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THE NUCLEOLUS ORGANIZER REGION of MAIZE (ZEA MAYS L.): TESTS for RIBOSOMAL GENE COMPENSATION OR MAGNIFICATION1~Z

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THE NUCLEOLUS ORGANIZER REGION of MAIZE (ZEA MAYS L.): TESTS for RIBOSOMAL GENE COMPENSATION OR MAGNIFICATION1~Z THE NUCLEOLUS ORGANIZER REGION OF MAIZE (ZEA MAYS L.): TESTS FOR RIBOSOMAL GENE COMPENSATION OR MAGNIFICATION1~z R. L. PHILLIPS, D. F. WEBERS, R. A. KLEESE4 AND S. S. WANG Department of Agronomy and Plant Genetics, University of Minnesota, Si. Paul, Minnesota 55101 Department of Biological Sciences, Illinois State University, Normal, Illinois 61761 Manuscript received December 6, 1973 ABSTRACT Ribosomal gene compensation and magnification that might be detected on a whole-plant basis was not found in maize. Plants monosomic for chromo- some 6 (the NOR chromosome) were compared with monosomic-8 and mono- somic-IO plants, disomic sibs, and parental lines. Assuming no rDNA com- pensation, monosomic-6 plants showed approximately the decrease expected in rRNA cistron number. Monosomic-8 had a normal ribosomal gene number, while monosomic-IO showed a decrease; but further documentation is needed. Besides demonstrating the absence of gene compensation, the results document our previous conclusion that maize chromosome 6 carries DNA complementary to ribosomal RNA. Further documentation was provided from studies with trisomic chromosome 6 plants showing proportional increases in ribosomal gene number. Progeny of the monosomic plants crossed as males to a standard singlecross hybrid possessed expected ribosomal gene numbers suggesting the lack of ribosomal gene magnification.-The ragged (rgd) mutant of maize, suspected of being deficient in rRNA cistrons, had a normal number. AMPLIFICATION of genes coding for ribosomal RNA occurs widely in the oocyte nuclei of animal species. Presumably the tremendous requirements for rRNA during early embryogenesis necessitates a mechanism for the rapid synthesis of large quantities of rRNA. In some cases rRNA is furnished by the nurse cells while the nucleus of the oocyte is relatively inactive (GALL1969). Multiple nucleoli are frequently present following amplification. Notable ex- amples are Triturus uiridescens and Xenopus Zaevis, in which the small nucleoli contain actively-synthesizing rDNA visualized by electron microscopy (MILLER and BEATTY1969). In Xenopus, for example, the redundancy of chromosomal rRNA cistrons is about 450 copies per haploid genome. Following amplification there is about 1500 times the original multiplicity, or 6.75 x IO5 extrachromo- somal copies per oocyte. The anucleolate mutant of Xenopus Zaeuis represents an apparent deletion of ' Paper No. 8522, Scientific Journal Series, Minnesota Agricultural Experiment Station, St. Paul, Minnesota. This work was partially supported by US. Atomic Energy Commission Contract AT(ll-1)-2121 to D. F. WEBER. Department of Biological Sciences, Illinois State University, Normal, Illinois 61761. *Present address: Department of Biology, Macalester College, St. Paul, Minnsota 55105. Genetics 77: 285-297 June, 1974. 286 R. L. PHILLIPS et al. the entire set of rRNA cistrons of the nucleolus organizer region (NOR). Such mutants are not expected in higher plant species due to the “gametophytic screen”; i.e., spores not receiving a nucleolus organizer are expected to abort. The special maize cytogenetic stock used in this study exhibits nondisjunction in post-meiotic divisions producing plants missing an entire nucleolus organizer. Bobbed (bb) mutants of Drosophila have been identified (RITOSSA.ATWOOD and SPIEGELMAN1966) as deletions of a portion of the rRNA cistrons in the nucleolus organizer. The common reversion of the bb phenotype to wild type is accompanied by a gain of rRNA cistrons (RITOSSA1968). This magnified condi- tion appears to occur in germ-line cells in males of a particular generation. In- dividuals of the subsequent generation receiving the magnified chromosome show reversion to wild type. Stability of the magnified condition depends upon association of the magnified chromosome with the other nucleolar chromosome. Instability ensues in individuals with a normal X chromosome but not in indi- viduals with a Y chromosome deficient in ribosomal genes. In 1971, TARTOFdiscovered that in an individual the absence of (1) a single nucleolus organizer segment (X/X,NO-) or (2) an entire wild-type NO chromo- some (X/O) results in an increase in the number of rRNA cistrons in the wild- type NO chromosome up to a multiplicity optimal for that particular strain (e.g., 250 rRNA cistrons amplified to 400) , This disproportionate replication. termed rDNA compensation (TARTOF1973), occurs in the X chromosome of somatic cells in both males and females. It involves more than a single cell type. TARTOF (1973) found the net increase in rDNA per NO inversely correlated with the number of rRNA cistrons in the opposite homolog. He also demonstrated that the rDNA of a Y chromosome is refractory to disproportionate replication. This does not appear to be the case for Y chromosomes carrying bobbed mutations (ATWOOD1969). Thus, the nucleolus organizers of the X and Y appear to respond differently to the lack of a second nucleolus organizer in the organism. Furthermore, TARTOF(1 973) found that when a bb+X was disproportionately replicated in bb+/O males, bb+/X, NO- females, or bb/Ybb- males, the increase in rDNA is generated at the level of somatic cells, and the increased rRNA gene numbers are not cumulatively inherited in subsequent generations. The rDNA of bb mutants is also capable of disproportionate replication in bb/X,NO- females and bb/Ybb- males. This increase occurs at the somatic cell level. The increased rRNA gene number in these cases is transmissible to subsequent generations. SPEARand GALL(1 973) reported that ribosomal gene compensation occurred only in polytene nuclei of Drosophila, and not in the diploid cells analyzed. Experiments reported here were designed: (1) to determine if ribosomal gene compensation and/or magnification occurs in maize; (2) to further document that chromosome 6 carries rRNA cistrons, as reported by PHILLIPS,KLEESE and WANG(1971); and (3) to determine if a ragged (rgd) mutant phenotype is the result of a partial deficiency of the rDNA. MATERIALS AND METHODS Strains: Monosomic progeny were generated at Illinois State University by crowing plants heterozygous for the r-Xi deficiency as the female parent to the Mangelsdorf Multiple Chromo- rDNA COMPENSATION IN MAIZE 287 some Tester strain. This strain carries a recessive marker in each of the ten maize chromosomes (bm,, lg,, a,, su,, pr, y, gll, jr, wx, g). Nondisjunction in r-XI deficiency heterozygotes occurs post-meiotically during divisions forming the eight-nucleated embryo sac. Singly, doubly, and triply monosomic maize plants were obtained at low frequencies by this method (WEBER1970 and 1973). The genetic background of the r-XI deficiency is that of inbred W22. The Mangels- dorf Tester has a different genetic background. The r-Xi-carrying line and Mangelsdorf Tester were originally obtained from K. SATYANARAYAMAof the University of Wisconsin. The single- cross W23 x L317 used as the female parent in crosses with the monosomic plants was obtained from the Maize Genetics Cooperative, University of Illinois, Urbana. Trisomic chromosome 6 plants were obtained from the Minnesota collection. They were iso- lated by K. E. MICHELby crossing triploid plants from 2n x 4n crosses with Mangelsdorf Tester. The resultant plants were backcrossed to the Mangelsdorf Tester for genetic identification of the trisomics. The trisomics were then crossed and backcrossed twice to inbred A188. Seed of the rgd mutant was obtained from ELLENDEMPSEY, Indiana University, Blooming- ton. DNA/rDNA hybridization: Procedures followed those reported by PHILLIPS,KLEESE and WANG(1971) with the following modifications: (a) plant material was ground in a Waring blender instead of a mortar; (b) weightjvolume ratio of fresh plant material to buffer was 1:2 instead of 1:l; (c) the pellet was shaken for 30 minutes in 0.05 M Tris, 0.015 M EDTA, 0.15 M NaC1, pH 9.0 instead of being homogenized with a Ten Broeck homogenizer; (d) the Bay- covin was omitted; and (e) the DNA extract was dissolved in 0.1 M NaCl in 0.05 M phosphate buffer, pH 6.7 instead of 0.1 x SSC before being placed on the MAK column. In certain cases DNA was extracted from single plants at the stage of development micro- sporocyte samples are normally taken for cytology. Approximately 100 grams fresh weight was obtained by using the upper portion of the plant, but excluding the tassel in the experiments involving monosomics (the tassel was included in experiments involving trisomics) . The pro- cedure otherwise followed that described above except that the extract was centrifuged at 8,000 X g instead of 5,000 x g for 10 minutes after it was shaken in two volumes of chlorofom- isoamyl alcohol (24: 1). Just prior to placing the sample on the MAK column, the DNA extract was reprecipitated in cold ethanol and redissolved in 0.1 x SSC several times to free the DNA solution from the pigment these samples usually contained. Two radioactivity determinations were always made per DNA sample per concentration of 3H-rRNA. The background level, determined by using filters not impregnated with DNA but otherwise treated like those with DNA, was subtracted from each determination before plotting. The t test was used to test the significance of the various comparisons. The estimates of rRNA cistron number (see PHILLIPS,KLEESE and WANG1971) are based on a DNA/2C-nucleus value for maize of 10.07 x 10-12 g. This value is derived by altering MCLEISHand SUNDERLAND’S(1961) chemically measured value of 15.5 x 10-12 g with VAN’T HOF’S(1 965) formula. This corrects for the fact that some cells are 2C and some 4C in a popula- tion of somatic cells. The durations of the various parts of the maize nuclear cycle needed for VAN’THOF’S formula were taken from an extensive study by VERMA(1970). Estimates of rRNA cistron number are a function of the amount of DNA/2C-nucleus. The particular DNA value assumed when attempting to compare rRNA cistron numbers reported in various papers must be noted.
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