund complete adjuvont and injected intra-murcularly into rabbits over a period of 2 weeks. Booster injections contained approximately I mg of antigen in incomplete odjuvant. A maximum tihe was obtained in the third week and the animals were then bled by cardiac Puncture or from an ear vein.
The 20-30 ml of blood of blood obtained was oIlwed to clot in a Petri dish and, after standing overnight cl+ 4'C. the serum which had separated was decanted, centrifuged for 20 min at 3,000 x g to remove cells, and stored at -2O'C.

The Ouchterlony dwble diffusion method was used to screen extracts.
Degreased gloss slides (2 in x 2 in ) were first dipped in 0, .~%Ionagar"Z (in deionized water), drained and permitted to dry. 2 ml of 1% lonag& 12 (in 0.04 M veronol/HCI buffer, pH 7.S, + 0.003% methyl orange) war then poured to form an even foyer on one side of each slide: After storage at 4-C for I2 hr, one central and four peripheral wells were cut in each slide wing o template and cork-borer.
The ontisewm was placed in the central well and peripheral wells contained extracts of undesignated phenotypes alternoting with sampler of the electrophoretially identified irozymes "fart" or "slow". The extracts were prepared from acetone powdered mycelium os described previously for electropharerir (Yu et 01. 1971 Genetics 68:473) and IO pl sampler of ontirero and ontigeos were used throughout.
--Clear-cut differences between extracts of known "fast" and "slow" strains were obtained using antiserum prepared clgainrt trehalcrre from the wall fmction of "fast" strains, and such differences were onnulled by obrorption of the antiserum with purified "fort" trehalore. A total of 574 tre+recombinonts were screened by this method and only one yielded on ambiguous result on Ouchterlony plates. Electrophor~wor used to check 10% of the samples selected ot random and in alI cares the expected result WOI obtained. This work wal carried wt whilst the author was o Research Associate in the Deportment of Botany a+ the University of Michigan, and was supported by NSF grant GB 6811X to A. S. Surrmon, to whoni I om indebted for support and focilitier.
The cooperation of S. Liu and T. Jepron in the preporotion of antirero is gratefully acknowledged. ---Deportment of Botany, The Queen's University, Belfast, Northern Ireland.