PHASE VARIATION OF REGULATORY ELEMENTS IN MAIZE

PETER A. PETERSON

Agronomy Department, Iowa State University, Ames

Received March 4, 1966

HASE variation of regulatory elements in maize refers to changes in function- ing of these elements from periods of activity to periods of inactivity or vice

versa (PETERSON 1965a). For example, a case of pattern variation representing alternative phenotypes (presence or absence of mutability) was observed recently in aleurone tissue. This paper deals with an analysis of the “flow” and ‘‘cr(yw11’’ phenotypic patterns of mutability associated with the mutable a, allele, which is known to be a part of the two-element regulator-controller system that includes the regulatory element, Enhancer, E n (PETERSON 1960,1961).

In studies of the Ac-Ds system, MCCLINTOCK (1947, 1951) showed that the expression of alternative phenotypes (representing a mutation event) during growth and development of a tissue reflected the transposition of the inhibitory controlling element Ds away from the affected locus. Such mutational changes associated with the movement of a transposable element have been reported for M p at the P locus (BRINK and NILAN 1952; BRINK 1954), for Spm (MCCLINTOCK 1956, 1957,1961), E n (PETERSON 1965c) and Dt (NUFFER 1961) at the aI locus, and for a factor affecting the Ab allele (NEUFFER 1963). In Salmonella, the move- ment of an episomic element, comparable to the transposition of controlling ele- ments in maize, is also associated with a mutation event (DAWSON and SMITH- KEARY 1963).

In the above cited cases, the mutation event is associated with transposition of the element which controls the expression of the affected locus. Phase variation implies a phenotypic change that is not to be ascribed to transposition of the inhibitory element but to the inception or termination of its activity. Further examination of this concept will follow the analysis of the “flow” and “crown” pattern types (PETERSON 1965b).

MATERIALS A N D METHODS

Description of the alleles, phem:’ypes and testers: Each of the alleles discussed, the associated symbols and hypothetical designations are listed and described in Table 1.

The a,m(frow) allele: This allele originated as a variant of the original mutable a, allele- a,m(denre) (Figure lG)-in the E n system (PETERSON 1956, 1961). The mutable allele, ~ , m ( d c n s e ) , has been the source of most of the pattern types that represent different states of mutability (Figure 1, A-J). These exceptions include patterns differing in the time and frequency of mu- tation (Figure 1). Examples of the uniformity in the expression of states in two ears are shown in Figure 2. The mutability pattern of a l m ( f z o w ) is characterized by a high frequency of late- occurring mutations. This allele would be designated as ~ ~ m ( 1 . h . ) because of the fine or small size (late occurrence) of the colored spots and their high frequency were it not that mutations of this

Genehcs 54: 249-266 July 1966.

250 P. A. PETERSON

- c

?

L

7 - L L c

% f

II II I I I I I I I I I / 1 1

J ~

!

FIGURE 1 .-Comparison of niutablr a, alleles wprewnting states of mutability. Small-sized, colored dots represent late occurrences of the mutation evcnt. Mutation frequency is indicated by number of dots. A us. B, C us. D. E 11s. F are paired comparisons showing the effect of the Same En state on different a,"(*) alleles. G-J represent additional states of mutahility.

252 P. A. PETERSON

FIGURE 2.-T\vo ears sho\ving uniformity in expression of distinct stiitrs of mutiil)ility.

FIGURE 3.-The u,m(/lOlo) pattern.

PIIASE V A R I A T I O S IS 11A17.E 253

allrlr arr rrstrictrtl to thr tiazal ~iortions of thr kwnrl and thrrr arr na ralorrcl dots in thr crown (I:igiirr 3 ) . I n zoni? piirs. thr rxtent of this niutahility a t thr hnsr rarirs. Variants of thr original ( ~ ~ ' ~ l ~ / l ~ j ~ J rangcl from thosc that arr rolorlrsz without clots to those that arr "drnsr" (IGgurr 4). 1:uIly "clrnw" rxrrptions (dark typrs in Icigiirr 4) h a w hrrn trstrcl. antl most of thrni continue to srgrrgatr "flow" (Tahlr 2 R ) . 'I'hosr that c!o not rrgrrgatr arc similer to t h r original "tlrnsr" that is rharartrrizrrl hy a high ratr of changi~ to n l " " ' ~ r ~ (Tahlr 2D and PETERSON 1061).

T/rc "croiiv" p / t m o / y p c : 'I'hr stiitr of niiitahility of "crown" is. l i k r "flow." rharartrrizrtl by a high frrqiiwcy of Iiitr orcuring rnutat:on

P. A. PETERSOh.

rarirgatrd (no En) and rarirgatctl (En) krmrls). The second En trstrr. aI"I-J. givrs a stahle pnlr rolorrd alruronr (Figure fi. shrunken. full-colorrd) (M&I.INTOCK 1956) in the ahsrnccs of En. hut prnrlurrs alruronr with dots of rolor on a colorlrss harkgmund in the prrsrncr of En 1 Figurr 6. shrunkrn. rarirRatrtl ) . IVhm ri thrr stork is outcrossed antl niutahility rrsults. the trstrtl strain carries En.

Thc "En /inc'*-n rriutnhili~y tcstrr: T h r "En linr" with thr grnotypr. nld"sh/nld"sh, En/-. is usrtl to tcst rxcrptional colorlcsc krinrls for thcir ahility to rrcponcl to En. Since En caiisrs no rhangr in the phrnotypr of thr krrrirls of thr "En linr." contimiation of its presencr in all "En linr'' plants is madr hy crossrs to En trstrr plants. T h r numhrr antl location of En rlrmrnts in a girrn strain are uncertain owing to frrqurnt transpositions. and thrrrforr the constitution of thr En linr will hr drsipintrtl as En/-. Thr i r prrsrncr in an En line. howrrer. is undrniahlr in crossrs to an En trstrr (rxamplr. Figiir? 6 ) . T h r o l d f nllrlr is colorlrss and dotlcss (shrunken krrnrls in Figurr 2A) in thr prrsrncr of En sinrc i t wspontls only to DI (RIIOADFS 1938. 1Wl). Thr asso- riatrd shrunkrn ( sh , ) markcr. hrrraftrr d?signatrd ( sh) . is closrly linkrd to o f .

T h r tpstcross parent: T h r grnotypr of thr rrrurrent trstcross parrnt is n,~"sh/nIdfsh (without En). Any changes nrrurring in thr progeny of trstcrossrs of "flow" and "crown" will he revrnlrd sinre thr nld' nllrlr. as pnintrcl out ahorr. rrsponds only to DI. which is nhsrnt in all cmsws.

RESULTS

The ''flou~'' pottern. ( a ) . Inheritance of the allele governing the "flow" pattern: In the testcross progeny of the "flow" heterozygote. oJ"l("ntrJ Slt/oJ"'sh. the range

PHASE VARIATION I N MAIZE 235

FIGURE 6.-An ear culture from the cross n,”t‘r)Sh/nI’’’-I~h. En/- x nIdfdz/a,df showing the effect of En on u,”’(r) and a,”I-’ alleles. Nonvariegated, non-shrunhen--cl,”’“)Sh - no En; variegated, non-shrunken-d,m(rjSh - En; full colored. shrunken-a,’*+’sh - no En; varie- gated. shrunken-u,”’-Jsh - En.

of mutability expressions among the non-shrunken progeny of the different crosses (Table 2A and Figure 4) includes a very dark type similar to “dense,” “flow,” and colorless kernels without any dots. In order to test whether or not an independent En could be involved in the origin of these three types. 115 plants from nonvariegated, shrunken kernels (number tested from each cross is in parenthesis in Table 2A) were crossed to an En tester. If the diversity in pheno- typic expression was due to an independent En, one half or more of the crosses would give a positive test for the presence of En. In four of the tests, a “weak” regulatory element En’ (see Table 1) was detected ( in Table 2A), but no true En action was evident in the remainder. The absence of an independent En

25 6 P. A. PETERSON

TABLE 2

Segregations and tests of the “flow” allele

Crosses

Kernel phenotypes

Variegated - Non-shrunken

Nonvariegated ~ “dense” or

non-shrunken shrunken “near-dense’’ “flow” Shrunken

A. ‘‘flow” non-shrunken testcrossed. a l m f f I o w ) Sh/a,dtsh 1 2 127(12)1 2 2 82(12)* 3 0 159(16) 4 0 176(15)* 5 0 155(14) 6 12 166(30)+ 7 8 122 8 9 176 9 1 115(5)

10 2 138(4) 11 1 168(7)

B.

C.

D.

Testcross of selected types from A. alm(frow) Sh/a,dtsh 1. A6 non-shrunken, nonvariegated 18 8 2. AI non-shrunken, nonvariegated 3 8 3. A2 non-shrunken, nonvariegated 2 8 4. A6“flow” 8 2 5. Ae‘dense” 2 $ 6. A10 “dense” 0 8 7. A10 ‘Ldense” 0 8 8. A l l “dense” 0 8 Tests of selected progeny phenotypes from crosses listed in A 1. “flow” (A7) x nonvariegated

2. ‘‘flow’’ (A8) x nonvariegated shrunken (A9) 13 153

shrunken (A10) 3 179

20 62 83 80 0

67 2 1

98 127 174

31 7 6

13 75

112 132 1 04

3

2 ~I

Four changes to alm(dense) derived from the cross of aIm(frow) hybrids by En lines (a,m(dense)Sh/aldtsh X a,dtsh/aldtsh En/--, En/-) 1 19(4) $ 162 2 21(5) 2 184 3 18(3) 8 133 4 24(6) 2 130

112 21 84

1 02 1 62 97

117 174 21 13 3

102 115 138 92 28 42 29 11

152

187

0 0 0 0

0 0 0

0 0 0 0 0 0 0

1 ll

$ $ 2 $ 2 $ 2 $

0

0

8 8 $ 8

* or t indicate the source of independent En arising in “flow” hybrids. $ Shrunken not counted.

f I The variegated shrunken class is a minimal count since the loose nature of the shrunken kernel along with the infe- Numbers in parentheses include those crossed by an En tester and found not to contain En. qnency of dots makes the detection of this class difficult.

indicates that the control o€ the Ylow” phenotype resides at or near the a, locus. From the progeny of the same crosses, individual kernel phenotypes were selected and the resulting plants testcrossed to determine the heritable features of these phenotypes. In testcrossing non-shrunken colorless segregants from crosses 1, 2, and 6 in Table 2A, the resulting progeny included “flow” and “dense” phenotypes (crosses 1,2 and 3 in Table 2B). Crosses 4,5, 6, 7 and 8 in Table 2B demonstrate the kinds of segregations that result from tests of “flow” and “dense.” In order

PHASE VARIATION IN MAIZE 25 7

to test the effect of genetic modifiers on the propensity for the C C f l ~ ~ 7 ’ expression to become “dense,” plants containing the C ‘ f l ~ ~ ’ 7 allele from crosses that gave only a few “dense” (cross 7, Table 2A) were crossed by the nonvariegated, shrunken segregants of crosses that gave a large number of “dense” (cross 9, Table 2A). These crosses (1 and 2, ?able QC) result in a segregation pattern similar to the segregation of the “flow” allele used in the cross. In 22 other similar tests that are not listed, the same result is evident. This series of crosses demonstrates that the control of mutability in a lm( f zow) resides at or near the locus. It is likely that genetic modifiers, rather than changes in the “flow” allele, exist that accentuate or limit expression. It is possible that vacillation in the action of the “flow” allele due to “leakiness” (HAYES 1964, p. 294) results in a “ f l o ~ ” pattern diversity ranging from “near-dense’’ to colorless. It will be shown later that this allele is readily affected by a wide assortment of E n states including En’.

(b). The composition of the alm(fzOlo) allele: Since the factor(s) primarily governing the “flow” expression resides at the a, locus, tests were made to deter- mine the identity of the units at the locus. In the E n system it is hypothesized (PETERSON 1960, 1961 ) that a mutable allele consists of three components: (1 ) the dominant structural gene (in this case A , ) whose action is suppressed by (2) a controlling element, the inhibitor ( I ) , which is itself regulated by (3) the regulatory element, Enhancer ( E n ) (PETERSON 1965a). The presence of the I component is tested by crossing with an En-line. The kernels from such a cross, a,”(f zozo)Sh/a,dtsh x aIdtsh/aldtsh, En/-, show a very late and frequent type of mutability that is distributed evenly throughout the kernel, including the crown portions. In a similar cross of aIm(fzow) x En’ line, utilizing an En’ which results in only a few dots when crossed to the E n tester, the resulting kernels exhibit a high frequency of late mutations evenly distributed throughout the kernel. The test with En’ indicates that the “flow” allele is easily “tripped” and the I component must be very “active.” On the basis of these tests, it is concluded that the I component of aIm( f low) is Z v . f . h . (v.f. = very fine, due to very late muta- tions, and h = high in frequency).

The En component of the alm(f hetero- zygote, alm(f‘ozo)Sh/aldtsh, to the E n tester, alm(r). Non-shrunken kernels from such a cross show the typical “flow” pattern with the clear crown area. Testcrosses of these heterozygotes, a lmf f zozo)/alm(r) , result in segregation of “flow” and colorless types which confirms the genotype and indicates that the E n component of the “flow” allele does not noticeably affect the a,“(‘) allele.

These tests indicate that the “flow” pattern of the a lm( f zow) allele is due to the restricted activity of the regulatory element, En, which is, therefore, designated E n ( f low) . The a lm( f zow) allele may be hypothetically depicted as AIZv.f.h.En(f (Table 1 ) .

(c) . Transposition of En( f zow) : As discussed in a previous section, sib shrunken kernels from the crosses in Table 2A were grown and tested for En. This cross is as follows: aldtsh/aldtsh X alm(r)Sh/alm(r)Sh. In four of the 115 tests of shrunken kernels, mutable non-shrunken (Sh) kernels appear in numbers that indicate the presence of an independent En. The dots are infrequent and represent the effect

allele is tested by crossing the

25 8 P. A. PETERSON

of a very weak En’ on a standard alm(“j allele. Since an independent En was not present in the original almffZow) hybrids, these four ears suggest the origin of an independent En’ by transposition from the “flow” allele. These observations show that the En(fzowj effect on a standard almfr) allele (standard in that a fine- high pattern of mutability results when combined with En) produces a very weak mutability response. The detection of the “flow” pattern is made possible by the interaction of the very active Z v . f . h . component with Enrf l ow) . The pattern produced by En’ combined with standard would not readily be detected since only a few dots would be scattered on the kernels.

The above test does not identify transpositions into the long arm of chromosome 3 near the a, locus which are known to occur preferentially with the mutations of “dense” alleles to almlnr) (PETERSON 1 9 6 5 ~ ) .

(d). The effect of En on almffzow): In section (b) above, the cross of heterozy- gous Y~ow” kernels by an En line (alm(fzow)Sh/aldtsh x alatsh/aldtsh, En/-) results in the appearance of “dense” kernels. From testcrosses of 39 plants coming from these “dense” kernels, four ears did not have the expected “flow” kernels. They may be considered to represent permanent changes to “dense” (Table 2D, crosses I to 4). Eighteen nonvariegated, non-shrunken derivatives from the prog- eny of these crosses were then crossed either by an En-tester (12/18) or by an En line (6/18). The En-tester cross showed that each of these kernels was hetero- zygous for En and, in the cross with the En-line none of the kernels responded to the presence of En. This is evidence that these nonvariegated kernels represent changes from a “dense” allele to almfnr) originating from an En effect on alm(flozu). It has previously been shown that the original ‘‘dense” allele changes to almlnT) at a high frequency (PETERSON 1961). En caused a change in state of the allele from “flow” to “dense” and the latter, in turn, mutates to alm(nr) as is character- istic of “dense” alleles.

The “crown” pattern. Factors gouerning the inheritance of “crown”: Progeny of the original selfs and testcrosses (Table 3A, crosses 1 4 ) were testcrossed in order to determine the inheritance of “crown” pattern. Only the “crown” pheno- type appeared among the variegated progeny (Table 3B, crosses 1 - 4 ) . In order to determine the basis of the non-shrunken, nonvariegated class among the prog- eny in Table 3A, 15 nonvariegated types were crossed by an En-line (Table 3C). All the nonvariegated segregants responded to En which indicates that they are almfr) types. The phenotypic pattern resulting from this cross is a fine (late), high frequency mutable type with dots distributed evenly throughout the kernel. It may be concluded that the colorless segregants are a,”(‘) types characterized by a fine, high-frequency mutability.

The above results imply that an independently segregating En affects the almfr) allele. This was tested by crossing the shrunken sibs from cross 4 in Table 3A by an En tester (Table 3D). The resulting mutability in 22 of the 524 shrunken kernel progenies tested indicates the presence of an independently segregating En. The pattern type is late and concentrated around the crown (Figure 7) but is not as striking as the original “C~OWTI” (Figure 5 ) . This indicates that the “crown” phenotype is primarily dependent on a segregating regulatory element

PHASE VARIATION I N MAIZE 259

TABLE 3

Segregations in “crown” selfs and testcrosses and varied tests on the components of “crown”

Crosses

Nonvariegated Variegated

non-shrunken shrunken non-shrunken shrunken

A. The segregation of “crown” in selfs and testcrosses of “crown” kernels a, nl f 7 ) Sh/a, m f r)Sh, En f crown) /-

1 34 0 155 0 2 44 0 91 0

3. En(crown)/- in heterozygous

4. En(crown)/En(crolon) in heterozygous

a,n’(r)Sh/a,dtsh x a,dtsh/a,dfsh

(for a,) parent 86 159 82 0

(for a , ) parent 8 3 136 3

1. En(crown)/Enfcrotun) 15 0 21 0 0 2 Enfcrown)/En(crolon) 5 0 132 0 3. Enfcrown)/Enfcl.olon) 12 0 156 0 4. Enfcrown)/-, EnfCrOWn)/- 42 0 86 0

B. Testcross of “crown” kerncls from ear culture A-I (assumed En constltution shown for each) aI’)l(r)Sh/a,m(r)Sh, x a,dtsh/a,dtsh

C. Nonvariegated non-shrunken segregants from A crossed by an En line a,n7(7)Sh/aIm(7)Sh x a,dtsh/a,dfsh, En/- En/-

fine, late 1. (A-I) 20 8 12.8 8 2 (A-I) 5 3 116 $ 3. (A-2) 11 8 138 3 4. (A-2) 18 8 107 3

Eleven others from A-I and A-2, not listed, gave similar results. D. Shrunken sibs from progeny of cross 4 in A crossed by an En tester

a,“sh/a,dt sh, Enf‘rolun) x aln’fr)Sh/alm(r) Sh 24 ear cultures In 22 of the 24, a fine, late type of mutability

resulted, though not as concentrated in the crown of the kernel as the original “crown”.

1 Shmnhen not counted.

whose activity is limited to the crown region of the kernel. It is designated E n f c r o w n ) . The lack of response in two of the 24 cultures arising from a putative homozygous En parent (cross 4, Table 3A) suggests that En was lost or trans- posed and was subsequently not included in the meiotic products. Losses of En have been reported in ear sectors (PETERSON 1961). This loss probably also explains the appearance of the colorless segregants in crosses 1, 2, 3 and 4 in Table 3B. The following hypothetical representation is suggested for the “crown” phenotype: AIZ(u.f.hJ plus an independent En(croton) (Table 1 ) .

The interaction of En(Crotun) and En(f tow): The progeny from the cross of “crown” and Y~OW’’ plants included non-shrunken kernels which were pheno- typically “dense,” “crown,” “flow,” and colorless (Table 4A). Ratios were not obtained since the variation in expression tends to obscure precise classification (i.e. a very heavy “flow” could be mistaken for “dense”). Distinct “dense” and “CI -OW~’~ types were selected and testcrossed (Table 4B). From 14 crosses with

260 P. A. PETERSON

TABLE 4

Thp internrtion of "flow" nnd "crou*n"

Yiirirg.iird -~

XoiivarirEatd _____ - __ (:I.# *..e. nim.~liriinLrn sltiiinhcn tinti-4iriinhrii Ariinlirn - ~~ ~ - .~ __ ~ -

A. "Crown" x "flow". nr""r'.~h/n,"'s/l. En'"r"lrnj x n l ~ ~ ~ ( l l ~ ~ r ) . ~ h / a l f ' l . ~ h "tlrnsc"+ "crown"+ "flow"

R. Trstcrowrs of progrny typrs from cross in A "crown" "crown"

1 3 5 140 2 27 I26 3- 1.1. 12 othrr progrnirs of "crown" krrnrls testrd

gave siniilar rrsiilts "tlrnsc" "tlrnse" "flow.'

1 5 0 163 6 7 XH 0 16 0 95 43 39 0 17-36 20 othrr proprnirs of "tlrnsc" kernrls trstrd;

all segregatrrl "flow"

PHASE VARIATION I N MAIZE 261

plants coming from “crown” kernels, the variegated progeny included only crown.’’ In addition. 22 “dense” kernels were selected and testcrossed and in all

of these. “flow” kernels segregated. Among the 22 “dense” kernels, the genotype of the a, and sh loci was as follows: 12 were a,ttl(/lntc)Sh/a,dfsh and ten were a,”’f~ln“~Sh/a,nlf “Sh. was phenotypically obvious in the latter group. In the former, Enfrrnsn) results in “dense” types (crosses 15 and 16, Table 4B), although in some kernels a combination of EnlrrnlrnJ with weak Enllln“) results in the clear expression of both (Figure 8). Thesz tests indicate that Enrrmtr’l’ caused the ‘‘flow*’ allele in most cases to become phenotypically “dense.” Since it has been postulated that the I component of the “flow” allele is very active ( I w . / . * . ) and that activity is concentrated at the base of the kernel while Enrrrnrm) activity is concentrated near the crown. it is not surprising that the mutability of the hybrid, “flow”/”crown.” is evident throughout the kernel. The question that must now be answered is, ‘What is the basis of the colorless area in the crown and base of the kernel resulting from the activity of the “flow” and “crown*’ alleles. respectively?’

Phnse vnriation: In previous sections, it was shown that the two phenotypes “flow” and “crown” are dependent on differences in activity of the regulatory elements. Enrlrn’r) and EnfCrnnn) . On this basis, the “flow” phenotype could be interpreted as indicating that En(lrnlr) is active only in tissue forming the base of the kernel and is inactive or switches off in the crown tissue. The situation in Edrrn t rn’ is the reverse-i.e. En(rrn1rn) is not active until the time of development of the crown tissue of the aleurone. Such an hypothesis could be tested by utiliz-

“

262 P. A. PETERSON

ing another allele (a,"'-', MCCLINTOCK 1956. 1961) that signals the presence or absence of En action (PETERSON 1965a). The a,"' allele, which normally pro- duces colored aleurone with different levels of anthocyanin pigmentation depend- ing on the particular state of the allele, gives colorless kernels in the presence of En. In the hybrids, aln'-Jsh/a,ll'n'r'Sh, or arm-r/aln'f r), Enrrotrn . any change in the activity of En could he recognized from known responses of the allele. If En is active, the distinguishable pale-purple expression of the am'-' allele should he inhibited since En suppresses the anthocyanin coloration of this allele until a mutation event occurs (PETERSON 1965a).

In the aleurone of the hybrid. aln~-'.~h/a,n'flro'e)Sh, dotting is present in a color- less background in the basal portion. This is the expected expression of In the crown of the aleurone, dotting is absent; however, in contrast to the usual almfr'o'r) expression. the background is not colorless but pale (Figure 9A). The pale coloration in the crown of the kernel is similar to the expression of the (I,"'-' allele in the absence of En.

These observations represent a consistent sequence of events. The dotting in the basal area of alm('lolr) represents En action and is correlated with the sup- pression of the pale coloration of the al'"-' allele by En. Alternatively, the crown portions of the kernel are free of dots which indicates lack of En action. Restora- tion of the pale aleurone color produced by the a,"'-' allele is also indicative of the absence of En activity. This set of observations is consistent with the hypothesis that Enflrolr' acts only in certain portions of a developing endosperm. That En has not been lost is indicated by the pattern itself, which lacks the sharp sectoring and abruptness associated with losses. Further, testcrossing (by ard'sh/aIdfsh) of 54 plants coming from these hybrid kernels produced both a,"'-'sh and Q , " ' ( / ~ ~ ~ ' ) Sh types. They were reisolated in every case and their presence demonstrated in the hybrid.

Similar tests with hybrids of "crown" and a,"'-' lead to the same conclusion. The mutability in kernels of the hybrid, a,'"-'sh/a,"(')Sh, En(rrotrn), is concen-

PHASE VARIATION IN MAIZE 263

trated in the crown while the noncrown areas are pale colored (Figure 9B). This supports the proposition that action of En(crown) is restricted to the crown of the kernel.

Heterozygous kernels of am-lsh/al m(r)En(standurd)Sh constitution possess a color- less background indicating that standard E n is active in all of the aleurone tissue and not restricted as in the case of the “phase” alleles, and En(flOW). From these observations, it is evident that the “ f l o ~ ” and “ c ~ o w ~ ” phenotypes are dependent on E n activity in localized areas of the endosperm. En( f1ow) is “switched off” in crown tissue although operative in the remainder of the endo- sperm, while En(crown) is active in crown tissue but inactive in the basal portion of the kernel. This does not explain the mechanism of the E n effect, or the muta- tion-inducing effect responsible for the numerous dots of the C L f l ~ ~ ’ 7 and LLcrown” patterns in their restricted areas. These observations only emphasize that there is a specific time of triggering, and that this is influenced by the part of the tissue in which the regulatory element is operative. The behavior of En(crown) and En(’ low) demonstrates that an apparently homogeneous tissue such as the aleurone is actually differentiated into regions which are recognized by different phases of En activity. Such differentiation of maize aleurone tissue has previously been shown by LAMPE (1931) in a study of differential enzyme activity in different portions of the aleurone.

DISCUSSION

In the E n system, the regulatory element, En, affects the action of a structural gene, A,, by causing changes in the controlling element, I. I suppresses the action of A , and resides adjacent to it. E n affects I by changing its suppressive function and this leads to expression of the structural gene. Such an event may occur at various times and in various frequencies to produce mutable areas of diverse size and pattern. Small dots indicate a late change and may be dependent on late receptivity of the I component to E n action or on late action of the En component. By the use of the alm-l allele, it is possible to show that the restriction of mutability observed in the YIOW’’ and “ C ~ O W I I ’ ~ phenotypes (the colorless nonvariegated areas) is dependent upon phase variation of the regulatory elements that undergo a cycle of activity and inactivity. The regulatory elements, En(fLozo) and En(crown), are active in different regions of the endosperm tissue. Phase variation, however, is not necessarily confined to specific tissue. MCCLINTOCK (1958) described an Spm element with cyclical behavior that changed from active to inactive to active and then back to an inactive phase within one seemingly homogeneous mass of aleurone tissue. In MCCLINTOCK’S study an aZm-l allele, that readily signals this alternating behavior of an S p m element undergoing phase changes, was utilized. The changes in S p m activity represent a “switching on” and “switching-off” of regulatory element activity during somatic divisions. With En(crown) and E n ( f Z o w ) this “switching” of activity occurs in specific areas of the aleurone possibly dis- tinguished by different times of development. LAMPE (1931) has shown that the crown area is differentiated from other areas of the endosperm in time of develop- ment.

264 P. A. PETERSON

The phenotypic expression of the state of a controlling or regulatory element is based on a distinct pattern of mutability. It results from the differences in the receptivity of the I element to En action or to differences in the timing of En action. In hybrids of and Enrstandard), alm-*sh/alm(r)Sh, En(standard), the lack of pale coloration in the background indicates that E n is always “switched on”. Further, the distribution of dots throughout the aleurone tissue supports this contention. With the phase variation En(Cr0Wn) and En( fZow) alleles, En is not al- ways “switched on” and is restricted in action. Phase variation of regulatory elements may represent changes in state of these elements.

A precocious “switching off” of gene activity, namely enzyme production in maize endosperm, has been described by SCHWARTZ (1962) for two alleles of the E, locus which specifies the pH 7.5 esterase enzyme. Though the enzyme is found in the endosperm in the presence of most of the E alleles for a period longer than 19 days following pollination, the EF’ and ES’ alleles cause a precocious cessation of gene action in the developing endosperm at approximately 16 days. These two alleles represent a class of mutants differing in the time of gene activity.

An additional case of localized differences in expression of gene activity is found at the R locus. WEATHERWAX (1923) described an R-Navajo (R”i) allele that was selected by South American Indians. This allele is characterized by a stable full coloration that is restricted to the crown of the kernel, as is the pheno- type associated with the active phase of En(cTown).

Phase variation is also evident in microorganisms. Two examples, one in Sal- monella and one in Tetrahymena will be discussed. LEDERBERG and IINO (1956) investigated the genetic and functional relationships of flagellar antigens in Sal- monella that alternate between two distinct antigenic structures (compare with variegated versus nonvariegated in the E n system). The alternative manifesta- tions of the two antigens represent changes in phase of a locus that is apart from the antigen producing determinants. The locus, H,, switches on and off in diphasic Salmonella and may become stabilized in one or the other phase. Though these stabilized strains may behave as if they were monophasic, they still carry the determinant of the suppressed phase since they can readily be transduced to other serotypes. Similarly in the En system in maize each of the phase variable regula- tory elements expresses a diphasic condition. When the elements are active, color of the alW1 allele is suppressed while changes occur at the almfr) allele. Alter- natively when these regulatory elements are in an inactive phase, alm-l color is expressed and mutability is not evident at the alm(r) allele. The two phases in Salmonella appear to parallel the active and inactive mutable phases described here for maize. The regulatory elements of the E n system alternate, as do the Salmonella phase alleles, between one phase and the other. It can also be pointed out that, like the regulatory elements, the H I and H , loci are not themselves the determinants of the flagellar antigens.

In studies of mating type, esterase and phosphatase iso-enzymes, and H sero- types in Tetrahymena, NANNEY (1960,1964) has reported differentiation of sub- nuclei; fixing of allelic expression occurs, with one of the alleles repressing the alternative allele. The repression is mutual and is termed “allelic repression.”

PHASE VARIATION IN MAIZE 265

With alleles regulated by En, the same result follows from a differential reaction of the two alleles, and alm(fLoza), to a single E n leading to the mutual ex- clusion of the phenotypes. Significantly, there is a specific timing to the allelic- repression associated with particular allelic combinations and this is consistent with the observations reported here with En(crown) and EnffLow). One or the other allele is expressed depending on the phase of En. In the Tetrahymena case, how- ever, no additional factor such as En is postulated.

Mutual exclusion of phenotypes has been discussed by BRINK (1962) in a review of an extensive series of observations and experiments dealing with pheno- typic changes occurring during the life cycle of higher plants. “Phase change” has been applied to the phenomenon that describes “the more or less abrupt switch in potential from a juvenile to an adult type of growth.” The pattern of control of gene action in these cases has not been elucidated.

SUMMARY

The distinctive behavior of the “flow” and ‘‘crown” phenotypes is due to phase variation of two different regulatory elements, En(fLow) and En(crown). Phase varia- tion refers to the changes in the functioning of these elements from periods of activity to periods of inactivity or vice versa. En(fLow) is active in the basal por- tions of the kernel while En(crown) is active in the crown portion. The mutability pattern of the “flow” and “crown” phenotypes is dependent on the active and inactive phases of these regulatory elements. It is hypothesized that these regula- tory elements “switch on” and “switch off” during the development of the endo- sperm.-Several parallels are drawn between the changes observed here and changes in gene activity involving other mutants in maize as well as examples in microorganisms.

LITERATURE CITED

BRINK, R. A., 1954 Very light variegated pericarp in maize. Genetics 39: 724-740. __ Phase change in higher plants and somatic cell heredity. Quart. Rev. Biol. 37: 1-22.

BRINK, R. A., and R. A. NILAN, 1952 The relation between light variegated and medium varie-

DAWSON, G. W. L., and P. F. SMITH-KFARY, 1963 Episomic control of mutation in Salmonella typhimurium. Heredity 18: 14%.

GREENBLATT, I. M., and R. A. BRINK, 1962 Twin mutations in medium variegated pericarp maize. Genetics 47 : 489-501.

HAYES, W., 1964 The Genetics of Bacteria and their Viruses. Blackwell Scientific Publications, Oxford.

LAMPE, L., 1931 A microchemical and morphological study of the developing endosperm of

LEDERBERG, J., and T. IINO, 1956 Phase variation in Salmonella. Genetics 41: 743-757. MCCLINTOCK, B., 1947

1962

gated pericarp in maize. Genetics 37: 519-544.

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Cytogenetic studies of maize and Neurospora. Carnegie Inst. Wash. Year- book 46: 146-152. - 1951 Chromosome organization and gen-tic expression. Cold Spring Harbor Symp. Quant. Biol 96: 13-47. - 1956 Intranuclear systems control- ling gene action and mutation. Brookhaven Symp. Biol. 8 : 58-74. - 1957 Genetic

266 P. A. PETERSON

and cytological studies of maize. Carnegie Inst. Wash. Yearbook 56: 393401. 1958 The suppressor-mutator system of control of gene action in maize. Carnegie Inst. Washington Yearbook 57: 415-429. - 1961 Some parallels between gene control systems in maize and in bacteria. Am. Naturalist 95: 265-277.

Microbiology, developmental genetics and evolution. Am. Naturalist 94: 167-179. - 1964 Macronuclear differentiation and subnuclear assortment in Ciliates. Symp. Soc. Study Develop. Growth 23: 253-273.

NEUFFER, M. G., 1963 Transposition of mutability among components of a compound allele a t the A , locus in maize. (Abstr.) Proc. 1 Ith Intern. Congr. Genetics 1 : 44-45.

NUFFER, M. G., 1961 Mutation studies at the A locus in maize, I. A mutable allele controlled by Dt. Genetics 46: 625-640.

PETERSON, P. A., 1956 An a, mutable arising in pgm stocks. Maize Genet. Coop. News Letter 30: 82. - 1960 The pale green mutable system in maize. Genetics 45: 115-133. - 1961 Mutable a, of the En system in maize. Genetics 46: 75%771. - 1965a A rela- lationship between the Spm and En control systems in maize. Am. Naturalist 99: 391-398. __ 1965b. Phase variation of controlling elements in maize. Genetics 52: 466. - 1965c Changes at the alm(p and p ) allele: The status of En. Maize Genet. Coop. News Letter 39: 102-103.

RHOADES, M. M., 1938 Effect of the Dt gene on the mutability of the a, alleles in maize. Genetics 23: 377-395. - 1941 The genic control of mutability in maize. Cold Spring Harbor Symp. Quant. Biol. 9 : 138-144.

SCIIWARTZ, D., 1962 Genetic studies on mutant enzymes in maize. 111. Control of gene action in the synthesis of pH 7.5 esterase. Genetics 47: 1609-1615.

WEATHERWAX, P., 1923 The Story of the Maize Plant. University of Chicago Press, Chicago,

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NANNEY, D. L., 1960

Illinios.