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Text of tems. HAWTHORNE


    G. R. FINK AND c. A. STYLES Genetics, Development and Physiology, Cornell University, Ithaca, New York 14850

    Manuscript received January 11, 1974


    A selection procedure has been devised which allows the recovery of dele- tions of the his4 region of Saccharomyces cerevisiae. Many deletions were obtained by this procedure and were characterized genetically and biochem- ically. These deletions permit the construction of a linear map of the his4A and his4B region. A deletion which was large on this genetic map reduced the size of the his4 gene product. Tetrad analyses revealed that deletions can affect the conversion frequency of other alleles at his4. Analysis of reciprocal re- combination in conversion tetrads showed that the order of genes on chromo- some I11 is: centromere-EeuZ-his4A-his4C. This means that in his4 transcription and translation proceed away from the centromere.

    IABLE haploid strains carrying deletions are rare in yeast. Among 550 mu- tations induced at the hid locus by EMS, ICR-170, ultraviolet light (u.v.) ,

    nitrous acid, and N-methyl-N'-nitro-N-nitrosoguanidine, none was a deletion (FINK, unpublished). Even in systems where large numbers of spontaneous mu- tations have been selected (ROMAN 1956; JONES 1972) deletions are rare. There have been several reports of deletion mutants obtained by special selection sys- tems. HAWTHORNE (1963) reported a deletion of the mating type locus which was lethal in the haploid. Various selective procedures have produced viable de- letions of the cycl gene ( SHERMAN and STEWART 1973).

    In this paper we report a positive selection procedure for viable haploid strains carrying deletions in the his4 region in yeast. Many of the deletions appear to involve the loss of several hundred nucleotides. Analysis of tetrads from crosses in which strains carrying these deletions were mated to wild type shows that even large deletions undergo gene conversion. The deletion can be converted to wild type and the wild type can be converted to the deletion. In this respect deletions appear to be no different from single nucleotide changes. A deletion wholly within the his4 region does not affect the conversion frequency of a distal site in the region, whereas a deletion through the proximal end of his4 ( SHAFFER, EDEL- STEIN and FINK 1972) lowers the conversion frequency of the distal site. The use of deletions has enormous advantages for fine-structure studies because deletion mapping is more rapid and more accurate than the other generally available methods. In addition deletions should provide an invaluable tool in yeast for the elucidation of regulatory elements adjacent to structural genes.

    Supported by NIH grant, No. GM 15.108-07.

    Genebcs i'i: 231-244. June, 1974.

  • 232 G . R. F I N K A N D C. A. STYLES


    Yeast strains: The strains used in this study are derived from (1: S288C, which was obtained from h. R. K. MORTIMER. As described in a previous publication (FINK 1966), histidine- requiring mutants of yeast are unable to grow on histidinol in place of histidine. Mutations per- mitting growth on histidinol have been obtained in a gene called HOLI. Double mutants hisHOLI can grow on histidinol if they have a functional his4C region. All eight HOLI mu- tations in our collection are dominant in diploids, segregate 2:2 in crosses by wild type, and are unlinked to any of the known histidine genes.

    Genetic analysis: All media, procedures for sporulation, tetrad dissection, and scoring of genetic markers have been described by HAWTHORNE and MORTIMER (1960).

    Hislidinol dehydrogenase assay: A sensitive assay for histidinol dehydrogenase has been developed with the use of 3H-histidinol. Radioactive histidinol was converted to histidine by histidinol dehydrogenase, the two imidazoles were separated, and the amount of radioactive histidine was used as the measure of enzyme activity. 2.5 nanomoles of 3H-histidinol (75 mCi/ mmole), 3 pmoles of Tris-HC1 pH 8.9 and 70 pg of protein from a crude yeast homogenate were placed in a total volume of 50 pl. The reaction was allowed to proceed at 30" for 30 mintes and was terminated by addition of 4 81 of chromatography solvent. 20 pl of the reaction mixture was spcltted on Whatman 1 paper. The paper sheet was then placed in a solvent system contain- ing isopropanol: ammonia:H,O (70:2O:IO, vo1:vol:vol). Histidine and histidinol were located using non-radioactive standards as described earlier (FINK 1965). The histidine spots were eluted and the eluate was counted in a Nuclear Chicago scintillation counter. The amount of histidine produced by the reaction is proportional to time and enzyme concentration.

    Nonsense mutations of his4: Nonsense mutations were identified by analysis of suppression patterns. Two procedures were used to determine whether a strain carried a nonsense mutation in the his4 region. Both tests showed that only polar his4A and B mutations are nonsense. In the first test, strains carrying the his4 allele in question were crossed to strains carrying known nonsense suppressors. The diploids were isolated, sporulated, and the segregation of the sup- pressor and the his4 allele determined by tetrad analysis. The appearance of 3: 1 or 4: 0 segrega- tion (h i s+:h i s ) was taken as preliminary evidence for suppression. The putative double mu- tant, SUP his4, was backcrossed to wild type and shown to segregate both the suppressor and the his4 allele. In these studies SUP3, SUP4-I, SUP5 and SUPII , known ochre suppressors, and SUP4-3 and SUP7-2, known amber suppressors, were used (SHERMAN et al. 1973; MESSENGUY and FINK 1973). In the second test, multiply-marked strains were constructed containing the histidine mutation in question together with known ochre (Zeu2-I. arg4-17, l y s l - I ) and amber (metd-I, trpl-I , tyr7-I) mutations. These strains were reverted for one of the requirements and then tested for the presence of the others. Simultaneous reversion of the histidine allele together with the previously identified nonsense alleles was taken as evidence that the his4 allele was a nonsense mutation of the same type as the co-reverting allele (MORTIMER and GILMORE 1968).

    For many his4 mutations the assignment to either the amber or ochre class could be made unambiguously. Some his4 mutations gave less clear-cut results. When strains carrying metd-I, leu2-I, and either his4-619 or his4-I20 were reverted for their histidine requirement, a few of the revertants grew extremely slowly without leucine and without methionine. We have been un- able to confirm the existence of a suppressor in these strains because of spore inviability.

    Leu2-I seems to be suppressed by a greater variety of nonsense suppressors than other ochre alleles. Strains which are leu2-I his4-I7 arg4-17 lysl-I or leu2-I his4-I66 arg4-17 lys l - I , when reverted for leucine, co-revert at high frequency for histidine or for arginine and lysine. HOW- ever, simultaneous reversion of leucine, histidine, and lysine or leucine, histidine, and arginine, or leucine. histidine, arginine and lysine are extremely rare.

    Selection of deletions: The strategy employed in the selection of deletions depended upon several unique featuies of the his4 region. The A, B, and C segments control three different enzyme activities (Figure 1). These segments have some degree of functional autonomy since missense mutations in one of the segments (in A, for example) do not effect the enzymes spe- cified by the other two. The his4 region is transcribed and translated from A to C (Figure 1).


    A I B I C


    FIGURE 1.-The reactions controlled by the his4 region. The A,B,C designations refer to seg- ments of his4 responsible for catalyzing the 3rd, 2nd and loth steps (respectively) in the path- way of histidine biosynthesis. The basis for the selection (see text) is that polar his4A mutations prevent growth on histidinol. PRATP = phosphoribosyl-ATP; PRAMP = Phosphoribosyl- AMP; BBMII = N- (5-phospho-D-ribulosylformimino) -5-amino-1- (5 phosphoribosyl) -4-imidi- zolecarboxamide.

    By virtue of the polarity of translation and transcription from 5 + 3, a nonsense mutation in his4A has no his4B or his4C activity. Whereas mutants carrying a missense mutation in his4A or his4B can grow on histidinol, a strain carrying a polar his4A o r his4B mutation will not grow at all on histidinol. Deletions were obtained by selecting revertants of polar his4A mutants on histidinol. In theory, an in-frame deletion of the polar mutation could abolish polarity. Such deletions should allow growth on the histidinol plates so long as they do not extend into his4C. The efficiency of the selection system is low and presumptive deletions must be tested further for ability to recombine and revert. Growth on histidinol is often the result of nonsense sup- pression of the polar mutation or reversion of the polar to wild type or to a his4A missense mu- tation. In a typical experiment approximately 107 cells of a strain containing a polar his4 muta- tion were spread on the surface of a minimal f histidinol plate. The colonies were irradiated with U.V. light to give a survival of 20% and stored in the dark at 30 until colonies appeared. The plates were replica-plated to a minimal plate to identify revertants which still required histidine. Colonies which grew on minimal + histidinol but not minimal were picked, purified by streaking, and retested by replica plating.

    Recombination testing: In deletion mapping and allele identification in the gene conversion study, we have relied on mitotic reversion to prototrophy as a measure of the ability of two alleles to