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 (GALL 1969). 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 BEATTY 1969). 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 t o 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 SPIEGELMAN 1966) 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 (RITOSSA 1968). 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, TARTOF discovered that in an individual the absence of (1) a single nucleolus organizer segment ( X / X , NO-) o r (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 (TARTOF 1973), 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 (ATWOOD 1969). 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, o r 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.

SPEAR and 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 - X i deficiency as the female parent to the Mangelsdorf Multiple Chromo-

r D N A COMPENSATION I N 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, j r , 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 (WEBER 1970 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. SATYANARAYAMA of 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. MICHEL by 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 ELLEN DEMPSEY, 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 a t 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 WANG 1971) are based on a DNA/2C-nucleus value for maize of 10.07 x 10-12 g. This value is derived by altering MCLEISH and 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’T HOF’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.

Estimates of rRNA cistron number for a particular strain are different in the various ex- periments reported in this paper. The major experiments utilized different extractions of labelled rRNA. There may be errors associated with determining the specific activity of the rRNA. This presents no problem in our interpretations, however, since we are interested in relative compari- sons (not necessarily the absolute values) in any one experiment.

RESULTS A N D DISCUSSION

Gene compensation The reported finding of ribosomal gene compensation in X / O Drosophila males

prompted us to investigate monosomic chromosome 6 maize plants. The origin

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FIGURE 1.-Saturation curves of DNA from monosomic and disomic adult plants. 0 = one Mangelsdorf Tester plant, A = one monosomic-8 plant, A = average of three disomic plants (en sibs) = one monosomic-10 plant, 0 = average of three monosomic-6 plants. Limited amounts of DNA of Mangelsdorf Tester, monosomic-8 and-IO and disomic plants were available. Only two concentrations of 3H-rRNA were used. Two determinations were made per plant per concentration of 3H-rRNA. Since monosomic plants carry one less chromosome per nucleus, the amount of DNA per nucleus was reduced 5 % in calculating the number of rRNA cistrons (with the exception of one monosomic-6 plant which was trisomic for an unidentified chromosome). Counting efficiency was approximately 56% and specific activity of 3H-rRNA (from inbred AI 88) was 2000 cpm/pg. Hybridization plateau of Mangelsdorf Tester is significantly different (.05 level) from monosomic-8, which is significantly different (.01 level) from the disomic (2n) progeny. The 2n plateau is not significantly different from monosomic-10. Monosomic-10 is sig- nificantly different (0.1 level) from monosomic-6.

r D N A COMPENSATION IN MAIZE 289

of chromosome 6 must be considered to derive the expected ribosomal gene num- ber. The monosomics were generated in crosses between R/r -XI heterozygotes as the female parent (since r-XI causes nondisjunction during embryo-sac devel- opment) and the Mangelsdorf Multiple Chromosome Tester. The Mangelsdorf Tester parent contributed chromosome 6 in the resultant monosomic-6 plants. Assuming that all of the ribosomal genes are on chromosome 6 and that there is no compensation, the monosomic plants should possess half the number of ribo- somal genes carried by the male Mangelsdorf Tester parent. This number may be somewhat different than half the number of a disomic sib if the two parents involved in the cross have different numbers of rRNA cistrons, as in this case. Agronomically desirable maize inbreds can vary in their rRNA cistron number over a range of 4700-8200 (PHILLIPS et aZ. 1973). The rRNA cistron number would be higher than expected if ribosomal gene compensation occurs in mono- somic-6 plants.

The average rRNA cistron numbers for the various strains may be summarized as follows:

Monosomic-8 8600 2n sibs (from r/r-XI kernels) 7800 Monosomic-I 0 6600 Monosomic-6’s 5100 Mangelsdorf Tester 9400 R/r kernels from [ (R / r -XI in W22) x Mangelsdorf Tester] 8800 (R / r -XI in W22) sib and ( R / r in W22) sib 7400

One would expect monosomic-8, -10, and the diploid sibs to be intermediate be- tween the two parents of the cross. The average of the two parents is 8400 (aver- age of 9400 and 7400). The actual F, plants (Figure 2) gave a value of 8800, slightly higher than expected. Therefore, we assume that the ribosomal gene number estimates for monosomic-8, -10, and the diploid sibs to be about 8400- 8800, considering the expected and observed results. The estimate for mono- somic-8 is within this range and the diploid sibs were only slightly lower. Monosomic-10 appears to be reduced in gene number, although not as dramat- ically as in monosomic-6 plants.

The results displayed in Figures 1 and 2 show that monosomic-6 plants possess approximately the number of ribosomal genes expected with no compensation. The observed number is only slightly above half that of Mangelsdorf Tester. This result provides further documentation that chromosome 6 carries DNA complementary to ribosomal RNA. The single monosomic-I 0 plant analyzed contained a reduced number of rRNA cistrons. It is unlikely that chromosome 10 carries DNA complementary to ribosomal RNA because the other data indicate that most or all rRNA cistrons are located in chromosome 6. One possible ex- planation for this apparent discrepancy could be that the monosomic-10 plant was a sectoral doubly monosomic plant, and although root-tip samples showed 19 chromosomes, chromosome 6 was lost from part of the plant.

Gene magnification If ribosomal genes in the monosomic-6 plants are increased in number in cells

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FIGURE 2.-Saturation curves of DNA from parental lines used in generating the monosomics, as well as the F, between the parents. A =Mangelsdorf Tester (single, adult plant)-same data as presented in Figure 1, =results averaged from t w o experiments, one using DNA from [ (R/r -XI in W22) x Mangelsdorf Tester] and one using DNA from [ ( R / r in W22) x Mangelsdorf Tester], 0 = results averaged from two experiments, one using DNA from ( R / r in W22) sib and one from (R/r -XI in W22) sib. DNA from Mangelsdorf Tester and the W22 line was limited in amount; therefore, only two concentrations of 3H-rRNA were used. Counting efficiency was approximately 56% and specific activity of 3H-rRNA was 2000 cpm/@g. Mangels- dorf Tester is significantly different (.05 level) from W22 x M. T., which is significantly dif- ferent (.01 level) from W22.

rDNA COMPENSATION I N MAIZE 29 1

giving rise to reproductive tissues and the condition is heritable, the progeny of a monosomic-6 x normal cross would carry a "magnified" nucleolus organizer chromosome and possess more than the expected number of ribosomal genes. This possibility was tested (Figure 3) by investigating progeny of monosomic-6, -8 and -10 plants crossed as males with a normal singlecross hybrid (W23 X L317). The progeny would all be 2n since only gametes from the monosomic parent carrying the monosomic chromosome are viable.

Estimates of rRNA cistron numbers of the various strains used in this experi- ment (Figure 2) may be summarized as follows:

Mangelsdorf Tester 8100 (R/r-XI in W22) sib 5300 (W23 X L317) F, 4800 (W23 X L317) X monosomic-6 5300 (W23 X L317) X monosomic-8 5300 (W23 X L317) X monosomic-10 5700

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FIGURE 3.-Saturation curves of DNA from progeny of crosses using monosomic plants, as well as parental lines. DNA obtained from 6-day-old seedlings. A = Mangelsdorf Tester, A = (W23 x L317) x monosomic-IO, = (W23 x L317) x monosomic-6, (W23 x L317) X mon- osimc-8, and ( R / r - X I in W22) sib, 0 = (W23 x L317) F,. Specific activity of 3H-rRNA (from inbred A188) was 3000 cpm/,ug at approximately 40% counting efficiency. All plateaus are significantly different (.01 level).

292 R . L. PHILLIPS et al.

Ribosomal gene magnification apparently did not occur since the progeny of crosses between monosomic-6, -8, and -10 plants all carried approximately equiv- alent levels of rRNA cistrons.

One can estimate the expected values of the progeny of various monosomics utilizing the parental values (see Figure 3 o r the above tabulation). Considering the (W23 x L317) x monosomic-6 progeny, the rRNA cistron contribution from (W23 X L317) is about 2400 copies and that from the monosomic-6 parent would be 4050, since only gametes with a chromosome 6 are viable and the chromosome 6 originally came from Mangelsdorf Tester (see Figure 3). The progeny should possess approximately 6450 ribosomal genes. The explanation for the lower ob- served value (5300) is unclear at this time. The deviation is opposite from any deviation that might be expected due to ribosomal gene magnification.

Considering the (W23 x L317) x monosomic-8 and x monosomic-10 prog- eny, (W23 x L317) is expected to contribute approximately 2400 ribosomal genes. Since the monosomic plants are heterozygous for a chromosome 6 from the Mangelsdorf Tester and one from the (R/r-XI in W22) parent, they should transmit. in equal frequencies, gametes carrying 4050 or 2650 ribosomal genes. Consequently, the rRNA cistron number in the progeny would be expected to average 5750 (average of 2400 1- 4050 and 2400 f 2650). The observed values m e close to this value for the monosomic-8 cross (5300) and extremely close for the monosomic-10 cross (5 700). No indication of ribosomal gene magnification in progeny of monosomic-6, -8, or -10 plants was evident.

Further documentation that chromosome 6 carries rRNA cistrons Trisomic chromosome 6 plants and their disomic sibs were compared to de-

termine if the number of rRNA cistrons increased in an additive fashion. This would provide further documentation that chromosome 6 carries ribosomal genes. The data displayed in Figure LE show that trisomic chromosome 6 plants carry additional copies of rRNA cistrons. The average number was approximately 11,000 fo r the trisomics and 6900 for the disomic sibs. These observed values closely fit a 50% increase in ribosomal gene multiplicity in the trisomics as com- pared with the disomics. The variation within the disomic group of plants and the trisomic group of plants is probably due to the fact that they were second- backcross progeny( to inbred A188). Since Mangelsdorf Tester was involved in the initial isolation of these trisomics and has a much higher ribosomal gene number than A188, segregation of a rather large magnitude would not be sur- prising, although trisomics would still be expected to have higher ribosomal gene numbers than disomics.

The proportional decreases and increases in monosomic and trisomic plants, respectively, as compared with controls, strongly support the conclusion that the NOR of maize contains DNA complementary to ribosomal RNA (PHILLIPS, KLEESE and WANG 1971 ) . I s ragged (rgd) a partial deficiency of ribosomal genes?

Several features of the recessive rgd mutant of maize suggested that i t may be the result of insufficient rRNA cistrons, comparable to the bobbed mutants of

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FIGURE 4.-Saturation curves of DNA from trisomic-6 and diploid adult plants. 0, A, and A = three different trisomic-6 plants, and 0 = two Werent diploid sibs. Amount of DNA per nucleus was increased 5% for the trisomic plants in calculating the rRNA cistron number. Specific activity of "-rRNA (from inbred A188) was 2000 cpm/pg at approximately 56% counting efficiency. All plateaus are significantly different a t .01 level, except for the two diploid plateaus which are significantly different at .05 level.

Drosophila. Firstly, the rgd gene maps as the most distal marker in the short arm of chromosome 6 and is expected to be close to if not in the NOR. Tests with the translocation TB-6a (PALMER and DEMPSEY 1968) suggested that the rgd gene is not in the distal portion of the NOR (i.e., beyond the center of the heterochro- matin). The locus could be in the proximal portion of the heterochromatin. Sec- ondly, homozygous rgd plants are weak. They usually do not emerge if planted in soil. The mutants may be observed if germinated in paper toweling or on filter paper in a petri dish. The first leaves appear as narrow strips of discon- nected tissue. These plants commonly survive only a few days. Thirdly, an oc- casional rgd plant will develop to maturity, but the tassel and ear are composed of abortive tissues.

294 R. L. PHILLIPS et al.

The three F, genotypes (considering the rgd gene) from a Y r g d / y + heterozy- gote can be separated by using a linked yellow endosperm ( Y ) genetic marker. The recombination frequency between Y and rgd was reported by KRAMER (1959) to be only 9.0%. Normal plants in the yellow (Y-) F2 seed class from self-pollinated Y t g d / y + heterozygotes were assumed to be heterozygotes, while the normal plants in the white ( y y ) class were assumed to be homozygous nor- mal. The results presented in Figure 5 show that rgd is not the result of a de- ficiency of ribosomal genes. A repeat of the original experiment yielded similar results, also showing that the rgd/rgd plants possess significantly more ribosomal gene than heterozygous or homozygous normal plants. These results indicate that

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FIGURE 5.-Saturation curves of D N A from rgd/rgd, r g d / f , +/+ 6-day-old seedlings. A = rgd/rgd, = rgd/+, 0 = +/+. Each curve is the average of two experiments each with two determinations per concentration of 3H-rRNA. D N A from rgd/+ seedlings was limiting; there- fore, only three concentrations of 3H-rRNA were used. Specific activity of 3H-rRNA was 5000 cpm/pg at approximately 40% counting efficiency. Hybridization plateau for rgd/rgd is sig- nificantly different from red/+ and +/+ at .05 and .01 levels, respectively.

r D N A COMPENSATION I N MAIZE 295

the chromosome 6 carrying the rgd allele has more ribosomal genes in its NOR than the one carrying the +rga allele. As a result of linkage between rgd and the ribosomal genes of the NOR, the rgd/rgd homozygote possesses more ribosomal genes than the other genotypes.

Cytology of root-tip cells possessing two nucleoli in plants heterozygous for rgd revealed two nucleoli of similar size. If the mutant resulted from a deficiency of ribosomal genes, unequal-sized nucleoli might be expected. Nucleoli in micro- sporocyte cells at mid-pachynema of two +/+ plants and two t+/rgd plants were measured (see PHILLIPS, KLEESE and WANG 1971 for technique). The average nucleolar diameter was 11.9 p in +/+ plants and 13.5 p in ,+Jrgd plants. These cytological observations support the conclusion that the ragged phenotype is not the result of a deficiency of ribosomal genes.

Hypo thesis

Ribosomal gene compensation and magnification phenomena that could be detected on a whole-plant basis did not occur in these studies. Why might maize be different than Drosophila? Firstly, compensation in a normal, complete nu- cleolus organizer may only occur in Drosophila cells with polytene chromosomes (SPEAR and GALL 1973). The only polytene cells reported in maize appear to be in the endosperm (TSCHERMAR-WOESS and ENZENBERG-KUNZ 1965) and this tissue was not used in our studies. Secondly, maize appears to have a very large number of rRNA cistrons per nucleus, which varies among lines. Our estimates of rRNA cistron number for various agronomically desirable inbred lines of maize range from at least 4700 to 8200. This large range in itself might suggest that several of the ribosomal genes are not needed. In addition, cytogenetic studies of (1 ) approximately 20 homozygous chromosomal interchanges with a break in the NOR and (2) duplications of the NOR suggest that the heterochromatic portion of the NOR observed at pachynema includes “inactive” rRNA cistrons (PHILLIPS et a2. 1973; GIVENS and PHILLIPS 1973) at least in terms of nucleolar organization. It is possible that maize normally carries in the NOR several thousand rRNA cistrons in an inactive state. If there is a need, these inactive genes can become active. Thus, there is no need for increasing the number of rRNA cistrons, only a need to activate already existing ones. This interpretation is consistent with X/ICCL~NTOCK’S (1 934) observation that the proximal two- thirds of the NOR heterochromatin when not accompanied by the distal-NOR portion can develop a normal-sized microspore nucleolus. The proximal portion developed a small nucleolus when accompanied by the distal portion at pachy- nema. At least the proximal two-thirds of the heterochromatin has the potential to become active under certain conditions. The large number of ribosomal genes in maize may have been generated by unequal crossing over.

INGLE and SINCLAIR (1972) checked for ribosomal gene increases in maize during seed germination, since there is such a rapid rate of RNA synthesis during this period. They found no change in rRNA cistron number in dormant embryos compared with embryos after 48 hours of germination.

Although certain cell types of maize still may contain increased numbers of

296 R. L. PHILLIPS et aE.

ribosomal genes, there is no evidence from our studies that gene compensation occurs at a level detectable on a whole-plant basis. The monosomic-6 plants studied had an average of 5100 rRNA cistrons, which is still as much as certain inbred lines of maize. Perhaps a more critical test for ribosomal gene compensa- tion would be to assay monosomics with considerably lower ribosomal gene Rum- bers. For the extraction o€ such a monosomic, the incorporation of a chromosome 6 seedling marker into a low gene number line would be required in our system. Such a stock is not yet available. This test may not be conclusive because we may need to be concerned with the number of active ribosomal genes. not the total number of ribosomal genes. No methods have been developed to date to dis- tinguish between the number of hypothetically active versus inactive ribosomal genes in normal stocks.

LITERATURE CITED

GALL, J. G., 1969

GIVENS, J. F. and R. L. PHILLIPS, 1973

The genes for ribosomal RNA during oSgenesis. Genetics (Suppl.) 61: 121- 132.

Localization and estimation of the number of active versus inactive rDNA cistrons within the nucleolus organizer region of Zen mays. Genetics 74: ~ 9 4 4 9 5 (abst.).

INGLE, J. and J. SINCLAIR, 1972 Ribosomal RNA genes and plant development. Nature 235: 30-32.

KRAMER, H. H., 1959 Gene order of y, ms-si, and rg on chromosome 6. Maize Genetics COOP. News Letter 33: 102-103.

MCCLINTOCK, B., 1934 The relation of a particular chromosomal element to the development of the nucleoli in Zea mays. Z. Zellforsch. Mikroskop. Anat. 21 : 294-328.

MCLEISH, J., and N. SUNDERLAND, 1961 Measurements of deoxyribosenucleic acid (DNA) in higher plants by Feulgen photometry and chemical methods. Exptl. Cell Res. 24: 527-540.

MILLER, 0. L., JR. and B. R. BEATTY, 1969 Extrachromosomal nucleolar genes in amphibian oocytes. Genetics (Suppl.) 61: 133-143.

PALMER, R. G. and E. DEMPSEY, 1968 Cytological location of rgd on chromosome 6. Maize Genetics Coop. News Letter 42 : 75-77.

PHILLIPS, R. L., R. A. KLEESE and S. S. WANG, 1971 The nucleolus organizer region of maize (Zea mays L.) : chromosomal site of DNA complementary to ribosomal RNA. Chromosoma 36: 79-88.

The nucleolus organizer region (NOR) of maize: a summary. Genetics 74: 5212 (abst.).

Unstable redundancy of genes for ribosomal RNA. Proc. Natl. Acad. Sci. U.S. 60: 509-516.

A molecular explanation of the bobbed mutants of Drosophila as partial deficiencies of “ribosomal” DNA. Genetics 54: 819-834.

Independent control of ribosomal gene replication in polytene chromosomes of Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S. 70: 1359-1363.

Increasing the multiplicity of ribosomal RNA genes in Drosophila melano- gaster. Science 171: 294-297. ~ , 1973 Regulation oE ribosomal RNA gene multi- plicity in Drosophili melanogaster. Genetics 73: 57-71. __ , 1973 Unequal mitotic sister chromatid exchange and disproportionate replication as mechanisms regulating ribo- somal RNA gene redundancy. Cold Spring Harbor Symp. Quant. Biol. 38: 49-500.

PHILLIPS, R. L., S. S. WANG, D. F. WEBER and R. A. KLEESE, 1973

RITOSSA, F. M., 1968

RITOSSA, F. M., K. C. ATWOOD and S. SPIEGELMAN, 1966

SPEAR, B. B. and J. G. GALL, 1973

TARTOF, K. D., 1971

r D N A COMPENSATION IN MAIZE 29 7

Die Struktur der hoch Endopolyploiden Kerne in Endosperm von Zea muys. Das auffallende Verhalten ihrer Nucleolen und ihr Endopolyploidiegrad. Planta. 6p: 149-1 69.

VAN’T HOF, J., 1965 Relationships between mitotic cycle duration, S period duration and the average rate of DNA synthesis in the root meristem cells of several plants. Exptl. Cell Res. 39: 48-58.

VERMA, R. S., 1970 Nuclear cycle in Zea mays L. root tips. Maize Genetics Coop. News Letter 4.4.: 192-195.

WEBER, D. F., 1970 Doubly and triply monosomic Zea mays. Maize Genetics Coop. News Letter 44: 203. -, 1973 A test of distributive pairing in Zeu msys utilizing doubly mono- somic plants. Theoret. and Appl. Genet. 43: 167-173.

Corresponding editor: B. D. HALL

TSCHERMAK-WOESS, E. and U. ENZENBERG-KUNZ, 1965